WO2020210392A1 - Trispecific binding proteins, methods, and uses thereof - Google Patents

Trispecific binding proteins, methods, and uses thereof Download PDF

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Publication number
WO2020210392A1
WO2020210392A1 PCT/US2020/027320 US2020027320W WO2020210392A1 WO 2020210392 A1 WO2020210392 A1 WO 2020210392A1 US 2020027320 W US2020027320 W US 2020027320W WO 2020210392 A1 WO2020210392 A1 WO 2020210392A1
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amino acid
seq
acid sequence
sequence
cdr
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PCT/US2020/027320
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French (fr)
Inventor
Zhi-Yong Yang
Joerg Birkenfeld
Gary J. Nabel
Huawei Qiu
Joerg Regula
Edward Seung
Ronnie WEI
Lan Wu
Zhen XING
Ling Xu
Catherine Prades
Tarik Dabdoubi
Béatrice Cameron
Cendrine Lemoine
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Sanofi
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Priority to MX2021012386A priority Critical patent/MX2021012386A/en
Priority to KR1020217036094A priority patent/KR20210149141A/en
Priority to AU2020272839A priority patent/AU2020272839A1/en
Priority to EP20722422.1A priority patent/EP3953388A1/en
Priority to CA3136821A priority patent/CA3136821A1/en
Priority to BR112021019915A priority patent/BR112021019915A2/en
Application filed by Sanofi filed Critical Sanofi
Priority to CN202080041550.1A priority patent/CN113950484A/en
Priority to JP2021559759A priority patent/JP2022527994A/en
Priority to SG11202111012QA priority patent/SG11202111012QA/en
Publication of WO2020210392A1 publication Critical patent/WO2020210392A1/en
Priority to IL286929A priority patent/IL286929A/en
Priority to CONC2021/0014918A priority patent/CO2021014918A2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the disclosure relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation.
  • the disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins.
  • Monoclonal antibody based biotherapeutics have become an important avenue for new drug development.
  • Monoclonal antibody technology offers specific targeting, precise signaling delivery and/or payload to specific cell population, and provides long lasting biological effect through its Fc functions.
  • Efforts in antibody engineering have allowed developing bispecific antibodies combining the specificities of two monoclonal antibodies for various biological applications, expanding the scope of antibody drug development.
  • Newly discovered neutralizing antibodies with improved breadth and potency may provide more options for developing biotherapeutics to treat complexed diseases such as cancer, arthritis, and/or inflammatory disorders.
  • Immuno-oncology is a promising, emerging therapeutic approach to disease management in cancer.
  • the immune system is the first line of defense against cancer development and progression.
  • T cells are able to control tumor growth and prolong the survival of cancer patients in both early and late stages of disease.
  • T cells specific for tumors can be limited in a number of ways preventing them from controlling the disease.
  • T cell immunity plays crucial role in controlling viral infection and cancer, possibly eliminating infected cells and malignant cells which result in clearance of viral infection or cure of cancer.
  • chronic infectious diseases such as Herpes viral infection (HSV, CMV, EBV, etc.), HIV, and HBV, viruses establish their persistence in humans by various mechanisms including immune
  • viral infection generally induces viral antigen specific immunity including antigen specific CD8 T cells that can readily recognize infected cells for controlling or killing through cytokine release or cytotoxic T cell (CTL) mediated killing processes.
  • CTL cytotoxic T cell
  • viral antigen specific T cell activation and/or amplification in vivo and/or ex vivo may provide therapeutic strategies against chronic viral infections.
  • trispecific binding proteins e.g ., antibodies
  • These binding proteins can specifically bind one, two, or three antigen targets or target proteins, such as CD28, CD3, and a tumor target protein.
  • Some tumors express specific antigens.
  • HER2 amplification and overexpression can be found in molecular subtypes of breast cancer, and also in gastric, ovarian, lung and prostate carcinomas.
  • Optimal activation of T cells requires two factors: 1. Antigen recognition and 2. Co-stimulation. Using the trispecific
  • HER2/CD28xCD3 trispecific binding proteins described herein Signal 1 is provided by an agonist anti-CD3 binding site, and Signal 2 is provided by an agonist anti-CD28 binding site.
  • the trispecific binding protein recruits T cells to the tumor via HER2, CD38, or a binding site recognizing another tumor target protein and activates the engaged T cells via anti-CD3 and -CD28. The resulting activation induces the killing potential of the immune cells against the nearby tumor cells.
  • anti-CD3 binding sites are described with high affinity binding to human CD3 polypeptides and potential manufacturing liabilities (e.g ., deamidation sites) removed.
  • anti-CD38/CD28xCD3 trispecific antibodies that were developed and evaluated for their potential in activating T cells, and subsequent proliferation and/or amplification of antigen specific T cells.
  • These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations, including antigen specific CD8 T central memory and effector memory cells in vitro.
  • CD4 and CD8 effector and memory populations including antigen specific CD8 T central memory and effector memory cells in vitro.
  • CD8 T central memory and effector memory cells were demonstrated.
  • the anti-CD38/CD28xCD3 trispecific antibodies described herein exhibited novel properties by engaging CD3/CD28/CD38, providing signaling pathways to stimulate and expand T cells, which may offer an effective strategy treating chronic infectious diseases such as HSV, CMV, EBV, HIV-1, and HBV infections.
  • binding proteins that bind CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins with at least one antigen binding site that binds a CD38 polypeptide.
  • these binding proteins have the ability to recruit T cells to the proximity of cancer cells, subsequently to activate T cells and promote the activated T cells killing of adjacent cancer cells through a Granzyme/Perforin mechanism, providing a different mode of action for anti -tumor activity from anti-CD38 antibodies such as DARZALEX® (daratumumab).
  • DARZALEX® daratumumab
  • the ability to bind both human and cynomolgus monkey CD38 polypeptides allows binding proteins to be readily tested in preclinical toxicological studies, e.g., to evaluate their safety profiles for later clinical use.
  • binding proteins comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and Cm domains; and L 1 , L 2 , L3 and L4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • V H1 and V L1 form a first antigen binding site
  • Vm and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of
  • QSLVHX1NX 2 X3TY wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and
  • VH 3 and VL 3 form a third antigen binding site.
  • the first binding site binds a CD28 polypeptide.
  • the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the V L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L 2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 54).
  • the Vm domain comprises the amino acid sequence of
  • V L1 domain comprises the amino acid sequence of
  • the CDR-L1 sequence of the V L2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62).
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain
  • a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).
  • the VH 2 domain comprises the amino acid sequence of
  • V L2 domain comprises an amino acid sequence selected from the group consisting of
  • DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK
  • the VH 2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD K SN S Y AT Y Y AD S VKGRFTI SRDD SKNTL YLQMN SLRAEDT A V Y Y CRGV Y Y AL SPF DYWGQGTLVTVSS (SEQ ID NO:93) or
  • V L2 domain comprises an amino acid sequence selected from the group consisting of
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:302, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the third antigen binding site binds a tumor target protein.
  • the tumor target protein is a CD38 polypeptide (e.g ., a human CD38 polypeptide).
  • the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide).
  • a tumor target protein of the present disclosure includes, without limitation, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL 2 (also known as MCP-1), CCL3 (also known as MIP-la), CCL4 (also known as MIP-lb), CCL5 (also known as RANTES),
  • CCL7 also known as MCP-3
  • CCL8 also known as mcp-2
  • CCL11 also known as eotaxin
  • CCL15 also known as MIP-ld
  • CCL17 also known as TARC
  • CCL19 also known as MIP-3b
  • CCL 2 0 also known as MIP-3a
  • CCL 2 1 also known as MIP-2
  • CCL 2 4 also known as MPIF-2/eotaxin-2
  • CCL 2 5 also known as TECK
  • CCL 2 6 also known as eotaxin-3
  • CD20 also known as FCER2
  • CD24 CD27, CD28, CD38, CD39, CD40
  • CD70, CD80 also known as B7-1
  • CD86 also known as B7-2
  • CD122 CD137
  • CD137L also known as 41BB
  • CD137L CD137L
  • CD 152 also known as CTLA4
  • CD 154 also known as CD40L
  • CD 160 CD272, CD273 (also known as PDL 2 ), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD- 1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR,
  • one or more of the above antigen targets are human antigen targets.
  • the third antigen binding site binds a human CD38 polypeptide.
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L 2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18).
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21), and the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L 2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24).
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27), and the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30).
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of
  • ARRFEGF YY SMD Y (SEQ ID NO:33), and the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36).
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39), and the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L 2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42).
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45), and the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L 2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48).
  • the VH3 domain comprises the amino acid sequence of
  • VL 3 domain comprises the amino acid sequence of
  • DIVLTQ SP ATLSL SPGERATISCRASQ S V S S YGQGFMHW YQQKPGQPPRLLI Y GAS S RAT GIP ARF S GS GS GTDFTLTI SPLEPEDF A V Y Y C QQNKEDPWTF GGGTKLEIK
  • the VH3 domain comprises the amino acid sequence of
  • VQL VQSGAEVKKPGAS VKVSCKVSGYTLTEF SIHWVRQ APGQGLEWMGGFDPE
  • DGETIY AQKF QGRVIMTEDT STDT AYMEMN SLRSEDT AIYYCTT GRFFDWF W GQG TLVTVSS (SEQ ID NO:81)
  • the VL 3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIP VRF SGSGSGTDF SLTIS SLQPEDC AVYYCQQDSNLPITF GQGTRLEIK (SEQ ID NO:82).
  • the VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPG SGSTNY AQKF QGRVTMT VDRS STT AYMEL SRLRSDDT AVYY CARY AY GYW GQG TLVTVSS (SEQ ID NO:83), and/or the VL 3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYT
  • the VH3 domain comprises the amino acid sequence of Q VQLVESGGGVVQPGRSLRLSC AASGFTF S S Y GMYWVRQAPGKGLEWVAVIWYD GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGG MDVWGQGTTVTVSS (SEQ ID NO:87), and/or the VL 3 domain comprises the amino acid sequence of
  • the VH3 domain comprises the amino acid sequence of
  • VL 3 domain comprises the amino acid sequence of
  • VL 3 domain comprises the amino acid sequence of
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.
  • the third antigen binding site binds a human HER2 polypeptide.
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), SRWGGEGFY AMDY (SEQ ID NO:7), or SRWGGSGFYAMDY (SEQ ID NO: 8), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO: 10), a CDR-L1 sequence comprising the amino acid sequence
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of
  • SRW GGDGF YAMD Y (SEQ ID NO:6), and the V L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7)
  • the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • the VH 3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8)
  • the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • the VH 3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8)
  • the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • the VH 3 domain comprises a CDR- H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR- H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), and the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of
  • the VL 3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • the VH3 domain comprises the amino acid sequence of
  • VL 3 domain comprises the amino acid sequence of
  • the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF Y AMD Y WGQGTLVTVSS (SEQ ID NO:72), and/or the VL 3 domain comprises the amino acid sequence of
  • the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:73), and/or the VL 3 domain comprises the amino acid sequence of
  • the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO:75), and/or the VL 3 domain comprises the amino acid sequence of
  • the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO:74), and/or the VL 3 domain comprises the amino acid sequence of
  • the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:76), and/or the VL 3 domain comprises the amino acid sequence of
  • the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF Y AMD Y WGQGTLVTVSS (SEQ ID NO:72), and/or the VL 3 domain comprises the amino acid sequence of
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: l 16;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.
  • At least one of Li, L 2 , L 3 or L 4 is independently 0 amino acids in length.
  • L 1 , L 2 , L 3 and L 4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71).
  • GGGGSGGGGS SEQ ID NO:69
  • GGGGSGGGGSGGGGS SEQ ID NO: 70
  • S RT, TKGPS
  • GQPKAAP SEQ ID NO: 67
  • GGSGSSGSGG SEQ ID NO: 71
  • L 1 , L 2 , L 3 and L 4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and
  • L 1 comprises the sequence GQPKAAP (SEQ ID NO: 67)
  • L 2 comprises the sequence TKGPS (SEQ ID NO:68)
  • L 3 comprises the sequence S
  • L 4 comprises the sequence RT.
  • at least one of L 1 , L 2 , L 3 or L 4 comprises the sequence DKTHT (SEQ ID NO:66).
  • L 1 , L 2 , L 3 and L 4 comprise the sequence DKTHT (SEQ ID NO:66).
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein the hinge-C H2 -C H3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L 2 35A.
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein the hinge-C H2 - CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L 2 35A, and a deletion at 236.
  • the hinge-C H2 -Cro domains of the second and the third polypeptide chains are human IgG4 hinge-C H2 -Cro domains, and wherein the hinge-C H2 -C H3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgGl hinge-C H2 -C H3 domains, and wherein the hinge-C H2 -C H3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L 2 34A, L 2 35A, and P329A.
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgGl hinge-C H2 -C H3 domains, and wherein the hinge-C H2 - CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S.
  • the hinge-C H2 -Cro domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-C H2 -C H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
  • the hinge-C H2 -C H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-C H2 -C H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
  • nucleic acid molecules comprising a nucleotide sequence encoding the binding protein of any one of the above embodiments.
  • expression vectors comprising the nucleic acid molecule of any one of the above embodiments.
  • isolated host cells comprising the nucleic acid molecule of any one of the above embodiments or the expression vector of any one of the above embodiments.
  • the host cell is a mammalian or insect cell.
  • compositions comprising the binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier.
  • provided herein are methods of preventing and/or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein or pharmaceutical composition of any one of the above embodiments.
  • a binding protein or pharmaceutical composition according to any one of the above embodiments for use in a method of preventing and/or treating cancer in a patient wherein the method comprises administering to the patient a therapeutically effective amount of the binding protein or pharmaceutical composition.
  • the at least one binding protein is co-administered with a chemotherapeutic agent.
  • the patient is a human.
  • the third antigen binding site binds a human CD38 polypeptide, and wherein cancer cells from the individual or patient express CD38.
  • the cancer is multiple myeloma.
  • the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma.
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • B cell lymphoma prior to administration of the binding protein, the patient has been treated with daratumumab without a wash-out period.
  • the third antigen binding site binds a human HER2 polypeptide, and wherein cancer cells from the individual or patient express HER2.
  • the cancer is breast cancer, colorectal cancer, gastric cancer, or non small cell lung cancer (NSCLC).
  • a method for expanding virus-specific memory T cells comprising contacting a virus-specific memory T cell with a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H3 domains; and L 1 , L 2 , L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • V H1 and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the VH 2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX 2 X3TY, wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and
  • a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH 3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH 3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and L 1 , L 2 , L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • V H1 and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the VH 2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX 2 X3TY, wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and
  • the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo. In some embodiments, contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus- specific memory T cells.
  • a method for expanding T cells comprising contacting a T cell with a binding protein in vitro or ex vivo , wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH 3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH3 is an immunoglobulin CH3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and L 1 , L 2 , L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • Vm and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the VH 2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX 2 X3TY, wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a
  • a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • Vm is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH 3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and L 1 , L 2 , L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • Vm and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the VH 2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX 2 X3TY, wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a
  • the T cell is a memory T cell or an effector T cell.
  • the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.
  • CAR chimeric antigen receptor
  • a method for treating chronic viral infection comprising administering to an individual or patient in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH 3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and L 1 , L 2 , L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • V H1 and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • theVH 2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX 2 X3TY, wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and
  • a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • VH 3 -C H1 -hinge-C H2 -C H3 [III] and a fourth polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH 3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH 3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and L 1 , L 2 , L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • V H1 and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the VH 2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX 1 NX 2 X 3 TY, wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and
  • the individual or patient is a human. In some embodiments, the individual or patient is a human.
  • the binding protein is administered to the individual or patient in
  • composition comprising the binding protein and a pharmaceutically acceptable carrier.
  • administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual or patient.
  • the memory T cells are CD8+ or CD4+ memory T cells.
  • the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).
  • the virus is a human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein- barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 1 (HSV-2).
  • HAV human immunodeficiency virus
  • influenza virus influenza virus
  • CMV cytomegalovirus
  • HBV hepatitis B virus
  • HPV human papillomavirus
  • HPV human papillomavirus
  • EBV Epstein- barr virus
  • HMV human foamy virus
  • HSV-1 herpes simplex virus 1
  • HSV-2 herpes simplex virus 1
  • the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.
  • a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein of any one of the above embodiments.
  • the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.
  • kits comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above
  • kits further comprise instructions for using the polypeptide chain or binding protein according to any of the methods or uses described herein, e.g. , supra.
  • kits comprising one, two, three, or four polynucleotides according to any one of the above embodiments.
  • kits of polynucleotides comprising one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189
  • FIG. 1A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and HER2.
  • a first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes HER2.
  • the trispecific binding protein shown in FIG. 1A uses a constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.
  • FIG. IB provides the fold change (vs. parental) in binding affinities of anti- CD28/CD3/HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3, and anti-CD28 binding domains.
  • Mutations 3233QQ to QEQ top to bottom refer to mutations introduced into residues 32-35 of the VL domain of the anti-CD3 binding site (indicated by *); the remaining mutations were introduced into the VH or VL domain of the trastuzumab anti-HER2 binding site (indicated by #; numbering according to Kabat).
  • mutation 30Q was introduced into the VL domain, and the remaining mutations were introduced into the VH domain.
  • the binding affinities were measured by ELISA, and the values provided are relative to parental trispecific antibody.
  • FIG. 1C provides binding curves for the indicated trispecific antibodies binding to human HER2, human CD28, and CD3, as determined by ELISA.
  • FIG. ID provides a proposed mechanism of action for HER2/CD28xCD3 trispecific antibody-mediated T cell activation and HER2+ cancer cell killing.
  • FIG. 2A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and CD38.
  • a first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes CD38.
  • the trispecific binding protein shown in FIG. 2A uses an IgG4 constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.
  • FIGS. 2B-2E show the binding affinities, as measured by ELISA, of
  • FIG. 3 shows SPR competition assays for binding to CD38 by Daratumumab and anti-CD38 monospecific antibodies with the indicated anti-CD38 binding domains. If an antibody recognized an epitope on CD38 which was different from that of
  • FIGS. 4A-4B show the in vitro cell killing activity of CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human multiple myeloma NCI-H929 cells (CD38+/CD28+).
  • the assays were carried out in the presence of 5 nM isotype control antibody (FIG. 4A) or Daratumumab (FIG. 4B). In the presence of daratumumab, the trispecific antibodies continued to exhibit cell killing activity.
  • FIGS. 5A-5B show the in vitro cell killing activity of CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human lymphoma OCI-Lyl9 cells (CD38+/CD28-).
  • the assays were carried out in the presence of 5 nM isotype control antibody (FIG. 5A) or
  • Daratumumab (FIG. 5B). Daratumumab caused a decrease in the cell killing activity of anti-CD38/CD28xCD3 trispecific antibodies.
  • FIGS. 6A-6J show the characterization of in vitro T cell subset expansion in
  • a trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA).
  • T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated
  • FIGS. 7A-7J show the characterization of in vitro T cell subset expansion in
  • CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains.
  • a trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA).
  • Antibodies shown in legend from top to bottom are shown in the graphs from left to right.
  • T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7).
  • the indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM, 1 nM, and 2 nM.
  • Flow cytometry was used to quantify CMV-specific CD8+ T cells (FIGS. 7A-7B), CMV-specific T Cm CD8+ cells (FIGS. 7C-7D), and CMV-specific Tem CD8+ cells (FIGS. 7E-7F).
  • the percentages of CMV-specific Tcm (FIGS. 7G- 7H) and Tem (FIGS. 7I-7J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in dose response manner.
  • FIGS. 8A-8J show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected Donor C in response to CD38/CD28sup x
  • a trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA).
  • T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated
  • EBV-specific CD8+ T cells FIGS. 8A-8B
  • CMV-specific T cm CD8+ cells FIGS. 8C-8D
  • CMV-specific Tem CD8+ cells FIGS. 8E-8F
  • the percentages of EBV-specific T cm FIGS. 8G-8H
  • T em FIGS. 8I-8J
  • FIGS. 9A-12 show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected Donor D in response to CD38/CD28sup x
  • a trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VHl/(DCD28sup x (DCD3mid IgG4 FALA).
  • T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated
  • EBV-specific CD8+ T cells FIGS. 9A-9B
  • EBV-specific T cm CD8+ cells FIGS. 9C-9D
  • EBV-specific Tem CD8+ cells FIGS. 9E-9F
  • the percentages of EBV-specific T cm FIGS. 9G-10
  • T em FIGS. 11-12
  • FIGS. 13A-13D show the change over time (days) in tumor volume (FIG. 13A) and body weight (FIG. 13B) in ZR-75-1 tumor bearing NSG mice engrafted with in vitro expanded human CD3+ T cells.
  • Groups of 10 mice were either treated with vehicle or Her2/CD28 x CD3 trispecific antibody at the indicated dosages.
  • Arrow heads indicate days of administration.
  • Tumor volume is depicted as mean ⁇ SEM, mm 3 .
  • Body weight change is depicted as % change, mean ⁇ SEM.
  • X-axis shows days after implantation with ZR-75-1 cells.
  • Tumor volume (mm 3 ) over time for individual mice in each treatment group are shown in FIG. 13C.
  • Tumor weight (mg) for each treatment group is shown in FIG. 13D.
  • FIGS. 14A-14C show the effect of Her2/CD28 x CD3 trispecific antibody treatment on T cells from whole blood.
  • FIG. 14A shows the analysis of hCD45+, CD8+, CD4+, and mCD45+ cells by flow cytometry.
  • FIG. 14B shows the effect of control or Her2/CD28 x CD3 trispecific antibody treatment (at the indicated doses) on hCD45+, CD8+, CD4+, and mCD45+ cell counts.
  • FIG. 14C shows the effect of control or
  • Her2/CD28 x CD3 trispecific antibody treatment (at the indicated doses) on human cell ratios (CD4+/CD45+ and CD8+/CD45+).
  • conditions are (left to right): control, lOOug/kg trispecific antibody, lOug/kg trispecific antibody, lug/kg trispecific antibody, and 0. lug/kg trispecific antibody.
  • Percentages shown in FIGS. 14B & 14C are based on control sample vs. lOOug/kg.
  • FIGS. 15A-15C show the effect of Her2/CD28 x CD3 trispecific antibody treatment on tumor infiltrating lymphocytes (TILs), as examined by immunohistochemistry (IHC). Arrows indicate tumor infiltrating T cells identified in ZR-75-1 breast tumors.
  • Tipper images are at IX magnification; lower images are at 20X magnification.
  • staining for human CD45, human CD4, and human CD8 are shown from left to right. Shown are tumors from mice treated with vehicle control (FIG. 15A), lOOug/kg trispecific antibody (FIG. 15B), or 0. lug/kg trispecific antibody (FIG. 15C).
  • FIGS. 17A-17F show in vitro cell lysis of HER2+ breast cancer target cells in the presence of human CD8+ T cells by Her2/CD28 x CD3 trispecific antibody with wild- type trastuzumab antigen binding domain and an anti-CD3 antigen binding domain without without 32/35 QQ mutations in the VL domain (“ctl”) as compared to a Her2/CD28 x CD3 trispecific antibodies having mutations in the anti-HER2 arm and the VL domain of the anti-CD3 arm (numbering as shown in Table 1).
  • Cell killing activities against cell lines with varying expression of HER2 are depicted: HCC1954 for high HER2 expression (FIG. 17A), BT20 for intermediate HER2 expression (FIG.
  • FIG. 17C Graphs depicting cell killing as a function of antibody concentration against target cells HCC1954 (FIG. 17B), BT20 (FIG. 17D), and MDA-MD- 231 (FIG. 17F) are shown, comparing binding protein #2 vs. ctl or binding protein #1 and #5 vs. ctl.
  • FIGS. 18A & 18B summarize the mean EC50 (pM) of in vitro cell killing by experimental or control Her2/CD28 x CD3 trispecific antibodies against the indicated breast cancer (FIG. 18A) or gastric cancer cell lines (FIG. 18B). Amino acid sequences of the indicated trispecific antibodies are provided in Table 1.
  • the disclosure provides trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation.
  • polynucleotide refers to single-stranded or double- stranded nucleic acid polymers of at least 10 nucleotides in length.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or
  • deoxyribonucleotides or a modified form of either type of nucleotide.
  • modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as
  • polynucleotide specifically includes single-stranded and double-stranded forms of DNA.
  • An "isolated polynucleotide” is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • An "isolated polypeptide” is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof.
  • the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • Naturally occurring antibodies typically comprise a tetramer.
  • Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light” chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • the terms "heavy chain” and “light chain” as used herein refer to any combination of polypeptide chains
  • each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • the carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function.
  • a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (C H1 , CH 2 , and C H3 ), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (C L ), wherein the VL domain is at the amino-terminus of the polypeptide and the C L domain is at the carboxyl-terminus.
  • Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4.
  • IgM has subclasses including, but not limited to, IgMl and IgM2.
  • IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2.
  • variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • the variable regions of each light/heavy chain pair typically form an antigen binding site.
  • the variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen.
  • the exact boundaries of these CDRs have been defined differently according to different systems.
  • the system described by Rabat (Rabat et al. , SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs.
  • These CDRs may be referred to as Rabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol.
  • the amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • the numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80.
  • Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.
  • Fc refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region.
  • the original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgGl and IgG2 are preferred.
  • Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent ( i.e ., disulfide bonds) and non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2).
  • class e.g., IgG, IgA, and IgE
  • subclass e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2
  • Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
  • native Fc as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • a F(ab) fragment typically includes one light chain and the VH and C H1 domains of one heavy chain, wherein the VH-C H1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide.
  • a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a C H1 domain.
  • a F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the C H1 and C H2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.
  • binding protein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen.
  • a trispecific binding protein of the present disclosure typically comprises four polypeptide chains that form at least three antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
  • a third polypeptide chain has a structure represented by the formula:
  • polypeptide chain has a structure represented by the formula:
  • VLI is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH 3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains
  • L 1 , L 2 , L 3 and L 4 are amino acid linkers
  • a "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.
  • One embodiment of the disclosure provides binding proteins having biological and immunological specificity to between one and three target antigens.
  • Another embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins.
  • Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins.
  • host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).
  • swapability refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity.
  • Full swapability refers to the ability to swap the order of both Vm and VH 2 domains, and therefore the order of V L1 and V L2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while
  • VH and VL refer only to the domain's location on a particular protein chain in the final format.
  • Vm and VH 2 could be derived from V L1 and V L2 domains in parent antibodies and placed into the V and VH 2 positions in the binding protein.
  • V L1 and V L2 could be derived from Vm and VH 2 domains in parent antibodies and placed in the Vm and VH 2 positions in the binding protein.
  • the VH and VL designations refer to the present location and not the original location in a parent antibody. VH and VL domains are therefore "swappable.”
  • antigen or "target antigen” or “antigen target” as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a target antigen may have one or more epitopes.
  • the binding protein is capable of competing with an intact antibody that recognizes the target antigen.
  • Her2 refers to human epidermal growth factor receptor 2 which is a member of the epidermal growth factor receptor family.
  • CD3 is cluster of differentiation factor 3 polypeptide and is a T-cell surface protein that is typically part of the T cell receptor (TCR) complex.
  • CD28 is cluster of differentiation 28 polypeptide and is a T-cell surface protein that provides co-stimulatory signals for T-cell activation and survival.
  • CD38 is cluster of differentiation 38 polypeptide and is a glycoprotein found on the surface of many immune cells.
  • T-cell engager refers to binding proteins directed to a host's immune system, more specifically the T cells' cytotoxic activity as well as directed to a tumor target protein.
  • the term "monospecific binding protein” refers to a binding protein that specifically binds to one antigen target.
  • the term "monovalent binding protein” refers to a binding protein that has one antigen binding site.
  • binding protein refers to a binding protein that specifically binds to two different antigen targets.
  • binding protein refers to a binding protein that has two binding sites.
  • trispecific binding protein refers to a binding protein that specifically binds to three different antigen targets.
  • trivalent binding protein refers to a binding protein that has three binding sites.
  • the trivalent binding protein can bind to one antigen target.
  • the trivalent binding protein can bind to two antigen targets.
  • the trivalent binding protein can bind to three antigen targets.
  • An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated binding proteins include the binding protein in situ within recombinant cells since at least one component of the binding protein's natural environment will not be present.
  • substantially pure refers to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present.
  • a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition.
  • the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • epitope includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody or binding protein.
  • a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is ⁇ 10 8 M, more preferably when the equilibrium dissociation constant is ⁇ 10 9 M, and most preferably when the dissociation constant is ⁇ 10 10 M.
  • the dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance.
  • surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ).
  • SPR surface plasmon resonance
  • Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen).
  • KD refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.
  • the term "specifically binds" as used herein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 10 6 M, 1 x 10 7 M, 1 x 10 8 M, 1 x 10 9 M, 1 x 10 10 M, 1 x 10 11 M, 1 x 10 12 M, or more, and/or to bind to an epitope with an affinity that is at least two fold greater than its affinity for a nonspecific antigen.
  • an antigen binding domain and/or binding protein of the present disclosure “cross reacts” with human and cynomolgus monkey CD38 polypeptides, e.g., CD38 extracellular domains, human CD38 isoform A, human CD38 isoform E, and cynomolgus monkey CD38.
  • a binding protein binding to antigen 1 (Agl) is“cross-reactive” to antigen 2 (Ag2) when the ECSOS are in a similar range for both antigens.
  • a binding protein binding to Agl is cross-reactive to Ag2 when the ratio of affinity of Ag2 to affinity of Agl is equal or less than 20, affinities being measured with the same method for both antigens.
  • linker refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins.
  • a linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level.
  • the transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood.
  • the precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction.
  • the linkers described herein are referred to as L 1 , which is located on the light chain between the C-terminus of the V L2 and the N-terminus of the V L1 domain; and L 2 , which is located on the light chain between the C-terminus of the V L1 and the N-terminus of the C L domain.
  • the heavy chain linkers are known as L 3 , which is located between the C- terminus of the Vm and the N-terminus of the VH 2 domain; and L 4 , which is located between the C-terminus of the VH 2 and the N-terminus of the C H1 domain.
  • vector refers to any molecule (e.g, nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell.
  • vector includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double- stranded DNA molecule into which additional DNA segments may be inserted.
  • viral vector Another type of vector, wherein additional DNA segments may be inserted into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” may be used interchangeably herein, as a plasmid is the most commonly used form of vector.
  • the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g ., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g ., replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • recombinant host cell refers to a cell into which a recombinant expression vector has been introduced.
  • a recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term "host cell” as used herein.
  • host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems).
  • a suitable bacterial expression vector is pUC19.
  • a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA.
  • a cell is transformed where it is genetically modified from its native state.
  • the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid.
  • a cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.
  • naturally occurring as used herein and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring.
  • non-naturally occurring refers to an object that is not found in nature or that has been structurally modified or synthesized by man.
  • the twenty conventional amino acids and their abbreviations follow conventional usage.
  • Stereoisomers e.g ., D-amino acids
  • unnatural amino acids and analogs such as a-, a-di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins.
  • Examples of unconventional amino acids include: 4-hydroxyproline, g-carboxyglutamate, e-N,N,N-trimethyllysine, e-N- acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.
  • Naturally occurring residues may be divided into classes based on common side chain properties:
  • Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • a skilled artisan will be able to determine suitable variants of the polypeptide chains of the binding proteins using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • patient includes human and animal subjects.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment include those having a disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.
  • binding proteins can be used to treat humans with cancer, or humans susceptible to cancer, or ameliorate cancer in a human subject.
  • the binding proteins can also be used to prevent cancer in a human patient.
  • the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, germinal center B-cell lympohoma or B-cell acute lymphoblastic leukemia,
  • the binding proteins can be used to treat humans with inflammatory disorders, or humans susceptible to inflammatory disorders, or ameliorate inflammatory disorders in a human subject.
  • composition or “therapeutic composition” as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a binding protein.
  • a therapeutically effective amount when used in reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo , factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.
  • One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.
  • Certain aspects of the present disclosure relate to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation and wherein a second pair of polypeptides forming the binding protein possess a single variable domain. Any of the CDRs or variable domains of any of the antigen binding proteins described herein may find use in a trispecific binding protein of the present disclosure.
  • each of the three antigen binding sites binds a different target (e.g ., polypeptide antigen).
  • the trispecific binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • VL 3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • VH 2 is a second immunoglobulin heavy chain variable domain
  • VH3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • CH 2 is an immunoglobulin C H2 heavy chain constant domain
  • CH3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and L 1 , L 2 , L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.
  • a trispecific binding protein of the present disclosure comprises a Vm and V L1 domain pair that form a first antigen binding site, a VH 2 and V L2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH 3 and VL 3 domain pair that form a third antigen binding site.
  • a trispecific binding protein of the present disclosure comprises a Vm and V L1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH 2 and V L2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL 3 domain pair that form a third antigen binding site.
  • a trispecific binding protein of the present disclosure comprises a Vm and V L1 domain pair that form a first antigen binding site, a VH 2 and V L2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH 3 and VL 3 domain pair that form a third antigen binding site that binds a tumor target protein.
  • a trispecific binding protein of the present disclosure comprises a Vm and V L1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH 2 and V L2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH 3 and VL 3 domain pair that form a third antigen binding site that binds a tumor target protein.
  • a trispecific binding protein of the present disclosure comprises a Vm and V L1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH 2 and V L2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH 3 and VL 3 domain pair that form a third antigen binding site that binds a CD38 polypeptide.
  • a trispecific binding protein of the present disclosure comprises a Vm and V L1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH 2 and V L2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL 3 domain pair that form a third antigen binding site that binds a HER2 polypeptide.
  • a binding protein of the present disclosure binds one or more tumor target proteins and one or more T cell target proteins.
  • the binding protein is capable of specifically binding one tumor target protein and two different epitopes on a single T cell target protein.
  • the binding protein is capable of specifically binding one tumor target protein and two different T cell target proteins (e.g., CD28 and CD3).
  • the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds a tumor target protein.
  • the target protein is CD38 or HER2. Additional tumor target proteins are provided infra.
  • the one or more T cell target proteins are one or more of CD3 and CD28. Exemplary and non-limiting polypeptides that may find use in any of the trispecific binding proteins described herein are provided in Table 1.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein .the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.
  • CD38 polypeptide is a human CD38 polypeptide, also known as ADPRC1.
  • Human CD38 polypeptides are known in the art and include, without limitation, the polypeptide represented by NCBI Accession Number NP_001766.2, or a polypeptide produced from NCBI Gene ID Number 952.
  • the antigen binding site binds a human CD38 polypeptide, a non-human primate (e.g ., cynomolgus monkey) CD38 polypeptide, or a human CD38 polypeptide and a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide.
  • a binding protein comprising an antigen binding site that binds a CD38 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
  • any of the CDRs and/or variable domains of the anti-CD38 binding sites described below can be used in a monospecific antibody.
  • any of the CDRs and/or variable domains of the anti-CD38 binding sites described below can be used in any binding site of a trispecific binding protein comprising four polypeptides that form three antigen binding sites, e.g, as described supra.
  • a binding protein that comprises an antigen binding site that binds a CD38 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites as described supra , wherein the VH3 and VL 3 domains pair and form a third antigen binding site that binds a CD38 polypeptide.
  • an anti-CD38 binding site cross-reacts with human CD38 (e.g, a human CD38 isoform A and/or isoform E polypeptide) and cynomolgus monkey CD38.
  • a binding protein comprising an anti-CD38 binding site induces apoptosis of a CD38+ cell.
  • a binding protein comprising an anti-CD38 binding site recruits a T cell to a CD38+ cell and optionally activates the T cell (e.g, though TCR stimulation and/or costimulation).
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L 2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L 2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L 2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L 2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L 2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L 2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L 2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L 2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48).
  • VH antibody heavy chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:79, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:80.
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:79, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:80.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:81, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:82.
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:81, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:82.
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:83, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:84.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:83, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:84.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VQLVESGGGVVQPGRSLRLSC AASGFTF S S Y GMYWVRQAPGKGLEWVAVIWYDG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMD VWGQGTTVTVSS (SEQ ID NO:87), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:87, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:88.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:87, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:88.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • amino acid sequence comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:89, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:90.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:89, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:90.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:85, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:86.
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:85, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:86.
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:277, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:278.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:277, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:278.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the VH and/or VL domains are humanized.
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:279, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:280.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:279, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:280.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the VH and/or VL domains are humanized.
  • the binding protein is a trispecific binding protein.
  • the trispecific binding protein comprising an antigen binding site that binds a CD38 polypeptide, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide.
  • the binding protein is a trispecific binding protein comprising four
  • polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair ( e.g ., as described herein).
  • the VH and VL domains of any of the anti-CD38 antigen binding sites described above represent VH3 and VL 3 and form a third antigen binding site that binds a CD38 polypeptide.
  • Vm and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the VH and VL domains of any of the anti-CD38 antigen binding sites described above and/or in Table 2 represent VH3 and VL 3 and form a third antigen binding site that binds a CD38 polypeptide.
  • a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD38 antibody described in Table 2.
  • a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD38 antibody described in Table 2.
  • antibodies e.g ., monospecific antibodies
  • antibodies comprising any of the anti-CD38 CDRs and/or variable domains described supra.
  • a binding protein of the present disclosure comprises an antigen binding site that binds an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide.
  • an antigen binding site that binds an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide.
  • Exemplary assays for determining whether an antigen binding site binds an antigen are described herein and known in the art, including (without limitation) ELISA, SPR, and flow cytometry assays.
  • the HER2 polypeptide is a human HER2 polypeptide, also known as NEU, NGL, ERBB2, TKR1, CD340, HER-2, MLN19, and HER-2/neu.
  • Human HER2 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP_024306411.1 , XP_024306410.1 , XP_024306409.1 , NP_001276867.1, NP_001276866.1, NP_001276865.1, NP_001005862.1, or NP_004439.2, or a polypeptide produced from NCBI Gene ID Number 2064.
  • a binding protein comprising an antigen binding site that binds a HER2 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
  • a binding protein that comprises an antigen binding site that binds a HER2 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites as described supra , wherein VH3 and VL 3 domain pair that form a third antigen binding site that binds a HER2 polypeptide.
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRW GGDGF Y AMD Y (SEQ ID NO: 6), SRWGGEGFY AMDY (SEQ ID NO:7), or SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO:
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NOG), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NOG), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), SRWGGEGFY AMDY (SEQ ID NOG), or SRWGGSGFYAMDY (SEQ ID NOG); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NOG), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and a CDR-H3 sequence comprising the amino acid sequence of
  • SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VL antibody light chain variable domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L 2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
  • VH antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO: 72),
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76; and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO:78.
  • VT antibody heavy chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76; and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO:78.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds HER2 comprises: : an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:73, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:73, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:75, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:75, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO:74), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:74, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:74, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:76, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:76, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:78.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:78.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • an anti-HER2 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of anti-HER2 antibody trastuzumab,
  • an anti-HER2 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of anti-HER2 antibody trastuzumab,
  • an anti-HER2 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-HER2 antibody described in Table 3.
  • an anti-HER2 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-HER2 antibody described in Table 3.
  • a binding protein of the present disclosures comprises an antigen binding site that binds a tumor target protein.
  • the tumor target protein is a CD38 polypeptide (e.g ., a human CD38 polypeptide).
  • the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide).
  • a tumor target protein of the present disclosure includes, without limitation, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL 2 (also known as MCP-1), CCL3 (also known as MIP-la), CCL4 (also known as MIP-lb), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL1 1 (also known as eotaxin), CCL15 (also known as MIP-ld), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL 2 0 (also known as MIP-3a), CCL 2 1 (also known as MIP-2), CCL 2 4 (also known as MPIF
  • CD28 polypeptide is a human CD28 polypeptide, also known as Tp44.
  • Human CD28 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP_011510499.1 , XP_011510497.1 , XP_011510496.1 ,
  • a binding protein comprising an antigen binding site that binds a CD28 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
  • a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites.
  • a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a CD38 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a HER2 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a tumor target protein.
  • a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51) and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L 2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 54).
  • VH antibody heavy chain variable
  • a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR- H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR- H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR- H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID
  • VL antibody light chain variable domain
  • a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L 2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54).
  • a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:91, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:92.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:91, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:92.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the binding protein is a trispecific binding protein.
  • the trispecific binding protein comprising an antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2), an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide.
  • the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain- heavy chain pair ( e.g ., as described herein).
  • the VH and VL domains of any of the anti-CD28 antigen binding sites described above represent Vm and V L1 and form a first antigen binding site that binds a CD28 polypeptide.
  • the VH and VL domains of any of the anti-CD28 antigen binding sites described above and/or in Table 4 represent Vm and V L1 and form a first antigen binding site that binds a CD28 polypeptide, VH 2 and V L2 form a second antigen binding site that binds a CD3 polypeptide, and VH3 and VL 3 and form a third antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2).
  • an anti-CD28 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD28 antibody described in Table 4.
  • an anti-CD28 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD28 antibody described in Table 4.
  • CD3 polypeptide is a human CD3 polypeptide, including CD3-delta (also known as T3D, IMD19, and CD3-DELTA), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-GAMMA).
  • CD3-delta also known as T3D, IMD19, and CD3-DELTA
  • CD3-epsilon also known as T3E, IMD18, and TCRE
  • CD3-gamma also known as T3G, IMD17, and CD3-GAMMA
  • Human CD3 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP 006510029.1 or NP_031674.1, or a polypeptide produced from NCBI Gene ID Numbers 915, 916, or 917.
  • a binding protein comprising an antigen binding site that binds a CD3 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a CD38 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a HER2 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a tumor target protein.
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX 2 X3TY, wherein X 1 is E or Q, X 2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:55), a CDR
  • the CDR-L1 sequence of the V L2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and Q SL VHENLRT Y (SEQ ID NO:62).
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).
  • VH antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).
  • VH antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L 2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of
  • DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK (SEQ ID NO:95), DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96),
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.
  • VT antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.
  • VET antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • VET antibody heavy chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
  • VT antibody heavy chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 302, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:302, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the binding protein is a trispecific binding protein.
  • the trispecific binding protein comprising an antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2), an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide.
  • the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain- heavy chain pair ( e.g ., as described herein).
  • the VH and VL domains of any of the anti-CD3 antigen binding sites described above represent VH 2 and V L2 and form a second antigen binding site that binds a CD3 polypeptide.
  • Vm and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • the VH and VL domains of any of the anti-CD3 antigen binding sites described above and/or in Table 5 represent VH 2 and V L2 and form a second antigen binding site that binds a CD3 polypeptide
  • VH 3 and VL 3 form a third antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2).
  • an anti-CD3 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD3 antibody described in Table 5.
  • an anti-CD3 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD3 antibody described in Table 5.
  • the linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids long.
  • L 1 , L 2 , L 3 , and L 4 in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.
  • linkers include, for example, GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), GGSGSSGSGG (SEQ ID NO: 71), and DKTHT (SEQ ID NO:66), as well as those disclosed in International Publication Nos. WO2017/074878 and W02017/180913.
  • linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the binding proteins.
  • the identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.
  • the length of L 1 is at least twice the length of L 3 .
  • the length of L 2 is at least twice the length of L 4 .
  • the length of L 1 is at least twice the length of L 3
  • the length of L 2 is at least twice the length of L 4 .
  • L 1 is 3 to 12 amino acid residues in length
  • L 2 is 3 to 14 amino acid residues in length
  • L 3 is 1 to 8 amino acid residues in length
  • L 4 is 1 to 3 amino acid residues in length.
  • L 1 is 5 to 10 amino acid residues in length
  • L 2 is 5 to 8 amino acid residues in length
  • L 3 is 1 to 5 amino acid residues in length
  • L 4 is 1 to 2 amino acid residues in length.
  • L 1 is 7 amino acid residues in length
  • L 2 is 5 amino acid residues in length
  • L 3 is 1 amino acid residue in length
  • L 4 is 2 amino acid residues in length.
  • L 1 , L 2 , L 3 and L 4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGS GGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71).
  • L 1 , L 2 , L 3 and L 4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGS GGGGS GGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and
  • L 1 comprises the sequence GQPKAAP (SEQ ID NO: 67)
  • L 2 comprises the sequence TKGPS (SEQ ID NO:68)
  • L 3 comprises the sequence S
  • L 4 comprises the sequence RT.
  • L 1 , L 2 , L 3 or L 4 comprises the sequence DKTHT (SEQ ID NO:66). In some embodiments, L 1 , L 2 , L 3 and L 4 comprise the sequence DKTHT (SEQ ID NO: 66).
  • a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a third polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3
  • immunoglobulin heavy chain constant domains are immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fc region.
  • the Fc region is a human Fc region, e.g ., a human IgGl, IgG2, IgG3, or IgG4 Fc region.
  • the Fc region includes an antibody hinge, C H1 , C H2 , C H3 , and optionally C H3 domains.
  • the Fc region is a human IgGl Fc region.
  • the Fc region is a human IgG4 Fc region.
  • the Fc region includes one or more of the mutations described infra.
  • the Fc region is an Fc region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4.
  • the heavy chain constant region is a constant region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4.
  • the light chain constant region is a constant region of one of the light chain polypeptides (e.g, polypeptide 1 or 4) of a binding protein shown in Table 4.
  • a binding protein of the present disclosure includes one or two Fc variants.
  • Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art.
  • Fc variant can comprise a molecule or sequence that is humanized from a non-human native Fc.
  • a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention.
  • Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody- dependent cellular cytotoxicity (ADCC).
  • ADCC antibody- dependent cellular cytotoxicity
  • a binding protein of the present disclosure (e.g ., a trispecific binding protein) comprises a“knob” mutation on the second polypeptide chain and a“hole” mutation on the third polypeptide chain.
  • a binding protein of the present disclosure comprises a“knob” mutation on the third polypeptide chain and a“hole” mutation on the second polypeptide chain.
  • the“knob” mutation comprises substitution(s) at positions corresponding to positions 354 and/or 366 of human IgGl or IgG4 according to EU Index.
  • the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y.
  • the“knob” mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index.
  • the amino acid substitutions are S354C and T366W.
  • the“hole” mutation comprises substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 and of human IgGl or IgG4 according to EU Index.
  • the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A.
  • the “hole” mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index.
  • the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution at position corresponding to position 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution at position corresponding to position 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitution is T366W.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
  • the first and/or second Fc regions are
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L 2 35A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L 2 35A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to
  • a binding protein of the present disclosure comprises one or more mutations to reduce effector function, e.g ., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody- dependent cellular cytotoxicity (ADCC).
  • ADCP Fc receptor-mediated antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • ADCC antibody- dependent cellular cytotoxicity
  • the second polypeptide chain further comprises a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgGl Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgGl according to EU Index, wherein the amino acid substitutions are L 2 34A and L 2 35A.
  • the Fc regions of the second and the third polypeptide chains are human IgGl Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgGl according to EU Index, wherein the amino acid substitutions are L 2 34A and L 2 35A.
  • the second polypeptide chain further comprises a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and CH3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgGl Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L 2 34A, L 2 35A, and P329A.
  • the Fc regions of the second and the third polypeptide chains are human IgGl Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L 2 34A, L 2 35A, and P329A.
  • the Fc regions of the second and the third polypeptide chains are human IgG4 Fc regions, and the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L 2 35A.
  • the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising
  • immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L 2 35A.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L 2 35A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an
  • immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L 2 35A, Y349C, T366S, L368A, Y407V, and R409K.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L 2 35A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 23
  • substitutions are S228P, F234A, L 2 35A, S354C, T366W, and R409K.
  • the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fey I and/or Fcyll binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fcyl and/or Fcyll binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228 and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P and /or R409K.
  • the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 234 and/or 235 of human IgG4 according to EU Index.
  • the amino acid substitutions are F234A and/or L 2 35A.
  • the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and/or 409 of human IgG4 according to EU Index.
  • the amino acid substitutions are S228P, F234A, L 2 35A, and /or R409K.
  • the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index.
  • the amino acid substitutions are E233P, F234V, L 2 35A, and a deletion at 236.
  • the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to EU Index.
  • the amino acid mutations are S228P; E233P, F234V, L 2 35A, and a deletion at 236; and /or R409K.
  • the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region (e.g ., Fc receptor- mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC)).
  • ADCP Fc receptor- mediated antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • the Fc region is a human IgGl Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgGl according to EU Index.
  • the amino acid substitutions are L 2 34A, L 2 35A, and/or P329A.
  • the Fc region is a human IgGl Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgGl according to EU Index.
  • the amino acid substitutions are S298N, T299A, and/or Y300S.
  • a binding protein of the present disclosure comprises one or more mutations to improve stability, e.g., of the hinge region and/or dimer interface of IgG4 ( See e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593).
  • the mutation comprises substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
  • the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
  • a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve stability.
  • the first and/or second Fc regions are human IgG4 Fc regions.
  • the Fc region is a human IgGl Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgGl according to EU Index.
  • the amino acid substitutions are L 2 34A, L 2 35A, and/or P329A.
  • the Fc region is a human IgGl Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgGl according to EU Index.
  • the amino acid substitutions are S298N, T299A, and/or Y300S.
  • nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein.
  • Exemplary and non-limiting nucleic acid sequences are provided in Table 5.
  • kits of polynucleotides e.g ., that encode one or more polypeptides of a binding protein as described herein.
  • a kit of polynucleotides of the present disclosure comprises one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193,
  • the vector system comprises one or more vectors encoding a first, second, third, and fourth polypeptide chain of any of the binding proteins described herein.
  • the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein, e.g ., as shown in the polynucleotides of Table 6.
  • the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein.
  • the one or more vectors of the vector system may be any of the vectors described herein.
  • the one or more vectors are expression vectors.
  • the first, second, third, and fourth polynucleotides are present on one or more expression vectors, e.g. , one, two, three, or four expression vectors.
  • polynucleotides that encode the polypeptides which form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Sambrook et al, 2001 , MOLECULAR C L ONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses,
  • the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of the binding protein-coding nucleic acid sequence.
  • a promoter may refer to nucleic acid control sequences which direct transcription of a nucleic acid.
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence.
  • promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), and the like), from heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, from heat-shock promoters, and the like), the CAG-promoter (Niwa et ah, Gene 108(2): 193-9, 1991), the phosphoglycerate kinase (PGK)-promoter, a tetracycline-inducible promoter (Masui et ah, Nucleic Acids Res.
  • viruses such as polyoma virus, fowlpox virus,
  • polynucleotides encoding binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or other suitable promoter that will be readily recognized by one skilled in the art.
  • the isolated nucleic acid is incorporated into a vector.
  • the vector is an expression vector.
  • Expression vectors may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed.
  • regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • Suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, a- fetoprotein, insulin and the like), and enhancer sequences from a eukaryotic cell virus (such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like).
  • mammalian genes such as globin, elastase, albumin, a- fetoprotein, insulin and the like
  • enhancer sequences from a eukaryotic cell virus such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like).
  • suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.).
  • Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art, and can be used to transfect any cell of interest.
  • a host cell comprising one or more isolated polynucleotides, vectors, and/or vector systems described herein.
  • an isolated host cell of the present disclosure is cultured in vitro.
  • the host cell is a bacterial cell (e.g, an E. coli cell).
  • the host cell is a yeast cell (e.g, an S. cerevisiae cell).
  • the host cell is an insect cell. Examples of insect host cells may include, for example, Drosophila cells (e.g, S2 cells), Trichoplusia ni cells (e.g, High Five TM cells), and
  • the host cell is a mammalian cell.
  • mammalian host cells may include, for example, human embryonic kidney cells (e.g, 293 or 293 cells subcloned for growth in suspension culture), Expi293TM cells, CHO cells, baby hamster kidney cells (e.g, BHK, ATCC CCL 10), mouse sertoli cells (e.g, TM4 cells), monkey kidney cells (e.g, CV1 ATCC CCL 70), African green monkey kidney cells (e.g, VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g, HELA, ATCC CCL 2), canine kidney cells (e.g, MDCK, ATCC CCL 34), buffalo rat liver cells (e.g, BRL 3A, ATCC CRL 1442), human lung cells (e.g, W138, ATCC CCL 75), human liver cells (e.g, Hep G
  • human embryonic kidney cells e.g, 293 or 293 cells subcloned for growth in suspension culture
  • the method includes a) culturing a host cell (e.g, any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g, any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell.
  • a host cell e.g, any of the host cells described herein
  • an isolated nucleic acid, vector, and/or vector system e.g, any of the isolated nucleic acids, vectors, and/or vector systems described herein
  • Methods of isolating proteins from cultured host cells are well known to one of ordinary skill in the art, including, for example, by affinity chromatography (e.g, two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography) .
  • affinity chromatography e.g, two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography
  • Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure.
  • Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or
  • hydroxypropyl-beta-cyclodextrin fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorb
  • compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly
  • binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions of the disclosure can be selected for parenteral delivery or subcutaneous. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally.
  • compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition can be formulated for inhalation.
  • a binding protein can be formulated as a dry powder for inhalation.
  • Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized.
  • binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxy ethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.
  • Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
  • compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes.
  • compositions are lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration can be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations can be stored either in a ready -to-use form or in a form (e.g, lyophilized) requiring reconstitution prior to administration.
  • kits for producing a single-dose administration unit can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g, liquid syringes and lyosyringes).
  • the effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g ., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed- release bolus, or continuous administration.
  • the pharmaceutical compositions can be used to prevent and/or treat HIV infection.
  • the pharmaceutical compositions can be used as a standalone therapy or in combination with standard anti-retroviral therapy.
  • the disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples.
  • reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • the kit comprises a composition comprising any binding protein,
  • polynucleotide, vector, vector system, and/or host cell described herein are examples of nucleotide, vector, vector system, and/or host cell described herein.
  • the kit comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition (e.g, HIV infection) and may have a sterile access port (for example the container may be an
  • the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice.
  • the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • Certain aspects of the present disclosure relate to methods for expanding virus- specific memory T cells.
  • the methods comprise contacting a virus- specific memory T cell with a binding protein of the present disclosure, e.g ., a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
  • a binding protein of the present disclosure e.g ., a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
  • the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo.
  • contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus-specific memory T cells.
  • the methods comprise contacting a T cell with a binding protein of the present disclosure, e.g. , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
  • a binding protein of the present disclosure e.g. , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
  • the T cell is a memory T cell or an effector T cell.
  • the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.
  • CAR chimeric antigen receptor
  • kits for treating chronic viral infection e.g. , in an individual in need thereof.
  • the methods comprise administering to an individual in need thereof an effective amount of a binding protein of the present disclosure, e.g. , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
  • a binding protein of the present disclosure e.g. , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
  • the individual is a human.
  • the binding protein is administered to the individual in pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier.
  • administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual.
  • memory T cells can be CD8+ or CD4+ memory T cells.
  • memory T cells can be central memory T cells (TCM) or effector memory T cells (TEM).
  • Certain aspects of the present disclosure relate to methods for preventing and/or treating cancer in a patient.
  • the methods comprise administering to the patient a therapeutically effective amount of a binding protein or pharmaceutical composition of the present disclosure.
  • a binding protein of the present disclosure is administered to a patient in need thereof for the treatment or prevention of cancer.
  • the present disclosure relates to a method of preventing and/or treating a proliferative disease or disorder (e.g ., cancer).
  • the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein.
  • the present disclosure relates to uses of at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein for preventing and/or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof.
  • the present disclosure relates to at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein for use in the manufacture of a medicament for preventing and/or treating a proliferative disease or disorder (e.g, cancer) in a patient in need thereof.
  • a proliferative disease or disorder e.g, cancer
  • the patient is a human.
  • the at least one binding protein is administered (or is to be administered) in combination with one or more anti-cancer therapies (e.g, any anti-cancer therapy known in the art, such as a chemotherapeutic agent or therapy).
  • anti-cancer therapies e.g, any anti-cancer therapy known in the art, such as a chemotherapeutic agent or therapy.
  • the at least one binding protein is administered (or is to be administered) before the one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) concurrently with the one or more anti- cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) after the one or more anti-cancer therapies.
  • the binding protein comprises one or two antigen binding site(s) that binds a T-cell surface protein and another antigen binding site that binds the extracellular domain of a human HER2 polypeptide.
  • the binding protein comprises an antigen binding site that binds the extracellular domain of a human HER2 polypeptide, an antigen binding site that binds a human CD28 polypeptide, and an antigen binding site that binds a human CD3 polypeptide.
  • cancer cells from the individual express HER2.
  • the patient is selected for treatment on the basis that the cells of the cancer express a human HER2 polypeptide.
  • Assays known in the art suitable for detecting HER2 expression by cancer cells include, without limitation, immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) assays.
  • the cancer e.g ., HER2-positive cancer
  • the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the binding protein comprises one or two antigen binding site(s) that binds a T-cell surface protein and another antigen binding site that binds the extracellular domain of a human CD38 polypeptide.
  • the binding protein comprises an antigen binding site that binds the extracellular domain of a human CD38 polypeptide, an antigen binding site that binds a human CD28 polypeptide, and an antigen binding site that binds a human CD3 polypeptide.
  • cancer cells from the individual express CD38.
  • cells of the cancer express a human CD38 isoform A polypeptide on their cell surface.
  • cells of the cancer express a human CD38 isoform E polypeptide on their cell surface.
  • the patient is selected for treatment on the basis that the cells of the cancer express a human CD38 isoform E polypeptide on their cell surface.
  • the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 and do not express CD28.
  • the cancer e.g., CD38-positive cancer
  • the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, germinal center B-cell lympohoma or B-cell acute lymphoblastic leukemia.
  • the cancer is multiple myeloma.
  • the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma.
  • the cancer is multiple myeloma.
  • Anti-CD38 antibodies have been tested for the treatment of multiple myeloma, such as daratumumab.
  • multiple myeloma is considered treatable, relapse is inevitable in almost all patients, leading to the development of treatment-refractory disease.
  • the cancer is relapsed or refractory multiple myeloma.
  • the patient has been treated with a prior multiple myeloma treatment.
  • a binding protein of the present disclosure is administered to the patient as a 1 st , 2 nd , or 3 rd line treatment for multiple myeloma.
  • an anti- CD38xanti-CD28xanti-CD3 binding protein of the present disclosure may be useful in treating multiple myeloma, e.g ., by recruiting T cells to tumor cells via anti-CD38 (or anti- CD28/anti-CD38), activation of engaged T cells via anti-CD3/anti-CD28, and/or killing of tumor cells through perforin/granzyme-based mechanisms.
  • CD28 has been reported as a novel cancer marker for multiple myeloma. See Nair, J.R. el al. (2011) J. Immunol.
  • binding proteins described herein may find use in the methods of the present disclosure.
  • the patient prior to administration of the binding protein, the patient has been treated with daratumumab.
  • the present disclosure provides anti-CD38 binding proteins and sites that do not compete for binding CD38 with daratumumab. Without wishing to be bound to theory, it is thought that this is advantageous because a patient previously treated with daratumumab can be treated with a binding protein of the present disclosure, e.g. , without a wash-out period prior to treatment.
  • the binding proteins can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantitation of one or more target antigens.
  • the binding proteins will bind the one or more target antigens with an affinity that is appropriate for the assay method being employed.
  • binding proteins can be labeled with a detectable moiety.
  • the detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, 125 I, "Tc, U1 ln, or 67 Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, b-galactosidase, or horseradish peroxidase.
  • binding proteins are also useful for in vivo imaging.
  • a binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host assayed.
  • the binding protein can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • binding proteins can be conjugated to a cytotoxic agent.
  • cytotoxic agents i.e., antibody-drug conjugates
  • Cytotoxic agents and linkers that conjugate the agents to an antibody are known in the art; see, e.g., Parslow, A.C. et al. (2016) Biomedicines 4: 14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11 :2265-2276.
  • Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure.
  • Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • These pharmaceutical compositions may find use in any of the methods and uses described herein (e.g, ex vivo, in vitro, and/or in vivo).
  • Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or
  • hydroxypropyl-beta-cyclodextrin fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorb
  • compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly
  • binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions of the disclosure can be selected for parenteral delivery or subcutaneous.
  • the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition can be formulated for inhalation.
  • a binding protein can be formulated as a dry powder for inhalation.
  • Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized.
  • binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when
  • bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.
  • Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
  • compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes.
  • compositions are lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration can be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations can be stored either in a ready -to-use form or in a form (e.g, lyophilized) requiring reconstitution prior to administration.
  • the disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples.
  • reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein.
  • the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice.
  • the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • kits for producing a single-dose administration unit can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g, liquid syringes and lyosyringes).
  • the effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g.
  • compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed- release bolus, or continuous administration.
  • Example 1 Development of trispecific HER2/CD28xCD3 antibodies and variant anti- CD3 binding sites
  • Immuno-oncology is a promising, emerging therapeutic approach to disease management in cancer.
  • the immune system is the first line of defense against cancer development and progression.
  • T cells are able to control tumor growth and prolong the survival of cancer patients in both early and late stages of disease.
  • T cells specific for tumors can be limited in a number of ways preventing them from controlling the disease.
  • novel antibodies were developed in the trispecific antibody format depicted in FIG. 1A to specifically activate the T cells to engage HER2 expressing cancer cells. These novel trispecific antibodies are able to bind to three targets: HER2, CD3, and CD28. Anti-HER2 and anti-CD3 binding sites were further optimized for high affinity binding and reduction in potential manufacturing liabilities.
  • HER2 amplification and overexpression can be found in molecular subtypes of breast cancer, and also in gastric, ovarian, lung and prostate carcinomas.
  • Optimal activation of T cells requires two factors: (1) Antigen recognition and (2) Co-stimulation.
  • Signal 1 is provided by an agonist anti-CD3 binding site
  • Signal 2 is provided by an agonist anti- CD28 binding site (see, e.g., FIG. ID). It is thought that the trispecific antibodies described in the subsequent Examples recruit T cells to the tumor via HER2 and activate the engaged T cells by binding to CD3 and CD28. The resulting activation induces the killing potential of the immune cells against the nearby tumor cells.
  • Trispecific antibody variants were produced by transient transfection of expression plasmids into Expi293 cells. 5 days after transfection, the supernatant from transfected cells was collected, quantified and normalized by absorbance at 280 nm on Nano Drop. The binding of supernatant to corresponding antigens were determined by ELISA and the absorbance of parental HER2 WT tri Ab was set as 1.0. The fold changes of other variants were calculated by dividing the corresponding absorbance to that of parental Ab.
  • Trispecific antibody variants were purified using protein A affinity purification followed by SEC purification. The binding of purified antibodies to corresponding antigens were determined by ELISA. The EC50 were determined based on the binding curve generated by Graphpad Prism7.
  • Trispecific Ab variants were produced with several mutations in the binding arms in order to mitigate potential manufacturing liabilities, e.g ., deamidation sites.
  • a binding ELISA assay was performed to assess binding of the indicated trispecific antibodies to each of the three targets: HER2, CD3, and CD28.
  • HER2/CD3xCD28 trispecific antibodies with the indicated anti-HER2 or anti-CD3 variants were compared to parental Trispecific Ab.
  • Introducing some sets of mutations e.g, 32/33 QQ and 33/35QQ) into the VL domain of the anti-CD3 binding site led to dramatically reduced binding to CD3, whereas 32/35 QQ mutations retained near wild-type binding.
  • binding curves for the indicated antibodies binding to human HER2, human CD28, and CD3 are provided in FIG. 1C.
  • the EC50 values of selected trispecific antibody variants are provided in Table E.
  • Table E A binding ELISA assay was performed on purified trispecific antibodies to determine their binding affinities for HER2, human CD3, and human CD28.
  • HER2/CD3/CD28 trispecific antibodies recruit T cells to cancer cells through the anti-HER2 and anti-CD3/CD28 arms. Further, it is believed that engaged T cells are activated by the anti-CD28/CD3 arms. Killing of cancer cells is believed, without wishing to be bound by theory, to occur through T cell mediated mechanisms (e.g ., Perforin, granzyme). Without wishing to be bound to theory, it is contemplated that similar mechanisms may allow for killing of other types of tumors by substituting antigen binding sites that recognize other tumor target proteins.
  • Trispecific CD38/CD3xCD28 antibodies were developed and characterized for binding to CD38, CD3 and CD28 polypeptides.
  • a panel of anti-CD38, anti-CD3, and anti-CD28 antibodies, as well as human IgG4 Fc domains were used to generate CD38/CD28xCD3 trispecific antibodies in the trispecific antibody format depicted in FIG. 2A.
  • Trispecific binding proteins were produced by transient transfection of 4 expression plasmids into Expi293 cells using ExpiFectamineTM 293 Transfection Kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Briefly, 25% (w/w) of each plasmid was diluted into Opti-MEM, mixed with pre-diluted ExpiFectamine reagent for 20- 30 minutes at room temperature (RT), and added into Expi293 cells (2.5xl0 6 cells/ml). An optimization of transfection to determine the best ratio of plasmids was often used in order to produce the trispecific binding protein with good yield and purity.
  • SEC size-exclusion chromatography
  • Binding affinities to each target antigen by the CD38/CD28xCD3 T cell engagers were measured by ELISA. Briefly, each antigen was used to coat the 96-well Immuno Plate (Thermo Fisher Scientific) overnight at 4°C using 200 ng/well in PBS(pH7.4) of each antigen. The coated plate was blocked using 5% skim milk+2% BSA in PBS for one hour at RT, followed by washing with PBS+0.25% Tween 20 three times (Aqua Max 400, Molecular Devices).
  • the analyzed antibody was captured at a flow rate of 10 mL/min with an adjusted RU value that would result in maximal analyte binding signal of typically 30 RU. Binding kinetics were measured against the trispecific antibodies. Assay buffer HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005 % Surfactant P20) was used at a flow rate of 30 m ⁇ /min. Chip surfaces were regenerated with the regeneration solution of the respective capture kit. Kinetic parameters were analyzed and calculated in the BIA evaluation program package v4.1 using a flow cell without captured antibody as reference and the 1 :1 Langmuir binding model with mass transfer.
  • the binding affinities of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD38 were determined by SPR.
  • the association rate constant (Kon), dissociation rate constant (Koff), and the KD of the selected trispecific antibodies are provided in Table A.
  • the selected trispecific antibodies showed various degrees of affinities against human CD38 antigen.
  • Table A Binding characteristics of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD38 determined by SPR.
  • the binding affinities of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD3, human CD28, human CD38 and cynomolgus monkey CD38 were then determined by ELISA as described above.
  • the selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains showed various affinities to human (FIG. 2B) and cynomolgus monkey CD38 (FIG. 2C), but similar affinity to human CD3 (FIG. 2D) and CD28 (FIG. 2E).
  • EC50 values were then calculated by GraphPad Prism 7.02 using variable slope model with four- parameter logistic curve.
  • the EC50 values of the selected trispecific antibodies for human CD3, human CD28, human CD38 and cynomolgus monkey CD38 are provided in Table B.
  • Control antibody was a human IgG4 isotype control.
  • Table B EC50 values of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD3, human CD28, human CD38 and cynomolgus monkey CD38.
  • Table B2 Summary of anti-CD38 binding characteristics to human or cynomolgus CD38.
  • Anti-CD38 antibodies were also tested for competitive binding to daratumumab in SPR assay.
  • daratumumab competition binding assay daratumumab was amine coupled to the active surface of CM5 chip. Reference surface was left blank and used to subtract any non-specific binding of injected molecules.
  • Recombinant CD38-His (Sino Biological, Part# 10818-H08H) was injected over the daratumumab surface followed by injection of test antibodies. If an antibody recognizes an epitope on CD38 which is different from that of daratumumab, injection of the antibody will result in an increased SPR signal. If an antibody recognizes an overlapping epitope as daratumumab, injection of the antibody will not increase SPR signal. According to the results of these assays the tested antibodies hhy992, hyb6284, hhyl 195 and hhyl370 did not compete with daratumumab.
  • Example 3 Trispecific CD38/CD3xCD28 antibodies promote lysis of human multiple myeloma and lymphoma tumor cells.
  • CD38/CD3xCD28 antibodies had anti -tumor cell activity using human multiple myeloma and lymphoma cells.
  • Peripheral blood mononuclear cells were isolated from normal human donors by Ficoll separation, and autologous CD8+ or pan-T cells were enriched using kits from Miltenyi Biotech (San Diego, CA).
  • the extent of cell lysis in the target cells was determined by staining with a LIVE/DEADTM Fixable Violet Dead Cell Stain Kit (Life Technologies) and measured by the number of dead cells in the labelled target cell population by running the samples on an LSRFortessa instrument (BD Biosciences) followed by analysis using the Flowjo software (Treestar).
  • CD38hhyl370 anti-CD38 binding domains were reduced by Daratumumab, while trispecific antibodies with the CD38hyb5739, CD38hyb6284, or CD38hhyl 195 anti-CD38 binding domains exhibited between 3-8 fold reductions in cell killing activity in the presence of Daratumumab (Table C).
  • Table C In vitro killing activity against human multiple myeloma cell line NCI-H929 (CD38+/CD28+) by CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains in the presence of Daratumumab.
  • an in vitro cell lysis assay was used to measure the cell killing activity of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains using a human lymphoma cell line OCI-LY19 that expresses CD38 but not CD28.
  • the assay was carried out in the presence of 5nM
  • CD38VH1 anti-CD38 binding domain was reduced by about 24 fold by Daratumumab, while trispecific antibodies with the CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhyl l95, or CD38hhyl370 anti-CD38 binding domains also exhibited reductions in cell killing activity in the presence of Daratumumab (Table D).
  • Table D In vitro killing activity against human lymphoma cell line OCI-LY19 (CD38+/CD28-) by selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains in the presence of Daratumumab.
  • T cell immunity plays a crucial role in controlling viral infection and cancer, possibly eliminating infected cells and malignant cells which result in clearance of viral infection or cure of cancer.
  • chronic infectious diseases such as Herpes viral infection (HSV, CMV, EBV, etc.)
  • HIV, and HBV viruses establish their persistence in humans by various mechanisms including immune suppression, T cell exhaustion, and latency establishment.
  • viral infection generally induces viral antigen specific immunity including antigen specific CD8 T cells that can readily recognize infected cells for controlling or killing through cytokine release or cytotoxic T cell (CTL) mediated killing processes.
  • CTL cytotoxic T cell
  • Anti-CD38/CD28xCD3 trispecific antibodies were developed and evaluated for their potential in activating T cells, and promoting proliferation and/or amplification of antigen specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations, including antigen specific CD8 T central memory and effector memory cells in vitro. Specifically, in vitro expansion of CMV and EBV specific CD8 central memory and effector memory cells were demonstrated.
  • the anti-CD38/CD28xCD3 trispecific antibodies described herein exhibited novel properties by engaging
  • CD3/CD28/CD38 providing signaling pathways to stimulate and expand T cells, which may offer an effective strategy treating chronic infectious diseases such as HSV, CMV, EBV, HIV-1, and HBV infections.
  • T cells were isolated from human PBMC donors by negative selection using a magnetic Pan T Cell Isolation Kit (Miltenyi Biotec GmbH, Germany). Antibodies were coated onto 96-well cell culture plates by preparing the antibodies in sterile PBS and dispensing 50 mL into each well (350 ng/well). The plates were then incubated at 37°C for at least 2 hours and then washed with sterile PBS. The untouched T cells were added to the antibody-coated plates (5 x 10 5 cells/mL) and incubated at 37°C for multiple days. The cells were passaged with new cell culture media onto fresh antibody-coated plates on Day 4. In certain experiments with 7 days incubation, only fresh medium was added without changing to fresh antibody-coated plate. The cells were collected at specific time points and cell numbers calculated using CountBrightTM counting beads.
  • Peripheral blood mononuclear cells were isolated from blood of healthy human donors collected by Research Blood Components, LLC (Boston, MA). The PBMCs were added to antibody-coated plates (350 ng/well) (5 x 10 5 cells/mL), as previously described above, and incubated at 37°C for 3 and 7days. The cells were collected at specific time points and analyzed by flow cytometry for T cell subsets: naive (CCR7+ CD45RO-), Tcm (CCR7+ CD45RO+), Tem (CCR7- CD45RO+), Tregs (CD4+ Foxp3+ CD25hi).
  • PBMC was obtained from HemaCare (Van Nuys, CA) for donors with known CMV or EBV infection. PMBC from donors negative for the restricting HLA type was used as negative control. Staining was done as per manufacturer’s protocol.
  • PBMCs Peripheral blood mononuclear cells
  • CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains DVH1CD38 (control), CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhyl l95, and CD38hhyl370 were tested as described above using PBMCs isolated from CMV-infected human donor D (FIGS. 6A-6J) and CMV- infected human donor E (FIGS. 7A-7J).
  • CD38 trispecific Abs activated and promoted the proliferation of CMV-specific T cells, leading to increases in CMV-specific CD8+ T cells (cells/well) with different potency and kinetics in a dose response manner over the 7 day experiment (CMV Donor D, FIGS. 6A-6B; CMV Donor E, FIGS. 7A-7B).
  • all tested CD38 trispecific Abs promoted the amplification (cells/well) of CMV- specific central memory (T cm ) (CMV Donor D, FIGS. 6C-6D; CMV Donor E, FIGS. 7C- 7D) and effector memory (T em ) CD8+ T cells (CMV Donor D, FIGS. 6E-6F; CMV Donor E, FIGS.
  • FIGS. 6G-6J (CMV Donor D) and FIGS. 7G-7J (CMV Donor E) provide time courses showing the percent of CMV-specific T cm and T em cells at days 0, 3, and 7 of the 7-day experiments described above.
  • CD38/CD28xCD3 trispecific antibodies promote activation and expansion of CMV-specific T cells, such as CMV-specific CD8+ T cells, CMV-specific effector memory (Tem) CD8+ T cells, and CMV-specific central memory (Tcm) CD8+ T cells.
  • CMV-specific T cells such as CMV-specific CD8+ T cells, CMV-specific effector memory (Tem) CD8+ T cells, and CMV-specific central memory (Tcm) CD8+ T cells.
  • Tcm CMV-specific central memory
  • PBMCs peripheral blood mononuclear cells
  • CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains DVH 1CD38 (control), CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhyl 195, and CD38hhyl370 were also tested as described above using PBMCs isolated from EBV-infected donor C (FIGS. 8A-8J) and EBV-infected donor D (FIGS. 9A-12).
  • CD38 trispecific Abs activated T cells and promoted the proliferation of EBV-specific T cells, leading to increases in EBV-specific CD8+ T cells (cells/well) with different potency and kinetics in a dose response manner over the 7 day experiment (EBV Donor C, FIGS. 8A-8B; EBV Donor D, FIGS. 9A-9B).
  • all tested CD38 trispecific Abs promoted the amplification (cells/well) of EBV- specific central memory (T cm ) (EBV Donor C, FIGS. 8C-8D; EBV Donor D, FIGS. 9C-9D) and effector memory (Tem) CD8+ T cells (EBV Donor C, FIGS.8E-8F; EBV Donor D,
  • FIGS. 9E-9F which were both amplified dramatically in 7 days.
  • FIGS. 8G-8J (EBV Donor C) and FIGS. 9G-12 (EBV Donor D) provide time courses showing the percent of EBV-specific Tcm and Tem cells at days 0, 3, and 7 of the 7-day experiments described above.
  • CD38/CD28xCD3 trispecific antibodies promote activation and expansion of EBV-specific T cells, such as EBV-specific CD8+ T cells, EBV-specific effector memory (Tem) CD8+ T cells, and EBV-specific central memory (Tcm) CD8+ T cells.
  • EBV-specific T cells such as EBV-specific CD8+ T cells, EBV-specific effector memory (Tem) CD8+ T cells, and EBV-specific central memory (Tcm) CD8+ T cells.
  • Tem EBV-specific effector memory
  • Tcm EBV-specific central memory
  • the Her2/CD28 x CD3 trispecific antibody was tested for anti tumor effects in a ZR-75-1 tumor bearing Nod scid gamma (NSG) mouse model engrafted with in vitro expanded T cells.
  • NSG Nod scid gamma
  • mice were divided into 5 groups of 10 mice each.
  • ZR-75-1 human breast cancer cells were implanted into the mammary fat pad with 50% matrigel into each mouse at 5 million cells/mouse.
  • Days 17/18 expansion of human CD3+ T cells was begun. Randomization of mice occurred on Day 24 when tumors were approximately 150mm 3 .
  • all mice were engrafted with in vitro expanded human CD3+ T cells at 10 million cells in 300mL/mouse (1QW, 1 IP injection).
  • mice received doses of vehicle alone (8% w/v sucrose, 0.05% w/v polysorbate 80, lOmM histidine, pH 5.5), while the other 4 groups received Her2/CD28 x CD3 trispecific antibody, both at lOmL/kg. Groups receiving trispecific antibody were dosed at 100, 10, 1, or 0.1 mg/kg. Antibody or vehicle was administered 1QW intravenously in 2 doses ( e.g ., Days 25 and 32). Blood and tumor tissue was collected on Day 38 or 39.
  • Her2/CD28 x CD3 trispecific antibody (binding protein #2 from Table 1, corresponding to SEQ ID Nos: 104-107) was compared to vehicle control for its effects on human breast tumor growth in the NSG mouse model engrafted with in vitro expanded human T cells described above.
  • Individual tumor volumes over time from each trispecific antibody treatment group are provided in FIG. 13C.
  • tumors from the low dose groups were generally of comparable size as the vehicle control group.
  • human TILs were increased in the group receiving the low dose of Her2/CD28 x CD3 trispecific antibody, but human TILs were sparse in the high dose group.
  • IHC images were also examined quantitatively (FIGS. 16A-16C). These results indicated significant reductions in CD45+ and CD8+ cells in the higher trispecific antibody dose groups (lOOug/kg and lOug/kg).
  • tumors from the high dose trispecific antibody treatment groups (lOOug/kg or lOug/kg) were characterized by sparse TILs. Moderate to large numbers of CD45+, CD4+, or CD8+ human TILs were observed in the lug/kg and 0. lug/kg trispecific antibody treatment groups. These TILs were mostly present at the tumor edges but occasionally extended deeper into the tumor core.
  • Example 7 Effect of anti-HER2 and anti-CD3 antigen binding domain sequences in Her2/CD28 x CD3 trispecific antibody on cancer cell killing
  • This Example describes the effect of anti-Her2 and anti-CD3 variable domain sequences on target cell killing.
  • a Her2/CD28 x CD3 trispecific antibody (“control”) with wild-type trastuzumab antigen binding domain and an anti-CD3 antigen binding domain without 32/35 QQ mutations in the VL domain (see Example 1) was compared with Her2/CD28 x CD3 trispecific antibodies #1-6 from Table 1, corresponding to SEQ ID Nos: 100-103, 104-107, 286-289, 290-293, 294-297, and 298-301, respectively.
  • CD8+ T cells were isolated from human PBMCs from healthy donor using a magnetic bead isolation kit (Miltenyi Biotec). The T cells were used as effector cells against breast cancer cell lines expressing various levels of HER2 at 3: 1 (EffectonTarget) ratio. The cells were incubated with experimental or control trispecific antibody for 2 days before flow cytometry acquisition using viability dye (Invitrogen) and PKH26 target cell staining
  • HCC1954 breast cancer cells were found to express high levels of HER2, as assessed by flow cytometry (up to -150,000 receptors/cell), IHC (3+), or the HercepTest HER2 expression assay (3+) (FIG. 17A).
  • BT20 breast cancer cells were found to express intermediate levels of HER2, as assessed by flow cytometry (-60,000 receptors/cell), IHC (1+), or the HercepTest HER2 expression assay (1+) (FIG. 17C).
  • MDA-MD-231 breast cancer cells were found to express low levels of HER2, as assessed by flow cytometry (-9,000 receptors/cell), IHC (0+), or the HercepTest HER2 expression assay (0) (FIG. 17E). Results of the cell killing assays targeting HCC1954, BT20, or MDA-MB-231 are shown in FIGS. 17B, 17D, and 17F, respectively, comparing binding protein #2 vs. control or binding proteins #1 and #5 vs. control.

Abstract

Provided herein are trispecific and/or tri valent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation, and wherein and a second pair of polypeptides possess a single variable domain forming a single antigen binding site. In some embodiments, the binding proteins comprise a binding site that binds a CD28 polypeptide, a binding site that binds a CDS polypeptide, and a binding site that binds a third polypeptide, such as a tumor target protein. The disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins.

Description

TRISPECIFIC BINDING PROTEINS, METHODS, AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 62/831,572, filed April 9, 2019, and EP Application No. EP19306311.2, filed October 8, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 183952032040SEQLIST.TXT, date recorded: April 6, 2020, size: 526 KB).
FIELD
[0003] The disclosure relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation. The disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins.
BACKGROUND
[0004] Monoclonal antibody based biotherapeutics have become an important avenue for new drug development. Monoclonal antibody technology offers specific targeting, precise signaling delivery and/or payload to specific cell population, and provides long lasting biological effect through its Fc functions. Efforts in antibody engineering have allowed developing bispecific antibodies combining the specificities of two monoclonal antibodies for various biological applications, expanding the scope of antibody drug development. Newly discovered neutralizing antibodies with improved breadth and potency may provide more options for developing biotherapeutics to treat complexed diseases such as cancer, arthritis, and/or inflammatory disorders.
[0005] Immuno-oncology is a promising, emerging therapeutic approach to disease management in cancer. The immune system is the first line of defense against cancer development and progression. There is now large evidence that T cells are able to control tumor growth and prolong the survival of cancer patients in both early and late stages of disease. However, T cells specific for tumors can be limited in a number of ways preventing them from controlling the disease.
[0006] As part of the human adaptive immunity, T cell immunity plays crucial role in controlling viral infection and cancer, possibly eliminating infected cells and malignant cells which result in clearance of viral infection or cure of cancer. In chronic infectious diseases such as Herpes viral infection (HSV, CMV, EBV, etc.), HIV, and HBV, viruses establish their persistence in humans by various mechanisms including immune
suppression, T cell exhaustion, and latency establishment. Nevertheless, viral infection generally induces viral antigen specific immunity including antigen specific CD8 T cells that can readily recognize infected cells for controlling or killing through cytokine release or cytotoxic T cell (CTL) mediated killing processes.
[0007] Thus, viral antigen specific T cell activation and/or amplification in vivo and/or ex vivo may provide therapeutic strategies against chronic viral infections.
[0008] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.
BRIEF SUMMARY
[0009] To meet these and other needs, provided herein are trispecific binding proteins ( e.g ., antibodies) that form three antigen binding sites. These binding proteins can specifically bind one, two, or three antigen targets or target proteins, such as CD28, CD3, and a tumor target protein. Some tumors express specific antigens. For example, HER2 amplification and overexpression can be found in molecular subtypes of breast cancer, and also in gastric, ovarian, lung and prostate carcinomas. Optimal activation of T cells requires two factors: 1. Antigen recognition and 2. Co-stimulation. Using the trispecific
HER2/CD28xCD3 trispecific binding proteins described herein, Signal 1 is provided by an agonist anti-CD3 binding site, and Signal 2 is provided by an agonist anti-CD28 binding site. The trispecific binding protein recruits T cells to the tumor via HER2, CD38, or a binding site recognizing another tumor target protein and activates the engaged T cells via anti-CD3 and -CD28. The resulting activation induces the killing potential of the immune cells against the nearby tumor cells. In addition, anti-CD3 binding sites are described with high affinity binding to human CD3 polypeptides and potential manufacturing liabilities ( e.g ., deamidation sites) removed.
[0010] Further provided herein are anti-CD38/CD28xCD3 trispecific antibodies that were developed and evaluated for their potential in activating T cells, and subsequent proliferation and/or amplification of antigen specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations, including antigen specific CD8 T central memory and effector memory cells in vitro. Specifically, in vitro expansion of CMV, EBV, HIV-1, Influenza specific CD8 central memory and effector memory cells were demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibited novel properties by engaging CD3/CD28/CD38, providing signaling pathways to stimulate and expand T cells, which may offer an effective strategy treating chronic infectious diseases such as HSV, CMV, EBV, HIV-1, and HBV infections.
[0011] To meet these and other needs, provided herein are binding proteins that bind CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins with at least one antigen binding site that binds a CD38 polypeptide. Advantageously, these binding proteins have the ability to recruit T cells to the proximity of cancer cells, subsequently to activate T cells and promote the activated T cells killing of adjacent cancer cells through a Granzyme/Perforin mechanism, providing a different mode of action for anti -tumor activity from anti-CD38 antibodies such as DARZALEX® (daratumumab). Moreover, the ability to bind both human and cynomolgus monkey CD38 polypeptides allows binding proteins to be readily tested in preclinical toxicological studies, e.g., to evaluate their safety profiles for later clinical use.
[0012] In some embodiments, provided herein are binding proteins comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-Cm-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein: VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and Cm domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VH1 and VL1 form a first antigen binding site;
wherein Vm and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of
QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and
wherein VH3 and VL3 form a third antigen binding site.
[0013] In some embodiments, the first binding site binds a CD28 polypeptide. In some embodiments, the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the VL1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 54). In some embodiments, the Vm domain comprises the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGN VNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDV WGKGTTVTVSS (SEQ ID NO:91), and/or the VL1 domain comprises the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHT GVPSRF SGSGSGTDFTLTIS SLQPEDIAT YYCQQGQTYP YTF GQGTKLEIK (SEQ ID NO: 92).
[0014] In some embodiments, the CDR-L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of
RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the VH2 domain comprises the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD K SN S Y AT Y Y AD S VKGRFTI SRDD SKNTL YLQMN SLR AEDT A V Y Y CRGV Y Y AL SPF DYWGQGTLVTVSS (SEQ ID NO:93), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK
(SEQ ID NO: 95),
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO: 96),
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVS NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO: 97), and
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO: 98). In some embodiments, the VH2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD K SN S Y AT Y Y AD S VKGRFTI SRDD SKNTL YLQMN SLRAEDT A V Y Y CRGV Y Y AL SPF DYWGQGTLVTVSS (SEQ ID NO:93) or
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD KSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPF DYWGQGTLVTVSS (SEQ ID NO:302), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK
(SEQ ID NO: 95),
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO: 96),
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVS NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO: 97), and
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO:98). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:302, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.
[0015] In some embodiments, the third antigen binding site binds a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide ( e.g ., a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, a tumor target protein of the present disclosure includes, without limitation, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-la), CCL4 (also known as MIP-lb), CCL5 (also known as RANTES),
CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-ld), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL2 0 (also known as MIP-3a), CCL2 1 (also known as MIP-2),
CCL24 (also known as MPIF-2/eotaxin-2), CCL2 5 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD 19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L,
CD 152 (also known as CTLA4), CD 154 (also known as CD40L), CD 160, CD272, CD273 (also known as PDL2 ), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD- 1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2 Rbeta, IL1, ILIA, IL1B, IL1F10, IL2 , IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILlO, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL2 5), IL18, IL22, IL23, IL2 5, IL2 7, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, 0X40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREMl, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as
GPR5/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.
[0016] In some embodiments, the third antigen binding site binds a human CD38 polypeptide. In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of
ARRFEGF YY SMD Y (SEQ ID NO:33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36). [0017] In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48). In some embodiments, the VH3 domain comprises the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPG QGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTY WGQGTLVTVSS (SEQ ID NO:79), and/or the VL3 domain comprises the amino acid sequence of
DIVLTQ SP ATLSL SPGERATISCRASQ S V S S YGQGFMHW YQQKPGQPPRLLI Y GAS S RAT GIP ARF S GS GS GTDFTLTI SPLEPEDF A V Y Y C QQNKEDPWTF GGGTKLEIK
(SEQ ID NO: 80). In some embodiments, the VH3 domain comprises the amino acid sequence of
Q VQL VQSGAEVKKPGAS VKVSCKVSGYTLTEF SIHWVRQ APGQGLEWMGGFDPE DGETIY AQKF QGRVIMTEDT STDT AYMEMN SLRSEDT AIYYCTT GRFFDWF W GQG TLVTVSS (SEQ ID NO:81), and/or the VL3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIP VRF SGSGSGTDF SLTIS SLQPEDC AVYYCQQDSNLPITF GQGTRLEIK (SEQ ID NO:82). In some embodiments, the VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPG SGSTNY AQKF QGRVTMT VDRS STT AYMEL SRLRSDDT AVYY CARY AY GYW GQG TLVTVSS (SEQ ID NO:83), and/or the VL3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYT
Figure imgf000013_0001
NO:84). In some embodiments, the VH3 domain comprises the amino acid sequence of Q VQLVESGGGVVQPGRSLRLSC AASGFTF S S Y GMYWVRQAPGKGLEWVAVIWYD GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGG MDVWGQGTTVTVSS (SEQ ID NO:87), and/or the VL3 domain comprises the amino acid sequence of
Figure imgf000013_0002
NO:88). In some embodiments, the VH3 domain comprises the amino acid sequence of
Figure imgf000013_0003
Figure imgf000013_0004
and/or the VL3 domain comprises the amino acid sequence of
Figure imgf000013_0005
Figure imgf000013_0006
(SEQ ID NO:85), and/or the VL3 domain comprises the amino acid sequence of
Figure imgf000013_0007
(SEQ ID NO: 86).
[0018] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.
[0019] In some embodiments, the third antigen binding site binds a human HER2 polypeptide. In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), SRWGGEGFY AMDY (SEQ ID NO:7), or SRWGGSGFYAMDY (SEQ ID NO: 8), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of
SRW GGDGF YAMD Y (SEQ ID NO:6), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR- H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR- H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of
SRW GGDGF YAMD Y (SEQ ID NO:6), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF YAMD Y WGQGTLVTVSS (SEQ ID NO: 72),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:73),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO: 74),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO: 75), or
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or
DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF Y AMD Y WGQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:73), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO:75), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO:74), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF Y AMD Y WGQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78).
[0020] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: l 16; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.
[0021] In some embodiments that may be combined with any other embodiments described herein, at least one of Li, L2, L3 or L4 is independently 0 amino acids in length.
In some embodiments, L1, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some
embodiments, L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and
GGSGSSGSGG (SEQ ID NO:71). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO:68), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, at least one of L1, L2, L3 or L4 comprises the sequence DKTHT (SEQ ID NO:66). In some embodiments, L1, L2, L3 and L4 comprise the sequence DKTHT (SEQ ID NO:66). [0022] In some embodiments that may be combined with any other embodiments described herein, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2- CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-CH2-Cro domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-Cro domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2- CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-CH2-Cro domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
[0023] In some embodiments, provided herein are isolated nucleic acid molecules comprising a nucleotide sequence encoding the binding protein of any one of the above embodiments. In some embodiments, provided herein are expression vectors comprising the nucleic acid molecule of any one of the above embodiments. In some embodiments, provided herein are isolated host cells comprising the nucleic acid molecule of any one of the above embodiments or the expression vector of any one of the above embodiments. In some embodiments, the host cell is a mammalian or insect cell.
[0024] In some embodiments, provided herein are pharmaceutical compositions comprising the binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier.
[0025] In some embodiments, provided herein are methods of preventing and/or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein or pharmaceutical composition of any one of the above embodiments. In some embodiments, provided herein is a binding protein or pharmaceutical composition according to any one of the above embodiments for use in a method of preventing and/or treating cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of the binding protein or pharmaceutical composition. In some embodiments, provided herein is a binding protein or pharmaceutical composition according to any one of the above embodiments for use in manufacturing a medicament for preventing and/or treating cancer in a patient.
[0026] In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the patient is a human.
[0027] In some embodiments, the third antigen binding site binds a human CD38 polypeptide, and wherein cancer cells from the individual or patient express CD38. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma. In some embodiments, prior to administration of the binding protein, the patient has been treated with daratumumab without a wash-out period.
[0028] In some embodiments, the third antigen binding site binds a human HER2 polypeptide, and wherein cancer cells from the individual or patient express HER2. In some embodiments, the cancer is breast cancer, colorectal cancer, gastric cancer, or non small cell lung cancer (NSCLC).
[0029] In some embodiments, provided herein is a method for expanding virus-specific memory T cells, comprising contacting a virus-specific memory T cell with a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH3 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide
[0030] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide for use in expanding virus-specific memory T cells.
[0031] In some embodiments, the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo. In some embodiments, contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus- specific memory T cells.
[0032] In some embodiments, provided herein is a method for expanding T cells, comprising contacting a T cell with a binding protein in vitro or ex vivo , wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
[0033] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
Vm is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide for use in a method for expanding T cells.
[0034] In some embodiments, the T cell is a memory T cell or an effector T cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.
[0035] In some embodiments, provided herein is a method for treating chronic viral infection, comprising administering to an individual or patient in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV] wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein theVH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
[0036] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III] and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide for use in a method for treating chronic viral infection, wherein said method comprises administering to an individual or patient in need thereof an effective amount of the binding protein.
[0037] In some embodiments, the individual or patient is a human. In some
embodiments, the binding protein is administered to the individual or patient in
pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier. In some embodiments, administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual or patient.
[0038] In some embodiments that may be combined with any other embodiments described herein, the memory T cells are CD8+ or CD4+ memory T cells. In some embodiments, the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).
[0039] In some embodiments that may be combined with any other embodiments described herein, the virus is a human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein- barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 1 (HSV-2).
[0040] In some embodiments that may be combined with any other embodiments described herein, the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.
[0041] In some embodiments, provided herein is a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein of any one of the above embodiments. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.
[0042] In some embodiments, provided herein are kits comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above
embodiments. In some embodiments, the kits further comprise instructions for using the polypeptide chain or binding protein according to any of the methods or uses described herein, e.g. , supra.
[0043] In some embodiments, provided herein are kits comprising one, two, three, or four polynucleotides according to any one of the above embodiments. In some
embodiments, provided herein are kits of polynucleotides comprising one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212; (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232; (1) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:236; (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240; (n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244; (o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268; (u) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; or (v) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276.
[0044] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and HER2. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes HER2. The trispecific binding protein shown in FIG. 1A uses a constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain. [0046] FIG. IB provides the fold change (vs. parental) in binding affinities of anti- CD28/CD3/HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3, and anti-CD28 binding domains. Mutations 3233QQ to QEQ (top to bottom) refer to mutations introduced into residues 32-35 of the VL domain of the anti-CD3 binding site (indicated by *); the remaining mutations were introduced into the VH or VL domain of the trastuzumab anti-HER2 binding site (indicated by #; numbering according to Kabat). For the mutations in the anti-HER2 binding site, mutation 30Q was introduced into the VL domain, and the remaining mutations were introduced into the VH domain. The binding affinities were measured by ELISA, and the values provided are relative to parental trispecific antibody.
[0047] FIG. 1C provides binding curves for the indicated trispecific antibodies binding to human HER2, human CD28, and CD3, as determined by ELISA.
[0048] FIG. ID provides a proposed mechanism of action for HER2/CD28xCD3 trispecific antibody-mediated T cell activation and HER2+ cancer cell killing.
[0049] FIG. 2A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and CD38. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes CD38. The trispecific binding protein shown in FIG. 2A uses an IgG4 constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.
[0050] FIGS. 2B-2E show the binding affinities, as measured by ELISA, of
CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains for the target antigens human CD38 (FIG. 2B), cynomolgus monkey CD38 (FIG. 2C), human CD3 (FIG. 2D), and human CD28 (FIG. 2E).
[0051] FIG. 3 shows SPR competition assays for binding to CD38 by Daratumumab and anti-CD38 monospecific antibodies with the indicated anti-CD38 binding domains. If an antibody recognized an epitope on CD38 which was different from that of
Daratumumab, injection of the antibody resulted in an increased SPR signal. If an antibody recognized an overlapping epitope as Daratumumab, injection of the antibody did not increase SPR signal. [0052] FIGS. 4A-4B show the in vitro cell killing activity of CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human multiple myeloma NCI-H929 cells (CD38+/CD28+). The assays were carried out in the presence of 5 nM isotype control antibody (FIG. 4A) or Daratumumab (FIG. 4B). In the presence of daratumumab, the trispecific antibodies continued to exhibit cell killing activity.
[0053] FIGS. 5A-5B show the in vitro cell killing activity of CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human lymphoma OCI-Lyl9 cells (CD38+/CD28-). The assays were carried out in the presence of 5 nM isotype control antibody (FIG. 5A) or
Daratumumab (FIG. 5B). Daratumumab caused a decrease in the cell killing activity of anti-CD38/CD28xCD3 trispecific antibodies.
[0054] FIGS. 6A-6J show the characterization of in vitro T cell subset expansion in
PBMCs collected from CMV-infected Donor D in response to CD38/CD28sup x
CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated alternative anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA). T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated
concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify CMV-specific CD8+ T cells (FIGS. 6A-6B), CMV-specific Tcm CD8+ cells (FIGS. 6C-6D), and CMV- specific Tem CD8+ cells (FIGS. 6E-6F). In addition, the percentages of CMV-specific Tcm (FIGS. 6G-6H) and Tem (FIGS. 6I-6J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in a dose-responsive manner.
[0055] FIGS. 7A-7J show the characterization of in vitro T cell subset expansion in
PBMCs collected from CMV-infected Donor E in response to CD38/CD28sup x
CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA).
Antibodies shown in legend from top to bottom are shown in the graphs from left to right.
T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM, 1 nM, and 2 nM. Flow cytometry was used to quantify CMV-specific CD8+ T cells (FIGS. 7A-7B), CMV-specific TCm CD8+ cells (FIGS. 7C-7D), and CMV-specific Tem CD8+ cells (FIGS. 7E-7F). In addition, the percentages of CMV-specific Tcm (FIGS. 7G- 7H) and Tem (FIGS. 7I-7J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in dose response manner.
[0056] FIGS. 8A-8J show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected Donor C in response to CD38/CD28sup x
CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated alternative anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA). T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated
concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (FIGS. 8A-8B), CMV-specific Tcm CD8+ cells (FIGS. 8C-8D), and CMV- specific Tem CD8+ cells (FIGS. 8E-8F). In addition, the percentages of EBV-specific Tcm (FIGS. 8G-8H) and Tem (FIGS. 8I-8J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in dose response manner.
[0057] FIGS. 9A-12 show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected Donor D in response to CD38/CD28sup x
CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated alternative anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VHl/(DCD28sup x (DCD3mid IgG4 FALA). T cell populations were measured at indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated
concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (FIGS. 9A-9B), EBV-specific Tcm CD8+ cells (FIGS. 9C-9D), and EBV- specific Tem CD8+ cells (FIGS. 9E-9F). In addition, the percentages of EBV-specific Tcm (FIGS. 9G-10) and Tem (FIGS. 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells with different potency and kinetics in dose response manner.
[0058] FIGS. 13A-13D show the change over time (days) in tumor volume (FIG. 13A) and body weight (FIG. 13B) in ZR-75-1 tumor bearing NSG mice engrafted with in vitro expanded human CD3+ T cells. Groups of 10 mice were either treated with vehicle or Her2/CD28 x CD3 trispecific antibody at the indicated dosages. Arrow heads indicate days of administration. Tumor volume is depicted as mean ± SEM, mm3. Body weight change is depicted as % change, mean ± SEM. X-axis shows days after implantation with ZR-75-1 cells. Tumor volume (mm3) over time for individual mice in each treatment group are shown in FIG. 13C. Tumor weight (mg) for each treatment group is shown in FIG. 13D.
** = p<0.001; *** = p<0.0003 (two-way ANOVA, control vs. 100 & lOug/kg).
[0059] FIGS. 14A-14C show the effect of Her2/CD28 x CD3 trispecific antibody treatment on T cells from whole blood. FIG. 14A shows the analysis of hCD45+, CD8+, CD4+, and mCD45+ cells by flow cytometry. FIG. 14B shows the effect of control or Her2/CD28 x CD3 trispecific antibody treatment (at the indicated doses) on hCD45+, CD8+, CD4+, and mCD45+ cell counts. FIG. 14C shows the effect of control or
Her2/CD28 x CD3 trispecific antibody treatment (at the indicated doses) on human cell ratios (CD4+/CD45+ and CD8+/CD45+). For each x-axis parameter shown in FIGS. 14B & 14C, conditions are (left to right): control, lOOug/kg trispecific antibody, lOug/kg trispecific antibody, lug/kg trispecific antibody, and 0. lug/kg trispecific antibody.
Percentages shown in FIGS. 14B & 14C are based on control sample vs. lOOug/kg.
[0060] FIGS. 15A-15C show the effect of Her2/CD28 x CD3 trispecific antibody treatment on tumor infiltrating lymphocytes (TILs), as examined by immunohistochemistry (IHC). Arrows indicate tumor infiltrating T cells identified in ZR-75-1 breast tumors.
Tipper images are at IX magnification; lower images are at 20X magnification. In both sets of images, staining for human CD45, human CD4, and human CD8 are shown from left to right. Shown are tumors from mice treated with vehicle control (FIG. 15A), lOOug/kg trispecific antibody (FIG. 15B), or 0. lug/kg trispecific antibody (FIG. 15C).
[0061] FIGS. 16A-16C show quantitation of the effect of Her2/CD28 x CD3 trispecific antibody treatment on TILs as measured by IHC. Each dot represents one tumor from an individual mouse; rectangles represent group means; and error bars indicate standard deviation. * = p<0.05 compared to vehicle control group (ANOVA). Numbers of CD45+ (FIG. 16 A), CD4+ (FIG. 16B), or CD8+ (FIG. 16C) cells are shown. In FIG. 16C, a $ area quantitation approach was used for CD8+ cells instead of cell counting algorithm due to excessive non-specific signal in the CD8 IHC slide.
[0062] FIGS. 17A-17F show in vitro cell lysis of HER2+ breast cancer target cells in the presence of human CD8+ T cells by Her2/CD28 x CD3 trispecific antibody with wild- type trastuzumab antigen binding domain and an anti-CD3 antigen binding domain without without 32/35 QQ mutations in the VL domain (“ctl”) as compared to a Her2/CD28 x CD3 trispecific antibodies having mutations in the anti-HER2 arm and the VL domain of the anti-CD3 arm (numbering as shown in Table 1). Cell killing activities against cell lines with varying expression of HER2 are depicted: HCC1954 for high HER2 expression (FIG. 17A), BT20 for intermediate HER2 expression (FIG. 17C), and MDA-MD-231 for low HER2 expression (FIG. 17E). Graphs depicting cell killing as a function of antibody concentration against target cells HCC1954 (FIG. 17B), BT20 (FIG. 17D), and MDA-MD- 231 (FIG. 17F) are shown, comparing binding protein #2 vs. ctl or binding protein #1 and #5 vs. ctl.
[0063] FIGS. 18A & 18B summarize the mean EC50 (pM) of in vitro cell killing by experimental or control Her2/CD28 x CD3 trispecific antibodies against the indicated breast cancer (FIG. 18A) or gastric cancer cell lines (FIG. 18B). Amino acid sequences of the indicated trispecific antibodies are provided in Table 1.
DETAILED DESCRIPTION
[0064] The disclosure provides trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation.
General Definitions
[0065] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0066] It is understood that aspects and embodiments of the disclosure described herein include“comprising,”“consisting,” and“consisting essentially of’ aspects and
embodiments.
[0067] The term "polynucleotide" as used herein refers to single-stranded or double- stranded nucleic acid polymers of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or
deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as
phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term
"polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.
[0068] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
[0069] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
[0070] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any
immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CH1, CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.
[0071] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See , e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
[0072] The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Rabat (Rabat et al. , SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Rabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al ., 1989, Nature 342: 877-83) found that certain sub-portions within Rabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2 , and L3 or HI, H2, and H3 where the "L" and the "H" designates the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Rabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, 1995, FASEB J. 9: 133- 39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp.
Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Diibel S., eds. Springer-Verlag, Berlin, p. 33-51 (2010). The amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.
[0073] The term "Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgGl and IgG2 are preferred. Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent ( i.e ., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.
[0074] A F(ab) fragment typically includes one light chain and the VH and CH1 domains of one heavy chain, wherein the VH-CH1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CH1 domain.
[0075] A F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CH1 and CH2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.
[0076] The term "binding protein" as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen.
A trispecific binding protein of the present disclosure, unless otherwise specified, typically comprises four polypeptide chains that form at least three antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
VL2- L1-VL1- L2-CL [I]
and a second polypeptide chain has a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain has a structure represented by the formula:
VH3-CH1 [III]
and a fourth polypeptide chain has a structure represented by the formula:
VLI-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is the immunoglobulin CH1 heavy chain constant domain; and
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains;
L1, L2, L3 and L4 are amino acid linkers;
nd wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.
[0077] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means. [0078] One embodiment of the disclosure provides binding proteins having biological and immunological specificity to between one and three target antigens. Another embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the disclosure provides host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).
[0079] The term "swapability" as used herein refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity. "Full swapability" refers to the ability to swap the order of both Vm and VH2 domains, and therefore the order of VL1 and VL2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while
maintaining full functionality of the binding protein as evidenced by the retention of binding affinity. Furthermore, it should be noted that the designations VH and VL refer only to the domain's location on a particular protein chain in the final format. For example, Vm and VH2 could be derived from VL1 and VL2 domains in parent antibodies and placed into the V and VH2 positions in the binding protein. Likewise, VL1 and VL2 could be derived from Vm and VH2 domains in parent antibodies and placed in the Vm and VH2 positions in the binding protein. Thus, the VH and VL designations refer to the present location and not the original location in a parent antibody. VH and VL domains are therefore "swappable."
[0080] The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes.
With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.
[0081] The term "Her2" refers to human epidermal growth factor receptor 2 which is a member of the epidermal growth factor receptor family.
[0082] "CD3" is cluster of differentiation factor 3 polypeptide and is a T-cell surface protein that is typically part of the T cell receptor (TCR) complex.
[0083] "CD28" is cluster of differentiation 28 polypeptide and is a T-cell surface protein that provides co-stimulatory signals for T-cell activation and survival. [0084] "CD38" is cluster of differentiation 38 polypeptide and is a glycoprotein found on the surface of many immune cells.
[0085] The term "T-cell engager" refers to binding proteins directed to a host's immune system, more specifically the T cells' cytotoxic activity as well as directed to a tumor target protein.
[0086] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.
[0087] The term "monovalent binding protein" refers to a binding protein that has one antigen binding site.
[0088] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.
[0089] The term "bivalent binding protein" refers to a binding protein that has two binding sites.
[0090] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.
[0091] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In particular embodiments the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.
[0092] An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated binding proteins include the binding protein in situ within recombinant cells since at least one component of the binding protein's natural environment will not be present.
[0093] The terms "substantially pure" or "substantially purified" as used herein refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
[0094] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is < 10 8 M, more preferably when the equilibrium dissociation constant is < 10 9 M, and most preferably when the dissociation constant is < 10 10 M.
[0095] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "KD," as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.
[0096] The term "specifically binds" as used herein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 10 6 M, 1 x 10 7 M, 1 x 10 8 M, 1 x 10 9 M, 1 x 10 10 M, 1 x 10 11 M, 1 x 10 12 M, or more, and/or to bind to an epitope with an affinity that is at least two fold greater than its affinity for a nonspecific antigen. [0097] In some embodiments, an antigen binding domain and/or binding protein of the present disclosure“cross reacts” with human and cynomolgus monkey CD38 polypeptides, e.g., CD38 extracellular domains, human CD38 isoform A, human CD38 isoform E, and cynomolgus monkey CD38. A binding protein binding to antigen 1 (Agl) is“cross-reactive” to antigen 2 (Ag2) when the ECSOS are in a similar range for both antigens. In the present application, a binding protein binding to Agl is cross-reactive to Ag2 when the ratio of affinity of Ag2 to affinity of Agl is equal or less than 20, affinities being measured with the same method for both antigens.
[0098] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction. The linkers described herein are referred to as L1, which is located on the light chain between the C-terminus of the VL2 and the N-terminus of the VL1 domain; and L2, which is located on the light chain between the C-terminus of the VL1 and the N-terminus of the CL domain. The heavy chain linkers are known as L3, which is located between the C- terminus of the Vm and the N-terminus of the VH2 domain; and L4, which is located between the C-terminus of the VH2 and the N-terminus of the CH1 domain.
[0099] The term "vector" as used herein refers to any molecule (e.g, nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double- stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein, as a plasmid is the most commonly used form of vector. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors ( e.g ., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
[0100] The phrase "recombinant host cell" (or "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To express a binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.
[0101] The term "transformation" as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transformation, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells. [0102] The term "naturally occurring" as used herein and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring. Similarly, "non-naturally occurring" as used herein refers to an object that is not found in nature or that has been structurally modified or synthesized by man.
[0103] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers ( e.g ., D-amino acids) of the twenty conventional amino acids; unnatural amino acids and analogs such as a-, a-di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins. Examples of unconventional amino acids include: 4-hydroxyproline, g-carboxyglutamate, e-N,N,N-trimethyllysine, e-N- acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.
[0104] Naturally occurring residues may be divided into classes based on common side chain properties:
(1) hydrophobic: Met, Ala, Val, Leu, lie, Phe, Trp, Tyr, Pro;
(2) polar hydrophilic: Arg, Asn, Asp, Gin, Glu, His, Lys, Ser, Thr ;
(3) aliphatic: Ala, Gly, lie, Leu, Val, Pro;
(4) aliphatic hydrophobic: Ala, He, Leu, Val, Pro;
(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(6) acidic: Asp, Glu;
(7) basic: His, Lys, Arg;
(8) residues that influence chain orientation: Gly, Pro;
(9) aromatic: His, Trp, Tyr, Phe; and
(10) aromatic hydrophobic: Phe, Trp, Tyr.
[0105] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. [0106] A skilled artisan will be able to determine suitable variants of the polypeptide chains of the binding proteins using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
[0107] The term "patient" as used herein includes human and animal subjects.
[0108] The terms "treatment" or "treat" as used herein refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those having a disorder as well as those prone to have the disorder or those in which the disorder is to be prevented. In particular embodiments, binding proteins can be used to treat humans with cancer, or humans susceptible to cancer, or ameliorate cancer in a human subject. The binding proteins can also be used to prevent cancer in a human patient. In particular embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, germinal center B-cell lympohoma or B-cell acute lymphoblastic leukemia, In other embodiments, the binding proteins can be used to treat humans with inflammatory disorders, or humans susceptible to inflammatory disorders, or ameliorate inflammatory disorders in a human subject.
[0109] The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
[0110] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a binding protein.
[0111] The terms "effective amount" and "therapeutically effective amount" when used in reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo , factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.
[0112] One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.
Trispecific and/or Trivalent Binding Proteins
[0113] Certain aspects of the present disclosure relate to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation and wherein a second pair of polypeptides forming the binding protein possess a single variable domain. Any of the CDRs or variable domains of any of the antigen binding proteins described herein may find use in a trispecific binding protein of the present disclosure.
[0114] In some embodiments, each of the three antigen binding sites binds a different target ( e.g ., polypeptide antigen). In some embodiments, the trispecific binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.
[0115] It is contemplated that any of the antigen binding sites described herein may find use in a trispecific binding protein of the present disclosure, e.g ., comprising four polypeptide chains having the structures described supra. For example, in some embodiments, a trispecific binding protein of the present disclosure comprises a Vm and VL1 domain pair that form a first antigen binding site, a VH2 and VL2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL3 domain pair that form a third antigen binding site. In some embodiments, a trispecific binding protein of the present disclosure comprises a Vm and VL1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH2 and VL2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL3 domain pair that form a third antigen binding site. In some embodiments, a trispecific binding protein of the present disclosure comprises a Vm and VL1 domain pair that form a first antigen binding site, a VH2 and VL2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL3 domain pair that form a third antigen binding site that binds a tumor target protein. In some embodiments, a trispecific binding protein of the present disclosure comprises a Vm and VL1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH2 and VL2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL3 domain pair that form a third antigen binding site that binds a tumor target protein. In some embodiments, a trispecific binding protein of the present disclosure comprises a Vm and VL1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH2 and VL2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL3 domain pair that form a third antigen binding site that binds a CD38 polypeptide. In some embodiments, a trispecific binding protein of the present disclosure comprises a Vm and VL1 domain pair that form a first antigen binding site that binds a CD28 polypeptide, a VH2 and VL2 domain pair that form a second antigen binding site that binds a CD3 polypeptide, and a VH3 and VL3 domain pair that form a third antigen binding site that binds a HER2 polypeptide.
[0116] In some embodiments, a binding protein of the present disclosure binds one or more tumor target proteins and one or more T cell target proteins. In some embodiments, the binding protein is capable of specifically binding one tumor target protein and two different epitopes on a single T cell target protein. In some embodiments, the binding protein is capable of specifically binding one tumor target protein and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds a tumor target protein. In some embodiments, the target protein is CD38 or HER2. Additional tumor target proteins are provided infra. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28. Exemplary and non-limiting polypeptides that may find use in any of the trispecific binding proteins described herein are provided in Table 1.
[0117] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159.
[0118] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163.
[0119] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167.
[0120] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171.
[0121] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175.
[0122] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179.
[0123] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184.
[0124] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.
[0125] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103.
[0126] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107.
[0127] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115.
[0128] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119. [0129] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123.
[0130] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127.
[0131] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131.
[0132] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135.
[0133] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139.
[0134] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143.
[0135] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147.
[0136] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151.
[0137] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155.
[0138] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289.
[0139] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein .the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293.
[0140] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297.
[0141] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.
Anti-CD38 Binding Sites
[0142] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a CD38 polypeptide. In some embodiments, the CD38 polypeptide is a human CD38 polypeptide, also known as ADPRC1. Human CD38 polypeptides are known in the art and include, without limitation, the polypeptide represented by NCBI Accession Number NP_001766.2, or a polypeptide produced from NCBI Gene ID Number 952. In some embodiments, the antigen binding site binds a human CD38 polypeptide, a non-human primate ( e.g ., cynomolgus monkey) CD38 polypeptide, or a human CD38 polypeptide and a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide. In some embodiments, a binding protein comprising an antigen binding site that binds a CD38 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
[0143] In some embodiments, any of the CDRs and/or variable domains of the anti-CD38 binding sites described below can be used in a monospecific antibody.
[0144] In other embodiments, any of the CDRs and/or variable domains of the anti-CD38 binding sites described below can be used in any binding site of a trispecific binding protein comprising four polypeptides that form three antigen binding sites, e.g, as described supra. In certain embodiments, a binding protein that comprises an antigen binding site that binds a CD38 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites as described supra , wherein the VH3 and VL3 domains pair and form a third antigen binding site that binds a CD38 polypeptide.
[0145] A variety of features of exemplary binding sites and binding proteins are described herein. For example, in some embodiments, an anti-CD38 binding site cross-reacts with human CD38 (e.g, a human CD38 isoform A and/or isoform E polypeptide) and cynomolgus monkey CD38. In some embodiments, a binding protein comprising an anti-CD38 binding site induces apoptosis of a CD38+ cell. In some embodiments, a binding protein comprising an anti-CD38 binding site recruits a T cell to a CD38+ cell and optionally activates the T cell (e.g, though TCR stimulation and/or costimulation).
[0146] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18).
[0147] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24).
[0148] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30).
[0149] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36).
[0150] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42).
[0151] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48).
[0152] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQ GGTNYNQKF QGRATLTADTS ASTAYMELS SLRSEDTAVYF C ARTGGLRRAYFTYW G QGTLVTVSS (SEQ ID NO:79), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVLTQ SP ATLSL SPGERATISCRASQ S V S S YGQGFMHW YQQKPGQPPRLLI Y GAS SR ATGIP ARE SGSGSGTDFTLTISPLEPEDF AVYYCQQNKEDPWTF GGGTKLEIK (SEQ ID NO:80). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:79, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:80. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:79, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:80.
[0153] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
Q VQL VQSGAEVKKPGAS VKVSCKVSGYTLTEF SIHWVRQ APGQGLEWMGGFDPED GETIY AQKFQGRVIMTEDT STDT AYMEMN SLRSEDT AIYYCTT GRFFDWF W GQGTL VTVSS (SEQ ID NO:81), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPV RF SGSGSGTDF SLTIS SLQPEDC AVYYCQQDSNLPITF GQGTRLEIK (SEQ ID NO:82).
In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:81, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:82. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:81, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:82.
[0154] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGS GSTNY AQKF QGRVTMTVDRS STT AYMEL SRLRSDDT AVYY CARY AY GYW GQGTL VTVSS (SEQ ID NO:83), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTG VPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO:84). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:83, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:84. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:83, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:84.
[0155] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
Q VQLVESGGGVVQPGRSLRLSC AASGFTF S S Y GMYWVRQAPGKGLEWVAVIWYDG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMD VWGQGTTVTVSS (SEQ ID NO:87), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVP SRF SGSGSGTEFTLTIS SLQPEDF ATYY CQQLN SFP YTF GQGTKLEIK (SEQ ID NO:88). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:87, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:88. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:87, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:88.
[0156] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDG SNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQG TLVTVSS (SEQ ID NO:89), and/or an antibody light chain variable (VL) domain
comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSG VP SRF SGSGSGTDFTLTISGLQPED S AT YY CLQD YI YYPTF GQGTKVEIK (SEQ ID NO:90). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:89, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:90. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:89, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:90.
[0157] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPY Y GDTT Y AQKF QGRVTMTVDKS S ST AYMEL SRLRSDDT AVYY C ARRFEGF YY SMD Y WGQGTLVTVSS (SEQ ID NO:85), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVS KRF S GVPDRF S GS GS GTDF TLKI SRVE AED V GV Y Y C S Q S THVPLTF GGGTK VEIK
(SEQ ID NO:86). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:85, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:86. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:85, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:86.
[0158] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGS TNYNEKFKGK ATLT VDRS STT AYMHL SGLT SDD S AVYFC ARY AY GYW GQGTTLT V SS (SEQ ID NO:277), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYT GVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK (SEQ ID NO:278). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:277, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:278. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:277, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:278. In some embodiments, the VH and/or VL domains are humanized.
[0159] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
Q V QLLQ S GAEL VRPGV S VKIS C TGS GY SFTN Y A VHW VKQ SHVK SLEWIGVI SP Y Y GD TT YN QKF T GK ATMT VDK S S S T A YMEL ARLT SED S AIYF C ARRFEGF Y Y SMD YW GQG TSVTVSS (SEQ ID NO:279), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSK RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPLTFGSGTQLEIK (SEQ ID NO:280). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:279, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:280. In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:279, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:280. In some embodiments, the VH and/or VL domains are humanized.
[0160] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprising an antigen binding site that binds a CD38 polypeptide, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four
polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair ( e.g ., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD38 antigen binding sites described above represent VH3 and VL3 and form a third antigen binding site that binds a CD38 polypeptide. In some embodiments, Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide, VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and the VH and VL domains of any of the anti-CD38 antigen binding sites described above and/or in Table 2 represent VH3 and VL3 and form a third antigen binding site that binds a CD38 polypeptide.
[0161] Sequences of exemplary anti-CD38 antigen binding sites are provided in Table 2. In some embodiments, a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD38 antibody described in Table 2. In some embodiments, a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD38 antibody described in Table 2.
Table 2. Anti-CD38 binding protein sequences.
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
[0162] Further provided herein are antibodies ( e.g ., monospecific antibodies) comprising any of the anti-CD38 CDRs and/or variable domains described supra.
[0163] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site that binds an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether an antigen binding site binds an antigen are described herein and known in the art, including (without limitation) ELISA, SPR, and flow cytometry assays.
Anti-HER2 Binding Sites
[0164] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a HER2 polypeptide. In some embodiments, the HER2 polypeptide is a human HER2 polypeptide, also known as NEU, NGL, ERBB2, TKR1, CD340, HER-2, MLN19, and HER-2/neu. Human HER2 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP_024306411.1 , XP_024306410.1 , XP_024306409.1 , NP_001276867.1, NP_001276866.1, NP_001276865.1, NP_001005862.1, or NP_004439.2, or a polypeptide produced from NCBI Gene ID Number 2064. In some embodiments, a binding protein comprising an antigen binding site that binds a HER2 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, a binding protein that comprises an antigen binding site that binds a HER2 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites as described supra , wherein VH3 and VL3 domain pair that form a third antigen binding site that binds a HER2 polypeptide.
[0165] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRW GGDGF Y AMD Y (SEQ ID NO: 6), SRWGGEGFY AMDY (SEQ ID NO:7), or SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NOG), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NOG), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), SRWGGEGFY AMDY (SEQ ID NOG), or SRWGGSGFYAMDY (SEQ ID NOG); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0166] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NOG), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0167] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0168] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0169] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0170] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0171] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0172] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and a CDR-H3 sequence comprising the amino acid sequence of
SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0173] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0174] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0175] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:l 1), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR- H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
[0176] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO: 72),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:73),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO: 74),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO: 75), or
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:76); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or
DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76; and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO:78. In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76; and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO:78. [0177] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTR Y AD S VKGRF TI S ADT SKNT A YLQMN SLRAEDT A V Y Y C SRW GGD GF Y AMD YW GQGTLVTVSS (SEQ ID NO:72), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VP SRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO:77). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77. In some embodiments, a binding site that binds HER2 comprises: : an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
[0178] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:73), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQ SP S SL S AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:73, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77. In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:73, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
[0179] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO:75), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VP SRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO:77). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:75, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77. In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:75, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
[0180] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO:74), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VP SRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO:77). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:74, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77. In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:74, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
[0181] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:76), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VP SRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO:77). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:76, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77. In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:76, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.
[0182] In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO:72), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSG VP SRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO:78). In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:78. In some embodiments, a binding site that binds HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:78.
[0183] In some embodiments, an anti-HER2 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of anti-HER2 antibody trastuzumab,
30R/55Q/102E, 30R/56A/102S, 30R/55Q/102S, 30R/56A/102E, or 30Q. In some
embodiments, an anti-HER2 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of anti-HER2 antibody trastuzumab,
30R/55Q/102E, 30R/56A/102S, 30R/55Q/102S, 30R/56A/102E, or 30Q.
[0184] Sequences of exemplary anti-HER2 antigen binding sites are provided in Table 3. In some embodiments, an anti-HER2 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-HER2 antibody described in Table 3. In some embodiments, an anti-HER2 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-HER2 antibody described in Table 3.
Table 3. Anti-HER2 binding protein sequences.
Figure imgf000086_0001
Figure imgf000087_0001
Other Anti-Tumor Target Binding Sites
[0185] In some embodiments, a binding protein of the present disclosures comprises an antigen binding site that binds a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide ( e.g ., a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, a tumor target protein of the present disclosure includes, without limitation, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-la), CCL4 (also known as MIP-lb), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL1 1 (also known as eotaxin), CCL15 (also known as MIP-ld), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL2 1 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL2 5 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD 19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7- 1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD 154 (also known as CD40L), CD 160, CD272, CD273 (also known as PDL2 ), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2 Rbeta, IL1, ILIA, IL1B, IL1F10, IL2 , IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILlO, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, 0X40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREMl, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.
Anti-CD28 Binding Sites
[0186] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a CD28 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide, also known as Tp44. Human CD28 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP_011510499.1 , XP_011510497.1 , XP_011510496.1 ,
NP_001230007.1, NP_001230006.1, or NP_006130.1, or a polypeptide produced from NCBI Gene ID Number 940. In some embodiments, a binding protein comprising an antigen binding site that binds a CD28 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a CD38 polypeptide. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a HER2 polypeptide. In some
embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a tumor target protein.
[0187] In some embodiments, a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51) and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 54). In some embodiments, a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR- H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID
NO:51); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54).
[0188] In some embodiments, a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNV NTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWG KGTTVTVSS (SEQ ID NO:91), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTG VPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92). In some embodiments, a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:91, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, a binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:91, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:92.
[0189] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprising an antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2), an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain- heavy chain pair ( e.g ., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD28 antigen binding sites described above represent Vm and VL1 and form a first antigen binding site that binds a CD28 polypeptide. In some embodiments, the VH and VL domains of any of the anti-CD28 antigen binding sites described above and/or in Table 4 represent Vm and VL1 and form a first antigen binding site that binds a CD28 polypeptide, VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and VH3 and VL3 and form a third antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2).
[0190] Sequences of exemplary anti-CD28 antigen binding sites are provided in Table 4. In some embodiments, an anti-CD28 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD28 antibody described in Table 4. In some embodiments, an anti-CD28 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD28 antibody described in Table 4.
Table 4. Anti-CD28 binding protein sequences.
Figure imgf000090_0001
Figure imgf000091_0001
Anti-CD3 Binding Sites
[0191] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a CD3 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide, including CD3-delta (also known as T3D, IMD19, and CD3-DELTA), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-GAMMA). Human CD3 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP 006510029.1 or NP_031674.1, or a polypeptide produced from NCBI Gene ID Numbers 915, 916, or 917. In some embodiments, a binding protein comprising an antigen binding site that binds a CD3 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a CD38 polypeptide. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a HER2 polypeptide. In some
embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds a tumor target protein.
[0192] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the CDR-L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and Q SL VHENLRT Y (SEQ ID NO:62).
[0193] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
[0194] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
[0195] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
[0196] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).
[0197] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:93), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK (SEQ ID NO:95), DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96),
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNR F SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97), and
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98. In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.
[0198] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:93), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
[0199] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:93), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:96). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96. In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96.
[0200] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:93), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNR F SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97.
[0201] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTL YLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:93), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRT YL S W YLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98. In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98. [0202] In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS NSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYW GQGTLVTVSS (SEQ ID NO:302), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S W YLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK (SEQ ID NO:95). In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 302, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, a binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:302, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95.
[0203] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprising an antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2), an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain- heavy chain pair ( e.g ., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD3 antigen binding sites described above represent VH2 and VL2 and form a second antigen binding site that binds a CD3 polypeptide. In some embodiments, Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide, the VH and VL domains of any of the anti-CD3 antigen binding sites described above and/or in Table 5 represent VH2 and VL2 and form a second antigen binding site that binds a CD3 polypeptide, and VH3 and VL3 form a third antigen binding site that binds a tumor target protein (including, without limitation, CD38 or HER2).
[0204] Sequences of exemplary anti-CD3 antigen binding sites are provided in Table 5. In some embodiments, an anti-CD3 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD3 antibody described in Table 5. In some embodiments, an anti-CD3 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD3 antibody described in Table 5.
Table 5. Anti-CD3 binding protein sequences.
Figure imgf000099_0001
Figure imgf000100_0001
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Linkers
[0205] In some embodiments, the linkers L1, L2, L3, and L4 range from no amino acids (length=0) to about 100 amino acids long, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. The linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids long. L1, L2, L3, and L4 in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.
[0206] Examples of suitable linkers include, for example, GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), GGSGSSGSGG (SEQ ID NO: 71), and DKTHT (SEQ ID NO:66), as well as those disclosed in International Publication Nos. WO2017/074878 and W02017/180913. The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the binding proteins.
[0207] The identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.
[0208] In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3, and the length of L2 is at least twice the length of L4. In some embodiments, L1 is 3 to 12 amino acid residues in length, L2 is 3 to 14 amino acid residues in length, L3 is 1 to 8 amino acid residues in length, and L4 is 1 to 3 amino acid residues in length. In some embodiments, L1 is 5 to 10 amino acid residues in length, L2 is 5 to 8 amino acid residues in length, L3 is 1 to 5 amino acid residues in length, and L4 is 1 to 2 amino acid residues in length. In some embodiments, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length.
[0209] In some embodiments, L1, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGS GGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some
embodiments, L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGS GGGGS GGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and
GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO:68), L3 comprises the sequence S, and L4 comprises the sequence RT.
[0210] In some embodiments, at least one of L1, L2, L3 or L4 comprises the sequence DKTHT (SEQ ID NO:66). In some embodiments, L1, L2, L3 and L4 comprise the sequence DKTHT (SEQ ID NO: 66).
Fc regions and constant domains
[0211] In some embodiments, a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. In some embodiments, a binding protein of the present disclosure comprises a third polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. In some embodiments, a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and CH3
immunoglobulin heavy chain constant domains.
[0212] In some embodiments, a binding protein of the present disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, e.g ., a human IgGl, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region includes an antibody hinge, CH1, CH2, CH3, and optionally CH3 domains. In some embodiments, the Fc region is a human IgGl Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some
embodiments, the Fc region includes one or more of the mutations described infra. In some embodiments, the Fc region is an Fc region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4. In some embodiments, the heavy chain constant region is a constant region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4. In some embodiments, the light chain constant region is a constant region of one of the light chain polypeptides (e.g, polypeptide 1 or 4) of a binding protein shown in Table 4.
[0213] In some embodiments, a binding protein of the present disclosure includes one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term "Fc variant" can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term "Fc variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody- dependent cellular cytotoxicity (ADCC). [0214] In some embodiments, a binding protein of the present disclosure ( e.g ., a trispecific binding protein) comprises a“knob” mutation on the second polypeptide chain and a“hole” mutation on the third polypeptide chain. In some embodiments, a binding protein of the present disclosure comprises a“knob” mutation on the third polypeptide chain and a“hole” mutation on the second polypeptide chain. In some embodiments, the“knob” mutation comprises substitution(s) at positions corresponding to positions 354 and/or 366 of human IgGl or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the“knob” mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the“hole” mutation comprises substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 and of human IgGl or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A. In some embodiments, the “hole” mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
[0215] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A.
[0216] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C.
[0217] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution at position corresponding to position 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V.
[0218] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution at position corresponding to position 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitution is T366W.
[0219] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the first and/or second Fc regions are human IgGl Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.
[0220] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K.
[0221] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L2 35A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L2 35A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L2 35A, S354C, and T366W.
[0222] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to reduce effector function, e.g ., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody- dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgGl Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgGl Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgGl Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgGl Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG4 Fc regions, and the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an
immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. [0223] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an
immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235,
354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid
substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.
[0224] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fey I and/or Fcyll binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fcyl and/or Fcyll binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228 and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P and /or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 234 and/or 235 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are F234A and/or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and /or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and /or R409K.
[0225] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region ( e.g ., Fc receptor- mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC)).
[0226] In some embodiments, the Fc region is a human IgGl Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgGl according to EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgGl Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgGl according to EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.
[0227] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve stability, e.g., of the hinge region and/or dimer interface of IgG4 ( See e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve stability. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.
[0228] In some embodiments, the Fc region is a human IgGl Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgGl according to EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgGl Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgGl according to EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.
Nucleic acids
[0229] Other aspects of the present disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. Exemplary and non-limiting nucleic acid sequences are provided in Table 5.
[0230] Other aspects of the present disclosure relate to kits of polynucleotides, e.g ., that encode one or more polypeptides of a binding protein as described herein. In some embodiments, a kit of polynucleotides of the present disclosure comprises one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212; (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232; (1) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:236; (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240; (n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244; (o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268; (u) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; or (v) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276.
[0231] Other aspects of the present disclosure relate to a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein, e.g ., as shown in the polynucleotides of Table 6. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system may be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors. In some embodiments, the first, second, third, and fourth polynucleotides are present on one or more expression vectors, e.g. , one, two, three, or four expression vectors.
[0232] Standard recombinant DNA methodologies are used to construct the
polynucleotides that encode the polypeptides which form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Sambrook et al, 2001 , MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses,
pharmaceutical preparation, formulation, delivery, and treatment of patients.
[0233] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of the binding protein-coding nucleic acid sequence. A promoter may refer to nucleic acid control sequences which direct transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence.
Examples of promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), and the like), from heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, from heat-shock promoters, and the like), the CAG-promoter (Niwa et ah, Gene 108(2): 193-9, 1991), the phosphoglycerate kinase (PGK)-promoter, a tetracycline-inducible promoter (Masui et ah, Nucleic Acids Res. 33:e43, 2005), the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the promoter for 3 -phosphoglycerate kinase, the promoters of yeast acid phosphatase, and the promoter of the yeast alpha-mating factors. Polynucleotides encoding binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or other suitable promoter that will be readily recognized by one skilled in the art.
[0234] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. Expression vectors may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, a- fetoprotein, insulin and the like), and enhancer sequences from a eukaryotic cell virus (such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.). Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art, and can be used to transfect any cell of interest.
Host cells
[0235] Other aspects of the present disclosure relate to a host cell (e.g., an isolated host cell) comprising one or more isolated polynucleotides, vectors, and/or vector systems described herein. In some embodiments, an isolated host cell of the present disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g, an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g, an S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, Drosophila cells (e.g, S2 cells), Trichoplusia ni cells (e.g, High Five™ cells), and
Spodoptera frugiperda cells (e.g, Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human embryonic kidney cells (e.g, 293 or 293 cells subcloned for growth in suspension culture), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g, BHK, ATCC CCL 10), mouse sertoli cells (e.g, TM4 cells), monkey kidney cells (e.g, CV1 ATCC CCL 70), African green monkey kidney cells (e.g, VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g, HELA, ATCC CCL 2), canine kidney cells (e.g, MDCK, ATCC CCL 34), buffalo rat liver cells (e.g, BRL 3A, ATCC CRL 1442), human lung cells (e.g, W138, ATCC CCL 75), human liver cells (e.g, Hep G2, HB 8065), mouse mammary tumor cells (e.g, MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, a human hepatoma line (e.g, Hep G2), and myeloma cells (e.g, NS0 and Sp2/0 cells).
[0236] Other aspects of the present disclosure relate to a method of producing any of the binding proteins described herein. In some embodiments, the method includes a) culturing a host cell (e.g, any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g, any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods of culturing host cells under conditions to express a protein are well known to one of ordinary skill in the art. Methods of isolating proteins from cultured host cells are well known to one of ordinary skill in the art, including, for example, by affinity chromatography (e.g, two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography) .
Pharmaceutical Compositions
[0237] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure. Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
[0238] Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.
[0239] The pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides - e.g ., sodium or potassium chloride - or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g,
REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).
[0240] The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.
[0241] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly
supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the disclosure, binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
[0242] The pharmaceutical compositions of the disclosure can be selected for parenteral delivery or subcutaneous. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the art.
[0243] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
[0244] When parenteral administration is contemplated, the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
[0245] In one embodiment, a pharmaceutical composition can be formulated for inhalation. For example, a binding protein can be formulated as a dry powder for inhalation. Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized.
[0246] It is also contemplated that certain formulations can be administered orally. In one embodiment of the disclosure, binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
[0247] Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
[0248] Additional pharmaceutical compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxy ethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
[0249] Pharmaceutical compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes.
Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
[0250] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready -to-use form or in a form (e.g, lyophilized) requiring reconstitution prior to administration.
[0251] The disclosure also encompasses kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g, liquid syringes and lyosyringes).
[0252] The effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
[0253] Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
[0254] The route of administration of the pharmaceutical composition is in accord with known methods, e.g ., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
[0255] The composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed- release bolus, or continuous administration.
[0256] The pharmaceutical compositions can be used to prevent and/or treat HIV infection. The pharmaceutical compositions can be used as a standalone therapy or in combination with standard anti-retroviral therapy.
The disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples. Such reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any binding protein,
polynucleotide, vector, vector system, and/or host cell described herein. In some
embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition (e.g, HIV infection) and may have a sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
Methods and Uses for Binding Proteins
Virus
[0257] Certain aspects of the present disclosure relate to methods for expanding virus- specific memory T cells. In some embodiments, the methods comprise contacting a virus- specific memory T cell with a binding protein of the present disclosure, e.g ., a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
[0258] In some embodiments, the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo.
[0259] In some embodiments, contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus-specific memory T cells.
[0260] Other aspects of the present disclosure relate to methods for expanding T cells. In some embodiments, the methods comprise contacting a T cell with a binding protein of the present disclosure, e.g. , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
[0261] In some embodiments, the T cell is a memory T cell or an effector T cell.
[0262] In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.
[0263] Other aspects of the present disclosure relate to methods for treating chronic viral infection, e.g. , in an individual in need thereof. In some embodiments, the methods comprise administering to an individual in need thereof an effective amount of a binding protein of the present disclosure, e.g. , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds a CD38 polypeptide.
[0264] In some embodiments, the individual is a human. [0265] In some embodiments, the binding protein is administered to the individual in pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier.
[0266] In some embodiments, administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual.
[0267] In any of the above methods, memory T cells can be CD8+ or CD4+ memory T cells. In any of the above methods, memory T cells can be central memory T cells (TCM) or effector memory T cells (TEM).
Cancer
[0268] Certain aspects of the present disclosure relate to methods for preventing and/or treating cancer in a patient. In some embodiments, the methods comprise administering to the patient a therapeutically effective amount of a binding protein or pharmaceutical composition of the present disclosure.
[0269] In some embodiments, a binding protein of the present disclosure is administered to a patient in need thereof for the treatment or prevention of cancer. In some embodiments, the present disclosure relates to a method of preventing and/or treating a proliferative disease or disorder ( e.g ., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein. In some embodiments, the present disclosure relates to uses of at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein for preventing and/or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the present disclosure relates to at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein for use in the manufacture of a medicament for preventing and/or treating a proliferative disease or disorder (e.g, cancer) in a patient in need thereof. In some embodiments, the patient is a human.
[0270] In some embodiments, the at least one binding protein is administered (or is to be administered) in combination with one or more anti-cancer therapies (e.g, any anti-cancer therapy known in the art, such as a chemotherapeutic agent or therapy). In some
embodiments, the at least one binding protein is administered (or is to be administered) before the one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) concurrently with the one or more anti- cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) after the one or more anti-cancer therapies.
[0271] In some embodiments, the binding protein comprises one or two antigen binding site(s) that binds a T-cell surface protein and another antigen binding site that binds the extracellular domain of a human HER2 polypeptide. In some embodiments, the binding protein comprises an antigen binding site that binds the extracellular domain of a human HER2 polypeptide, an antigen binding site that binds a human CD28 polypeptide, and an antigen binding site that binds a human CD3 polypeptide.
[0272] In some embodiments, cancer cells from the individual express HER2. In some embodiments, the patient is selected for treatment on the basis that the cells of the cancer express a human HER2 polypeptide. Assays known in the art suitable for detecting HER2 expression by cancer cells include, without limitation, immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) assays.
[0273] In some embodiments, the cancer ( e.g ., HER2-positive cancer) is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).
[0274] In some embodiments, the binding protein comprises one or two antigen binding site(s) that binds a T-cell surface protein and another antigen binding site that binds the extracellular domain of a human CD38 polypeptide. In some embodiments, the binding protein comprises an antigen binding site that binds the extracellular domain of a human CD38 polypeptide, an antigen binding site that binds a human CD28 polypeptide, and an antigen binding site that binds a human CD3 polypeptide.
[0275] In some embodiments, cancer cells from the individual express CD38. In some embodiments, cells of the cancer express a human CD38 isoform A polypeptide on their cell surface. In some embodiments, cells of the cancer express a human CD38 isoform E polypeptide on their cell surface. In some embodiments, the patient is selected for treatment on the basis that the cells of the cancer express a human CD38 isoform E polypeptide on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 and do not express CD28.
[0276] In some embodiments, the cancer (e.g., CD38-positive cancer) is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, germinal center B-cell lympohoma or B-cell acute lymphoblastic leukemia. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma.
[0277] In certain embodiments, the cancer is multiple myeloma. Anti-CD38 antibodies have been tested for the treatment of multiple myeloma, such as daratumumab. However, while multiple myeloma is considered treatable, relapse is inevitable in almost all patients, leading to the development of treatment-refractory disease. In some embodiments, the cancer is relapsed or refractory multiple myeloma. In some embodiments, the patient has been treated with a prior multiple myeloma treatment. In some embodiments, a binding protein of the present disclosure is administered to the patient as a 1st, 2nd, or 3rd line treatment for multiple myeloma. Without wishing to be bound to theory, it is thought that an anti- CD38xanti-CD28xanti-CD3 binding protein of the present disclosure may be useful in treating multiple myeloma, e.g ., by recruiting T cells to tumor cells via anti-CD38 (or anti- CD28/anti-CD38), activation of engaged T cells via anti-CD3/anti-CD28, and/or killing of tumor cells through perforin/granzyme-based mechanisms. CD28 has been reported as a novel cancer marker for multiple myeloma. See Nair, J.R. el al. (2011) J. Immunol.
187: 1243-1253.
[0278] Any of the binding proteins described herein may find use in the methods of the present disclosure.
[0279] In some embodiments of any of the methods of the present disclosure, prior to administration of the binding protein, the patient has been treated with daratumumab. As described herein, the present disclosure provides anti-CD38 binding proteins and sites that do not compete for binding CD38 with daratumumab. Without wishing to be bound to theory, it is thought that this is advantageous because a patient previously treated with daratumumab can be treated with a binding protein of the present disclosure, e.g. , without a wash-out period prior to treatment.
[0280] The binding proteins can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantitation of one or more target antigens. The binding proteins will bind the one or more target antigens with an affinity that is appropriate for the assay method being employed.
[0281] For diagnostic applications, in certain embodiments, binding proteins can be labeled with a detectable moiety. The detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety can be a radioisotope, such as 3H, 14C, 32P, 35S, 125I, "Tc, U1ln, or 67Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, b-galactosidase, or horseradish peroxidase.
[0282] The binding proteins are also useful for in vivo imaging. A binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host assayed. The binding protein can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
[0283] For clinical or research applications, in certain embodiments, binding proteins can be conjugated to a cytotoxic agent. A variety of antibodies coupled to cytotoxic agents (i.e., antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers that conjugate the agents to an antibody are known in the art; see, e.g., Parslow, A.C. et al. (2016) Biomedicines 4: 14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11 :2265-2276.
Binding protein therapeutic compositions and administration thereof
[0284] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure. Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. These pharmaceutical compositions may find use in any of the methods and uses described herein (e.g, ex vivo, in vitro, and/or in vivo).
[0285] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
[0286] The pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides - preferably sodium or potassium chloride - or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).
[0287] The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.
[0288] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly
supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the disclosure, binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
[0289] The pharmaceutical compositions of the disclosure can be selected for parenteral delivery or subcutaneous. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.
[0290] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
[0291] When parenteral administration is contemplated, the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
[0292] In one embodiment, a pharmaceutical composition can be formulated for inhalation. For example, a binding protein can be formulated as a dry powder for inhalation. Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized.
[0293] It is also contemplated that certain formulations can be administered orally. In one embodiment of the disclosure, binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
[0294] Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
[0295] Additional pharmaceutical compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
[0296] Pharmaceutical compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes.
Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
[0297] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready -to-use form or in a form (e.g, lyophilized) requiring reconstitution prior to administration.
[0298] The disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples. Such reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
[0299] The disclosure also encompasses kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g, liquid syringes and lyosyringes).
[0300] The effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
[0301] Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data. [0302] The route of administration of the pharmaceutical composition is in accord with known methods, e.g. , orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
[0303] The composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed- release bolus, or continuous administration.
Table 1. Trispecific binding protein polypeptide sequences
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Table 6. Trispecific binding protein polynucleotide sequences
Figure imgf000152_0002
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Figure imgf000191_0001
EXAMPLES
[0304] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the invention in any way.
Example 1: Development of trispecific HER2/CD28xCD3 antibodies and variant anti- CD3 binding sites
[0305] Immuno-oncology is a promising, emerging therapeutic approach to disease management in cancer. The immune system is the first line of defense against cancer development and progression. There is now large evidence that T cells are able to control tumor growth and prolong the survival of cancer patients in both early and late stages of disease. However, T cells specific for tumors can be limited in a number of ways preventing them from controlling the disease.
[0306] In order to remove the limitations on T cells induced by uncontrolled tumors, novel antibodies were developed in the trispecific antibody format depicted in FIG. 1A to specifically activate the T cells to engage HER2 expressing cancer cells. These novel trispecific antibodies are able to bind to three targets: HER2, CD3, and CD28. Anti-HER2 and anti-CD3 binding sites were further optimized for high affinity binding and reduction in potential manufacturing liabilities.
[0307] HER2 amplification and overexpression can be found in molecular subtypes of breast cancer, and also in gastric, ovarian, lung and prostate carcinomas. Optimal activation of T cells requires two factors: (1) Antigen recognition and (2) Co-stimulation. Using the trispecific HER2/CD28xCD3 trispecific binding proteins described herein, Signal 1 is provided by an agonist anti-CD3 binding site, and Signal 2 is provided by an agonist anti- CD28 binding site (see, e.g., FIG. ID). It is thought that the trispecific antibodies described in the subsequent Examples recruit T cells to the tumor via HER2 and activate the engaged T cells by binding to CD3 and CD28. The resulting activation induces the killing potential of the immune cells against the nearby tumor cells.
Materials and Methods
Production and characterization of antibodies
[0308] Trispecific antibody variants were produced by transient transfection of expression plasmids into Expi293 cells. 5 days after transfection, the supernatant from transfected cells was collected, quantified and normalized by absorbance at 280 nm on Nano Drop. The binding of supernatant to corresponding antigens were determined by ELISA and the absorbance of parental HER2 WT tri Ab was set as 1.0. The fold changes of other variants were calculated by dividing the corresponding absorbance to that of parental Ab.
[0309] Trispecific antibody variants were purified using protein A affinity purification followed by SEC purification. The binding of purified antibodies to corresponding antigens were determined by ELISA. The EC50 were determined based on the binding curve generated by Graphpad Prism7.
Results
[0310] Trispecific Ab variants were produced with several mutations in the binding arms in order to mitigate potential manufacturing liabilities, e.g ., deamidation sites. A binding ELISA assay was performed to assess binding of the indicated trispecific antibodies to each of the three targets: HER2, CD3, and CD28. In FIG. IB, HER2/CD3xCD28 trispecific antibodies with the indicated anti-HER2 or anti-CD3 variants were compared to parental Trispecific Ab. Introducing some sets of mutations (e.g, 32/33 QQ and 33/35QQ) into the VL domain of the anti-CD3 binding site led to dramatically reduced binding to CD3, whereas 32/35 QQ mutations retained near wild-type binding. MS peptide analyses showed that binding sites with the DNAQ mutations in CDR-L1 (SEQ ID NO:63) were still subject to greater than 15% deamidation, whereas ENLQ (SEQ ID NO:281), ENLF (SEQ ID NO:282), and ENLR (SEQ ID NO:283) led to less than 5% deamidation. Importantly, these variants also retained binding to CD3.
[0311] In addition, binding curves for the indicated antibodies binding to human HER2, human CD28, and CD3 are provided in FIG. 1C. The EC50 values of selected trispecific antibody variants are provided in Table E.
Table E. A binding ELISA assay was performed on purified trispecific antibodies to determine their binding affinities for HER2, human CD3, and human CD28.
Figure imgf000194_0001
Figure imgf000195_0001
[0312] Without wishing to be bound by theory, as depicted in FIG. ID, it is believed that HER2/CD3/CD28 trispecific antibodies recruit T cells to cancer cells through the anti-HER2 and anti-CD3/CD28 arms. Further, it is believed that engaged T cells are activated by the anti-CD28/CD3 arms. Killing of cancer cells is believed, without wishing to be bound by theory, to occur through T cell mediated mechanisms ( e.g ., Perforin, granzyme). Without wishing to be bound to theory, it is contemplated that similar mechanisms may allow for killing of other types of tumors by substituting antigen binding sites that recognize other tumor target proteins.
Example 2: Development of trispecific CD38/CD3xCD28 antibodies
[0313] Trispecific CD38/CD3xCD28 antibodies were developed and characterized for binding to CD38, CD3 and CD28 polypeptides.
Materials and Methods
Generation of CD38/CD28xCD3 trispecific antibodies
[0314] A panel of anti-CD38, anti-CD3, and anti-CD28 antibodies, as well as human IgG4 Fc domains were used to generate CD38/CD28xCD3 trispecific antibodies in the trispecific antibody format depicted in FIG. 2A.
[0315] Trispecific binding proteins were produced by transient transfection of 4 expression plasmids into Expi293 cells using ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Briefly, 25% (w/w) of each plasmid was diluted into Opti-MEM, mixed with pre-diluted ExpiFectamine reagent for 20- 30 minutes at room temperature (RT), and added into Expi293 cells (2.5xl06 cells/ml). An optimization of transfection to determine the best ratio of plasmids was often used in order to produce the trispecific binding protein with good yield and purity.
[0316] 4-5 days post transfection, the supernatant from transfected cells was collected and filtered through 0.45 pm filter unit (Nalgene). The trispecific binding protein in the supernatant was purified using a 3-step procedure. First, protein A affinity purification was used, and the bound Ab was eluted using“IgG Elution Buffer”(Thermo Fisher Scientific). Second, product was dialyzed against PBS (pH7.4) overnight with 2 changes of PBS buffer. Any precipitate was cleared by filtration through 0.45 pm filter unit (Nalgene) before next step. Third, size-exclusion chromatography (SEC) purification (Hiload 16/600 Superdex 200pg, or Hiload 26/600 Superdex 200pg, GE Healthcare) was used to remove aggregates and different species in the prep. The fractions were analyzed on reduced and non-reduced SDS-PAGE to identify the fractions that contained the monomeric trispecific binding protein before combining them. The purified antibody can be aliquoted and stored at -80°C long term.
ELISA binding assay
[0317] Binding affinities to each target antigen by the CD38/CD28xCD3 T cell engagers were measured by ELISA. Briefly, each antigen was used to coat the 96-well Immuno Plate (Thermo Fisher Scientific) overnight at 4°C using 200 ng/well in PBS(pH7.4) of each antigen. The coated plate was blocked using 5% skim milk+2% BSA in PBS for one hour at RT, followed by washing with PBS+0.25% Tween 20 three times (Aqua Max 400, Molecular Devices). Serial dilution of antibodies (trispecific and control Abs) were prepared and added onto the ELISA plates (100 pl/well in duplicate), incubated at room temperature (RT) for one hour, followed by washing 5 times with PBS+0.25% Tween 20. After washing, the HRP conjugated secondary anti-human Fab (1 :5000, Cat. No. 109-035-097, Jackson
ImmunoResearch Inc) was added to each well and incubated at RT for 30 minutes. After washing 5 times with PBS+0.25% Tween 20, 100 pi of TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA) was added to each well. The reaction was terminated by adding 50 pi 1M H2SO4, and OD450 was measured using SpectraMax M5 (Molecular Devices) and analyzed using SoftMax Pro6.3 software (Molecular Devices). The final data was transferred to GraphPad Prism software (GraphPad Software, CA, USA), and plotted. EC50 was calculated using the same software. Measurement of trispecific antibody binding using SPR
[0318] Human CD38-His antigens were used (Cambridge Biologies, Cambridge, MA) for full kinetic analysis. Kinetic characterization of purified antibodies was performed using SPR technology on a BIACORE 3000 (GE Healthcare). A capture assay using human IgGl specific antibody capture and orientation of the investigated antibodies was used. For capture of Fc containing protein constructs the human antibody capture kit (GE Healthcare) was used. For capture of His tagged antigen, anti-His antibody capture kit (GE Healthcare) was used. The capture antibody was immobilized via primary amine groups (11000 RU) on a research grade CM5 chip (GE Life Sciences) using standard procedures. The analyzed antibody was captured at a flow rate of 10 mL/min with an adjusted RU value that would result in maximal analyte binding signal of typically 30 RU. Binding kinetics were measured against the trispecific antibodies. Assay buffer HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005 % Surfactant P20) was used at a flow rate of 30 mΐ/min. Chip surfaces were regenerated with the regeneration solution of the respective capture kit. Kinetic parameters were analyzed and calculated in the BIA evaluation program package v4.1 using a flow cell without captured antibody as reference and the 1 :1 Langmuir binding model with mass transfer.
Daratumumab competition binding assay
[0319] For Daratumumab competition binding assay, Daratumumab was amine coupled to the active surface of CM5 chip. Reference surface was left blank and used to subtract any non-specific binding of injected molecules. Recombinant CD38-His (Sino Biological, Part# 10818-H08H) was injected over the Daratumumab surface followed by injection of test antibodies. If a monospecific anti-CD38 antibody recognized an epitope on CD38 which was different from that of Daratumumab, injection of the antibody resulted in an increased SPR signal. If an antibody recognized an overlapping epitope as Daratumumab, injection of the antibody did not increase SPR signal.
Results
[0320] The binding affinities of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD38 were determined by SPR. The association rate constant (Kon), dissociation rate constant (Koff), and the KD of the selected trispecific antibodies are provided in Table A. The selected trispecific antibodies showed various degrees of affinities against human CD38 antigen. Table A. Binding characteristics of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD38 determined by SPR.
Figure imgf000198_0001
[0321] The binding affinities of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD3, human CD28, human CD38 and cynomolgus monkey CD38 were then determined by ELISA as described above. As shown in FIGS. 2B-2E, the selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains showed various affinities to human (FIG. 2B) and cynomolgus monkey CD38 (FIG. 2C), but similar affinity to human CD3 (FIG. 2D) and CD28 (FIG. 2E). EC50 values were then calculated by GraphPad Prism 7.02 using variable slope model with four- parameter logistic curve. The EC50 values of the selected trispecific antibodies for human CD3, human CD28, human CD38 and cynomolgus monkey CD38 are provided in Table B. Control antibody was a human IgG4 isotype control.
Table B. EC50 values of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains for human CD3, human CD28, human CD38 and cynomolgus monkey CD38.
Figure imgf000198_0002
Figure imgf000199_0001
[0322] An SPR competition assay was carried out to determine whether anti-CD38 antibodies hhy6284, hhy992, hhy5379, or hhyl 195 (tested in monospecific antibody format) compete with Daratumumab for binding to CD38. Following CD38 injection over
Daratumumab (immobilized on SPR sensor chip), the test antibodies (or Daratumumab) were injected over the Daratumumab/CD38 complex. As shown in FIG. 3, injection of Hyb6264, hhy992, Hyb5379, and Hhyl 195 increased SPR signal, indicating that these antibodies recognized the epitopes on CD38 which are different from the epitope which Daratumumab recognizes. As expected, injection of free Daratumumab (a competitive binding control) did not increase the SPR signal.
[0323] Binding of anti-CD38 antibodies to human or cynomolgus CD38 polypeptides is summarized in Table B2.
Table B2. Summary of anti-CD38 binding characteristics to human or cynomolgus CD38.
Figure imgf000199_0002
[0324] Anti-CD38 antibodies were also tested for competitive binding to daratumumab in SPR assay. For daratumumab competition binding assay, daratumumab was amine coupled to the active surface of CM5 chip. Reference surface was left blank and used to subtract any non-specific binding of injected molecules. Recombinant CD38-His (Sino Biological, Part# 10818-H08H) was injected over the daratumumab surface followed by injection of test antibodies. If an antibody recognizes an epitope on CD38 which is different from that of daratumumab, injection of the antibody will result in an increased SPR signal. If an antibody recognizes an overlapping epitope as daratumumab, injection of the antibody will not increase SPR signal. According to the results of these assays the tested antibodies hhy992, hyb6284, hhyl 195 and hhyl370 did not compete with daratumumab.
Example 3: Trispecific CD38/CD3xCD28 antibodies promote lysis of human multiple myeloma and lymphoma tumor cells.
[0325] An in vitro cell lysis assay was used to determine whether trispecific
CD38/CD3xCD28 antibodies had anti -tumor cell activity using human multiple myeloma and lymphoma cells.
Materials and Methods
In vitro killing assay against tumor cells using human T cells
[0326] Target tumor cells were labeled with the membrane dye PKH-26 (Sigma) and co cultured for 24 hours with human PBMC or enriched CD8 T cells as effector cells at E:T ratio of 10: 1(E:T=3:1 using enriched CD8 T cells) in the presence of indicated concentrations of tri-specific or relevant control antibodies. Peripheral blood mononuclear cells were isolated from normal human donors by Ficoll separation, and autologous CD8+ or pan-T cells were enriched using kits from Miltenyi Biotech (San Diego, CA). The extent of cell lysis in the target cells was determined by staining with a LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Life Technologies) and measured by the number of dead cells in the labelled target cell population by running the samples on an LSRFortessa instrument (BD Biosciences) followed by analysis using the Flowjo software (Treestar).
In vitro killing assay against tumor cells using human T cells in the presence of Daratumumab
[0327] 5 nM Daratumumab or isotype control antibodies were pre-incubated with PKH-
26 labeled target tumor cells (105 cells/well) for 30 minutes, followed by addition of trispecific TCEs at indicated concentrations, and human PBMCs (E:T=10: 1). 24 hours later, the extent of cell lysis in the target cells was determined by staining with a LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Life Technologies) and measured by the number of dead cells in the labelled target cell population by running the samples on an LSRFortessa instrument (BD Biosciences) followed by analysis using the Flowjo software (Treestar).
Results
[0328] The in vitro cell killing activity of CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains was determined using a human multiple myeloma cell line NCI-H929 that expresses both CD38 and CD28. The assay was carried out in the presence of 5nM Daratumumab or isotype control antibodies (present during the assay period). As shown in FIGS. 4A-4B, all tested trispecific antibodies led to cell lysis in a concentration-dependent manner in the presence and absence of Daratumumab. The EC50 values were then calculated in the presence and absence of Daratumumab (Table C). The cell killing activities of trispecific antibodies CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA with the CD38VH1 or
CD38hhyl370 anti-CD38 binding domains were reduced by Daratumumab, while trispecific antibodies with the CD38hyb5739, CD38hyb6284, or CD38hhyl 195 anti-CD38 binding domains exhibited between 3-8 fold reductions in cell killing activity in the presence of Daratumumab (Table C).
Table C. In vitro killing activity against human multiple myeloma cell line NCI-H929 (CD38+/CD28+) by CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains in the presence of Daratumumab.
Figure imgf000201_0001
[0329] In addition, an in vitro cell lysis assay was used to measure the cell killing activity of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains using a human lymphoma cell line OCI-LY19 that expresses CD38 but not CD28. The assay was carried out in the presence of 5nM
Daratumumab or isotype control antibodies which were present in the assay period. As shown in FIGS. 5A-5B, all tested trispecific led to cell lysis in a concentration-dependent manner in the presence and absence of Daratumumab. The EC50 values were then calculated in the presence and absence of Daratumumab (Table D). The cell killing activity of CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with
CD38VH1 anti-CD38 binding domain was reduced by about 24 fold by Daratumumab, while trispecific antibodies with the CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhyl l95, or CD38hhyl370 anti-CD38 binding domains also exhibited reductions in cell killing activity in the presence of Daratumumab (Table D).
Table D. In vitro killing activity against human lymphoma cell line OCI-LY19 (CD38+/CD28-) by selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains in the presence of Daratumumab.
Figure imgf000202_0001
Example 4: CD38/CD28xCD3 trispecific antibodies promote CMV-specific immune response
[0330] As part of adaptive immunity, T cell immunity plays a crucial role in controlling viral infection and cancer, possibly eliminating infected cells and malignant cells which result in clearance of viral infection or cure of cancer. In chronic infectious diseases such as Herpes viral infection (HSV, CMV, EBV, etc.), HIV, and HBV viruses establish their persistence in humans by various mechanisms including immune suppression, T cell exhaustion, and latency establishment. Nevertheless, viral infection generally induces viral antigen specific immunity including antigen specific CD8 T cells that can readily recognize infected cells for controlling or killing through cytokine release or cytotoxic T cell (CTL) mediated killing processes. Thus, viral antigen specific T cell activation and/or amplification in vivo and/or ex vivo provide therapeutic strategies against chronic viral infections.
[0331] Anti-CD38/CD28xCD3 trispecific antibodies were developed and evaluated for their potential in activating T cells, and promoting proliferation and/or amplification of antigen specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations, including antigen specific CD8 T central memory and effector memory cells in vitro. Specifically, in vitro expansion of CMV and EBV specific CD8 central memory and effector memory cells were demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibited novel properties by engaging
CD3/CD28/CD38, providing signaling pathways to stimulate and expand T cells, which may offer an effective strategy treating chronic infectious diseases such as HSV, CMV, EBV, HIV-1, and HBV infections.
[0332] In this Example, the ability of CD38/CD28xCD3 trispecific antibodies to promote activation and expansion of CMV-specific T cells was determined.
Materials and Methods
In vitro T cell proliferation measurement
[0333] T cells were isolated from human PBMC donors by negative selection using a magnetic Pan T Cell Isolation Kit (Miltenyi Biotec GmbH, Germany). Antibodies were coated onto 96-well cell culture plates by preparing the antibodies in sterile PBS and dispensing 50 mL into each well (350 ng/well). The plates were then incubated at 37°C for at least 2 hours and then washed with sterile PBS. The untouched T cells were added to the antibody-coated plates (5 x 105 cells/mL) and incubated at 37°C for multiple days. The cells were passaged with new cell culture media onto fresh antibody-coated plates on Day 4. In certain experiments with 7 days incubation, only fresh medium was added without changing to fresh antibody-coated plate. The cells were collected at specific time points and cell numbers calculated using CountBright™ counting beads.
In vitro T cell proliferation assay and T cell subset determination
[0334] Peripheral blood mononuclear cells were isolated from blood of healthy human donors collected by Research Blood Components, LLC (Boston, MA). The PBMCs were added to antibody-coated plates (350 ng/well) (5 x 105 cells/mL), as previously described above, and incubated at 37°C for 3 and 7days. The cells were collected at specific time points and analyzed by flow cytometry for T cell subsets: naive (CCR7+ CD45RO-), Tcm (CCR7+ CD45RO+), Tem (CCR7- CD45RO+), Tregs (CD4+ Foxp3+ CD25hi). CMV pp65-specific and EBV BMLF-specific CD8+ T cells were detected using fluorescent-conjugated pentamer restricted to the PBMC donors’ HLA/viral peptide (A*02:01/NLVPMVATV, SEQ ID
NO:284), (A*02:01/GLCTLVAML, SEQ ID NO:285), respectively (Prolmmune, Oxford, UK). PBMC was obtained from HemaCare (Van Nuys, CA) for donors with known CMV or EBV infection. PMBC from donors negative for the restricting HLA type was used as negative control. Staining was done as per manufacturer’s protocol.
Quantification of CMV-specific T-Cells
[0335] As indicated above Peripheral blood mononuclear cells (PBMCs) were isolated from blood of known CMV-infected human donors and added to plates containing the trispecific antibody or control antibody. The plates were incubated at 37°C. The cells were collected at specific time points and analyzed by flow cytometry.
Results
[0336] CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains DVH1CD38 (control), CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhyl l95, and CD38hhyl370 were tested as described above using PBMCs isolated from CMV-infected human donor D (FIGS. 6A-6J) and CMV- infected human donor E (FIGS. 7A-7J). All tested CD38 trispecific Abs activated and promoted the proliferation of CMV-specific T cells, leading to increases in CMV-specific CD8+ T cells (cells/well) with different potency and kinetics in a dose response manner over the 7 day experiment (CMV Donor D, FIGS. 6A-6B; CMV Donor E, FIGS. 7A-7B). In addition, all tested CD38 trispecific Abs promoted the amplification (cells/well) of CMV- specific central memory (Tcm) (CMV Donor D, FIGS. 6C-6D; CMV Donor E, FIGS. 7C- 7D) and effector memory (Tem) CD8+ T cells (CMV Donor D, FIGS. 6E-6F; CMV Donor E, FIGS. 7E-7F), which were both amplified dramatically in 7 days. FIGS. 6G-6J (CMV Donor D) and FIGS. 7G-7J (CMV Donor E) provide time courses showing the percent of CMV-specific Tcm and Tem cells at days 0, 3, and 7 of the 7-day experiments described above.
[0337] Taken together, these data indicate that CD38/CD28xCD3 trispecific antibodies promote activation and expansion of CMV-specific T cells, such as CMV-specific CD8+ T cells, CMV-specific effector memory (Tem) CD8+ T cells, and CMV-specific central memory (Tcm) CD8+ T cells. Example 5: CD38/CD28xCD3 trispecific antibodies promote EBV-specific immune response
[0338] Next, the ability of CD38/CD28xCD3 trispecific antibodies to promote activation and expansion of Epstein-Barr virus (EBV)-specific T cells was determined.
Materials and Methods
Quantification of EBV-specific T-Cells
[0339] As indicated above, peripheral blood mononuclear cells (PBMCs) were isolated from blood of known EBV-infected human donors and added to plates containing the trispecific antibody or control antibody. The plates were incubated at 37°C for up to 11 days. The cells were collected at specific time points and analyzed by flow cytometry.
Results
[0340] CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains DVH 1CD38 (control), CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhyl 195, and CD38hhyl370 were also tested as described above using PBMCs isolated from EBV-infected donor C (FIGS. 8A-8J) and EBV-infected donor D (FIGS. 9A-12). All tested CD38 trispecific Abs activated T cells and promoted the proliferation of EBV-specific T cells, leading to increases in EBV-specific CD8+ T cells (cells/well) with different potency and kinetics in a dose response manner over the 7 day experiment (EBV Donor C, FIGS. 8A-8B; EBV Donor D, FIGS. 9A-9B). In addition, all tested CD38 trispecific Abs promoted the amplification (cells/well) of EBV- specific central memory (Tcm) (EBV Donor C, FIGS. 8C-8D; EBV Donor D, FIGS. 9C-9D) and effector memory (Tem) CD8+ T cells (EBV Donor C, FIGS.8E-8F; EBV Donor D,
FIGS. 9E-9F), which were both amplified dramatically in 7 days. FIGS. 8G-8J (EBV Donor C) and FIGS. 9G-12 (EBV Donor D) provide time courses showing the percent of EBV- specific Tcm and Tem cells at days 0, 3, and 7 of the 7-day experiments described above.
[0341] Taken together, these data indicate that CD38/CD28xCD3 trispecific antibodies promote activation and expansion of EBV-specific T cells, such as EBV-specific CD8+ T cells, EBV-specific effector memory (Tem) CD8+ T cells, and EBV-specific central memory (Tcm) CD8+ T cells. Example 6: Anti-tumor effects of Her2/CD28 x CD3 trispecific antibody in tumor-bearing mice
[0342] In this Example, the Her2/CD28 x CD3 trispecific antibody was tested for anti tumor effects in a ZR-75-1 tumor bearing Nod scid gamma (NSG) mouse model engrafted with in vitro expanded T cells.
Materials and Methods
[0343] NSG mice were divided into 5 groups of 10 mice each. On Day 0, ZR-75-1 human breast cancer cells were implanted into the mammary fat pad with 50% matrigel into each mouse at 5 million cells/mouse. On Days 17/18, expansion of human CD3+ T cells was begun. Randomization of mice occurred on Day 24 when tumors were approximately 150mm3. On Day 25, all mice were engrafted with in vitro expanded human CD3+ T cells at 10 million cells in 300mL/mouse (1QW, 1 IP injection).
[0344] Starting on Day 25, one group of mice received doses of vehicle alone (8% w/v sucrose, 0.05% w/v polysorbate 80, lOmM histidine, pH 5.5), while the other 4 groups received Her2/CD28 x CD3 trispecific antibody, both at lOmL/kg. Groups receiving trispecific antibody were dosed at 100, 10, 1, or 0.1 mg/kg. Antibody or vehicle was administered 1QW intravenously in 2 doses ( e.g ., Days 25 and 32). Blood and tumor tissue was collected on Day 38 or 39.
Results
[0345] Her2/CD28 x CD3 trispecific antibody (binding protein #2 from Table 1, corresponding to SEQ ID Nos: 104-107) was compared to vehicle control for its effects on human breast tumor growth in the NSG mouse model engrafted with in vitro expanded human T cells described above. Treatment with Her2/CD28 x CD3 trispecific antibody at the highest dose (lOOug/kg) led to the most significant inhibition of tumor growth and regression, although the lOug/kg dose also showed anti -tumor effects (FIGS. 13A & 13D). No significant body weight loss was observed (FIG. 13B). Individual tumor volumes over time from each trispecific antibody treatment group are provided in FIG. 13C.
[0346] Next, the effect of trispecific antibody treatment on individual immune cell subsets was examined. Human CD45+, human CD8+, and human CD4+ cell populations were measured by flow cytometry, as well as mouse CD45+ cells (FIGS. 14A-14C). Highest dose (100ug/kg) of trispecific antibody led to depletion of human CD4+ cells, and this effect was dose dependent (FIGS. 14B & 14C). Counts of human CD8+ cells were largely unaffected by trispecific antibody administration. [0347] The effect of trispecific antibody treatment on tumor infiltrating lymphocytes (TILs) was also assessed by immunohistochemical (IHC) staining for human CD45, CD4, and CD8. Using H&E staining, tumors from the low dose groups (lug/kg or 0. lug/kg trispecific antibody) were generally of comparable size as the vehicle control group. As shown in FIGS. 15A-15C, human TILs were increased in the group receiving the low dose of Her2/CD28 x CD3 trispecific antibody, but human TILs were sparse in the high dose group. IHC images were also examined quantitatively (FIGS. 16A-16C). These results indicated significant reductions in CD45+ and CD8+ cells in the higher trispecific antibody dose groups (lOOug/kg and lOug/kg).
[0348] Compared to vehicle control, tumors from the high dose trispecific antibody treatment groups (lOOug/kg or lOug/kg) were characterized by sparse TILs. Moderate to large numbers of CD45+, CD4+, or CD8+ human TILs were observed in the lug/kg and 0. lug/kg trispecific antibody treatment groups. These TILs were mostly present at the tumor edges but occasionally extended deeper into the tumor core.
[0349] In conclusion, these results demonstrate that treatment of ZR-75-1 breast tumor bearing NSG mice engrafted with in vitro activated T cells using 2 intravenous doses of HER2 -targeting, T cell-engaging trispecific antibody at lOOug/kg or lOug/kg resulted in significant reductions in tumor volume and, concomitantly, a significant decrease in TILs. At the lug/kg trispecific antibody dose, there was a marginal and inconsistent trend for increased TILs as compared to vehicle control.
Example 7: Effect of anti-HER2 and anti-CD3 antigen binding domain sequences in Her2/CD28 x CD3 trispecific antibody on cancer cell killing
[0350] This Example describes the effect of anti-Her2 and anti-CD3 variable domain sequences on target cell killing. In this Example, a Her2/CD28 x CD3 trispecific antibody (“control”) with wild-type trastuzumab antigen binding domain and an anti-CD3 antigen binding domain without 32/35 QQ mutations in the VL domain (see Example 1) was compared with Her2/CD28 x CD3 trispecific antibodies #1-6 from Table 1, corresponding to SEQ ID Nos: 100-103, 104-107, 286-289, 290-293, 294-297, and 298-301, respectively.
Materials and Methods
[0351] CD8+ T cells were isolated from human PBMCs from healthy donor using a magnetic bead isolation kit (Miltenyi Biotec). The T cells were used as effector cells against breast cancer cell lines expressing various levels of HER2 at 3: 1 (EffectonTarget) ratio. The cells were incubated with experimental or control trispecific antibody for 2 days before flow cytometry acquisition using viability dye (Invitrogen) and PKH26 target cell staining
(Sigma). Mean EC50 for target cell lysis was calculated from 2-3 PBMC donors for each trispecific Ab.
Results
[0352] All trispecific antibodies were characterized for in vitro cell lysis of three HER2+ breast cancer target cell lines: HCC1954, BT20, and MDA-MB-231. HCC1954 breast cancer cells were found to express high levels of HER2, as assessed by flow cytometry (up to -150,000 receptors/cell), IHC (3+), or the HercepTest HER2 expression assay (3+) (FIG. 17A). BT20 breast cancer cells were found to express intermediate levels of HER2, as assessed by flow cytometry (-60,000 receptors/cell), IHC (1+), or the HercepTest HER2 expression assay (1+) (FIG. 17C). MDA-MD-231 breast cancer cells were found to express low levels of HER2, as assessed by flow cytometry (-9,000 receptors/cell), IHC (0+), or the HercepTest HER2 expression assay (0) (FIG. 17E). Results of the cell killing assays targeting HCC1954, BT20, or MDA-MB-231 are shown in FIGS. 17B, 17D, and 17F, respectively, comparing binding protein #2 vs. control or binding proteins #1 and #5 vs. control. The results demonstrated that the Her2/CD28 x CD3 trispecific antibodies having 30R/55Q/102E mutations in the anti-HER2 arm and 32/35 QQ mutations in the VL domain of the anti-CD3 arm showed improved target cell killing against all three cell lines, particularly at lower antibody concentrations.
[0353] Mean EC50 (pM) for in vitro cell killing was determined for all trispecific antibodies targeting the three breast cancer cell lines noted above (HCC1954, BT20, and MDA-MB-231) as well as the gastric cancer cell lines OE19 (high HER2 expression) and GSU (intermediate HER2 expression). Generally, the Her2/CD28 x CD3 trispecific antibodies having mutations in the anti-HER2 arm and in the VL domain of the anti-CD3 arm showed a lower EC50 (and thus superior cell killing) against all three breast cancer cell lines (FIG. 18A) and both gastric cancer cell lines (FIG. 18B). These results demonstrate that, while all trispecific antibodies are able to induce cell killing of HER2+ cells, the mutated trispecific antibodies consistently displayed improved cell killing efficacy against multiple target cell types.

Claims

CLAIMS What is claimed is:
1. A binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein VH1 and VL1 form a first antigen binding site; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and wherein VH3 and VL3 form a third antigen binding site.
2. The binding protein of claim 1, wherein the first binding site binds a CD28 polypeptide.
3. The binding protein of claim 2, wherein the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the VL1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54).
4. The binding protein of claim 3, wherein the Vm domain comprises the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNV NTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWG KGTTVTVSS (SEQ ID NO:91), and/or the VL1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTG VPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92).
5. The binding protein of any one of claims 1-4, wherein the CDR-L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of Q SLVHQNAQT Y (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and Q SL VHENLRT Y (SEQ ID NO:62).
6. The binding protein of claim 5, wherein the VH2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:93) or
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AS S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD YW GQGTLVTVSS (SEQ ID NO:302), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95),
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96),
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNR F SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97), and
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98).
7. The binding protein of any one of claims 1-6, wherein the third antigen binding site binds a tumor target protein.
8. The binding protein of any one of claims 1-6, wherein the third antigen binding site binds a human CD38 polypeptide.
9. The binding protein of claim 8, wherein:
(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18); (b) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24);
(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30);
(d) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36);
(e) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42); or
(f) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48).
10. The binding protein of claim 9, wherein:
(a) the VH3 domain comprises the amino acid sequence of
Figure imgf000213_0001
(b) the VH3 domain comprises the amino acid sequence of
Figure imgf000213_0002
(c) the VH3 domain comprises the amino acid sequence of
Figure imgf000213_0003
(d) the VH3 domain comprises the amino acid sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPY Y GDTT Y AQKF QGRVTMTVDRS S ST AYMEL SRLRSDDT AVYY C ARRFEGF YY SMD Y WGQGTL VTVSS (SEQ ID NO:85), and/or the VL3 domain comprises the amino acid sequence of
Figure imgf000213_0004
Figure imgf000214_0001
(e) the VH3 domain comprises the amino acid sequence of
Figure imgf000214_0002
sequence of
Figure imgf000214_0003
or
(f) the VH3 domain comprises the amino acid sequence of
Figure imgf000214_0004
1 1. The binding protein of claim 8, wherein:
(a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175; (f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179;
(g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184; or
(h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.
12. The binding protein of any one of claims 1-7, wherein the third antigen binding site binds a human HER2 polypeptide.
13. The binding protein of claim 12, wherein: (a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRW GGDGF YAMD Y (SEQ ID NO: 6), SRW GGEGF YAMD Y (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12);
(b) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12);
(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12);
(d) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (e) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12);
(f) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12); or
(g) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
14. The binding protein of claim 13, wherein:
(a) the VH3 domain comprises the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO: 72),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:73),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG
YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO: 74),
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO: 75), or
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or
DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78);
(b) the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);
(c) the VH3 domain comprises the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:73), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);
(d) the VH3 domain comprises the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO:75), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGKAPKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);
(e) the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO:74), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGKAPKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);
(f) the VH3 domain comprises the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGKAPKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77); or
(g) the VH3 domain comprises the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO:78).
15. The binding protein of claim 12, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123;
(f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127;
(g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131; (h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135;
(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139;
(j) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143;
(k) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147;
(l) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151;
(m) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155;
(n) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289; (o) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293;
(p) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297; or
(q) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.
16. The binding protein of any one of claims 1-15, wherein at least one of L1, L2, L3 or L4 is independently 0 amino acids in length.
17. The binding protein of any one of claims 1-15, wherein (a) L1, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71); or (b) L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO:70), S, RT, TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO:71).
18. The binding protein of any one of claims 1-15, wherein L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO:68), L3 comprises the sequence S, and L4 comprises the sequence RT.
19. The binding protein of any one of claims 1-15, wherein at least one of L1, L2, L3 or L4 comprises the sequence DKTHT (SEQ ID NO:66).
20. The binding protein of claim 19, wherein L1, L2, L3 and L4 comprise the sequence DKTHT (SEQ ID NO:66).
21. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.
22. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.
23. The binding protein of any one of claims 1-22, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
24. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.
25. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S.
26. The binding protein of any one of claims 1-25, wherein the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-CH2- CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
27. The binding protein of any one of claims 1-25, wherein the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
28. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein of any one of claims 1-27.
29. An expression vector comprising the nucleic acid molecule of claim 28.
30. An isolated host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29.
31. The isolated host cell of claim 30, wherein the host cell is a mammalian or insect cell.
32. A pharmaceutical composition comprising the binding protein of any one of claims 1- 27 and a pharmaceutically acceptable carrier.
33. A method of preventing and/or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of claims 1-27 or the pharmaceutical composition of claim 32.
34. The method of claim 33, wherein the at least one binding protein is co-administered with a chemotherapeutic agent.
35. The method of claim 33 or claim 34, wherein the patient is a human.
36. The method of any one of claims 33-35, wherein the third antigen binding site binds a human CD38 polypeptide, and wherein cancer cells from the patient express CD38.
37. The method of claim 36, wherein the cancer is multiple myeloma.
38. The method of claim 36, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma.
39. The method of any one of claims 36-38, wherein, prior to administration of the binding protein, the patient has been treated with daratumumab without a wash-out period.
40. The method of any one of claims 33-35, wherein the third antigen binding site binds a human HER2 polypeptide, and wherein cancer cells from the patient express HER2.
41. The method of claim 40, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).
42. The binding protein of any one of claims 7-27 or the pharmaceutical composition of claim 32 for use in preventing and/or treating cancer in a patient.
43. The binding protein for use or the composition for use of claim 42, wherein the at least one binding protein is to be co-administered with a chemotherapeutic agent.
44. The binding protein for use or the composition for use of claim 42 or claim 43, wherein the patient is a human.
45. The binding protein for use or the composition for use of any one of claims 42-44, wherein the third antigen binding site binds a human CD38 polypeptide, and wherein cancer cells from the patient express CD38.
46. The binding protein for use or the composition for use of claim 45, wherein the cancer is multiple myeloma.
47. The binding protein for use or the composition for use of claim 45, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma.
48. The binding protein for use or the composition for use of any one of claims 45-47, wherein, prior to administration of the binding protein, the patient has been treated with daratumumab without a wash-out period.
49. The binding protein for use or the composition for use of any one of claims 42-44, wherein the third antigen binding site binds a human HER2 polypeptide, and wherein cancer cells from the patient express HER2.
50. The binding protein for use or the composition for use of claim 49, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).
51. A method for expanding T cells, comprising contacting a T cell with a binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
52. The method of claim 51, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.
53. The method of claim 51 or claim 52, wherein the T cell is a memory T cell or an effector T cell.
54. A method for expanding virus-specific memory T cells, comprising contacting a virus- specific memory T cell with a binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
55. The method of claim 54, wherein the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo.
56. The method of claim 54 or claim 55, wherein contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus-specific memory T cells.
57. A method for treating chronic viral infection, comprising administering to a patient in need thereof an effective amount of a binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
58. The method of claim 57, wherein the patient is a human.
59. The method of claim 57 or claim 58, wherein the binding protein is administered to the patient in pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier.
60. The method of any one of claims 57-59, wherein administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the patient.
61. The method of any one of claims 53-56 and 60, wherein the memory T cells are CD8+ or CD4+ memory T cells.
62. The method of any one of claims 53-56, 60, and 61, wherein the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).
63. The method of any one of claims 54-62, wherein the virus is a human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 1 (HSV-2).
64. The method of any one of claims 51-63, wherein the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.
65. A binding protein for use in expanding T cells, wherein the binding protein is to be contacted with a T cell, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
66. The binding protein for use of claim 65, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.
67. The binding protein for use of claim 65 or claim 66, wherein the T cell is a memory T cell or an effector T cell.
68. A binding protein for use in expanding virus-specific memory T cells, wherein the binding protein is to be contacted with a virus-specific memory T cell, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
Vm is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
69. The binding protein for use of claim 68, wherein the virus-specific memory T cell is to be contacted with the binding protein in vitro or ex vivo.
70. The binding protein for use of claim 68 or claim 69, wherein contacting the virus- specific memory T cell with the trispecific binding protein causes activation and/or proliferation of virus-specific memory T cells.
71. A binding protein for use in treating chronic viral infection, wherein the binding protein is to be administered to a patient in need thereof, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
72. The binding protein for use of claim 71, wherein the patient is a human.
73. The binding protein for use of claim 71 or claim 72, wherein the binding protein is to be administered to the patient in pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier.
74. The binding protein for use of any one of claims 71-73, wherein administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the patient.
75. The binding protein for use of any one of claims 67-70 and 74, wherein the memory T cells are CD8+ or CD4+ memory T cells.
76. The binding protein for use of any one of claims 67-70, 74, and 75, wherein the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).
77. The binding protein for use of any one of claims 68-76, wherein the virus is a human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 1 (HSV-2).
78. The binding protein for use of any one of claims 65-77, wherein the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.
79. The method or binding protein for use of any one of claims 51-78, wherein the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the VL1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54).
80. The method or binding protein for use of claim 79, wherein the Vm domain comprises the amino acid sequence of
Figure imgf000239_0002
81. The method or binding protein for use of any one of claims 51-80, wherein the CDR- L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and Q SL VHENLRT Y (SEQ ID NO:62).
82. The method or binding protein for use of claim 81, wherein the VH2 domain comprises the amino acid sequence of
Figure imgf000239_0001
RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98).
83. The method or binding protein for use of any one of claims 51-82, wherein:
(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18);
(b) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24);
(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30);
(d) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36); (e) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42); or
(f) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45), and the VL3 domain comprises a CDR- L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48).
84. The method or binding protein for use of claim 83, wherein:
(a) the VH3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQ GGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWG QGTLVTVSS (SEQ ID NO:79), and/or the VL3 domain comprises the amino acid sequence of DIVLTQ SP ATLSL SPGERATISCRASQ S V S S YGQGFMHW YQQKPGQPPRLLI Y GAS SR ATGIP ARE SGSGSGTDFTLTISPLEPEDF AVYYCQQNKEDPWTF GGGTKLEIK (SEQ ID NO:80);
(b) the VH3 domain comprises the amino acid sequence of
Q VQL VQSGAEVKKPGAS VKVSCKVSGYTLTEF SIHWVRQ APGQGLEWMGGFDPED GETIY AQKFQGRVIMTEDT STDT AYMEMN SLRSEDT AIYYCTT GRFFDWF W GQGTL VTVSS (SEQ ID NO:81), and/or the VL3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPV RF SGSGSGTDF SLTIS SLQPEDC AVYYCQQDSNLPITF GQGTRLEIK (SEQ ID NO:82);
(c) the VH3 domain comprises the amino acid sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGS GSTNY AQKF QGRVTMTVDRS STT AYMEL SRLRSDDT AVYY CARY AY GYW GQGTL
Figure imgf000242_0001
(d) the VH3 domain comprises the amino acid sequence of
Figure imgf000242_0002
sequence of
S
Figure imgf000242_0003
(e) the VH3 domain comprises the amino acid sequence of
Figure imgf000242_0004
sequence of
Figure imgf000242_0005
or
(f) the VH3 domain comprises the amino acid sequence of
Figure imgf000242_0006
85. The method or binding protein for use of any one of claims 51-82, wherein:
(a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175;
(f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179;
(g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184; or
(h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.
86. A vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein of any one of claims 1-27.
87. The vector system of claim 86, wherein the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.
88. A kit of polynucleotides, comprising:
(a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192;
(b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196;
(c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200;
(d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204;
(e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208;
(f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212;
(g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216;
(h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220;
(i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224;
(j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228;
(k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232;
(l) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:236;
(m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240;
(n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244;
(o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248;
(p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252;
(q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256;
(r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260;
(s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264;
(t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268;
(u) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; or
(v) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276.
89. The kit of claim 88, wherein the first, second, third, and fourth polynucleotides are present on one or more expression vectors.
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