WO2019226658A1 - Compositions multispécifiques de liaison à l'antigène et procédés d'utilisation - Google Patents

Compositions multispécifiques de liaison à l'antigène et procédés d'utilisation Download PDF

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WO2019226658A1
WO2019226658A1 PCT/US2019/033325 US2019033325W WO2019226658A1 WO 2019226658 A1 WO2019226658 A1 WO 2019226658A1 US 2019033325 W US2019033325 W US 2019033325W WO 2019226658 A1 WO2019226658 A1 WO 2019226658A1
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antigen
binding
antibody
cancer
cell
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Michael March SCHMIDT
Rachel Rennard
Jennifer Watkins
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Compass Therapeutics Llc
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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
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • Cancer is one of the leading causes of death, accounting for almost one in six deaths worldwide in 2015, according to the American Association for Cancer Research (AACR) Cancer Progress Report of 2017.
  • the molecular mechanisms involved in cancer are highly complex.
  • immune cells such as T cells, natural killer cells, and macrophages, exhibit anti-tumor activity and can effectively control the development and/or progression of tumors.
  • the immune cells recognize tumor-specific or tumor-associated antigens and eliminate cells expressing the antigens.
  • tumors also constitute highly suppressive microenvironments and can downregulate the function of infiltrating immune cells.
  • the patient’s immune system may not recognize cancer cells as foreign or may not be strong enough to destroy the cancer cells.
  • the cancer cells may produce substances that allow the cancer cells to evade the patient’s innate immune response.
  • Immunotherapeutic approaches in particular, have been developed in recent years to utilize the patient’s endogenous immune system cells (e.g., T cells, natural killer cells, and macrophages) to inhibit tumor formation and progression.
  • endogenous immune system cells e.g., T cells, natural killer cells, and macrophages
  • the present disclosure is based, at least in part, on the discovery of multispecific antigen-binding constructs that are capable of enhancing an immune response against cancer cells. While the disclosure is not bound by any particular theory or mechanism of action, the constructs described herein, when bound to a tumor antigen expressed by a cancer cell, are capable of agonizing CD226 activity on adjacent immune effector cells and thereby enhancing an immune response (e.g., increasing NK effector function, T cell proliferation, IFNy production and secretion, and/or the cytolytic activity of T cells) toward the cancer cells to which the constructs are bound.
  • multispecific antigen-binding constructs are provided that agonize CD226 function and enhance the immune response with respect to tumor cells that express the tumor antigen (e.g., B cell maturation antigen (BCMA or Her2)).
  • the disclosure provides multispecific (e.g., bispecific) antigen-binding constructs that include at least two linked antigen-binding units, wherein a first antigen-binding unit specifically binds a tumor antigen, and wherein a second antigen-binding unit specifically binds a CD226 antigen.
  • the tumor antigen is associated with a carcinoma, sarcoma, myeloma, leukemia, lymphoma, or combination thereof.
  • the tumor antigen is an epithelial cancer antigen, a prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a bladder cancer antigen, a lung cancer (e.g., small cell lung) antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, a colorectal cancer antigen, a lymphoma (e.g., non-Hodgkin’s lymphoma or Hodgkin’s lymphoma) antigen, a B-cell lymphoma cancer antigen, a leukemia antigen, a myeloma (e.g., multiple myeloma or plasma cell myeloma) antigen, an acute lymphoblastic leukemia antigen, a chronic lympho
  • the first antigen-binding unit is a polypeptide, small molecule, or an aptamer.
  • the first antigen-binding unit is an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding portion thereof of the first antigen-binding unit is optionally a monoclonal antibody or antigen-binding portion thereof.
  • the antibody or antigen-binding portion thereof of the first antigen-binding unit can be a humanized antibody, a fully human antibody, or an antigen-binding portion of either.
  • the antibody or antigen-binding portion thereof of the first antigen-binding unit is optionally an scFv or Fab antibody fragment.
  • the multispecific antigen-binding construct binds to Her2. In some embodiments, the multispecific antigen-binding construct binds to BCMA.
  • the second antigen-binding unit is a polypeptide, small molecule, or an aptamer.
  • the second antigen-binding unit is an antibody or antigen-binding fragment thereof.
  • the second antigen-binding unit is optionally a monoclonal antibody or antigen-binding portion thereof.
  • the second antigen-binding unit can be a humanized antibody or a fully human antibody.
  • the second antigen-binding unit is optionally a scFv or Fab antibody fragment.
  • the antigen-binding construct optionally further comprises a third antigen-binding unit that binds specifically to a molecule expressed by an effector immune cell.
  • the molecule expressed by the effector immune cell is CD 16, CDl6a, CDl6b,
  • CD32a, CD64, or CD89 are examples of CD32a, CD64, or CD89.
  • the two or more antigen-binding units disclosed herein may be the same or different structure within the same antigen-binding construct.
  • all antigen-binding units can be antibodies or antigen-binding portions thereof or a subset can be antibodies or antigen binding portions thereof.
  • the second antigen-binding unit is optionally capable of agonizing CD226.
  • the constructs described herein are capable of agonizing CD226 expressed by an immune effector cell when the constructs are bound to a cell (e.g., a cancer cell) expressing the tumor antigen.
  • any of the constructs described herein are capable of modulating (e.g., increasing, stimulating, enhancing) the activity of T cells. In some embodiments, any of the constructs described herein are capable of modulating (e.g., increasing, stimulating, enhancing) the activity of NK cells.
  • any of the constructs described herein comprise any of the anti-CD226 antibodies or antigen-binding portions or fragments described herein.
  • compositions that include a multispecific antigen binding construct (e.g., a bispecific antigen-binding construct) disclosed herein and a pharmaceutically acceptable carrier.
  • the antigen-binding construct optionally further comprises a third antigen-binding unit that specifically binds to a molecule expressed by an effector immune cell.
  • the composition may include more than one multispecific antigen-binding construct disclosed herein.
  • the disclosure further provides methods for treating cancer in a subject, including the step of administering to the subject having cancer an effective amount of a multispecific antigen-binding construct described herein or a composition including a multispecific antigen-binding construct described herein.
  • the subject may be a human or other mammal.
  • the multispecific antigen-binding construct may enhance the subject’s immune response by agonizing CD226 function.
  • the cancer is optionally selected from the group consisting of a hematological cancer, neurological cancer, breast cancer, prostate cancer, skin cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, and vascular cancer.
  • the methods for treating cancer may further include the step of administering a second anticancer therapy to the subject.
  • the anticancer therapy is chemotherapy, immunotherapy, hormone therapy, cytokine therapy, radiotherapy, cryotherapy, or surgical therapy.
  • the multispecific antigen-binding construct or composition is administered, for example, subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.
  • the disclosure also provides methods of enhancing an immune response in a subject in need thereof, comprising administering to the subject (e.g., a human or other mammal) a therapeutically effective amount of a multispecific antigen-binding construct described herein or a composition that includes the disclosed multispecific antigen-binding construct.
  • the enhanced immune response includes one or more of enhanced T cell function, enhanced NK cell function, or enhanced macrophage function.
  • the multispecific antigen-binding construct or composition is administered subcutaneously, intravenously, intradermally,
  • intraperitoneally orally, intramuscularly, or intracranially.
  • an isolated antibody or antigen-binding portion thereof that binds to human CD226, wherein the antibody or antigen-binding portion thereof comprises a heavy chain polypeptide comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 20, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 21, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO:22; and a light chain polypeptide comprising a CDRL1 having the amino acid sequence set forth in SEQ ID NO:23, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:24, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:25.
  • the heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the heavy chain polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the light chain polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:
  • an isolated antibody or antigen-binding portion thereof that binds to human CD226, wherein the antibody or antigen-binding portion thereof comprises a heavy chain polypeptide comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 26, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 27, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO:28; and a light chain polypeptide comprising a CDRL1 having the amino acid sequence set forth in SEQ ID NO:29, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:30, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:3 l.
  • the heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the heavy chain polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the light chain polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15.
  • any antibody or antigen-binding portion thereof described herein is a humanized antibody or a fully human antibody (or antigen-binding fragments thereof).
  • any antibody or antigen-binding portion thereof described herein is an scFv or a Fab antibody fragment.
  • any antibody or antigen-binding portion thereof described herein is an IgGl, IgG2, IgG3, IgG4, IgA, IgE, or IgM isotype antibody.
  • composition comprising any isolated antibody or antigen-binding portion thereof described herein and a pharmaceutically acceptable carrier.
  • nucleic acid comprising a nucleotide sequence encoding any antibody or antigen-binding portion thereof or any multispecific antigen-binding construct described herein.
  • the disclosure further provides a vector comprising the nucleic acid and operably linked to an expression control sequence.
  • a cell comprising the vector.
  • the cell can be a mammalian cell, e.g., a rodent cell (e.g., a CHO or NS0 cell), a non human primate cell or a human cell.
  • a method for producing a protein the method comprising culturing the cell under conditions suitable for expression of the antibody, antigen-binding portion thereof, or multispecific antigen-binding construct.
  • the method can also include isolating the antibody, antigen-binding fragment, or multispecific antigen binding construct from the cultured cell or cells and/or the media in which the cell or cells are cultured.
  • any of the antibodies or antigen-binding portions thereof described herein are capable of modulating (e.g., increasing, stimulating, enhancing) the activity of T cells.
  • any of the constructs described herein are capable of modulating (e.g., increasing, stimulating, enhancing) the activity of NK cells.
  • T cell function or activity, or activation of T cells refers to a cellular process in which mature T cells, which express antigen-specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cell- based effector functions.
  • T cell activation typically requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second by subsequent engagement of co- stimulatory molecules (e.g., CD28).
  • TCR T cell antigen-specific receptor
  • MHC antigen-major histocompatibility complex
  • enhanced T cell function encompasses increased T cell function or activity and/or enhanced survival and/or enhanced proliferation of the T cell.
  • T cell-mediated response refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells, including subsets thereof, such as THI , TH2, TH3, TH17, TH9, and TFH cells).
  • T cell-mediated responses include, for example, T cell cytotoxicity, T cell cytokine secretion, and proliferation.
  • NK cell function or activity, or activation of NK cells refers to a cellular process by which NK cells respond to cognate ligands or to the multispecific antigen-binding constructs described herein by entering the cell cycle (i.e., proliferating), increasing cell-surface expression of one or more activation markers, secreting or increasing secretion of cytokines or chemokines (including IFN-g, TNF-a, IL-17A, and IL-22) or lytic enzymes (e.g., perforin and granzymes), and initiating or becoming competent to perform cell-based effector functions.
  • Methods for measuring such activities are routine and known in the art, and exemplary methods are described herein, such as in the Examples.
  • a cytokine assay such as an ELISA or cytokine bead array assay, can be used to determine the increase.
  • an increase in cytokine production in the presence of the multispecific antigen-binding constructs is at least l-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more compared to a control or reference antibody.
  • the assay comprises detecting an increase in surface expression of at least one activation marker on the NK cells, for example using flow cytometry.
  • the disclosure further provides methods for treating cancer in a subject, including the step of administering to the subject having cancer an effective amount of an anti-CD226 antibody or antigen-binding portion thereof described herein or a composition including the antibody or portion thereof.
  • the subject can be a human or other mammal.
  • the antibody or antigen-binding portion thereof can enhance the subject’s immune response by agonizing CD226 function.
  • the cancer is optionally selected from the group consisting of a
  • the methods for treating cancer can further include the step of administering a second anticancer therapy to the subject.
  • the anticancer therapy is chemotherapy, immunotherapy, hormone therapy, cytokine therapy, radiotherapy, cryotherapy, or surgical therapy.
  • the antibody, antigen-binding portion thereof, or composition can be administered, for example, subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.
  • the disclosure also provides methods of enhancing an immune response in a subject in need thereof, comprising administering to the subject (e.g., a human or other mammal) a therapeutically effective amount of an antibody or antigen-binding portion thereof described herein or a composition that includes the disclosed antibody or portion thereof.
  • the enhanced immune response includes one or more of enhanced T cell function, enhanced NK cell function, or enhanced macrophage function.
  • FIG. 1 is an illustrative representation of an exemplary antibody format (e.g., as described in Kontermann and Brinkmann (2015) Drug Discovery Today 20(7)).
  • FIG. 2 is a bar graph depicting the effect of multispecific antigen-binding constructs on the proliferation of target cells.
  • the Y-axis represents the cell index as a measure of the proliferation of SK-BR3 target cells in the presence of (i) CD8+ T cells, and (ii) the Control Construct, Construct 3 (C3), or Construct 4 (C4), at 24 hours and 48 hours incubation.
  • the immune system has the capability of recognizing and eliminating tumor cells; however, tumor cells can use multiple strategies to evade the immune system.
  • Blockade of immune checkpoints is one of the approaches to activating or reactivating therapeutic antitumor immunity.
  • Another approach to inhibit cancer formation or progression is by enhancing the function of various effector molecules and interactions involved in native immune responses.
  • the present disclosure provides multispecific antigen-binding constructs that enhance the native immune response.
  • the disclosure provides multispecific (e.g., bispecific) antigen-binding constructs that include at least two linked antigen-binding units, wherein a first antigen-binding unit specifically binds a tumor antigen, and wherein a second antigen-binding unit specifically binds a CD226 antigen.
  • the tumor antigen is a tumor-specific antigen (TSA).
  • TSA tumor-specific antigen
  • a TSA is an antigen that is unique to tumor cells and does not occur on other cells in the body.
  • the tumor antigen is a tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • a TAA is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • a TAA is expressed on normal cells during fetal development when the immune system is immature and unable to respond or is normally present at extremely low levels on normal cells but which is expressed at much higher levels on tumor cells.
  • the TAA is determined by sequencing a patient’s tumor cells and identifying mutated proteins only found in the tumor. These antigens are referred to as “neoantigens.” Once a neoantigen has been identified, therapeutic antibodies can be produced against it and used in the methods described herein.
  • the tumor antigen is an epithelial cancer antigen, a prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a bladder cancer antigen, a lung cancer antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, a colorectal cancer antigen, a lymphoma antigen, a B-cell lymphoma cancer antigen, a leukemia antigen, a myeloma antigen, an acute lymphoblastic leukemia antigen, a chronic myeloid leukemia antigen, or an acute myelogenous leukemia antigen.
  • PSA prostate specific cancer antigen
  • PSMA prostate specific membrane antigen
  • BCMA B cell maturation antigen
  • TNF receptor superfamily The B cell maturation antigen (BCMA) is an example of a tumor antigen that may be targeted by the disclosed multispecific antigen-binding constructs.
  • BCMA is a cell surface receptor of the TNF receptor superfamily and is normally expressed on mature B cells.
  • BCMA naturally occurs in various isoforms.
  • human BCMA comprises the amino acid sequence of SEQ ID NO:6.
  • BCMA is also known as BCM and tumor necrosis factor receptor superfamily 17 (TNFRSF17).
  • BCMA binds to its ligand B cell activating factor (BAFF), which is a member of the TNF family and is expressed by T cells and dendritic cells for the purpose of B cell costimulation.
  • BAFF ligand B cell activating factor
  • BCMA is detected on certain cancer cells, for example, multiple myeloma cells.
  • Additional tumor antigens that may be targeted by the disclosed multispecific antigen binding constructs include, but are not limited to, 1GH-IGK, 43-9F, 5T4, 79lTgp72, 9D7, acyclophilin C-associated protein, a-fetoprotein (AFP), a-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, BCR-ABL, b-catenin, b-HCG, BrE3-antigen, BCA225, BING-4, BRCA1/2, BTAA, CA125, CA l5-3 ⁇ CA 27.29VBCAA, CA195, CA242, CA-50, calcium activated chloride channel 2, CAGE, CAM43, CAMEL, CAP-l, carbonic anhydrase IX, c-Met, CA19-9, CA72-4, CAM 17.1, CASP-8/m, CCCL19, CCCL21, CD1, CD la, CD2, CD3, CD4, CD5, CD8, CD
  • the tumor antigen is a viral antigen derived from a virus associated with a human chronic disease or cancer (such as cervical cancer).
  • the viral antigen is derived from Epstein-Barr virus (EBV), HPV antigens E6 and/or E7, hepatitis C virus (HCV), hepatitis B virus (HBV), or cytomegalovirus (CMV).
  • CD226 Cluster of differentiation 226
  • CD226 is an approximately 65 kDa polypeptide found on the surface of natural killer cells, platelets, monocytes, and some T cells.
  • the amino acid sequence of human CD226 comprises SEQ ID NO:7, although at least one other naturally occurring variant occurs.
  • CD226 also known as DNAX accessory molecule-l (DNAM-l)
  • DNAM-l DNAX accessory molecule-l
  • CD155 also known as poliovirus receptor (PVR), nectin-like protein 5 (Necl-5), Tage4, HVED, PVS).
  • CD155 is a transmembrane glycoprotein with three extracellular immunoglobulin-like domains, is highly expressed on dendritic cells (DC), FDC, fibroblasts, and endothelial cells, and is also found on some tumor cells. Binding of CD226 to CD155 may allow immune system cells to attack tumor cells, and CD155 is believed to play a role in NK cell adhesion and triggering NK cell effector functions.
  • DC dendritic cells
  • FDC fibroblasts
  • endothelial cells and is also found on some tumor cells. Binding of CD226 to CD155 may allow immune system cells to attack tumor cells, and CD155 is believed to play a role in NK cell adhesion and triggering NK cell effector functions.
  • the present disclosure provides compositions and methods for enhancing an immune response to tumor cells by agonizing certain immune response effector molecules and interactions.
  • Multispecific antigen-binding constructs that include at least two linked antigen binding units that recognize specific target antigens are provided.
  • multispecific antigen-binding construct as used herein includes bispecific, trispecific, tetraspecific, or multispecific antigen-binding constructs.
  • a multispecific antigen-binding construct can be a single multifunctional polypeptide, small molecule, or aptamer, or it can be a multimeric complex of two or more molecules that are covalently or non-covalently associated with one another.
  • Multispecific antigen-binding constructs include antibodies (or antigen-binding fragments thereof) that may be linked to or co-expressed with another functional molecule, e.g., another peptide, protein, and/or aptamer.
  • another functional molecule e.g., another peptide, protein, and/or aptamer.
  • an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as a protein or fragment thereof to produce a multispecific antigen binding construct with a second binding specificity.
  • an antibody or antigen-binding fragment thereof is functionally linked to one or more antibody or antigen binding fragment thereof having a different binding specificity to produce a multispecific antigen-binding construct.
  • Each antibody or antigen-binding portion thereof of the construct may have one or more antigen-binding specificities.
  • an antigen-binding unit refers to a domain, region, or the like, of the multispecific antigen-binding construct that forms an area of the construct that binds to an antigen.
  • a first antigen-binding unit forms a separate binding area of the multispecific antigen-binding construct from a second antigen-binding unit of the construct, each unit forming a separate region of antigen-binding.
  • one unit (first unit) is distinct from the other unit (second unit) in its antigen-binding.
  • one antigen-binding unit of the construct is monovalent for and binds to a tumor antigen (e.g., BCMA) whereas the other antigen-binding unit of the construct is monovalent for and binds to CD226.
  • a tumor antigen e.g., BCMA
  • the other antigen-binding unit of the construct is monovalent for and binds to CD226.
  • one antigen-binding unit of the construct binds to a tumor antigen whereas the other arm of the construct binds to CD226 or a related molecule (cross-reacts with two antigens due to, e.g., similarity in structure). See, e.g., U.S. Pat. No. 9,845,356.
  • the multispecific construct is tetravalent.
  • one antigen-binding unit is bivalent for a tumor antigen, each binding the same epitope on the tumor antigen, whereas the other antigen-binding unit is bivalent for CD226, each binding the same epitope on CD226.
  • the multispecific construct is tetravalent, wherein one antigen-binding unit is bivalent for a tumor antigen, each binding two different epitopes on the tumor antigen.
  • the multispecific construct is tetravalent, wherein one antigen-binding unit is bivalent for CD226, each binding two different epitopes on a ligand of CD226.
  • the multispecific construct is tetravalent, wherein one antigen-binding unit is bivalent for a tumor antigen, each binding two different but overlapping epitopes of the tumor antigen. In some embodiments, the multispecific construct is tetravalent, wherein one antigen-binding unit is bivalent for CD226, each binding two different but overlapping epitopes of CD226.
  • valency when used to describe an antigen-binding construct or protein, refers to the number of recognition (binding) sites in the antigen-binding construct or protein. Each recognition site specifically recognizes, and is therefore capable of binding, one epitope (binding site) on an antigen.
  • an antigen-binding protein comprises more than one recognition site (e.g., when an antigen-binding protein is an IgG, which has two recognition sites in its variable regions), each recognition site can specifically recognize the same epitope on the same antigen, or different epitopes, whether on the same or different antigens.
  • the multispecific antigen-binding constructs comprise a first antigen-binding unit that specifically binds a tumor antigen and a second antigen-binding unit that specifically binds CD226.
  • first antigen-binding unit that specifically binds a tumor antigen
  • second antigen-binding unit that specifically binds CD226.
  • specific binding specifically binds to, specific for, selectively binds, selective for, and the like as related to a particular target antigen or molecule (e.g., a polypeptide target) or an epitope on a particular target antigen or molecule mean binding that is measurably different from a non-specific or non-selective interaction.
  • Specific binding can be measured, for example, by determining binding of a target molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.
  • epitope means a component of an antigen capable of specific binding to an antigen-binding construct or unit. Epitopes frequently consist of surface-accessible amino acid residues and/or sugar side chains and can have specific three- dimensional structural characteristics, as well as specific charge characteristics.
  • Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • An epitope can comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding.
  • the epitope to which an antigen binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen-binding protein binding to antigen variants with different point mutations.
  • At least one antigen-binding unit has a KD of at least 1 x 10 7 M, at least 1 x 10 8 M, at least 1 x 10 9 M, at least 1 x 10 10 M, at least 1 x 10 11 M, at least 1 x 10 12 M, or at least 1 x 10 13 M.
  • the antigen-binding units of the construct have the same or similar KD.
  • KD refers to the dissociation equilibrium constant of a particular antigen-binding unit / antigen interaction.
  • KD kd/k a .
  • kd (sec -1 ) refers to the dissociation rate constant of a particular antigen-binding unit/antigen interaction. This value is also referred to as the k 0ff value.
  • the binding of one antigen-binding unit (e.g., the first antigen binding unit) of the multispecific antigen-binding construct to its target does not block or sterically hinder the binding of other antigen-binding unit(s) (e.g., the second antigen-binding unit) to its target.
  • the second or subsequent antigen-binding unit is free to bind a CD226 antigen.
  • the first antigen-binding unit and second antigen-binding unit bind to their respective targets concurrently.
  • binding of the first antigen-binding unit and the second antigen-binding unit to their respective targets bridges the immune cell and the second cell together, bringing the two cells in close proximity.
  • bridge refers to the joining of two cell types (e.g., one immune cell that expresses CD226 and a second cell that expresses a tumor antigen) or bringing of the two cells together in close proximity, such that the two cells need not be in physical contact.
  • the multispecific antigen-binding construct acts as a connector (e.g., a bridge) to the two cells, each one expressing either CD226 or a tumor antigen.
  • the bridging of the immune cell and the second cell is determined by, e.g., flow cytometry, FRET,
  • the constructs of the present disclosure are capable of binding to one or more immune cells that expresses CD226 and one or more cells that expresses a tumor antigen.
  • the type of immune cell depends on the context of the disease to be treated, and the particular type of immune cell can be determined by one of skill in the art depending on the disorder under consideration.
  • the immune cell is a T cell, including a CD8+ T cell and CD4+ T cell.
  • the immune cell is a natural killer (NK) cell.
  • the immune cell is a B cell.
  • the immune cell is a macrophage.
  • the constructs are capable of binding one or more tumor cells (a solid or non-solid tumor cell).
  • tumor cell is sometimes used as used herein.
  • the tumor cell is a cancer cell that can be treated by enhancing CD226 function, while bridging the immune cell and the cell expressing a tumor antigen (e.g., BCMA-expressing tumor cell).
  • a tumor antigen e.g., BCMA-expressing tumor cell
  • the tumor cell is selected from the group consisting of a hematological cancer, a lymphoma, a myeloma, a leukemia, a neurological cancer, skin cancer, breast cancer, a prostate cancer, a colorectal cancer, lung cancer, head and neck cancer, a gastrointestinal cancer, liver cancer, pancreatic cancer, a genitourinary cancer, a bone cancer, renal cancer, and a vascular cancer.
  • the tumor cell (and specific tumor antigens associated with such tumor cells, but not exclusively) is selected from the group consisting of Kaposi’s sarcoma, leukemia, acute lymphocytic leukemia (etv6, amll, cyclophilin b), acute myelocytic leukemia, myeloblasts promyelocyte myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia (cyclophilin b), mantle cell lymphoma, primary central nervous system lymphoma, Burkitt’s lymphoma, marginal zone B cell lymphoma (Ig- idiotype), Polycythemia vera Lymphoma, Hodgkin’s disease (Imp-l, EBNA-l), non- Hodgkin’s disease, myeloma (MUC family, p2lras), multiple myel,
  • Wilm s tumor, cervical cancer, uterine cancer, testicular tumor (NY-ESO-l), lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma (HER2/neu, c-erbB-2), bladder carcinoma, epithelial carcinoma, glioma (E-cadherin, a-catenin, b-catenin, g-catenin, pl20ctn), astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal oma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma (p5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides, Mel an- A/M ART- 1, cdc27, MAGE-3, p2lras, gplOO), neuroblastoma,
  • the disclosed multispecific antigen-binding constructs include bispecific, trispecific, tetraspecific, or multispecific antibodies or antigen-binding fragments or portions thereof.
  • immunoglobulin or antibody refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an intact immunoglobulin, all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul (2013) Fundamental Immunology 7th ed., Ch. 5, Lippincott Williams & Wilkins, Philadelphia, PA.
  • each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region typically comprises three domains, CHI, Cm, and Cm.
  • Each light chain typically comprises a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region typically comprises one domain, abbreviated CL.
  • An antibody as used herein, can refer to intact antibodies (e.g, intact
  • Antigen-binding fragments can be used interchangeably with an intact antibody.
  • Antigen-binding fragments comprise at least one antigen-binding domain.
  • One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer.
  • Antibodies and antigen-binding fragments can be described by the antigen to which they specifically bind.
  • a CD226 antibody, or anti-CD226 antibody is an antibody that specifically binds to CD226.
  • the VH and VL regions can be further subdivided into regions of hypervariability (hypervariable regions (HVRs), also called complementarity determining regions (CDRs)) interspersed with regions that are more conserved.
  • the more conserved regions are called framework regions (FRs).
  • Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the CDRs are involved in antigen-binding and confer antigen specificity and binding affinity to the antibody.
  • the three heavy chain CDRs can be referred to as CDRH1, CDRH2, and CDRH3, and the three light chain CDRs can be referred to as CDRL1, CDRL2, and CDRL3.
  • Rabat also referred to as“numbered according to Rabat,” “Rabat numbering”, “Rabat definitions”, and “Rabat labeling,” provides an unambiguous residue numbering system applicable to any variable domain of an antibody, and provides precise residue boundaries defining the three CDRs of each chain.
  • CDRs are referred to as Kabat CDRs and comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 31-35 (CDR1), 50-65 (CDR2) and 95- 102 (CDR3) in the heavy chain variable domain.
  • the light chain FR residues are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues.
  • the "EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.
  • CDRs can be referred to as“Chothia CDRs,”“Chothia numbering,” or“numbered according to Chothia,” and comprise about residues 24-34 (CDR1), 52-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-32 (CDR1), 50-56 or 52-56 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain.
  • CDR1 Cyclona CDRs
  • CDR2 residues 24-34
  • CDR2 52-56
  • CDR3 89-97
  • MacCallum also referred to as“numbered according to MacCallum,” or“MacCallum numbering” comprises about residues 30-36 (CDR1), 46-55 (CDR2) and 89-96 (CDR3) in the light chain variable domain, and 30-35 (CDR1), 47-58 (CDR2) and 93-101 (CDR3) in the heavy chain variable domain.
  • MacCallum et al. ((1996) J. Mol. Biol. 262(5):732-745).
  • AbM The system described by AbM, also referred to as“numbering according to AbM,” or “AbM numbering” comprises about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-35 (CDR1), 50-58 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain.
  • the IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering of variable regions can also be used, which is the numbering of the residues in an immunoglobulin variable heavy or light chain according to the methods of the IMGT, as described in Lefranc, M.-P.,“The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains,” The Immunologist, 7, 132-136 (1999), and is expressly incorporated herein in its entirety by reference.
  • “IMGT sequence numbering” or“numbered according to IMTG” refers to numbering of the sequence encoding a variable region according to the IMGT.
  • the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3.
  • the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3.
  • the CDRs recited herein comprise about residues 24-34 (CDR1), 49-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 27-35 (CDR1), 49-60 (CDR2) and 93-102 (CDR3) in the heavy chain variable domain, when numbered according to Chothia numbering.
  • CDR2 in the light chain variable domain can comprise amino acids 49-56, when numbered according to Chothia numbering.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g ., 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%, or at least 99% identical) to SEQ ID NO: 12 or 14.
  • the antibody or antigen binding portion thereof that specifically binds human CD226 comprises a light chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., 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%, or at least 99% identical) to SEQ ID NO: 13 or 15.
  • the heavy chain variable region comprises an amino acid sequence that differs by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less,
  • the light chain variable region comprises an amino acid sequence that differs by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less,
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 12, and three light chain CDRs of the light chain variable region of SEQ ID NO: 13.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 12, and three light chain CDRs of the light chain variable region of SEQ ID NO: 13, wherein the heavy and light chain CDR residues are numbered according to Rabat.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 12, and three light chain CDRs of the light chain variable region of SEQ ID NO: 13, wherein the heavy and light chain CDR residues are numbered according to Chothia.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 12, and three light chain CDRs of the light chain variable region of SEQ ID NO: 13, wherein the heavy and light chain CDR residues are numbered according to MacCallum.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 12, and three light chain CDRs of the light chain variable region of SEQ ID NO: 13, wherein the heavy and light chain CDR residues are numbered according to AbM.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 12, and three light chain CDRs of the light chain variable region of SEQ ID NO: 13, wherein the heavy and light chain CDR residues are numbered according to IMGT.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 14, and three light chain CDRs of the light chain variable region of SEQ ID NO: 15.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 14, and three light chain CDRs of the light chain variable region of SEQ ID NO: 15, wherein the heavy and light chain CDR residues are numbered according to Rabat.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 14, and three light chain CDRs of the light chain variable region of SEQ ID NO: 15, wherein the heavy and light chain CDR residues are numbered according to Chothia.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 14, and three light chain CDRs of the light chain variable region of SEQ ID NO: 15, wherein the heavy and light chain CDR residues are numbered according to MacCallum.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 14, and three light chain CDRs of the light chain variable region of SEQ ID NO: 15, wherein the heavy and light chain CDR residues are numbered according to AbM.
  • the antibody or antigen-binding portion thereof that specifically binds human CD226 comprises three heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 14, and three light chain CDRs of the light chain variable region of SEQ ID NO: 15, wherein the heavy and light chain CDR residues are numbered according to IMGT.
  • chimeric antibody or antibody fragment refers to an antibody or antibody fragment in which a component of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • Humanized forms of non-human antibodies or antibody fragments are chimeric antibodies or antibody fragments that contain minimal sequence derived from the non-human antibody or antibody fragment.
  • a humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
  • the donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
  • selected framework region residues of the recipient antibody are replaced by the
  • Humanized antibodies or antibody fragments can also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. (See Jones et al. (1986) Nature 321 :522-525; Riechmann et al. ( 1988) Nature, 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol. 2:593-596).
  • a human antibody or antibody fragment is one that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies or antibody fragments specifically exclude humanized antibodies or antibody fragments.
  • an antibody molecule comprises a diabody and a single-chain molecule, as well as an antigen-binding fragment of an antibody (e.g., Fab, F(ab')2, and Fv).
  • an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL).
  • VH heavy chain variable domain sequence
  • VL light chain variable domain sequence
  • an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody).
  • an antibody molecule in another example, includes two heavy chain variable domain sequences and two light chain variable domain sequence, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen.
  • Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies.
  • the preparation of antibody molecules can be monoclonal or polyclonal.
  • An antibody molecule can also be a human, humanized, CDR-grafted, or an in vitro generated antibody.
  • the antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4.
  • the antibody can also have a light chain chosen from either kappa or lambda light chains.
  • Antigen-binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH,
  • a F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;
  • a Fd fragment consisting of the VH and CH1 domains;
  • a Fv fragment consisting of the VL and VH domains of a single arm of an antibody,
  • a diabody (dAb) fragment which consists of a VH domain;
  • a single chain Fv (scFv) see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.
  • Antibody molecules can also be single domain antibodies.
  • Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be any known in the art or any future single domain antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are disclosed in WO 94/04678, for example.
  • variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
  • VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca, and guanaco) or other species besides Camelidae.
  • constructs and antigen-binding units described herein can comprise, in part, scaffold domains, proteins, or portions, e.g., molecules which do not provide target receptor binding activity, but which can provide a portion or domain of the construct which provides spatial organization, structural support, a means of linking of multiple receptor-binding units, or other desired characteristics, e.g, improved half-life.
  • Various scaffold technologies and compositions are known in the art and can be readily linked or conjugated to the antigen binding units described herein.
  • the scaffold domain, protein, or portion can be derived from an antibody or not derived from an antibody.
  • Such scaffold proteins, and domains thereof are, generally, obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins.
  • Non-antibody protein scaffolds can be considered to fall into two structural categories, domain-sized constructs (in the range of 6 to 20 kDa), and constrained peptides (in the 2-4 kDa range).
  • Domain-sized non-antibody scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, pronectins and OBodies.
  • Peptide-sized non-antibody scaffolds include, for example, avimers, bicyclic peptides and cysteine knots.
  • non antibody scaffolds and the underlying proteins or peptides on which they are based or from which they have been derived are reviewed by, e.g, Simeon and Chen, Protein Cell 9(1): 3- 14 (2016); Vazquez-Lombardi et al, Drug Discovery Today 20: 1271-1283 (2015), and by Binz et al, Nature Biotechnol. 23: 1257-1268 (2005), the contents of each of which are herein incorporated by reference in their entireties.
  • Advantages of using non-antibody scaffolds include increased affinity, target neutralization, and stability.
  • Various non-antibody scaffolds also can overcome some of the limitations of antibody scaffolds, e.g, in terms of tissue penetration, smaller size, and thermostability.
  • non-antibody scaffolds can also permit easier construction, not being hindered, for example, by the light chain association issue when bispecific constructs are desired.
  • Methods of constructing constructs on a non antibody scaffold are known to those of ordinary skill in the art. While not formally on an antibody scaffold, such constructs often include antibody binding domains, whether in the form of single-domain antibodies, scFvs or other antibody binding-domain variants that provide specific target-binding capabilities.
  • a construct can comprise a non-antibody scaffold protein.
  • at least one of the receptor-binding units can comprise a non-antibody scaffold protein.
  • the scaffold portion of a non antibody scaffold protein can include, in some embodiments, e.g ., an adnectin scaffold or a portion derived from human tenth fibronectin type III domain (l0Fn3); an anticalin scaffold derived from human lipocalin (e.g, such as those described in, e.g, W02015/104406); an avimer scaffold or a protein fragment derived from the A-domain of low density -related protein (LRP) and/or very low density lipoprotein receptor (VLDLR); a fynomer scaffold or portion of the SH3 domain of FYN tyrosine kinase; a kunitz domain scaffold or portion of Kunitz-type protease inhibitors, such as a human trypsin inhibitor, aprotinin (bovine pancreatic trypsin inhibitor), Alzheimer’s amyloid precursor protein, and tissue factor pathway inhibitor; a knottin scaffold (cysteine knot miniprotein
  • elateriunr an affibody scaffold or all or part of the Z domain of S. aureus protein A; a b-Hairpin mimetic scaffold; a Designed ankyrin repeat protein (DARPin) scaffold or artificial protein scaffolds based on ankyrin repeat (AR) proteins; or any scaffold derived or based on human transferrin, human CTLA-4, human crystallin, and human ubiquitin.
  • DARPin Designed ankyrin repeat protein
  • AR kyrin repeat
  • the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al. (1999) J. Biol. Chem.
  • the portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites.
  • the libraries are then screened, as an antibody library is, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol. 23(l0):514-522.
  • the scaffold portion of the non-antibody scaffold protein can include, e.g., all or part of the Z domain of S. aureus protein A, human transferrin, human tenth fibronectin type III domain, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins, such as those described in, e.g., WO2015/104406), human crystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.
  • the multispecific antigen-binding construct comprises a bispecific antibody, having specificity for at least two antigens but optionally having more than two binding sites.
  • a bispecific antibody molecule is characterized by a first
  • immunoglobulin variable domain sequence that has binding specificity for a first antigen (e.g., BCMA or other tumor antigen) and a second immunoglobulin variable domain sequence that has binding specificity for a second antigen (e.g., CD226).
  • a first antigen e.g., BCMA or other tumor antigen
  • a second immunoglobulin variable domain sequence that has binding specificity for a second antigen (e.g., CD226).
  • a bispecific antibody molecule comprises a scFv or fragment thereof having binding specificity for a first antigen and a scFv or fragment thereof having binding specificity for a second antigen (See, e.g., Kontermann and Brinkmann (2015) Drug
  • bispecific antibody formats are known in the art, including, for example, a bispecific IgG, a bispecific antibody fragment, a bispecific fusion protein, an appended IgG, and a bispecific antibody conjugate, described herein.
  • Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgGl /lgG2, dual acting Fab (DAF)-lgG, and Mab 2 bispecific formats (see, e.g., Klein et al. (2012) mAbs 4:6, 1-11, and references cited therein, for a review
  • the multispecific antigen-binding constructs disclosed herein comprise an antibody that comprises a common light chain.
  • the antibody or antigen-binding fragment thereof of the second antigen-binding unit comprises known antibodies or the CDRs of known CD226 antibodies, for example, anti-CD226 [DX11] ab33397 (Abeam, Cambridge, MA). See Lozano et al. (2013) J. Immunol. 191 :3673-80.
  • CD226 antibodies useful in this regard include, by way of example, antibodies described in ET.S. Pat. Nos. 9,034,324, 9,273,141, and 9,243,058 and PCT Publication WO2016090327.
  • the antibody or antigen-binding fragment thereof of the first antigen-binding unit comprises known antibodies or the CDRs of known antibodies to a tumor antigen.
  • trademarked and non-proprietary names of exemplary tumor antigen-targeting antibodies currently approved for use in cancer therapy by the U.S. Food and Drug Administration and/or the European Medicines Agency include, but are not limited to: LEMTRADA® (alemtuzumab, SanofiGenzyme; see e.g., Keating et al. (2002) Blood 99(l0):3556-356l; Hillmen et al. (2007) J. Clin. Oncol.
  • PERJETA® pertuzumab, Genentech; Baselga et al. (2012) N. Engl. J. Med. 366(2): 109-119; Agus et al. (2005) J. Clin. Oncol. 23(11):2534-2543
  • RFFUXAN® rituximab, Biogen; see e.g., Feugier (2015) Future Oncol. 11(9): 1327-1342
  • ARZERRA® (ofatumumab, Novartis; see e.g., Teeling et al. (2006) J. Immunol. l77(l):362-37l; Teeling et al.
  • PORTRAZZA® necitumumab, Eli Lilly; see e.g., Divmann and Tabemero (2010) Curr. Opin. Investig. Drugs 11(12): 1434-1441
  • HERCEPTIN® tacuzumab, Genentech; see e.g., Slamon et al. (2001) N. Engl. J. Med. 344(11):783-792; Maximiano et al. (2016) BioDrugs 30(2):75-86
  • KADCYLA® ado-trastuzumab emtansine, Genentech; see e.g., Verma et al. (2012) N. Engl. J.
  • Additional exemplary tumor antigen-targeting antibodies include, but are not limited to, I-131-BC8 (alternatively known as Iomab-B, Actinium Pharmaceuticals), talacotuzumab (alternatively known as JNJ-56022473, Janssen), vadastuximab talirine (Seattle Genetics), ublituximab (TG Therapeutics), moxetumomab pasudotox (AstraZeneca/Medlmmune), XMAB-5574 (alternatively known as MOR208, Xencor), oportuzumab monatox (Viventia Bio), margetuximab (MacroGenics), MM-302 (Merrimack Pharmaceuticals), sacituzumab govitecan (Immunomedics, Inc.), glembatumumab vedotin (Celldex Therapeutics), andecaliximab (Gilead Sciences
  • the antibody or antigen-binding fragment thereof of the first antigen-binding unit comprises a heavy chain variable region comprising a CDRH1, a CDRH2, and a CDRH3 of SEQ ID NO:2 and a light chain variable region comprising a CDRL1, a CDRL2, and a CDRL3 of SEQ ID NO: 1, optionally with one or more
  • the antibody or antigen-binding fragment thereof of the first antigen-binding unit comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 and a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
  • the multispecific antigen-binding construct comprises at least one heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.
  • the multispecific antigen-binding construct comprises at least one heavy chain comprising an amino acid sequence of SEQ ID NO: 2.
  • the multispecific antigen-binding construct comprises at least one light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
  • the multispecific antigen-binding construct comprises at least one light chain comprising an amino acid sequence of SEQ ID NO: 1.
  • Identity or similarity with respect to a sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • at least 90% identity includes 95%, 98%, and 99% identity, and every percentage between 90% and 100%, inclusively.
  • the antibody or antigen-binding fragment thereof of the second antigen-binding unit comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3 of a CD226 antibody selected from anti-CD226 [DX11] ab33397 (Abeam, Cambridge, MA) as described in Lozano et al. (2013) J. Immunol. 191 :3673-80; U.S. Pat. Nos. 9,034,324, 9,273,141, or 9,243,058; or PCT Publication W02016090327.
  • the antibody or antigen-binding fragment thereof of the second antigen-binding unit comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to, and a light chain comprising an amino acid sequence that is at least 90% identical to, the sequence of any one of the antibodies described in Lozano et al. (2013) J. Immunol. 191 :3673-80; U.S. Pat. Nos. 9,034,324, 9,273,141, or 9,243,058; or PCT
  • the antigen-binding construct comprises at least one copy of a heavy chain that includes both CDRH sequences for the tumor antigen and CDRH sequences for CD226.
  • the present disclosure also encompasses antibodies or fragments thereof that bind to the same epitope of CD226 or the tumor antigen as the antibodies disclosed herein.
  • Such antibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.
  • formats and methods are known in the art that can be used to generate the multivalent and/or multispecific constructs described herein, such as multivalent and/or multispecific antibody formats of both asymmetric and symmetric architectures.
  • Non-limiting examples of such formats include (i) Fc-less bispecific antibody formats, such as tandem single-chain variable fragments (scFv2, taFv) and triplebodies, including bi-specific T cell engager (BiTE) and bispecific killer cell engagers (BiKE) molecules; bispecific single-domain antibody fusion proteins comprising single domain antibodies, such as VH or VL domains, VHH, VNAR and Nanobodies; diabodies and diabody derivatives, including tandem diabody and dual-affinity retargeting (DART) proteins; Fab fusion proteins; and other Fc-less fusion proteins, through the use of heterodimerizing peptides or miniantibodies from various proteins, e.g., leucine zippers with a coiled coil structure; (ii)
  • bispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., ET.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab' fragments cross-linked through sulfhdryl reactive groups, as described in, e.g.,
  • biosynthetic binding proteins e.g., pair of scFvs cross-linked through C-terminal tails preferably through disulfide or amine-reactive chemical cross- linking, as described in, e.g., U.S. Pat. No. 5,534,254
  • bifunctional antibodies e.g., Fab fragments with different binding specificities dimerized through leucine zippers (e.g., c-fos and c-jun) that have replaced the constant domain, as described in, e.g., U.S. Pat. No.
  • bispecific and oligospecific mono- and oligo-valent receptors e.g., VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody typically with associated light chains, as described in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA- antibody conjugates, e.g., crosslinking of antibodies or Fab fragments through a double stranded piece of DNA, as described in, e.g., U.S. Pat. No.
  • bispecific fusion proteins e.g., an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a full constant region, as described in, e.g., U.S. Pat. No. 5,637,481; multivalent and multispecific binding proteins, e.g., dimer of polypeptides having first domain with binding region of Ig heavy chain variable region, and second domain with binding region of Ig light chain variable region, generally termed diabodies (higher order structures are also encompassed creating for bispecific, trispecific, or tetraspecific molecules, as described in, e.g., U.S. Pat. No.
  • crosslinkable groups at the C-terminus further associated with VL domains to form a series of FVs (or scFvs), as described in, e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptides with both a VH and a VL domain linked through a peptide linker are combined into multivalent structures through non-covalent or chemical crosslinking to form, e.g., homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFV or diabody type format, as described in, e.g., U.S. Pat. No. 5,869,620. Additional exemplary
  • W02009021754A2 W02009068630A1, WO9103493A1, W09323537A1, WO9409131A1, W09412625A2, WO9509917A1, W09637621A2, WO9964460A1.
  • the multispecific antigen-binding construct of the present disclosure selectively binds a tumor antigen (e.g., BCMA) and CD226.
  • the antigen-binding units can be generated by standard techniques, as disclosed herein.
  • any known antibodies against a tumor antigen or CD226 can be used to generate a bispecific antibody according to the present disclosure, or the multispecific antigen-binding constructs can be generated using antibodies known in the art.
  • the multispecific antigen-binding construct (e.g., a trispecific antigen-binding construct) further comprises a third antigen-binding unit that binds specifically to a molecule expressed by an effector immune cell.
  • the third antigen-binding unit is an immunoglobulin Fc domain.
  • the molecule expressed by the effector immune cell can be, for example, CD 16, CDl6a, CDl6b, CD32a, CD64, or CD89.
  • the antigen-binding construct does not comprise an immunoglobulin Fc domain.
  • the first antigen-binding unit or second antigen-binding unit, or both can comprise a heavy chain comprising one or more immunoglobulin Fc modifications.
  • third or subsequent antigen-binding units also can comprise a heavy chain comprising one or more immunoglobulin Fc modifications.
  • the immunoglobulin Fc domain of the heavy chain comprises one or more amino acid mutations that, e.g., promote
  • the mutation is present in a CH3 domain of the heavy chain (See, e.g., Xu et al. (2015) mAbs 7(1): 231-42).
  • Methods to obtain desired pairing of Fc- containing polypeptides include, but are not limited to, charge-based pairing (electrostatic steering),“knobs-into-holes” steric pairing, SEEDbody pairing, and leucine zipper-based pairing (See, for example, Ridgway et al. (1996) Protein Eng. 9:617-621; Merchant et al. (1998) Nat. Biotech. 16:677-681; Davis et al. (2010) Protein Eng. Des. Sel. 23: 195-202; Gunasekaran et al. (2010) J. Biol. Chem. 285: 19637-19646; Wranik et al. (2012) J. Biol. Chem. 287:43331-43339; U.S. Pat. No. 5,932,448; and PCT Publication Nos. WO
  • protuberance-into-cavity knock-into-holes
  • protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., a first interaction pair) with larger side chains (e.g., tyrosine or tryptophan).
  • Complementary“cavities” of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide (e.g., a second interaction pair) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • a suitably positioned and dimensioned protuberance or cavity exists at the interface of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface.
  • protein complexes disclosed herein make use of the attractive interactions for promoting heteromultimer formation (e.g., heterodimer formation), and optionally repulsive interactions for hindering homodimer formation (e.g., homodimer formation) by carrying out site directed mutagenesis of charged interface residues.
  • the IgGl CH3 domain interface comprises four unique charge residue pairs involved in domain-domain interactions: Asp356-Lys439', Glu357-Lys370', Lys392- Asp399', and Asp399-Lys409' [residue numbering in the second chain is indicated by (')].
  • the numbering scheme used here to designate residues in the IgGl CH3 domain conforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction is represented twice in the structure (e.g., Asp-399-Lys409' and Lys409-Asp399').
  • K409- D399' favors both heterodimer and homodimer formation.
  • a single mutation switching the charge polarity (e.g., K409E; positive to negative charge) in the first chain leads to unfavorable interactions for the formation of the first chain homodimer. The unfavorable interactions arise due to the repulsive interactions occurring between the same charges (negative-negative; K409E-D399' and D399-K409E’).
  • a similar mutation switching the charge polarity (D399K'; negative to positive) in the second chain leads to unfavorable interactions (K409'-D399K' and D399K-K409') for the second chain homodimer formation. But, at the same time, these two mutations (K409E and D399K') lead to favorable
  • the electrostatic steering effect on heterodimer formation and homodimer discouragement can be further enhanced by mutation of additional charge residues which may or may not be paired with an oppositely charged residue in the second chain including, for example, Arg355 and Lys360 (See, e.g., PCT Publication No. WO 2016/164089).
  • the multispecific antigen-binding constructs described herein can comprise a constant domain of an immunoglobulin, including, for example, the Fc portion of an immunoglobulin.
  • a first antigen-binding unit may comprise an amino acid sequence that is derived from an Fc domain of an IgG (IgGl, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM immunoglobulin.
  • a second antigen binding unit and/or subsequent antigen-binding units may comprise an amino acid sequence that is derived from an Fc domain of an IgG (IgGl, lgG2, lgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM.
  • immunoglobulin domains may comprise one or more amino acid modifications (e.g., deletions, additions, and/or substitutions) that promote heterodimer formation.
  • a first antigen-binding unit and a second antigen-binding unit comprise Fc domains derived from the same immunoglobulin class and subtype.
  • a first and second antigen-binding unit comprise Fc domains derived from different immunoglobulin classes or subtypes.
  • a first and/or a second antigen binding unit e.g., an asymmetric pair or an unguided interaction pair
  • One or more of subsequent antigen-binding units are optionally from the same class and subtype or different than the first and/or second antigen-binding units. Methods of generating Fc modifications having the desired heterodimer formation are known in the art.
  • the Fc domain can be modified to enhance serum half-life of the multispecific antigen-binding construct disclosed herein.
  • Fc domain comprising one or more mutations which enhance or diminish antibody binding to the Fc receptor, e.g., at acidic pH as compared to neutral pH, are known in the art.
  • the constructs disclosed herein may comprise one or mutations in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the construct when administered to an animal.
  • Methods of modifying the Fc domain for desired characteristics, such as enhanced serum half-life are known in the art.
  • the constructs described herein comprise an altered heavy chain constant region that has reduced (or no) effector function relative to its corresponding unaltered constant region. Effector functions involving the constant region of the constructs described herein may be modulated by altering properties of the constant or Fc region.
  • Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and pro-inflammatory responses.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • apoptosis binding to one or more Fc-receptors
  • Fc-receptors Fc-receptors
  • pro-inflammatory responses include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and pro-inflammatory responses.
  • Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody or antigen-binding fragment thereof containing an altered constant region as compared to the activity of the unaltered form of the
  • An altered constant region with altered FcR binding affinity and/or ADCC activity and/or altered CDC activity is a polypeptide that has either an enhanced or diminished FcR binding activity and/or ADCC activity and/or CDC activity compared to the unaltered form of the constant region.
  • An altered constant region that displays increased binding to an FcR binds at least one FcR with greater affinity than the unaltered polypeptide.
  • An altered constant region which displays decreased binding to an FcR binds at least one FcR with lower affinity than the unaltered form of the constant region.
  • Such variants that display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32,
  • an altered constant region that displays modulated ADCC and/or CDC activity may exhibit either increased or reduced ADCC and/or CDC activity compared to the unaltered constant region.
  • the antibody or antigen-binding fragment thereof comprising an altered constant region can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48, 47, 46,
  • a multispecific antigen-binding construct described herein comprising an altered constant region displaying reduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDC activity.
  • the multispecific antigen-binding constructs described herein exhibit reduced or no effector function.
  • the multispecific antigen binding constructs comprise a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (See e.g., Burton et al. ( 1992) A civ. Immun. 51 : 1-18; Canfield et al.
  • the multispecific antigen-binding constructs contain an altered constant region exhibiting enhanced or reduced complement dependent cytotoxicity (CDC).
  • Modulated CDC activity may be achieved by introducing one or more amino acid
  • Antibodies or fragments thereof can be further selected for binding to more than one species.
  • antibodies or fragments that bind both mouse and human can be selected by screening with both mouse and human target cells.
  • the methods for producing the disclosed constructs include methods for preparing an antibody and antigen-binding fragments thereof. Such methods are well-known in the art and include, e.g., immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen.
  • a skilled artisan immunizes a suitable subject (e.g., a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate) with full-length tumor antigen or CD226 polypeptide or a variant or fragment thereof.
  • a suitable subject e.g., a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate
  • Antigenic fragments of a polypeptide can be selected to generate antibodies based on known structural features of the polypeptide.
  • regions within a tumor antigen or CD226, based on receptor/ligand interface information available in the art, can be used to design a suitable antigenic fragment to generate antibodies having desirable properties.
  • Resulting antibodies or antigen-binding constructs can then be screened for desired binding properties (e.g., binding affinities for the tumor antigen and CD226 and capacity to bridge cells on which the tumor antigen and CD226 are expressed).
  • a suitable subject e.g., a non-human mammal
  • the immunogen can be administered to a subject (e.g., a non-human mammal) with an adjuvant.
  • Adjuvants useful in producing an antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum or Salmonella minnesota ) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus , complete or incomplete Freund’s adjuvant; viral adjuvants; and chemical adjuvants, e.g., aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate.
  • protein adjuvants e.g., whole bacteria (BCG, Corynebacterium parvum or Salmonella minnesota ) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus , complete or incomplete Freund’s
  • the methods include preparing a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen.
  • a suitable mammal such as a laboratory mouse is immunized with a polypeptide (e.g., a tumor antigen or CD226) or antigenic fragment as described above.
  • Antibody-producing cells e.g., B cells of the spleen
  • the immunized mammal can be isolated two to four days after at least one booster immunization of the immunogen and then grown briefly in culture before fusion with cells of a suitable myeloma cell line.
  • the cells can be fused in the presence of a fusion promoter, such as, e.g., vaccinia virus or polyethylene glycol.
  • a fusion promoter such as, e.g., vaccinia virus or polyethylene glycol.
  • the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected.
  • spleen cells of Balb/c mice immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14.
  • the cells are expanded in suitable culture medium, which is supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.
  • the obtained hybridoma cells are then screened for secretion of the desired antibodies, e.g., an antibody that binds to the desired antigen.
  • an antibody specific for a tumor antigen or CD226 is selected from a non-immune biased library as described in, e.g., U.S. Pat. No. 6,300,064 and
  • the methods described herein involve, or can be used in conjunction with, e.g., phage display technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosomal display) antibody screening techniques (See, e.g., Etz et al. (2001) J. Bacteriol. 183:6924-6935;
  • phage display methods functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them.
  • Such phage can be utilized to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-bond stabilized Fv antibody fragments, expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage used in these methods are typically filamentous phage such as fd and Ml 3.
  • the antigen-binding domains are expressed as a recombinantly-fused protein to any of the phage coat proteins pill, pVIII, or pIX. (See, e.g., Shi et al. (2010) JMB 397:385-396.) Examples of phage display methods that can be used to make the
  • immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995) J Immunol. Methods 182:41-50; Ames et al. (1995) J Immunol. Methods 184: 177-186; Kettleborough et al. (1994) Eur. J. Immunol. 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994 ) Advances in Immunology 57: 191-280; and PCT publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO 95/20401.
  • Suitable methods are also described in, e.g., U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.
  • the phage display antibody libraries can be generated using mRNA collected from B cells from the immunized mammals.
  • a splenic cell sample comprising B cells can be isolated from mice immunized with a tumor antigen or CD226 polypeptide as described above.
  • mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques (See, e.g., Green and Sambrook (2012 ) Molecular Cloning— A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York; Harlow and Lane (1988 ) Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y; Lo (2004) Antibody Engineering:
  • the cDNA coding for the variable regions of the heavy chain and light chain polypeptides of immunoglobulins are used to construct the phage display library. Methods for generating such a library are described in, e.g., Merz et al. (1995) J. Neurosci. Methods 62(l-2):2l3-9; Di Niro et al. (2005) Biochem. ./. 388(Pt 3):889- 894; and Engberg et al. (1995) Methods Mol. Biol. 51 :355-376.
  • a combination of selection and screening can be employed to identify an antibody of interest from, e.g., a population of hybridoma-derived antibodies or a phage display antibody library.
  • Suitable methods are known in the art and are described in, e.g., Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995) J.
  • phagemid vectors each encoding a fusion protein of a bacteriophage coat protein (e.g., pill, pVIII, or pIX of M13 phage) and a different antigen combining region are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli).
  • a bacteriophage coat protein e.g., pill, pVIII, or pIX of M13 phage
  • a different antigen combining region are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli).
  • Expression of the bacteriophage in bacteria can, in some embodiments, require use of a helper phage. In some embodiments, no helper phage is required (see, e.g., Chasteen et al. (2006 ) Nucleic Acids Res. 34(2l):el45). Phage produced from the bacteria are recovered and then contacted to, e.g., a target antigen bound to a solid support (immobilized). Phage may also be contacted to antigen in solution, and the complex is subsequently bound to a solid support.
  • a subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular antigen (e.g., human tumor antigen or CD226) using any immunological or biochemical based method known in the art.
  • a particular antigen e.g., human tumor antigen or CD2266
  • specific binding of an antibody to the tumor antigen or CD226 may be determined, for example, using immunological or biochemical based methods such as, but not limited to, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above.
  • Immunoassays that can be used to analyze immunospecific binding and cross-reactivity of the antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays,
  • immunoprecipitation assays immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. It is understood that the above methods can also be used to determine if an antibody to the tumor antigen or CD226 does not bind to the human tumor antigen or CD226 proteins.
  • nucleic acids encoding the CDRs can be chemically synthesized as described in, e.g., Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1): 129-130 and U.S. Pat. No. 6,995,259.
  • nucleic acid sequence encoding an acceptor antibody the region of the nucleic acid sequence encoding the CDRs can be replaced with the chemically synthesized nucleic acids using standard molecular biology techniques.
  • the 5’ and 3’ ends of the chemically synthesized nucleic acids can be synthesized to comprise sticky end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody.
  • fragments of chemically synthesized nucleic acids, together capable of encoding an antibody can be joined together using DNA assembly techniques known in the art (e.g. Gibson
  • Any antibody of choice can be further modified to generate an antigen-binding fragment, as described herein, and/or manipulated using known techniques in the art to generate the multispecific antigen-binding constructs as described herein.
  • cross- linking methods can be used to generate a bispecific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl reactive group as described in, e.g., U.S. Pat. No. 4,433,059
  • bispecific antibody determinants can be generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycles of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., U.S. Pat. No. 4,444,878
  • trifunctional antibodies e.g., three Fab' fragments can be cross- linked through sulfhdryl reactive groups, as described in, e.g., U.S. Pat. No. 5,273,743.
  • Methods of generating multispecific constructs include, e.g., methods of generating multispecific constructs having common light chains.
  • the multispecific antigen-binding constructs can be modified as a single
  • modifications can be covalent or non-covalent modifications.
  • modifications can be introduced into the antibodies or antigen-binding fragments by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments.
  • the antibodies or antigen-binding fragments thereof can be conjugated to a heterologous moiety.
  • the heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin.
  • Suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDDK) (SEQ ID NO:3), polyhistidine (6-His; HHHHHH) (SEQ ID NO:4), hemagglutinin (HA; YPYDVPDYA)
  • heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT).
  • Suitable radioactive labels include, e.g., 32 P, 33 P, 14 C, 125 I, 131 1, 35 S, and 3 H.
  • Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLightTM 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7.
  • Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates.
  • suitable europium chelates include the europium chelate of di ethylene triamine pentaacetic acid (DTP A) or tetraazacyclododecane-l,4,7, l0-tetraacetic acid
  • Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.
  • Two proteins can be cross-linked using any of a number of known chemical cross linkers.
  • cross linkers are those that link two amino acid residues via a linkage that includes a“hindered” disulfide bond.
  • a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase.
  • SMPT 4- succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene
  • cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., l,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m- maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).
  • reagents which link two amino groups e.g., N-5-azido-2-nitrobenzoyloxysuccinimide
  • two sulfhydryl groups e.g
  • a radioactive label can be directly conjugated to the amino acid backbone of the antibody.
  • the radioactive label can be included as part of a larger molecule (e.g., 125 I in meta-[ 125 I]iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS), which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997) J. Nucl. Med. 38: 1221-1229) or chelate (e.g., to DOTA or DTP A), which is in turn bound to the protein backbone.
  • Methods of conjugating the radioactive labels or larger molecules/chelates containing them to the antibodies or antigen-binding fragments described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt
  • fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., of cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores.
  • the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC.
  • Suitable conjugation methods involve incubating an antibody protein or fragment thereof with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) Handbook of Radiopharmaceuticals: Radiochemistry and Applications , John Wiley and Sons.
  • the antibodies or fragments can be modified, e.g., with a moiety that improves the stabilization and/or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues.
  • the antibody or fragment can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug. Chem. 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476, or HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisic et al. (2010) Int. J. Pharm. 387(1-2): 110-119).
  • the stabilization moiety can improve the stability or retention of the antibody (or fragment) by at least, e.g., 1.5 (or at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.
  • the antibodies or antigen-binding fragments thereof described herein can be glycosylated.
  • an antibody or antigen-binding fragment thereof described herein can be subjected to enzymatic or chemical treatment or produced from a cell, such that the antibody or fragment has reduced or absent glycosylation.
  • Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993 )Mol. Immunol. 30: 1361.
  • compositions comprising a multispecific antigen-binding construct of the present disclosure and a pharmaceutically acceptable carrier are also provided.
  • the compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and/or adjuvant to be used with the methods disclosed herein.
  • Such compositions can be used in a subject having cancer or other condition that would benefit from the multispecific antigen-binding constructs described herein.
  • acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the formulation material(s) are for s.c. and/or I.V. administration.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, 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, b-cyclodextrin or hydroxypropyl- b- 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 polyethylene glycol
  • sugar alcohols such as mannitol or sorbitol
  • suspending agents such as mannitol or sorbitol
  • surfactants or wetting agents such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal);
  • the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NaOAC, pH 5.2, 9% Sucrose.
  • the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Allen (2012) Remington The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in vivo clearance of the multispecific antigen-binding construct.
  • composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • the saline comprises isotonic phosphate-buffered saline.
  • neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • 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 therefore.
  • a composition comprising the multispecific antigen-binding constructs disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Allen (2012) Remington The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments,
  • compositions comprising the multispecific antigen-binding construct disclosed herein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • the pharmaceutical composition can be selected for parenteral delivery.
  • the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such
  • compositions is within the ability of one skilled in 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.
  • a therapeutic composition when parenteral administration is contemplated, can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a multispecific antigen-binding construct, in a pharmaceutically acceptable vehicle.
  • a vehicle for parenteral injection is sterile distilled water in which a multispecific antigen-binding construct is formulated as a sterile, isotonic solution, and properly preserved.
  • the 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 can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide 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 can have the effect of promoting sustained duration in the circulation.
  • implantable drug delivery devices can be used to introduce the desired molecule.
  • a pharmaceutical composition can be formulated for inhalation.
  • a multispecific antigen-binding construct can be formulated as a dry powder for inhalation.
  • an inhalation solution comprising a multispecific antigen-binding construct can be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized. Pulmonary
  • formulations can be administered orally.
  • a multispecific antigen-binding construct that is administered in this fashion can be formulated with or without carriers customarily used in compounding 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.
  • at least one additional agent can be included to facilitate absorption of a multispecific antigen-binding construct.
  • diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • a pharmaceutical composition can involve an effective quantity of a multispecific antigen-binding construct in a mixture with non-toxic excipients 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.
  • Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving a multispecific antigen-binding construct 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. See for example, PCT Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.
  • sustained-release preparations can include
  • semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and European Pat. No. EP 058,481), copolymers of L-glutamic acid and g-ethyl-L- glutamate (Sidman et al. (1993 ) Biopolymers 22:547-556), poly (2-hydroxyethyl- methacrylate) (Langer et al. (1981) ./. Biomecl. Mater. Res. 15: 167-277; and Langer (1982) Chem. Tech.
  • sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art (See, e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA
  • the pharmaceutical composition to be used for in vivo administration typically is sterile.
  • sterilization is accomplished by filtration through sterile filtration membranes.
  • 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.
  • 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.
  • a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition 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. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
  • kits are provided for producing a single-dose administration unit.
  • the kit can contain both a first container having a dried protein and a second container having an aqueous formulation.
  • kits containing single and multi-chambered pre-filled syringes are included.
  • the effective amount of a pharmaceutical composition comprising a multispecific antigen-binding construct to be employed therapeutically depend, for example, upon the therapeutic context and objectives.
  • the appropriate dosage levels for treatment vary depending, in part, upon the molecule delivered, the indication for which a multispecific antigen-binding construct is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the clinician also selects the frequency of dosing, taking into account the
  • the composition can therefore be administered as a single dose or 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, for example, 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. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical is in certain embodiments.
  • composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular,
  • compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • individual elements of the combination therapy may be administered by different routes.
  • the composition can be administered locally, e.g., during surgery or topically.
  • local administration is via implantation of a membrane, sponge, or another 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.
  • a pharmaceutical composition comprising a multispecific antigen-binding construct in an ex vivo manner.
  • cells, tissues (including, e.g., blood) and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising a multispecific antigen-binding construct after which the cells, tissues and/or organs are subsequently implanted back into the patient.
  • the antigen-binding construct can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides.
  • such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic.
  • the cells can be immortalized.
  • the cells in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient’s immune system or by other detrimental factors from the surrounding tissues.
  • the present disclosure provides a method of treating a
  • the present disclosure provides a method of enhancing an immune response (e.g., enhanced T cell function; enhanced T cell-mediated response; increased IFNy secretion and/or production from T cells; enhanced NK cell function; enhanced macrophage function) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multispecific antigen-binding construct or composition comprising the construct of the present disclosure.
  • an immune response e.g., enhanced T cell function; enhanced T cell-mediated response; increased IFNy secretion and/or production from T cells; enhanced NK cell function; enhanced macrophage function
  • the enhancement of the immune response is greater upon administration of the multispecific antigen-binding construct as compared to an agent that has a single target.
  • the enhancement of the immune response is greater by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more as compared to an agent that binds either the tumor antigen alone or CD226 alone.
  • compositions described herein are useful in, inter alia , methods for treating or preventing a variety of cancers in a subject.
  • the compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration.
  • the route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), intradermal injection (ID), oral, intracranial injection, or intrathecal injection (IT).
  • IV intravenous injection or infusion
  • SC subcutaneous injection
  • IP intraperitoneal
  • IM intramuscular injection
  • ID intradermal injection
  • oral intracranial injection
  • intrathecal injection intrathecal injection
  • the injection can be in a bolus or a continuous infusion.
  • the term subject means a mammalian subject.
  • exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats and sheep.
  • the subject is a human.
  • the subject has or is suspected to have a disease or condition that can be treated with a
  • the disease or condition is a cancer.
  • the subject is a human with a cancer that can be treated with a multispecific antigen-binding construct provided herein.
  • the subject is a human that is suspected of having cancer that can be treated with a
  • Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject.
  • ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof.
  • treating or treatment includes ameliorating at least one physical parameter or symptom.
  • Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both.
  • Treating or treatment includes delaying or preventing metastasis.
  • administer or administration refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a multispecific antigen- binding construct provided herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • Administration can be achieved by, e.g., topical administration, local infusion, injection, or by means of an implant.
  • the implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Pat. Nos. 5,501,856; 4,863,457; and 3,710,795; and European Pat. Nos. EP488401 and EP 430539.
  • composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.
  • a multispecific antigen-binding construct of the present disclosure is therapeutically delivered to a subject by way of local administration.
  • the term enhanced T cell function or activation of T cells refers to a cellular process in which mature T cells, which express antigen-specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cell- based effector functions.
  • T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second by subsequent engagement of co- stimulatory molecules (e.g., CD28).
  • TCR T cell antigen-specific receptor
  • MHC antigen-major histocompatibility complex
  • enhanced T cell function also encompasses enhanced survival and/or enhanced proliferation of the T cell. Methods for measuring such activities are routine and known in the art.
  • T cell-mediated response refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8 + cells) and helper T cells (e.g ., CD4 + cells).
  • T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.
  • the term therapeutically effective amount or effective amount refers to an amount of a multispecific antigen-binding construct that, when administered to a subject, is effective to treat a disease or disorder.
  • a suitable dose of an antibody or fragment thereof described herein, which dose is capable of treating or preventing cancer in a subject, can depend on a variety of factors including the particular construct used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a whole multispecific antigen-binding construct may be required to treat a subject with cancer as compared to the dose of a fragment of the multispecific antigen-binding construct (e.g., single antigen-binding unit) required to treat the same subject.
  • a subject having metastatic melanoma may require administration of a different dosage of multispecific antigen-binding construct than a subject with glioblastoma.
  • Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of
  • a pharmaceutical composition can include a therapeutically effective amount of a multispecific antigen-binding construct described herein. Such effective amounts can be readily determined by one of ordinary skill in the art as described above. Considerations include the effect of the administered multispecific antigen-binding construct or the combinatorial effect of the multispecific antigen-binding construct with one or more additional active agents, if more than one agent is used in or with the pharmaceutical composition.
  • Suitable human doses of any of the multispecific antigen-binding constructs described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008 ) Am. J Transplantation 8(8): 1711-1718; Hanouska et al. (2007) Clin. Cancer Res. 13(2, part 1): 523-531 ; and Hetherington et al. (2006) Antimicrobial Agents and
  • Toxicity and therapeutic efficacy of such multispecific antigen-binding constructs can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50.
  • a multispecific antigen-binding construct that exhibits a high therapeutic index is preferred. While constructs that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of an antigen-binding construct described herein lies generally within a range of circulating concentrations of the antigen binding construct that include the ED5 0 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC5 0 (i.e., the
  • concentration of the construct - e.g., antibody - which achieves a half-maximal inhibition of symptoms) as determined in cell culture can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.
  • the antigen-binding construct described herein can be administered to a subject as a monotherapy.
  • the antigen-binding construct can be administered in conjunction with other therapies for cancer.
  • the composition can be administered to a subject at the same time, prior to, or after, radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplant therapy, precision medicine, genome editing therapy, or other pharmacotherapy .
  • Chemotherapeutic agents suitable for co-administration with compositions of the present disclosure include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g.
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine
  • alkylating agents e.g.
  • the multispecific antigen-binding construct and the one or more additional active agents are administered at the same time.
  • the multispecific antigen-binding construct is administered first in time and the one or more additional active agents are administered second in time.
  • the one or more additional active agents are administered first in time and the multispecific antigen binding construct is administered second in time.
  • the multispecific antigen binding construct and the one or more additional agents are administered simultaneously in the same or different routes.
  • a composition comprising the antigen-binding construct optionally contains one or more additional agents.
  • a multispecific antigen-binding construct described herein can replace or augment a previously or currently administered therapy. For example, upon treating with a multispecific antigen-binding construct, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels or dosages. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy is maintained until the level of the multispecific antigen-binding construct reaches a level sufficient to provide a therapeutic effect.
  • Monitoring a subject for an improvement in a cancer, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in tumor growth or size.
  • the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks, or more, after an administration.
  • the subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered.
  • Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a cancer described herein.
  • a therapeutically effective amount of a multispecific antigen binding construct, or a composition comprising the construct, described herein is
  • the enhanced immune response includes one or more of enhanced T cell function, enhanced NK cell function, or enhanced macrophage function.
  • the multispecific antigen-binding construct enhances the subject’s immune response by agonizing CD226 function by bridging an immune cell that expresses CD226 with a second cell (e.g., a tumor cell) that expresses a tumor antigen.
  • compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions.
  • NK cells are primed with 10 ng/mL IL-15 and incubated overnight (in the presence of 10 IU/mL IL-2) with BCMA-expressing target cells of the multiple myeloma cell line H929 and 10 nM of a bispecific antibody described herein having a format (see FIG. 1) comprising a whole antibody (IgGl) in which each arm specifically binds to BCMA and at the C- terminus of each heavy chain is an scFv that specifically binds to human CD226.
  • a format comprising a whole antibody (IgGl) in which each arm specifically binds to BCMA and at the C- terminus of each heavy chain is an scFv that specifically binds to human CD226.
  • the first antigen-binding domain comprises the heavy chain (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: l) of the exemplary CA8 antibody described in U.S. Pat. No. 9,273,141.
  • the scFv comprises the heavy chain and light chain variable regions of the DX11 antibody (commercially available from Abeam), which is an agonist of CD226 in assays in which the antibody is plate bound.
  • the BCMA and CD226 bispecific antibodies enhance NK effector function as compared to control BCMA antibody alone.
  • Construct 1 is a bispecific antibody (which is bivalent for both Her2 and CD226) and comprises:
  • trastuzumab (as a full IgGl antibody) in which the heavy chain is a fusion protein where the carboxy -terminus of the trastuzumab heavy chain is joined by way of a linker to the amino- terminus of the heavy chain variable region a Fab domain of an anti-CD226 antibody.
  • the linker includes several G4S repeats and is approximately 100 amino acids in length.
  • the heavy chain amino acid sequence for trastuzumab is set forth in SEQ ID NO: 10 and the heavy chain amino acid sequence of the anti-CD226 antibody for Construct 1 is set forth in SEQ ID NO: 12.
  • the light chain amino acid sequence for trastuzumab is set forth in SEQ ID NO: 11 and the light chain amino acid for the anti-CD226 antibody of Construct 1 is set forth in SEQ ID NO: 13.
  • Construct 2 is a bispecific antibody (which is bivalent for both Her2 and CD226) and comprises: trastuzumab (as a full IgGl antibody) in which the heavy chain is a fusion protein where the carboxy -terminus of the trastuzumab heavy chain is joined by way of a linker to the amino-terminus of the heavy chain variable region a Fab domain of an anti-CD226 antibody. (The linker includes several G4S repeats and is approximately 100 amino acids in length.)
  • the heavy chain amino acid sequence for trastuzumab is set forth in SEQ ID NO: 10 and the heavy chain amino acid sequence of the anti-CD226 antibody for Construct 1 is set forth in SEQ ID NO: 14.
  • the light chain amino acid sequence for trastuzumab is set forth in SEQ ID NO: 11 and the light chain amino acid for the anti-CD226 antibody of Construct 1 is set forth in SEQ ID NO: 15.
  • Construct 3 is a bispecific antibody (which is bivalent for both BCMA and CD226) and comprises: an anti-BCMA antibody (as a full IgGl antibody) in which the heavy chain is a fusion protein where the carboxy -terminus of the anti-BCMA heavy chain is joined by way of a linker to the amino-terminus of the heavy chain variable region a Fab domain of an anti- CD226 antibody. (The linker includes several G4S repeats and is approximately 100 amino acids in length.)
  • the heavy chain amino acid sequence for the anti-BCMA antibody is set forth in SEQ ID NO: 18 and the heavy chain amino acid sequence of the anti-CD226 antibody for Construct 3 is set forth in SEQ ID NO: 12.
  • the light chain amino acid sequence for the anti-BCMA antibody is set forth in SEQ ID NO: 19 and the light chain amino acid for the anti-CD226 antibody of Construct 3 is set forth in SEQ ID NO: 13.
  • Construct 4 is a bispecific antibody (which is bivalent for both BCMA and CD226) and comprises: an anti-BCMA antibody (as a full IgGl antibody) in which the heavy chain is a fusion protein where the carboxy -terminus of the anti-BCMA heavy chain is joined by way of a linker to the amino-terminus of the heavy chain variable region a Fab domain of an anti- CD226 antibody. (The linker includes several G4S repeats and is approximately 100 amino acids in length.)
  • the heavy chain amino acid sequence for the anti-BCMA antibody is set forth in SEQ ID NO: 18 and the heavy chain amino acid sequence of the anti-CD226 antibody for Construct 4 is set forth in SEQ ID NO: 14.
  • the light chain amino acid sequence for the anti-BCMA antibody is set forth in SEQ ID NO: 19 and the light chain amino acid for the anti-CD226 antibody of Construct 4 is set forth in SEQ ID NO: 15.
  • the Control Construct comprises an anti-BCMA antibody (as a full IgGl antibody) in which the heavy chain is a fusion protein where the carboxy-terminus of the anti-BCMA heavy chain is joined by way of a linker to the amino-terminus of the heavy chain variable region a Fab domain of trastuzumab.
  • the linker includes several G4S repeats and is approximately 100 amino acids in length.
  • the heavy chain amino acid sequence for the anti-BCMA antibody is set forth in SEQ ID NO: 18 and the heavy chain amino acid sequence of trastuzumab for the Control Construct is set forth in SEQ ID NO:8.
  • the light chain amino acid sequence for the anti-BCMA antibody is set forth in SEQ ID NO: 19 and the light chain amino acid for trastuzumab is set forth in SEQ ID NO:9.
  • Constructs 1-4 and/or the Control Construct can be prepared in alternative multispecific formats, such as the format set forth in Fig. 1 in which the c-terminal anti-CD226 Fab units are scFv antibody fragments, could also be generated from such sequences and are useful as described herein. It is understood that for any one of Constructs 1-4 or the Control Construct, the IgGl isotype can contain the N297A substitution as described in U.S. Patent Nos.
  • constructs can contain a wild-type IgGl amino acid sequence, e.g., wild-type human IgGl amino acid sequence, such as the amino acid sequence in SEQ ID NO: 10 without the N297 substitution.
  • NK cells Primary human NK cells isolated from PBMCs by negative selection were thawed and recovered overnight at 2 x 10 6 cells/ml in SCGM media (CellGenix) supplemented with 10% FBS, 10 ng/ml IL-2, 100 pg/ml IL-15 (“SCGM-NK”), and 2 pg/ml DNase. Recovered NK cells were incubated with CFSE-stained MM.1 S target cells in SCGM-NK media at a 2.5:1 E:T ratio, and in the presence of the Control Construct, Construct 3, or Construct 4, each at 10 nM. After an overnight incubation, IFN-g accumulation in the culture supernatant was assessed by Meso Scale Discovery (MSD) assay. NK cells produced more IFN-g in the presence of Construct 3 and 4 as compared to the Control Construct.
  • MSD Meso Scale Discovery
  • Previously expanded T cells restricted against HL A- A0201 with CMV peptide NLVPMVATV were thawed and recovered overnight at 2 x 10 6 cells/ml in X-VIVO 15 media (Lonza) supplemented with 10% FBS, 5 ng/ml IL-2, 2.5 ng/ml IL-7 (“hX-lO”), and 2 pg/ml DNase.
  • Recovered CMV-specific T cells were incubated with CFSE-stained EG266 cells that had been pulsed with 1 nM NLVPMVATV peptide in hX-lO media at a 2.5: 1 E:T ratio, and in the presence of the Control Construct, Construct 3, or Construct 4, each at 10 nM.
  • SK-BR3 cells a human breast cancer cell line that overexpresses the Her2 gene product, were modified to overexpress anti-CD3 scFv on the cell surface. They were stained with INCUCYTE

Abstract

La présente invention concerne des compositions immunothérapeutiques et des procédés d'amélioration d'une réponse immunitaire et de traitement du cancer chez un sujet. Selon certains aspects, l'invention concerne des constructions multispécifiques de liaison à l'antigène qui reconnaissent un antigène tumoral et CD226.
PCT/US2019/033325 2018-05-21 2019-05-21 Compositions multispécifiques de liaison à l'antigène et procédés d'utilisation WO2019226658A1 (fr)

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WO2022262678A1 (fr) * 2021-06-15 2022-12-22 盛禾(中国)生物制药有限公司 Protéine de liaison à un antigène multispécifique et son utilisation
WO2022256739A3 (fr) * 2021-06-04 2023-01-05 Nonagen Therapeutics Corporation Anticorps spécifique pour bcl-6 et méthodes d'utilisation
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