WO2022228545A1 - Anticorps et variants de ceux-ci contre la 4-1bb humaine - Google Patents

Anticorps et variants de ceux-ci contre la 4-1bb humaine Download PDF

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WO2022228545A1
WO2022228545A1 PCT/CN2022/090184 CN2022090184W WO2022228545A1 WO 2022228545 A1 WO2022228545 A1 WO 2022228545A1 CN 2022090184 W CN2022090184 W CN 2022090184W WO 2022228545 A1 WO2022228545 A1 WO 2022228545A1
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amino acid
acid sequence
seq
antibody
hcdr2
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PCT/CN2022/090184
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Gang Liu
Zhongdao LI
Zhihui ZHAO
Cuiying SHAO
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Nanjing GenScript Biotech Co., Ltd.
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Priority to CN202280031805.5A priority Critical patent/CN117255804A/zh
Publication of WO2022228545A1 publication Critical patent/WO2022228545A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to antibodies or antigen-binding fragments thereof specifically bind to 4-1BB (alternatively known as CD137 or TNFRSF9) .
  • the present disclosure also relates to use of the antibodies in the treatment of cancer.
  • Immune cells such as T cells, macrophages, and natural killer cells, can exhibit anti -tumor activity and effectively control the occurrence and growth of malignant tumors.
  • 4-1BB (alternatively known as TNFRSF9, CD137, and ILA) is a transmembrane co-stimulatory receptor protein belonging to the tumor necrosis factor superfamily [1] .
  • 4-1BB is a T cell co-stimulatory receptor induced upon TCR activation.
  • 4-1BB is also expressed on CD4 + CD25 + regulatory T cells, activated natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells [2-5] .
  • 4-1BB Upon interaction with its ligand, 4-1BB leads to increased TCR-induced T cell proliferation, cytokine production, functional maturation, and prolonged CD8 + T cell survival.
  • the potential of 4-1BB co-stimulation using various agonists in enabling the immune system to attack tumors has been documented in numerous models [6, 7] .
  • Clinical trial results of Urelumab [8] and Utomilumab [9] highlight that patients with diseases and conditions, including cancer, that are amenable to treatment with a 4-1BB agonist.
  • novel agonistic antibodies that bind to human 4-1BB and exhibit characteristics sufficient for the development of safe and efficacious therapeutics.
  • the present invention provides isolated antibodies or antigen-binding fragment thereof ( “an anti-4-1BB antibody or an antigen-binding fragment thereof” ) , that specially bind to 4-1BB dependent or independent on CD32b (Fc ⁇ IIb) -mediated cross-linking to activate 4-1BB/NF- ⁇ B signaling pathway.
  • CD32b (Fc ⁇ IIb) -mediated cross-linking dependent antibody or antigen-binding fragment thereof indicated no or negligible activity until cross-linked with CD32b positive accessory cells, evidenced by the fact that it activated 4-1BB/NF- ⁇ B signaling pathway in a clear Fc ⁇ IIb (CD32b) receptor-induced cross-linking dependent manner.
  • Once engaging 4-1BB receptor it triggers cytokine production of primary human CD8 + T cells much stronger than reference antibodies used. Therefore, it may achieve anti-tumor efficacy while minimizing adverse effects (e.g. liver toxicity) .
  • the present disclosure provides an isolated antibody or an antigen-binding fragment thereof, that specifically binds to 4-1BB or a fragment thereof ( “an anti-4-1BB antibody or an antigen-binding fragment thereof” ) comprising a heavy chain variable region (VH) , wherein the VH comprise: HCDR1 having the amino acid sequence of SEQ ID NO: 2, HCDR2 having the amino acid sequence of SEQ ID NO: 3, and HCDR3 having the amino acid sequence of SEQ ID NO: 4; HCDR1 having the amino acid sequence of SEQ ID NO: 10, HCDR2 having the amino acid sequence of SEQ ID NO: 11, and HCDR3 having the amino acid sequence of SEQ ID NO: 12; HCDR1 having the amino acid sequence of SEQ ID NO: 18, HCDR2 having the amino acid sequence of SEQ ID NO: 19, and HCDR3 having the amino acid sequence of SEQ ID NO: 20; HCDR1 having the amino acid sequence of SEQ ID NO: 26, HCDR2 having the amino acid sequence of SEQ ID NO: 26
  • the anti-4-1BB antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL) , wherein: the VH comprises HCDR1 having the amino acid sequence of SEQ ID NO: 2, HCDR2 having the amino acid sequence of SEQ ID NO: 3, and HCDR3 having the amino acid sequence of SEQ ID NO: 4; the VL comprises LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 8; the VH comprises HCDR1 having the amino acid sequence of SEQ ID NO: 10, HCDR2 having the amino acid sequence of SEQ ID NO: 11, and HCDR3 having the amino acid sequence of SEQ ID NO: 12; the VL comprises LCDR1 having the amino acid sequence of SEQ ID NO: 14, LCDR2 having the amino acid sequence of SEQ ID NO: 15, and LCDR3 having the amino acid sequence of SEQ ID NO: 16; the VH comprises HCDR1 having the amino acid sequence
  • the VH comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81 , 89, 97, 105, 113, 121, 129, 137, and 145, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the VH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto
  • the VL comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto;
  • the VH comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto
  • the VL comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence
  • the anti-4-1BB antibody or antigen-binding fragment thereof is a 4-1BB agonist.
  • the 4-1BB is a human 4-1BB or cynomolgus monkey 4-1BB.
  • the anti-4-1BB antibody or antigen-binding fragment thereof is a mouse, chimeric, humanized or human antibody.
  • the humanized antibody comprises a VH comprising an amino acid sequence of any one of SEQ ID NOs: 153-158, 165-170, 177-182, 189-193, 198-200, 205-208, and 214-219 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto.
  • the humanized antibody comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 153-158 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 159-164 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto; a VH comprising the amino acid sequence of any one of SEQ ID NOs: 165-170 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto, and a VH comprising
  • the humanized antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto; a VH comprising the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence at
  • the anti-4-1BB antibody or antigen-binding fragment thereof is a crosslinking-dependent 4-1BB agonist.
  • the anti-4-1BB antibody or antigen-binding fragment thereof is a CD32b-dependent 4-1BB agonist.
  • the anti-4-1BB antibody or antigen-binding fragment thereof is of the type of IgG4.
  • the binding of the anti-4-1BB antibody or antigen-binding fragment thereof to 4-1BB has a lower EC50 than that of the reference antibody GS020 or CTX-471.
  • the anti-4-1BB antibody or antigen-binding fragment thereof comprising a heavy chain constant region having amino acid sequence of SEQ ID NO: 225 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto, and/or a light chain constant region having an amino acid sequence of SEQ ID NO: 226 or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical thereto.
  • the present disclosure provides a bispecific molecule comprising the antibody, or antigen-binding fragment thereof, of the disclosure, linked to a second functional moiety (e.g., a second antibody or antigen-binding fragment thereof) having a different binding specificity than the antibody, or antigen-binding fragment thereof, such as Claudin18.2, B7-H3, Her2, CD30.
  • a second functional moiety e.g., a second antibody or antigen-binding fragment thereof
  • the present disclosure provides an isolated polynucleotide encoding any one of the VHs and/or any one of the VLs described above.
  • the present disclosure provides a host cell comprising the isolated polynucleotide acid.
  • the present disclosure provides a host cell expressing the anti-4-1BB antibody or antigen-binding fragment thereof.
  • a method for preparing the antibody or the antigen-binding fragment thereof of the disclosure using the host cell is also provided, which may comprise steps of (i) expressing the antibody or the antigen-binding portion thereof in the host cell and (ii) isolating the antibody or the antigen-binding portion thereof from the host cell or its cell culture.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-4-1BB antibody or antigen-binding fragment thereof, or the bispecific molecule, of the disclosure and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition furthering comprises one or more other anti-cancer agents.
  • the present disclosure provides use of the anti-4-1BB antibody or antigen-binding fragment thereof or the pharmaceutical composition in the manufacture of a medicament for the treatment of a cancer.
  • the present disclosure provides a method of treating cancer, which comprises administrating to a subject in need thereof a therapeutically effective amount of the anti-4-1BB antibody or antigen-binding fragment thereof or the pharmaceutical composition.
  • the cancer is solid cancer or hematologic malignant. In some embodiments, the cancer is gastric cancer, lung cancer or blood malignancy.
  • the anti-4-1BB antibody or antigen-binding fragment thereof is administrated in simultaneous, separate, or sequential combination with a therapeutically effective amount of one or more other anti-cancer agents.
  • the present disclosure provides a method of preparing 4-1BB-expressing cells, which comprises culturing the 4-1BB-expressing cells in a medium supplemented with the anti-4-1BB antibody.
  • the 4-1BB-expressing cells are primary human T cells.
  • the anti-4-1BB antibodies provided herein may show a higher activity of 4-1BB/NF- ⁇ B signaling pathway triggering and primary T cell activation while minimizing hepatoxicity than current anti-4-1BB reference antibodies.
  • Figure 1 shows binding curves of the murine anti-4-1BB antibodies to HEK293 cells expressing human 4-1BB.
  • Figure 2 shows binding curves of the murine anti-4-1BB antibodies to HEK293 cells expressing cynomolgus monkey 4-1BB.
  • Figure 3 shows the results of the 4-1BB signaling pathway activation by the murine anti-4-1BB antibodies in the presence or absence of CD32b-expressing cells.
  • Figure 4 shows the curves of the 4-1BB signaling pathway activation by the murine anti-4-1BB antibodies in different concentrations in the presence of CD32b-expressing cells.
  • Figure 5 shows the results of the 4-1BB signaling pathway activation by the humanized anti-4-1BB antibodies in the presence or absence of CD32b-expressing cells.
  • Figure 6 shows the curves of the 4-1BB signaling pathway activation by the humanized anti-4-1BB antibodies in different concentrations in the presence of CD32b-expressing cells.
  • Figure 7 shows the results of the cytokine production triggering by the indicated humanized anti-4-1BB antibodies.
  • Figure 8 shows binding curves of the indicated humanized anti-4-1BB antibodies to HEK293 cells expressing cynomolgus monkey 4-1BB.
  • 4-1BB refers to TNF receptor superfamily member 9 (also known as TNFRSF9 and CD137) . In humans, it is encoded by the TNFRSF9 gene. It is expressed on activated T cells, Tregs, NK cells, monocytes, DCs, and tumor endothelial cells as a cell surface glycoprotein. Upon interaction with its cognate ligand, CD137L, 4-1BB forms stable homotrimers that recruit the TRAF-1/2 signaling adaptors to stimulate downstream activation of the NF- ⁇ B transcriptional pathway. Activation of 4-1BB delivers potent costimulatory signals to CD8 + cytotoxic T cells, promoting cell proliferation, facilitating differentiation into memory cells, and delivering important survival signals.
  • TNF receptor superfamily member 9 also known as TNFRSF9 and CD137
  • 4-1BB intracellular signaling domain of 4-1BB has improved the clinical activity of the second generation of CAR (chimeric antigen receptor) T cell therapies, indicating an important role of 4-1BB signaling in effective antitumor immunity.
  • 4-1BB stimulation also enhances NK cell proliferation and IFN- ⁇ production, and it increases the ability of NK cells to perform antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells, demonstrating potential of 4-1BB agonists to invoke and bridge innate and adaptive immunity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • 4-1BB refers to a 4-1BB protein encoded by a wild-type 4-1BB gene or an extracellular domain of such protein.
  • an antibody includes full-length antibodies, and antigen-binding fragments of full-length antibodies.
  • an antibody molecule is comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1) .
  • CH1 first constant domain of the heavy chain
  • antibodies include, without limitation, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies) , human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv) , camelized antibodies, Fab fragments, F (ab') 2 fragments, disulfide-linked Fvs (sdFv) , and antigen-binding fragments of any of the above.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY) , any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) , or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule.
  • antibodies described herein are IgG antibodies or a class thereof (e.g. IgG4) .
  • the antibodies are monoclonal antibodies.
  • the antibodies are humanized antibodies.
  • the antibodies are chimeric antibodies. In other embodiment, the antibodies are human antibodies.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDR regions can be determined using the Kabat or Chothia numbering systems, both of which are well known to those of skill in the art.
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • the term “antibody” may also refer to an antibody derivative that is capable of binding to the same antigen (e.g., 4-1BB) and comprises an amino acid sequence of the antibody linked to an additional molecular entity.
  • the amino acid sequence of the antibody that is contained in the antibody derivative may be a full-length heavy chain, a full-length light chain, any portion or portions of a full-length heavy chain, any portion or portions of the full-length light chain of the antibody, any other fragment (s) of an antibody, or the complete antibody.
  • the additional molecular entity may be a chemical or biological molecule. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies) , and chemical compounds.
  • the additional molecular entity may have any utility, such as for use as a detection agent, label, marker, pharmaceutical or therapeutic agent.
  • the amino acid sequence of an antibody may be attached or linked to the additional molecular entity by chemical coupling, genetic fusion, non-covalent association, or otherwise.
  • antibody derivative also encompasses chimeric antibodies, humanized antibodies, and molecules that are derived from modifications of the amino acid sequences of a 4-1 BB antibody, such as conservation amino acid substitutions, additions, and insertions.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., 4-1BB) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include, but not limited to, (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a V H domain; (vi) an isolated complementarity determining region (CDR) ; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains.
  • a Fab fragment a monovalent fragment
  • the two domains of the Fv fragment, V L and V H are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) .
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody.
  • single chain variable fragment refers to a fusion protein of the heavy chain variable region and light chain variable region of immunoglobulins, connected with a short linker peptide of ten to twenty-five amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility.
  • the scFv retains the specificity of the original immunoglobulin.
  • the scFvs can be linkered by linkers of different lengths to form di-scFvs, diabodies, tri-scFvs, triabodies, or tetrabodies, which may show specificity to one or more antigens.
  • telomere binding portion as used herein, means that an antibody or an antigen-binding portion thereof interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to an antigen, or an epitope than alternative substances, including related and unrelated proteins.
  • An antibody or an antigen-binding fragment thereof that specifically binds a target molecule may be identified, for example, by immunoassays, ELISAs, SPR (e.g., Biacore) , or other techniques known to those of skill in the art.
  • a specific reaction will be at least twice background signal or noise and can be more than 10 times background.
  • an antibody or an antigen-binding fragment thereof that specifically binds a target molecule may bind the target molecule at a higher affinity than its affinity for a different molecule.
  • an antibody or an antigen-binding fragment thereof that specifically binds a target molecule may bind the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different molecule.
  • an antibody or an antigen-binding fragment thereof that specifically binds a particular target molecule may bind a different molecule at such a low affinity that the binding cannot be detected using an assay described herein or otherwise known in the art.
  • “specifically binds” means, for instance, that an antibody or an antigen-binding portion thereof binds a molecule target with a K D of about 5.0E-08 or less. Because of the sequence identity between homologous proteins in different species, an antibody or an antigen-binding portion thereof that specifically recognizes a target molecule may cross-react with the target in other species.
  • an antibody or an antigen-binding portion thereof that specifically binds a first target may or may not specifically bind a second target.
  • “specific binding” does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target.
  • an antibody or an antigen-binding portion thereof in some embodiments, specifically bind more than one target.
  • an antibody or an antigen-binding portion thereof may, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds a same epitope.
  • mouse antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences.
  • the mouse antibodies of the disclosure can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
  • the term “mouse antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.
  • humanized antibody refers to a chimeric antibody that contains sequences from a non-human (e.g., murine) antibody as well as sequences from a human antibody.
  • a humanized antibody may contain some or all of the CDRs from a non-human animal antibody while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.
  • the humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • chimeric antibody refers to an antibody made by combining genetic material from a nonhuman source with genetic material from a human being. Or more generally, a chimeric antibody is an antibody having genetic material from a certain species with genetic material from another species.
  • a “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) . Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE TM technology) . See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • variant refers to a different antibody or antigen-binding fragment thereof that comprises one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to a reference antibody or antigen-binding fragment thereof, but remains the antigen binding affinity/capacity as the reference antibody or antigen-binding portion thereof has.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma.
  • cancers include squamous cell carcinoma, lung adenocarcinoma, head/neck squamous cell cancer, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute myeloid leukemia (AML) , multiple myeloma, gastrointestinal (tract) cancer, rectal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, uterine cancer, ovarian cancer, and head
  • host cell refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest.
  • Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, and insect cells, and cells comprised within a transgenic animal or cultured tissue.
  • rodents rats, mice, guinea pigs, or hamsters
  • rodents rats, mice, guinea pigs, or hamsters
  • rodents rats, mice, guinea pigs, or hamsters
  • rodents rats, mice, guinea pigs, or hamsters
  • rodents rats, mice, guinea pigs, or
  • isolated polynucleotide refers to the purification status, and in such context means the named molecule is substantially free of other biological molecules such as, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media.
  • agonist antibody refers to an antibody molecule, which upon binding to 4-1 BB, (1) stimulates or activates 4-1 BB/NK- ⁇ B signaling pathway, (2) enhances, increases, promotes, induces, or prolongs an activity or function of 4-1 BB, or (3) enhances, increases, promotes, or induces the expression of 4-1 BB.
  • crosslinking-dependent 4-1BB agonist refers to that the activity of the 4-1BB agonist relies on crosslinking (or clustering) of the 4-1BBs in the 4-1BB-expressing cell. It is known in the art that receptors of the TNFR superfamily, such as 4-1BB, requires receptor trimerization and clustering for efficient signaling. In some embodiments where the ani-4-1BB antibody is used as the agonist, cells expressing Fc receptors can facilitate the crosslinking. In a specific embodiment, the crosslinking is medicated by a cell expressing CD32b (aFc receptor, also known as Fc ⁇ RIIb) .
  • aFc receptor also known as Fc ⁇ RIIb
  • the activity of the 4-1BB agonist relies on the presence of a cell expressing CD32b, or the activity of the 4-1BB agonist is enhanced by a CD32b-expressing cells.
  • the 4-1BB agonist is also referred to as “CD32b -dependent 4-1BB agonist” .
  • EC 50 also known as half maximal effective concentration, refers to the concentration of an antibody or an antigen-binding portion thereof that gives half-maximal response, e.g., in a test by FACS, ELISA, or LDH.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid) , solvent or encapsulating material, involved in carrying or transporting a therapeutic compound for administration to a subject.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent or encapsulating material involved in carrying or transporting a therapeutic compound for administration to a subject.
  • Each excipient should be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • an effective amount refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific dose may vary depending on one or more of: the dosing regimen to be followed, whether it is administered in combination with other therapeutics, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • phase “other anti-cancer agent” as used herein refers to an anti-cancer agent different from the anti-4-1BB antibody disclosed herein.
  • the other anti-cancer agent include chemotherapeutic agent, such as 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, doxorubicin, etoposide, carboplatin, cisplatin, cyclophosphamide, melphalan, dacarbazine, taxol, camptothecin, FOLFIRI, FOLFOX, docetaxel, daunorubicin, paclitaxel, oxaliplatin, and combinations thereof; biotherapeutic agent, such as antibodies against PD-L1, PD-1, CTLA-4, CCR4, OX40; ionizing radiation; cellular therapeutics, such as chimeric antigen receptor (CAR) modified T cells or NK cells.
  • CAR chimeric antigen receptor
  • sequence identity in the context of two or more peptides, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference peptide or antibody sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum correspondence over a comparison window or designated region. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In a specific embodiment, the sequence identity is acquired through BLAST software that is publicly available on the worldwide web at ncbi. nlm. nih. gov, with default parameters.
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
  • the antibodies of the disclosure may contain a heavy chain constant region, such as a human IgG4 heavy chain constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 225, and/or a light chain constant region such as human kappa constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 226.
  • the antibodies or antigen-binding portions thereof of the disclosure may also contain other appropriate complete or partial heavy chain constant regions and/or complete or partial light chain constant regions.
  • the antibody of the disclosure may be a full-length antibody, a heavy chain antibody (HCAb) , a Fab, a F (ab’) 2 , a Fv, a scFv, or a (scFv) 2 .
  • the antibody of the disclosure may be an IgA, IgD, IgE, IgG, or IgM antibody.
  • the IgG antibody may be an IgG1, IgG2, IgG3 or IgG4 antibody, with reduced or no FcR and/or complement system protein binding affinity.
  • VH and VL sequences (or CDR sequences) of other antibodies which bind to the 4-1BB protein can be "mixed and matched" with the VH and VL sequences (or CDR sequences) of the antibody of the present disclosure.
  • VH and VL chains or the CDRs within such chains
  • a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence.
  • a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence.
  • Antibodies of the disclosure can be prepared using an antibody having one or more of the VH/VL sequences of the antibody or antigen-binding portion thereof of the present disclosure as starting material to engineer a modified antibody.
  • An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL) , for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region (s) , for example to alter the effector function (s) of the antibody.
  • the anti-4-1BB antibody provided herein is of murine origin. In some embodiments, the anti-4-1BB antibody provided herein is able to specifically bind both human 4-1BB and cynomolgus monkey 4-1BB. In some embodiments, the anti-4-1BB antibody provided herein is 4-1BB agonist antibody.
  • the anti-4-1BB antibody provided herein is a CD32b-dependent 4-1BB agonist (e.g, clones 355B8E9, 351F10A6, 360B9A8, 382A3E3, 387B1E3, 355B8A4, 362F6A1, 367C3A3, 357E4C5, 380G9-2A6, 379E7A6, 360A2A12, 360B3G8, 383F11C5F11, which are described below) .
  • a CD32b-dependent 4-1BB agonist e.g, clones 355B8E9, 351F10A6, 360B9A8, 382A3E3, 387B1E3, 355B8A4, 362F6A1, 367C3A3, 357E4C5, 380G9-2A6, 379E7A6, 360A2A12, 360B3G8, 383F11C5F11, which are described below
  • the anti-4-1BB antibody provided herein is a CD32b-independent 4-1BB agonist (e.g, clones 380H10D5, 352G2A10, 387A4A3, 392H11C12, 384B2A1, which are described below) .
  • the anti-4-1BB antibody provided herein is humanized (see, Table 1 below) .
  • the anti-4-1BB antibody provided herein is a humanized and CD32b-dependent 4-1BB agonist (e.g., 382A3E3-VH1-VL2, 382A3E3-VH1-VL3, 382A3E3-VH1-VL4, 382A3E3-VH1-VL5, 382A3E3-VH2-VL5, 379E7A6-VH1-VL1, 379E7A6-VH1-VL2, 379E7A6-VH3-VL1, 367C3A3-VH1-VL2, 367C3A3-VH2-VL1 and 367C3A3-VH2-VL2, which are described below) .
  • 4-1BB agonist e.g., 382A3E3-VH1-VL2, 382A3E3-VH1-VL3, 382A3E3-VH1-VL4, 382A3E3-VH
  • the anti-4-1BB antibody provided herein is used as a stimulus in a cell medium to promote the activation and/or expansion of 4-1BB-expressing cells, such as T cells, NK cells, monocytes, DCs.
  • 4-1BB-expressing cells such as T cells, NK cells, monocytes, DCs.
  • the anti-4-1BB antibody provided herein is supplemented to a CD8 + T cell culture to achieve a better yield in terms of the numbers of lymphocytes and their anti-cancer activity.
  • the disclosure provides nucleic acid molecules that encode heavy and/or light chain variable regions, or CDRs, of the antibodies of the disclosure.
  • the nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid of the disclosure can be, e.g., DNA or RNA and may or may not contain intronic sequences.
  • Nucleic acids of the disclosure can be obtained using standard molecular biology techniques.
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • antibodies obtained from an immunoglobulin gene library e.g., using phage display techniques
  • a nucleic acid encoding such antibodies can be recovered from the gene library.
  • Preferred nucleic acids molecules of the disclosure include those encoding the VH and VL sequences of the antibody or the CDRs.
  • DNA fragments encoding VH and VL segments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a VL-or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operatively linked” is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3) .
  • heavy chain constant regions CH1, CH2 and CH3 .
  • the sequences of mouse/human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably may be an IgG4 constant region in the present disclosure.
  • the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of mouse/human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • the VH-and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., (1990) Nature 348: 552-554) .
  • the monoclonal antibodies of the disclosure may be isolated from phage display libraries expressing variable domains or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art. For example, a human B-cell antibody library in scFv format or a human Fab library may be screened for antibodies binding to the 4-1BB protein by solution panning with colorimetric plates coated with 4-1BB protein over several rounds of selection with increasing stringency.
  • Isolates may be first expressed as scFv or Fab and screened for binding to the receptor binding domain by ELISA, and the selected isolates may then be cloned and expressed as IgG, reanalyzed for binding to 4-1BB protein by ELISA and/or SPR and for functional activity in neutralization assays, and transfected in a CHO mammalian cell line for expression of the full IgG antibodies.
  • the monoclonal antibodies of the disclosure may be also prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a 4-1BB protein or a fragment thereof. Following immunization, lymphocytes may be isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells may be selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process.
  • Hybridomas that produce monoclonal antibodies directed to a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore) , and radioimmunoassay) .
  • in vitro binding assays e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore)
  • the clones may be subcloned by limiting dilution or other techniques.
  • the hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal.
  • the monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dia
  • Antibodies of the disclosure also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202) .
  • DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences.
  • the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • promoters e.g., promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • enhancers e.g., polyadenylation signals
  • polyadenylation signals e.g., polyadenylation signals
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • non-viral regulatory sequences can be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • regulatory elements composed of sequences from different sources, such as the SR ⁇ promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al. (1988) Mol. Cell. Biol. 8: 466-472) .
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors.
  • the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment (s) within the vector and the VL segment is operatively linked to the CL segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
  • the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017) .
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .
  • DHFR dihydrofolate reductase
  • the expression vector (s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • transfection are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159: 601-621) , NSO myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220
  • a DHFR selectable marker e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159: 601-621
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338, 841.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • composition e.g, a pharmaceutical composition
  • a pharmaceutical composition may comprise any number of excipients.
  • Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams &Wilkins 2003) , the disclosure of which is incorporated herein by reference.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) .
  • the antibody dose may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg of the active gradient.
  • the antibody dose may be 0.3 to 30 mg/kg, such as 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg.
  • An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
  • the anti-4-1BB antibody or the pharmaceutical composition provided herein is administrated to a subject for the treatment of a disorder, such as a cancer.
  • a disorder such as a cancer
  • the anti-4-1BB antibody or antigen-binding fragment provided herein is administrated to a subject in combination with other therapeutics (e.g, anti-cancer agents) for the treatment of a disorder.
  • the cancer can be solid cancer or hematologic malignant.
  • the solid cancer is gastric cancer or lung cancer.
  • mice were immunized with human TNFRSF9-His proteins (Acro Biosystems; catalog#: 41B-H5227) under current animal welfare regulations.
  • the antigen was administrated in PBS solution or formulated as an emulsion with CFA (Complete Freund's adjuvant; primary immunization) or IFA (incomplete Freund's adjuvant; boost immunizations) .
  • CFA Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • boost immunizations boost immunizations
  • anti-4-1BB antibody patent No.: US 2017/0247455 A1
  • Utomilumab Patent No.: US 10,023,649 B2; in some cases it was written as GS020 as an in-house product
  • CTX-471 Compass; patent No.: US 10,279,038 B2
  • normal IgG either human IgG or mouse IgG1, or both
  • splenocytes from the selected mouse were extracted and fused with sp2/0 cells following standard hybridoma generation protocol in sterile environment.
  • the fused cells were cultured in 1X HAT (hypoxanthine-aminopterin-thymidine) containing DMEM media, supplemented with 10%FBS for 7 days.
  • the contents in the supernatant were analyzed for their ability of binding to 4-1BB by ELISA.
  • the positive parental clones were subcloned by limited dilution and cultured in 1X HT (hypoxanthine-thymidine) containing DMEM media, supplemented with 10%FBS. In 200 ⁇ l media, 1-3 cells/well.
  • Antibodies were purified with Protein-A magnetic beads, eluted by 0.5M Sodium-citrate solution (pH3.5) , neutralized with 0.5M Tris-HCl (pH9.0) .
  • the storage buffer was changed into PBS to determine concentration with Nanodrop (DeNovix; catalog#: DS11) .
  • HEK293 cells 100,000/well expressing human 4-1BB (constructed by Genscript) were harvested and incubated with serially diluted anti-4-1BB mAbs (max. concentration: 100nM, 3-fold dilution) , followed by staining with 1.0ug/ml fluorophore (iFluor 647) -labeled goat anti-mouse IgG (H+L) secondary antibodies (Jackson ImmunoResearch; catalog#: 115-605-062) . The samples were then analyzed with flow cytometry.
  • Raw data were plotted with GraphPad Prism v6.02 software with four parameters, best-fit values program to analyze the EC 50 (FIG. 1) , and the results indicated that most of the molecules hold strong or moderate affinity with the cell surface human 4-1BB antigen.
  • Anti-human 4-1BB antibody (Urelumab, BMS) and GS020 (Utomilumab, Pfizer) were used as reference antibodies, while mouse IgG as isotype control.
  • HEK293 cells 100,000/well expressing cynomolgus monkey 4-1BB (constructed by Genscript; NCBI Reference Sequence: NC_022272.1) were harvested and incubated respectively with serially diluted anti-4-1BB mAbs (max. concentration: 100nM, 3-fold dilution) , followed by fluorophore (iFluor 647) -labeled goat anti-mouse IgG (H+L) secondary antibodies (Jackson ImmunoResearch; catalog#: 115-605-062) . The samples were then analyzed with flow cytometry.
  • Raw data was plotted with GraphPad Prism v6.02 software with four parameters, best-fit values program to analyze the EC 50 (FIG. 2) . Those that show significant cross-reaction with cynomolgus monkey 4-1BB protein, 37 mAbs in total, were chosen for further verification.
  • Anti-human 4-1BB antibody (Urelumab, BMS) and GS020 (Utomilumab, Pfizer) were used as reference antibodies, while mouse IgG as isotype control.
  • Bioluminescence signal was measured with Envision microplate reader and histogram was generated with GraphPad Prism v6.02 software. As showed in FIG. 3, the mAbs whose potency were better than GS020 (Pfizer) in 4-1BB signaling pathway activation, 19 in total, were selected for further verification.
  • Anti-human 4-1BB antibody (Urelumab, BMS) , CTX-471-mIgG1 (Compass) and GS020 (Utomilumab, Pfizer) were used as reference antibodies, while mouse IgG and human IgG as isotype controls.
  • the EC 50 values were measured via the coculture system as described above in the single-dose luciferase experiment, with serially diluted mAbs added to the solution (4-time dilution, max. 20.0 ⁇ g/ml) . Bioluminescence was detected as described above and plotted with GraphPad Prism v6.02 software (FIG. 4) .
  • CTX-471-mIgG1 (Compass) and GS020 (Utomilumab, Pfizer) were used as reference antibodies, while mouse IgG and human IgG as isotype controls. All the candidates indicated potency similar to or better than the reference antibodies, GS020 and CTX-471.
  • Variable regions of the 19 mAbs were sequenced. The results indicated that, among the 14 human CD32b-dependent mAbs, 355B8E9 and 355B8A4 were of same CDRs regions to each other, and the same happened to mAbs 360B9A8 and 383F11C5F11. Besides, 362F6A1 /379E7A6 and 360A2A12 /360B3G8 are of minor CDR regions difference.
  • CD32b-dependent agonistic mAbs that manifested 4-1BB/NF- ⁇ B signaling pathway activation potency similar to or better than that of CTX-471-mIgG1, namely 351F10A6, 360B9A8, 382A3E3, 387B1E3, 367C3A3, and 379E7A6 were acquired for further verification. Meanwhile, among the five CD32b-independent mAbs, 380H10D5 and 392H11C12 shared the same CDR sequences while the other three molecules are unique ones in variable region, so 392H11C12 was removed from further verification.
  • variable domain sequences, the CDRs, HV loops and FRs were analyzed and homology modeling was performed to obtain the modeled structure of the mAbs of 351F10A6, 360B9A8, 382A3E3, 387B1E3, 367C3A3, 379E7A6, 380H10D5, 352G2A10, 387A4A3 and 384B2A1.
  • identify framework residues that are buried i.e. with solvent accessible surface area of ⁇ 15%) .
  • Select human acceptors for VH and VL that share the top sequences identical to the mouse counterparts.
  • the humanized heavy chain and light chain of antibodies were combined for antibody production. For each heavy chain and light chain combination, the detailed information is shown in Table 1.
  • DNA sequences encoding the humanized light and heavy chains were synthesized, and antibodies were expressed in CHO-3E7 cells transfected with antibody heavy chain/light pair plasmids using PEImax 40,000 (polysciences) . Twenty-four hours later, the expression/secretion was boosted with Tryptone N-1 supplement. After 6 days of shaking culture in 37 °C and 5%CO 2 , supernatants were collected and the humanized antibodies were purified with Protein-A beads as described above. The humanized antibodies were kept in PBS for analysis.
  • Table 1 humanized antibody production by heavy chain and light chain pairing
  • the molecules are of better potency and/or lower EC50 values than the reference antibodies, GS020 and CTX-471 (FIG. 6) .
  • Urelumab (BMS) , CTX-471 (Compass) and GS020 (Utomilumab, Pfizer) were used as reference antibodies, while normal human IgG4 as isotype control.
  • Interferon gamma is critical cytokines for cellular immune responses.
  • 100,000 primary human CD8 + T cells freshly purified from human peripheral blood mononuclear cells (PBMCs; StemCell, catalog #: 2001414009) were co-cultured in 96-well plate with 50,000 CHO/K1-CD32b-Ile cells (constructed by Genscript) , with the presence of each of the indicated antibodies (the above 11 humanized antibodies and 4 additional humanized antibodies: 382A3E3-VH2-VL1, 382A3E3-VH2-VL2, 382A3E3-VH2-VL4, and 382A3E3-VH6-VL1) or control samples and suboptimal dose of human OKT3 CD3 antibody (eBioscience; catalog#: 16-0037-81) in medium, and were incubated at 37°C/5%CO2 for 48h.
  • PBMCs peripheral blood mononuclear cells
  • the cell-free supernatant was collected for IFN- ⁇ release detection with ELISA kit (Cisbio; catalog#: 62HIFNGPEH, 62HIL02PEH, and 62HTNFAPEH, respectively) .
  • a histogram was generated with GraphPad Prism v6.02 software (FIG. 7) .
  • Urelumab (BMS) , CTX-471 (Compass) and GS020 (Utomilumab, Pfizer) were used as reference antibodies, while normal human IgG4 as isotype control. The results showed intermediate to robust, compared to reference antibodies, activity of the molecules in triggering the cytokine production.
  • HEK293 cells 100,000/well expressing either human or cynomolgus monkey 4-1BB (constructed by Genscript) were harvested and incubated respectively with serially diluted anti-4-1BB mAbs (max. concentration: 100nM, 3-fold dilution) , followed by fluorophore (iFluor 647) -labeled goat anti-human IgG (H+L) secondary antibodies (Jackson ImmunoResearch; catalog#: 109-001-008) . The samples were then analyzed with flow cytometry. Raw data was plotted with GraphPad Prism v6.02 software with four parameters, best-fit values program to analyze the EC 50 (FIG.
  • the underlined amino acid sequences are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively.
  • CDRH1 GYTFTDYSMH (SEQ ID NO: 2)
  • CDRH2 WINTETGEPTYADDFKG (SEQ ID NO: 3)
  • CDRL1 KASQDVSSAVA (SEQ ID NO: 6)
  • CDRL2 WASIRHT (SEQ ID NO: 7)
  • CDRL3 QQRYTTPPT (SEQ ID NO: 8)
  • CDRH1 GYTFTDFNIH (SEQ ID NO: 10)
  • CDRL1 RASESADSYGNSFVH (SEQ ID NO: 14)
  • CDRL2 RASNLES (SEQ ID NO: 15)
  • CDRL3 QQSNEDPYT (SEQ ID NO: 16)
  • CDRH1 GFSLTTYGVH (SEQ ID NO: 18)
  • CDRH2 AIWSGGSTDYNAAFIS (SEQ ID NO: 19)
  • DDGSFAN SEQ ID NO: 20
  • CDRL1 KASENVGTYVS (SEQ ID NO: 22)
  • CDRL2 GASNRYT (SEQ ID NO: 23)
  • CDRL3 GQTYSYPLT (SEQ ID NO: 24)
  • CDRH1 GYTFTSYWIN (SEQ ID NO: 26)
  • CDRH2 NIYPSDTYTNYNQKFKD (SEQ ID NO: 27)
  • CDRL1 RASQDISNYLN (SEQ ID NO: 30)
  • CDRL2 YTSRLHS (SEQ ID NO: 31)
  • CDRL3 QQGHTLPYT (SEQ ID NO: 32)
  • CDRH1 GFTFSDYGMS (SEQ ID NO: 34)
  • CDRH2 FISNVAYGIFYADTVTG (SEQ ID NO: 35)
  • CDRL1 HVSQNINIWLS (SEQ ID NO: 38)
  • CDRL2 KASNLHT (SEQ ID NO: 39)
  • CDRL3 QQGQTFPRT (SEQ ID NO: 40)
  • CDRH1 GFTFSDYGMS (SEQ ID NO: 42)
  • CDRL1 HASHNINVWVS (SEQ ID NO: 46)
  • CDRL2 KASNLHT (SEQ ID NO: 47)
  • CDRL3 QQGQTYPRT (SEQ ID NO: 48)
  • CDRH1 GYTFTDFNIH (SEQ ID NO: 50)
  • CDRL1 RASESADSYGNSFVH (SEQ ID NO: 54)
  • CDRL2 RASNLES (SEQ ID NO: 55)
  • CDRL3 QQSNEDPYT (SEQ ID NO: 56)
  • CDRH1 GFTFSDYGMA (SEQ ID NO: 58)
  • CDRH2 FISNVAYSIFYADTVTG (SEQ ID NO: 59)
  • CDRH3 DELGRFAY (SEQ ID NO: 60)
  • CDRL1 HVSHNINIWLS (SEQ ID NO: 62)
  • CDRL2 KASNLHT (SEQ ID NO: 63)
  • CDRL3 QQGQTFPRT (SEQ ID NO: 64)
  • CDRH2 VIWAGGGTNYNSAFMS (SEQ ID NO: 67)
  • CDRH3 DELGRFAY (SEQ ID NO: 68)
  • CDRL1 RSSQSLVHSNGNTYLH (SEQ ID NO: 70)
  • CDRL2 KVSNRFS (SEQ ID NO: 71)
  • CDRL3 SQSTHVPWT (SEQ ID NO: 72)
  • CDRH1 GFSLSIYGVH (SEQ ID NO: 74)
  • CDRH2 AIWSGGSTDYNAAFIS (SEQ ID NO: 75)
  • CDRH3 DDGSFAY (SEQ ID NO: 76)
  • CDRL1 KASENVGIYVS (SEQ ID NO: 78)
  • CDRL2 GATNRYT (SEQ ID NO: 79)
  • CDRL3 GQSYSYPLT (SEQ ID NO: 80)
  • CDRH1 GFTFSDYGMA (SEQ ID NO: 82)
  • CDRH2 FISNVAYSKFYVDTVTG (SEQ ID NO: 83)
  • CDRH3 DELGRFPY (SEQ ID NO: 84)
  • CDRL1 HVSHNINIWLN (SEQ ID NO: 86)
  • CDRL2 KASNLHT (SEQ ID NO: 87)
  • CDRL3 QQGQTFPRT (SEQ ID NO: 88)
  • CDRH1 GYTFTDFNMH (SEQ ID NO: 90)
  • CDRL1 RASESVDNYGNSFMH (SEQ ID NO: 94)
  • CDRL2 RASNLES (SEQ ID NO: 95)
  • CDRL3 QQSNEDPYT (SEQ ID NO: 96)
  • CDRH1 GYTFTDFNMH (SEQ ID NO: 98)
  • CDRL1 RASESVDNYGNSFMH (SEQ ID NO: 102)
  • CDRL2 RASNLES (SEQ ID NO: 103)
  • CDRL3 QQSNEDPYT (SEQ ID NO: 104)
  • CDRH1 GYTFTSYWIN (SEQ ID NO: 106)
  • CDRH2 NIYPSDTYTNYNQKFKD (SEQ ID NO: 107)
  • CDRL1 RASQDISNYLN (SEQ ID NO: 102)
  • CDRL2 RASNLES (SEQ ID NO: 103)
  • CDRL3 QQSNEDPYT (SEQ ID NO: 104)
  • CDRH2 MIWTVRGPNYNSAFMS (SEQ ID NO: 115)
  • CDRL1 HASQNINVWLS (SEQ ID NO: 118)
  • CDRL2 KASNLHT (SEQ ID NO: 119)
  • CDRL3 QQVQTYPRT (SEQ ID NO: 120)
  • CDRH1 GFSLTSYDIS (SEQ ID NO: 122)
  • CDRH2 MIWTVRGTNYNSAFMS (SEQ ID NO: 123)
  • CDRL1 HASQHINVWLS (SEQ ID NO: 126)
  • CDRL2 KASNLHT (SEQ ID NO: 127)
  • CDRL3 QQVQTYPRT (SEQ ID NO: 128)
  • CDRH1 GFSLTTYDIS (SEQ ID NO: 130)
  • CDRH2 MIWTVRGTFYNSAFMS (SEQ ID NO: 131)
  • CDRL1 HASQNINVWLS (SEQ ID NO: 134)
  • CDRL2 KASNLHT (SEQ ID NO: 135)
  • CDRL3 QQVQSYPRT (SEQ ID NO: 136)
  • CDRH1 GFSLTNYDIS (SEQ ID NO: 138)
  • CDRH2 MIWTVRGPNYNSAFMS (SEQ ID NO: 139)
  • CDRL1 HASQNINVWLS (SEQ ID NO: 142)
  • CDRL2 KASNLHT (SEQ ID NO: 143)
  • CDRL3 QQVQSYPRT (SEQ ID NO: 144)
  • CDRH2 MIWTVRGPNYNSAFMS (SEQ ID NO: 147)
  • CDRL1 HASQNINVWLS (SEQ ID NO: 150)
  • CDRL2 KASNLHT (SEQ ID NO: 151)
  • CDRL3 QQVQSYPRT (SEQ ID NO: 152)
  • Human IgG4 heavy chain constant region (SEQ ID NO: 225) :
  • NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies.
  • 4-1BB CDw137
  • Kienzle, G. &von Kempis, J. CD137 (ILA/4-1BB) expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes. Int. Immunol. 12, 73–82 (2000) .

Abstract

L'invention concerne des molécules de liaison qui se lient spécifiquement à 4-1BB exprimée sur la surface des lymphocytes T humains, ainsi que l'utilisation de telles molécules de liaison dans la stimulation et l'activation spécifiques et puissantes de lymphocytes T humains primaires. L'invention concerne en outre des polynucléotides codant pour de telles molécules de liaison.
PCT/CN2022/090184 2021-04-29 2022-04-29 Anticorps et variants de ceux-ci contre la 4-1bb humaine WO2022228545A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
EP3470428A1 (fr) * 2017-10-10 2019-04-17 Numab Innovation AG Anticorps dirigés contre cd137 et leurs procédés d'utilisation
US20200308293A1 (en) * 2017-11-20 2020-10-01 Compass Therapeutics Llc Cd137 antibodies and tumor antigen-targeting antibodies and uses thereof
CN112010974A (zh) * 2019-05-30 2020-12-01 山东博安生物技术有限公司 一种特异性结合人4-1bb的抗体及其应用
CN112538116A (zh) * 2020-12-24 2021-03-23 东大生物技术(苏州)有限公司 一组4-1bb单克隆抗体及其医药用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3470428A1 (fr) * 2017-10-10 2019-04-17 Numab Innovation AG Anticorps dirigés contre cd137 et leurs procédés d'utilisation
US20200308293A1 (en) * 2017-11-20 2020-10-01 Compass Therapeutics Llc Cd137 antibodies and tumor antigen-targeting antibodies and uses thereof
CN112010974A (zh) * 2019-05-30 2020-12-01 山东博安生物技术有限公司 一种特异性结合人4-1bb的抗体及其应用
CN112538116A (zh) * 2020-12-24 2021-03-23 东大生物技术(苏州)有限公司 一组4-1bb单克隆抗体及其医药用途

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Title
GALAND CLAIRE, VIGNESH VENKATRAMAN, MARILYN MARQUES, JAMES STRAUSS, RICHARD CARVAJAL, MIN LIM, BENJAMIN MORIN, OLGA IGNATOVICH, MA: "AGEN2373 IS A CD137 AGONIST ANTIBODY DESIGNED TO LEVERAGE OPTIMAL CD137 AND FCγR CO-TARGETING TO PROMOTE ANTITUMOR IMMUNOLOGIC EFFECTS", J IMMUNOTHER CANCER, vol. 8, no. 3, 10 December 2020 (2020-12-10), XP055981999 *
QI XINYUE, LI FANLIN, WU YI, CHENG CHEN, HAN PING, WANG JIEYI, YANG XUANMING: "Optimization of 4-1BB antibody for cancer immunotherapy by balancing agonistic strength with FcγR affinity", NATURE COMMUNICATIONS, vol. 10, no. 1, 1 December 2019 (2019-12-01), XP055920548, DOI: 10.1038/s41467-019-10088-1 *

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