WO2019199896A9 - Anticorps agonistes contre le cd137 humain dans le cancer exprimant le cmh i - Google Patents

Anticorps agonistes contre le cd137 humain dans le cancer exprimant le cmh i Download PDF

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WO2019199896A9
WO2019199896A9 PCT/US2019/026675 US2019026675W WO2019199896A9 WO 2019199896 A9 WO2019199896 A9 WO 2019199896A9 US 2019026675 W US2019026675 W US 2019026675W WO 2019199896 A9 WO2019199896 A9 WO 2019199896A9
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seq
amino acid
nos
monoclonal antibody
antigen binding
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WO2019199896A1 (fr
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Ugur ESKIOCAK
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Compass Therapeutics Llc
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K16/28—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
    • C07K16/3061—Blood cells
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • 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
    • C07K2317/70—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • T cells such as T cells, macrophages, and natural killer cells
  • Tumor-specific or -associated antigens can induce immune cells to recognize and eliminate malignancies (Chen & Mellman, (2013) Immunity 39(1): 1-10).
  • CD137 (alternatively known as “tumor necrosis factor receptor superfamily member 9” (TNFRSF9), 4-1BB, and“induced by lymphocyte activation” (ILA)) is a transmembrane co stimulatory receptor protein belonging to the tumor necrosis factor superfamily.
  • CD 137 is a T cell co-stimulatory receptor induced upon TCR activation (Nam et ah, (2005) Curr Cancer Drug Targets 5:357-363; Watts et ah, (2005) Annu Rev Immunol 23:23-68).
  • CD 137 is also expressed on CD4+CD25+ regulatory T cells, activated natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells.
  • CD137 is ligated by CD137 ligand (CD137L), an agonist membrane molecule present on antigen-presenting cells including B cells, monocytes, macrophages, and dendritic cells (Watts et ah, (2005) Annu Rev Immunol 23:23-68).
  • CD137L CD137 ligand
  • B cells B cells
  • monocytes macrophages
  • dendritic cells dendritic cells
  • agonistic antibodies, recombinant CD 137L protein, and CD 137- specific aptamers in enabling the immune system to attack tumors has been documented in numerous models (Dharmadhikari et ah, (2016) Oncoimmunology 5(4):el 113367 and references therein).
  • a recent report on the clinical evaluation of an agonistic CD 137 antibody (Urelumab, BMS-663513; Bristol-Myers Squibb) documented the observation of treatment- related adverse events in human subjects, including indications of severe hep ato toxicity (transaminitis) correlating with antibody dose (Segal et ah, (2016) Clin Cancer Res 23(8): 1929- 1936).
  • the present disclosure is based, in part, on the discovery that cancer cells expressing MHC I are more responsive to anti-cancer therapy using anti-CDl37 agonist antibodies, and antigen-binding fragments thereof, disclosed herein.
  • the present discovery can be applied to identify a cancer patient population that would be particularly amenable to anti-cancer therapy using the anti-CD 137 agonist antibodies disclosed herein.
  • the anti-CD 137 monoclonal antibodies of the disclosure were found to agonize CD 137 and induce protective anti-tumor immunity in vivo with a concomitant reduction in the potential for toxicity-related events.
  • the anti-CDl37 antibodies described herein are efficacious against diverse tumor types, and over a wide dose range.
  • the anti-CD 137 agonist antibodies of the disclosure were also found to induce and/or enhance cytokine production, expansion of CD8+ T cells, and protective anti-tumor immunity and thus are particularly effective in treating cancer.
  • the anti-CD 137 agonist antibodies of the disclosure were found to bind a unique epitope on human CD 137.
  • the disclosure also features agonist anti- CD 137 antibodies having an affinity (KD) that is optimal for maximizing anti-tumor immunity while avoiding toxicity-related events associated with CD137 agonism.
  • the antibodies described herein are therapeutically effective against very large tumors. For example, treatment of tumor-bearing mice with agonist anti-CD 137 antibodies described herein resulted in complete regression of tumors as large as 1,800 mm 3 . As set forth in FIG. 15, treatment of such mice also resulted in protective immunity.
  • agonism of CD 137 has been associated with certain adverse events, including hepatotoxicity-related deaths in humans (see, e.g., Segal et al. (2017) Clin Cancer Res 23(8): 1929-1935). Similar toxicities resulting from treatment with agonist anti-CDl37 antibodies (such as the 3H3 antibody) have also been observed in animal models (see, e.g., Bartkowiak et al. (2016) Clin Cancer Res 24(5): 1138- 1151). Yet, the agonist anti-CDl37 antibodies described herein have minimal effects on the liver, as determined by, e.g., plasma levels of liver enzymes (e.g., alanine aminotransferase (ALT)) and immune cell infiltration. For example, there was no evidence of increased intrahepatic or intrasplenic immune cell infiltration in mice treated with the antibodies. Thus, the CD137 binding antibodies described herein are not only highly efficacious, but also sparing of certain toxicities associated with CD 137 agonism.
  • ALT alanine aminotransfer
  • the superior therapeutic and toxicity-sparing properties of the CD 137 binding antibodies described herein are believed to derive in part from one or both of their affinity and the novel epitope to which they bind. That is, the antibodies described herein share a common, novel epitope that is distinct from that of other agonist anti-CD 137 antibodies. And, as exemplified in the working examples, engagement of this epitope by the antibodies described herein gives rise to differentiated in vitro activity, such as effects on regulatory T cell proliferation, cytokine production by CD8 + T cells and macrophages, and intracellular signaling, as compared to agonist antibodies that bind to different epitopes of CD 137. Furthermore, it has been demonstrated that an affinity range (a“sweet spot”) for CD 137 binding antibodies is particularly optimal for anti tumor activity. For example, antibodies of intermediate affinity were shown to be more efficacious against large tumors as compared to antibodies with higher or lower affinity.
  • the disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agonist monoclonal antibody, or antigen-binding fragment thereof, that specifically binds human CD137, wherein the subject has cancer cells that express major histocompatibility complex I (MHC I).
  • MHC I major histocompatibility complex I
  • the MHC I expression is detected by the presence of any one or more markers selected from the group consisting of: MHC I protein, MHC I mRNA, beta-2- microglobulin chain (b2M) protein, b2M chain mRNA, MHC class I alpha chain domain protein (e.g., ocl, oc2, or oc3), and MHC class I alpha chain domain mRNA.
  • MHC I protein MHC I mRNA
  • beta-2- microglobulin chain (b2M) protein b2M chain mRNA
  • MHC class I alpha chain domain protein e.g., ocl, oc2, or oc3
  • MHC class I alpha chain domain protein e.g., ocl, oc2, or oc3
  • MHC class I alpha chain domain protein e.g., ocl, oc2, or oc3
  • MHC class I alpha chain domain mRNA MHC class I alpha chain domain
  • the class I alpha chain is an HLA-A alpha chain. In some aspects, the class I alpha chain is a HLA-B alpha chain. In some embodiments, the class I alpha chain is an HLA-C alpha chain.
  • MHC I expression is detected with an agent directed to any one or more of the markers.
  • the agent is an antibody or a nucleic acid probe.
  • the antibody or probe comprises a detectable label.
  • Those skilled in the art can readily determine the appropriate agent, e.g., antibody or probe, depending on the particular MHC I (e.g., HLA-A, HLA- B, or HLA-C) being detected. While the HLA-A, -B, and -C genes encoding the alpha chains of the MHC I complex are known to be polymorphic, those skilled in the art can readily determine their sequences, and select and/or design a suitable detecting agent directed to the alpha chain and/or the b2M chain.
  • HLA-A An exemplary amino acid sequence of MHC I (HLA-A) is shown in SEQ ID NO: 129 (its nucleotide sequence is shown in SEQ ID NO: 130); the amino acid sequence of b2M is shown in SEQ ID NO: 131 (its nucleotide sequence is shown in SEQ ID NO: 132).
  • the marker is detected using a method selected from any one or more of the following: reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, nucleic acid microarray using DNA, western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence- activated cell sorting (FACS), mass spectrometry, magnetic bead-antibody immunoprecipitation, or protein chip.
  • RT-PCR reverse transcription polymerase chain reaction
  • competitive RT-PCR real-time RT-PCR
  • RNase protection assay RNase protection assay
  • northern blotting nucleic acid microarray using DNA
  • western blotting enzyme-linked immunosorbent assay (ELISA), radio
  • the agonist monoclonal antibody or antigen binding portion (fragment) thereof is any one or more of the anti-CD 137 antibody disclosed herein. In any of the foregoing or related aspects, the agonist monoclonal antibody or antigen binding portion thereof binds human CD137 with an affinity (KD) of about 40-100 nM.
  • the agonist monoclonal antibody or antigen binding portion comprises a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid. In some aspects, X is any amino acid except for alanine.
  • the agonist monoclonal antibody or antigen binding portion comprises a heavy chain CDR3 comprising the amino acid sequence DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X 2 is a non-polar amino acid, wherein X 3 is a non-polar amino acid, wherein X 4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X 7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid.
  • X 2 is proline
  • X 3 is phenylalanine or tryptophan
  • X5 is aspartic acid or glutamic acid
  • X 8 is tyrosine
  • X9 is tyrosine.
  • the agonist monoclonal antibody or antigen binding portion binds to an epitope on human CD137 comprising Kl 14 of SEQ ID NO: 3.
  • the epitope comprises residues El 11, Tl 13, and Kl 14 of SEQ ID NO: 3.
  • the epitope comprises residues El 11, T113, K114, N126, 1132 and P135 of SEQ ID NO: 3.
  • the epitope comprises one or more residues El 11, T113, K114, N126, 1132 and P135 of SEQ ID NO: 3.
  • the agonist monoclonal antibody or antigen binding portion binds to an epitope comprising a sequence of one or more amino acid residues corresponding to amino acid positions 111 to 135 of SEQ ID NO: 3.
  • the epitope comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues corresponding to amino acid positions 111 to 135 of SEQ ID NO: 3.
  • the agonist monoclonal antibody or antigen binding portion binds to an epitope comprising ELTK (corresponding to amino acid residues 111- 114 of SEQ ID NO: 3).
  • the epitope further comprises one or more residues N 126, 1132 and P135 of SEQ ID NO: 3.
  • the agonist monoclonal antibody or antigen binding portion specifically binds to an epitope, wherein said epitope is a non-linear epitope.
  • the agonist monoclonal antibody or antigen binding portion has abrogated binding when a mutation of residue K114 of SEQ ID NO: 3 is present.
  • the agonist monoclonal antibody or antigen binding portion comprises a heavy chain CDR3 comprising the amino acid sequence DXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X is any amino acid. In some aspects, X is any amino acid except for alanine.
  • the agonist monoclonal antibody or antigen binding portion has a loss of binding to human CD137 as a result of the mutation of residues P97, F98, D100A, Y100D, Y100F, or combinations thereof to alanine.
  • the agonist monoclonal antibody or antigen binding portion has increased binding to human CD137 as a result of the mutation of residues P97, F98, D100A, Y 100D, Y 100F, or combinations thereof to any residue except alanine.
  • the agonist monoclonal antibody or antigen binding portion binds to a non-ligand binding region of the extracellular domain of human CD137.
  • the agonist monoclonal antibody or antigen binding portion binds human CD137 with an affinity (K D ) of about 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain CDRs, wherein heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68.
  • the agonist monoclonal antibody or antigen binding portion :
  • (iii) binds to an epitope on human CD137 comprising Kl 14, of SEQ ID NO: 3.
  • the non-ligand binding region spans cysteine rich domain (CRD) III and CRD IV.
  • the agonist monoclonal antibody or antigen binding portion :
  • (iii) binds to an epitope on human CD137 comprising Kl 14 of SEQ ID NO: 3.
  • the non-ligand binding region spans cysteine rich domain (CRD) III and CRD IV.
  • the agonist monoclonal antibody or antigen binding portion does not inhibit the interaction between CD 137 and CD137L.
  • the agonist monoclonal antibody or antigen binding portion does not inhibit the formation of a trimer of CDl37:CDl37L monomers.
  • the agonist monoclonal antibody or antigen binding portion comprises heavy and light chain CDRs selected from the group consisting of: (a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively; and (b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 51, 108 and 68, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 101; and wherein the light chain variable region comprises an amino acid sequence of SEQ ID NO: 6.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions comprising amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 4 and 6, respectively; and (b) SEQ ID NO: 101 and 6, respectively.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 101; and wherein the light chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 6.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions comprising amino acid sequences at least 90% identical to the amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 4 and 6, respectively; and (b) SEQ ID NO: 101 and 6, respectively.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chains comprising amino acid sequences selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain CDRs selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 101 and 103; and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 105.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions encoded by nucleotide sequences selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions encoded by nucleotide sequences having at least 90% identity to the nucleotide sequences selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions encoded by nucleotide sequences having at least 90% identity to SEQ ID NOs: 5 and 7, respectively.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions encoded by nucleotide sequences selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions comprising amino acid sequences selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 101 and 103; and wherein the light chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 105.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain variable regions comprising amino acid sequences at least 90% identical to the amino acid sequences selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain sequences comprising amino acid sequences selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain sequences having amino acid sequences set forth in SEQ ID NOs: 129 and 133, respectively.
  • the agonist monoclonal antibody or antigen binding portion thereof comprises heavy and light chain sequences having amino acid sequences set forth in SEQ ID NOs: 131 and 133, respectively.
  • the present disclosure also provides a method of inducing or enhancing one or more of the following in a cancer cell: (a) dimerization of CD 137 trimers; (b) multimerization of CD137 trimers; (c) human CDl37-mediated T cell activation; (d) human CDl37-mediated cytotoxic T cell response; (e) human CDl37-mediated T cell proliferation; and (f) human CDl37-meduated cytokine production, comprising contacting the cancer cell with an effective amount of an agonist monoclonal antibody that specifically binds human CD137, or antigen binding portion thereof, as disclosed herein, and wherein the cancer cell expresses MHC I.
  • the agonist monoclonal antibody or antigen binding portion thereof induces or enhances dimerization of CD 137 trimers in a manner that is not Fc receptor binding dependent.
  • the agonist monoclonal antibody or antigen binding portion thereof induces or enhances dimerization of CD 137 trimers in a manner which is enhanced by Fc receptor binding.
  • the agonist monoclonal antibody or antigen binding portion thereof cross-reacts with cynomolgus CD137 and/or mouse CD137.
  • the agonist monoclonal antibody or antigen binding portion thereof exhibits at least one or more of the following properties selected from the group consisting of:
  • the agonist monoclonal antibody or antigen binding portion thereof exhibits at least one or more of the following properties relative to a reference antibody that binds human CD137, selected from the group consisting of:
  • the reference antibody is urelumab.
  • the agonistic antibody or antigen-binding fragment induces or enhances human CDl37-mediated T cell activation in the tumor microenvironment, but does not significantly induce or enhance human CDl37-mediated T cell activation in the spleen and/or liver.
  • the agonistic antibody or antigen-binding fragment induces or enhances human CDl37-mediated cytotoxic T cell response in the tumor microenvironment, but does not significantly induce or enhance human CDl37-mediated cytotoxic T cell response in the spleen and/or liver.
  • the agonistic antibody or antigen-binding fragment induces human CDl37-mediated T cell proliferation in the tumor microenvironment, but does not significantly induce human CDl37-mediated T cell proliferation in the spleen and/or liver. In some aspects, the agonistic antibody or antigen binding fragment induces or enhances human CDl37-mediated cytokine production in the tumor microenvironment, but does not significantly induce or enhance human CDl37-mediated cytokine production in the spleen and/or liver. In some aspects, the properties of the agonistic antibody or antigen-binding fragment are not Fc gamma receptor binding dependent. In some aspects, the properties of the agonistic antibody or antigen-binding fragment are enhanced by Fc gamma receptor binding.
  • the agonistic antibody or antigen-binding fragment cross-reacts with cynomolgus CD137 and/or mouse CD137.
  • the agonistic antibody is selected from the group consisting of an IgGl, an IgG2, and IgG3, an IgG4, and IgM, and IgAl, and IgA2, and IgD, and an IgE antibody.
  • the agonistic antibody is an IgGl antibody or IgG4 antibody.
  • the isolated agonistic monoclonal antibody comprises an IgGl heavy chain constant region or an IgG4 heavy chain constant region. In some aspects, the isolated agonistic monoclonal antibody comprises an IgGl heavy chain constant region. In some aspects, the IgGl heavy chain constant region is a wild-type human IgGl heavy chain constant region. In some aspects, the IgGl heavy chain constant region comprises an amino acid substitution relative to a wild-type human IgGl heavy chain constant region. In some aspects, the isolated agonistic monoclonal antibody comprises an IgG4 heavy chain constant region. In some aspects, the IgG4 heavy chain constant region is a wild-type human IgG4 heavy chain constant region.
  • the IgG4 heavy chain constant region comprises an amino acid substitution relative to a wild-type human IgG4 heavy chain constant region.
  • the amino acid substitution is an amino acid substitution at position Ser228 according to EU numbering.
  • the amino acid substitution at position Ser228 is S228P.
  • the IgG4 heavy chain constant region comprises an amino acid substitution relative to a wild-type human IgG4 heavy chain constant region, wherein the amino acid substitution is an amino acid substitution at position Ser228 according to EU numbering, and wherein amino acid substitution at position Ser228 is S228P.
  • the agonist monoclonal antibody or antigen binding portion thereof induces or enhances T cell activation in the subject.
  • the T cell activation occurs in a tumor microenvironment.
  • the agonist monoclonal antibody or antigen binding portion thereof induces or enhances a cytotoxic T cell response in the subject.
  • the cytotoxic T cell response occurs in a tumor microenvironment.
  • the agonist monoclonal antibody or antigen binding portion thereof induces or enhances cytokine production in the subject.
  • the cytokine produced is IL-2, TNFoc, IL-13, IFNy, or combinations thereof.
  • the cytokine produced is IF-2.
  • the cytokine produced is TNFoc.
  • the cytokine produced is IF-13.
  • the cytokine produced is IFNy.
  • the cytokine produced is IF-2 and TNFoc.
  • the cytokine produced is IF-2 and IF-13.
  • the cytokine produced is IF-2 and IFNy.
  • the cytokine produced is TNFoc and IF-13. In some aspects, the cytokine produced is TNFoc and IFNy. In some aspects, the cytokine produced is IF-13 and IFNy. In some aspects, the cytokine produced is IF-2, TNFoc and IF-13. In some aspects, the cytokine produced is IF-2, TNFoc and IFNy. In some aspects, the cytokine produced is IFNy TNFoc and IF-13. In other aspects, cytokine production occurs in a tumor microenvironment. In yet other aspects, cytokine production does not significantly occur in the spleen and/or liver of the subject.
  • the agonist monoclonal antibody or antigen binding portion thereof induces or enhances T cell proliferation in the subject.
  • the T cell proliferation occurs in a tumor microenvironment.
  • the agonist monoclonal antibody or antigen binding portion thereof reduces or inhibits tumor growth in the subject.
  • the agonistic isolated monoclonal antibody or antigen binding fragment thereof increases infiltration of immune cells into a tumor microenvironment in the subject.
  • the immune cells express CD45
  • the agonist monoclonal antibody or antigen binding portion thereof reduces the quantity of T regulatory (Treg) cells in a tumor microenvironment in the subject.
  • the Treg cells express CD4, FOXP-3 and CD25.
  • the agonist monoclonal antibody or antigen binding portion thereof reduces the quantity of macrophages is reduced in a tumor microenvironment in the subject.
  • the macrophages express CD45 and CDl lb.
  • the isolated agonistic monoclonal antibody or antigen-binding portion thereof reduces T cell exhaustion in a tumor microenvironment in the subject.
  • reduction of T cell exhaustion comprises a decrease in expression of TIGIT, PD-l, LAG-3, or combinations thereof.
  • reduction of T cell exhaustion comprises a decrease in expression of a combination of TIGIT and PD-l.
  • the isolated agonistic monoclonal antibody or antigen-binding portion thereof induces an anti-tumor memory immune response in the subject.
  • the isolated agonistic monoclonal antibody or antigen-binding portion thereof binds Fc gamma receptor.
  • depletion of CD4+ T cells, CD8+ T cells, Natural Killer cells, or combinations thereof reduces the efficacy of the isolated agonistic monoclonal antibody or antigen-binding portion thereof.
  • the agonist monoclonal antibody or antigen binding portion thereof reduces or inhibits tumor (cancer) growth.
  • the agonist monoclonal antibody or antigen binding portion thereof treats a disorder mediated by human CD 137 in a subject.
  • the agonist monoclonal antibody or antigen binding portion thereof treats cancer in a subject.
  • the cancer is selected from the group consisting of melanoma, glioma, renal, colon, lung, prostate, breast, and head and neck cancer, among others as disclosed herein.
  • the agonist monoclonal antibody or antigen binding portion thereof binds Fc gamma receptor.
  • the agonist monoclonal antibody does not significantly induce or enhance intrahepatic and/or intrasplenic T cell activation and/or T cell proliferation.
  • the agonist monoclonal antibody binds to human CD137 with an equilibrium dissociation constant K D of 1 X 10 6 or less.
  • the agonist monoclonal antibody is selected from the group consisting of: human antibody, humanized antibody, chimeric antibody, and deimmunized antibody.
  • the agonist monoclonal antibody is selected from the group consisting of: a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab’ fragment, an F(ab’) 2 fragment, and a bispecific antibody.
  • the present disclosure also provides a method of detecting MHC I in a subject having cancer, comprising: (i) contacting a biological sample comprising cancer cells from the subject with an agent directed to MHC I; and (ii) detecting the agent bound to MHC I; and (iii) administering to the subject an agonist anti-CD 137 antibody, or antigen -binding fragment thereof, as disclosed herein.
  • the agent is an antibody or a nucleic acid probe.
  • the antibody or probe comprises a detectable label. Such detectable labels are commonly known in the art.
  • the present disclosure also provides a method of determining whether a subject having cancer would be amenable to agonist CD 137 antibody, or antigen-binding fragment thereof, therapy.
  • the method comprises contacting a biological sample comprising cancer cells from the subject with an agent directed to MHC I, and detecting the agent bound to MHC I according to known methods, and as described herein, wherein the presence of MHC I indicates that the cancer is amenable to treatment with an agonist CD 137 antibody, or an antigen binding fragment thereof.
  • detecting MHC I expression is performed by detecting the presence of any one or more markers selected from the group consisting of: MHC I protein, MHC I mRNA, beta-2-microglobulin chain (b2M) protein, b2M chain mRNA, MHC class I alpha chain domain protein (e.g., ocl, oc2, or oc3), and MHC class I alpha chain domain mRNA.
  • MHC I protein MHC I mRNA
  • beta-2-microglobulin chain (b2M) protein b2M chain mRNA
  • MHC class I alpha chain domain protein e.g., ocl, oc2, or oc3
  • MHC class I alpha chain domain protein e.g., ocl, oc2, or oc3
  • MHC class I alpha chain domain protein e.g., ocl, oc2, or oc3
  • MHC class I alpha chain domain mRNA e.g.,
  • any one or more of the class I alpha chains and/or their specific domains can be suitable for detecting MHC I expression.
  • MHC I expression is detected with an agent directed to any one or more of the markers.
  • the agent is an antibody or a nucleic acid probe.
  • the antibody or probe comprises a detectable label.
  • Those skilled in the art can readily determine the appropriate agent, e.g., antibody or probe, depending on the particular MHC I (e.g., HLA-A, HLA-B, or HLA-C) being detected.
  • HLA-A HLA-A
  • -B HLA-B
  • -C genes encoding the alpha chains of the MHC I complex are known to be polymorphic, those skilled in the art can readily determine their sequences, and select and/or design a suitable detecting agent directed to the alpha chain and/or the b2M chain.
  • An exemplary amino acid sequence of MHC I (HLA-A) is shown in SEQ ID NO: 129 (its nucleotide sequence is shown in SEQ ID NO: 130); the amino acid sequence of b2M is shown in SEQ ID NO: 131 (its nucleotide sequence is shown in SEQ ID NO: 132).
  • the detecting is performed by any method known in the art and include, e.g., a reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, nucleic acid microarray using DNA, western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting (FACS), mass spectrometry, magnetic bead-antibody immunoprecipitation, protein chip, or any combination thereof.
  • RT-PCR reverse transcription polymerase chain reaction
  • competitive RT-PCR real-time RT-PCR
  • RNase protection assay RNase protection assay
  • northern blotting nucleic acid microarray using DNA
  • western blotting enzyme-linked immuno
  • the method further comprises determining that the cancer is amenable to treatment with an agonist CD 137 antibody, or antigen -binding fragment thereof (by the presence of MHC I), and administering to the subject a therapeutically effective amount of an agonist anti- CD 137 antibody, or antigen-binding fragment thereof.
  • the present disclosure provides a method of identifying a cancer patient likely to respond to treatment with an antibody that specifically binds to CD137, the method comprising contacting a tumor sample from the patient with an antibody that specifically binds to MHC I, or an antigen -binding fragment thereof; and detecting the presence of the MHC I, wherein the presence of MHC I indicates the patient is likely to respond to treatment.
  • the method comprises detecting the presence of MHC I by determining an amount of MHC I in the sample, wherein the amount of MHC I relative to a control indicates the subject is likely to respond to treatment with the agent. In some aspects, if the amount of MHC I in the sample is increased relative to the control indicates the patient is likely to respond to treatment with the agent.
  • the present disclosure provides a method of identifying a cancer patient likely to respond to treatment with an antibody that specifically binds to CD137, the method comprising contacting a tumor sample from the patient with an antibody that specifically binds to beta-2-microglobulin chain (b2M), or an antigen-binding fragment thereof; and detecting the presence of the b2M, wherein the presence of b2M indicates the patient is likely to respond to treatment.
  • the method comprises detecting the presence of b2M by determining an amount of b2M in the sample, wherein the amount of b2M I relative to a control indicates the subject is likely to respond to treatment with the agent. In some aspects, if the amount of b2M in the sample is increased relative to the control indicates the patient is likely to respond to treatment with the agent.
  • the present disclosure provides method of identifying a cancer patient likely to respond to treatment with an antibody that specifically binds to CD137, the method comprising contacting a tumor sample from the patient with an antibody that specifically binds to MHC class I alpha chain protein, or an antigen-binding fragment thereof; and detecting the presence of the MHC class I alpha chain, wherein the presence of MHC class I alpha chain indicates the patient is likely to respond to treatment.
  • the method comprises detecting the presence of MHC class I alpha chain protein by determining an amount of MHC class I alpha chain in the sample, wherein the amount of MHC class I alpha chain relative to a control indicates the subject is likely to respond to treatment with the agent. In some aspects, if the amount of MHC class I alpha chain protein in the sample is increased relative to the control indicates the patient is likely to respond to treatment with the agent.
  • the disclosure provides a method of detecting a tumor susceptible to treatment with an antibody that specifically binds to CD137, the method comprising determining an expression level of a panel of polypeptides comprising MHC I protein, beta-2-micro globulin chain (b2M) protein, and MHC class I alpha chain protein, in a tumor sample, wherein the expression level of the panel of polypeptides in the tumor sample indicates the tumor is susceptible to treatment with the agent.
  • determining an expression level of the panel of polypeptides comprises comparing the expression levels of the panel of polypeptides in the tumor sample to an expression level of the panel of polypeptides in a reference sample. In some aspects, if the expression level of MHC I in the tumor sample is increased relative to the reference sample indicates the tumor is susceptible to treatment with the agent.
  • the disclosure provides a kit comprising a container comprising one or more reagents for detecting the presence of MHC I, optionally beta-2-microglobulin chain (b2M) and/or MHC class I alpha chain in a tumor sample, and a package insert comprising instructions for determining an amount or expression level of MHC I, optionally b2M and/or MHC class I alpha chain in the tumor sample.
  • a kit comprising a container comprising one or more reagents for detecting the presence of MHC I, optionally beta-2-microglobulin chain (b2M) and/or MHC class I alpha chain in a tumor sample, and a package insert comprising instructions for determining an amount or expression level of MHC I, optionally b2M and/or MHC class I alpha chain in the tumor sample.
  • b2M beta-2-microglobulin chain
  • the kit comprises a reagent for detecting the presence of b2M in the sample and instructions for determining an amount or expression level of b2M in the tumor sample
  • the kit comprises a reagent for detecting the presence of MHC class I alpha chain in the sample and instructions for determining an amount or expression level of MHC class I alpha chain in the tumor sample.
  • determining an amount or expression level of any one or more of MHC I, b2M, and MHC class I alpha chain, or a combination thereof comprises comparing the amount or expression level of MHC I, b2M, or MHC class I alpha chain, or a combination thereof in a tumor sample relative to the amount or expression level in a reference sample.
  • the kit comprises an antibody specifically reactive with MHC I. In some embodiments, the kit comprises an antibody specifically reactive with b2M. In some embodiments, the kit comprises an antibody specifically reactive with MHC class I alpha chain. In some embodiments, the kit comprises an antibody specifically reactive with one or more of MHC I, b2M, and MHC class I alpha chain.
  • the one or more antibodies is covalently attached to a label.
  • the label is a fluorescent molecule, a chromogenic molecule, or a radioactive molecule.
  • FIG. 1 provides graphs depicting the distribution of binding affinities of affinity matured clones of the parental anti-CD 137 antibody mAbl.
  • FIG. 2 provides a schematic showing the results of mAbl CDRH3 alanine scanning, as measured by binding affinity (K D ) to human or mouse CD 137.
  • FIG. 3A shows the amino acid sequence of human CD137 (residues 24-159 of SEQ ID NO: 3) wherein residues comprising an epitope bound by mAbl, mAb4 or mAb5 are indicated in bold.
  • FIG. 3B is a graph depicting kinetic binding data of mAbl to the extracellular domain of mouse and rat CD 137 as determined by surface plasmon resonance.
  • FIG. 3C provides x-ray crystallography images of human CD 137 bound to CDl37L( shown in grey) and residues El 11, Tl 13, Kl 14 and P135 shown as spheres.
  • FIG. 3D provides x-ray crystallography images of human CD 137 bound to CD137L (shown in grey) in trimeric formation, and residues E111, T113, K114 and P 135 shown as spheres.
  • FIG. 4A provides a scatterplot of flow cytometric data depicting an increase in TIGIT (top) or PD-l (bottom) expression on CD44+ T cells in response to anti-CDl37 antibodies.
  • FIG. 4B provides graphs depicting the quantification of CD8+ CD44+ T cells expressing TIGIT (top) or PD-l (bottom) in the spleen of mice after treatment with anti-CDl37 antibodies.
  • FIG. 4C provides graphs depicting the quantification of CD8+ T cells in the spleen of mice after treatment with anti-CD 137 antibodies, as percentage of CD45+ cells (left) or cell number per spleen (right).
  • FIG. 5A provides graphs showing individual CT26 tumor volumes in mice after treatment with anti-CDl37 antibodies at indicated dosages.
  • FIG. 5B is a graph showing the mean tumor volumes provided in FIG. 5A.
  • FIG. 5C is a Kaplan-Meier graph showing overall survival of mice with tumors after treatment with anti-CD 137 antibodies.
  • FIG. 5D is a graph showing tumor volume in mice re-challenged with tumorigenic CT26 cells.
  • FIG. 6A provides graphs showing individual CT26 tumor volumes in mice after treatment with parental and affinity-matured anti-CD 137 antibodies.
  • FIG. 6B is a graph providing the mean tumor volumes provided in FIG. 6A.
  • FIG. 7 provides graphs depicting the percentage of CD8+ or CD4+ T cells, from splenic T cells (top) and tumor infiltrating leukocytes (bottom) after treatment with anti-CD 137 antibodies at indicated dosages.
  • FIG. 8 provides graphs showing individual tumor volumes when mice were treated with mAbl, with or without lymphocyte depleting antibodies.
  • CD4+ T cells were depleted with GK1.5 (middle graph)
  • CD8+ T cells were depleted with YTS 169.4 (second graph from the right)
  • NK cells were depleted with an anti-asialo-GMl antibody (last graph on the right).
  • FIG. 9 provides graphs showing individual tumor volumes in mice having either CT26 tumors (colon carcinoma), EMT-6 tumors (breast carcinoma), A20 tumors (B cell lymphoma), or MC38 tumors (colon carcinoma) and treated with mAb8 or isotype control antibody.
  • FIGs. 10A-10C show the in vivo anti-tumor efficacy of anti-CD 137 antibodies administered at 150 mg/mouse. Individual tumor volumes are shown in 10A, mean tumor volumes are shown in 10B and percent survival is shown in 10C.
  • FIGs. 11A-11C show the in vivo anti-tumor efficacy of anti-CDl37 antibodies administered at 20 mg/mouse. Individual tumor volumes are shown in 11A, mean tumor volumes are shown in 11B and percent survival is shown in 11C.
  • FIG. 12 provides graphs showing individual tumor volumes in mice having CT26 tumors and treated with varying doses of mAbl ( i.e 12.5, 25, 50, 100 or 200mg) or isotype control.
  • FIGs. 13A and 13B show the contribution of Fc binding in the anti-tumor efficacy of mAbl.
  • FIG. 13A shows mAbl as an IgG4 isotype or an IgG4 aglycosylated isotype. Mean tumor volumes are shown on the top and individual tumor volumes are shown on the bottom.
  • FIG. 13B shows mAbl as an IgG4 isotype or an IgGl aglycosylated isotype. Mean tumor volumes are shown on the top and individual tumor volumes are shown on the bottom.
  • FIGs. 14A-14D show the in vivo anti-tumor efficacy of anti-CD 137 antibodies in mice with large established tumors (i.e., 500mm 3 ) prior to receiving treatment. Individual tumor volumes are shown in 14A and 14D, mean tumor volumes are shown in 14B and percent survival is shown in 14C.
  • FIG. 15 provides a Kaplan-Meier survival graph showing protective anti-tumor immunity in mice previously treated with mAbl, mAb8 or isotype control from FIGs. 14A-14C and considered cured, re-challenged with CT26 cells in an opposing flank.
  • FIG. 16A provides scatterplots of flow cytometric data showing the expansion of CD45+ intrahepatic T cells following treatment with anti-CDl37 antibodies at indicated dosages.
  • FIG. 16B provides graphs depicting the quantification of intrahepatic CD8+ T cells (left) and CD4+ T cells (right) following treatment with anti-CDl37 antibodies at indicated dosages.
  • FIG. 17A provides graphs depicting the percentage of CD3+, CD4+, or CD8+ T cells, from splenic T cells after treatment of mice with affinity-matured anti-CDl37 antibodies.
  • FIG. 17B provides graphs depicting the percentage of CD3+, CD4+, or CD8+ T cells from liver T cells after treatment of mice with affinity-matured anti-CDl37 antibodies.
  • FIG. 18A provides graphs depicting the percentage of splenic CD8+CD44+ T cells expressing TIGIT, PD-l, or LAG3 after treatment of mice with affinity-matured anti-CD 137 antibodies.
  • FIG. 18B provides graphs depicting the percentage of liver CD8+CD44+ T cells expressing TIGIT, PD-l, or LAG3 after treatment of mice with affinity-matured anti-CD 137 antibodies.
  • FIG. 19A provides graphs depicting the percentage of splenic CD4+CD44+ T cells expressing TIGIT, PD-l, or LAG3 after treatment of mice with affinity-matured anti-CD 137 antibodies.
  • FIG. 19B provides graphs depicting the percentage of liver CD4+CD44+ T cells expressing TIGIT, PD-l, or LAG3 after treatment of mice with affinity-matured anti-CD 137 antibodies.
  • FIGs. 20A-20C provide graphs of in vivo indicators of toxicity resulting from multiple administrations of anti-CD 137 antibodies mAbl, mAb8 or 3H3 at varying doses.
  • FIG. 20A is a graph showing percentage of CD8+ T cells in the liver after administration of the anti-CD 137 antibodies.
  • FIG. 20B is a graph showing alanine aminotransferase (ALT) activity in the plasma of mice administered anti-CDl37 antibodies.
  • FIG. 20C is a graph showing the levels of TNFoc in the plasma of mice administered anti-CDl37 antibodies.
  • FIG. 21 provides representative images of sectioned livers stained with hematoxylin and eosin (H&E) from mice treated with mAbl, mAb8, 3H3 or isotype control as described in FIGs. 20A-20C. Arrows indicate infiltration of immune cells.
  • H&E hematoxylin and eosin
  • FIGs. 22A-22D provide representative FACS plots showing immune cell reprogramming in the tumor microenvironment. Mice having CT26 tumors were administered multiple doses of mAb8 or isotype control (days 0, 3, 6 and 9).
  • FIG. 22A shows overall immune cell infiltration based on CD45 expression.
  • FIG. 22B shows reduction in Treg cells as measured by FOXP-3 and CD25 expression.
  • FIG. 22C shows reduction of T-cell exhaustion as measured by PD-l and TIGIT expression.
  • FIG. 22D shows reduction of tumor-associated macrophages as measured by F4/80 and CDl lb expression.
  • FIG. 23 shows immunophenotyping analysis of spleens from mice having CT26 tumors and treated with either anti-CD 137 antibodies mAbl and 3H3, or isotype control.
  • FIG. 24 is a graph showing the concentration of IL-2 (pg/ml) produced by murine T cells in an OVA stimulation assay, when stimulated with the anti-CDl37 antibodies indicated.
  • IL-2 pg/ml
  • Atezolizumab anti-PD-Ll antibody
  • RMP1-14 murine anti-PD-l
  • FIGs. 25A and 25B are graphs showing the percentage of murine CD8+ T cells expressing either CD25 (25A) or TIGIT (25B) when stimulated with the anti-CD 137 antibodies indicated, in an OVA stimulation assay.
  • Atezolizumab anti-PD-Ll antibody
  • RMP1-14 murine anti-PD-l
  • 3H3 murine anti-CDl37
  • FIG. 26 provides bar graphs depicting the quantification of cytokines (IL-2, TNFa, IL-13, and IFNy) produced by CD3+ T cells following incubation with plate-bound anti-CD 137 antibodies. Cytokine levels are shown as fold increase over baseline activation by an anti-CD3 antibody.
  • cytokines IL-2, TNFa, IL-13, and IFNy
  • FIGs. 27A-27C provide graphs depicting the dose-response of IFNy production in a mixed lymphocyte reaction following treatment with anti-CDl37 antibodies.
  • An anti-PDl antibody Keytruda; Merck was used as a control.
  • FIG. 28 is a graph showing IFNy production from human T cells co-cultured with CHO cells engineered to express CD32 (CHO-CD32 cells) in the presence of anti-CD 137 antibodies mAbl, mAb8, mAb4 or mAb5, or isotype control.
  • FIG. 29 is a graph showing proliferation of Treg cells when co-cultured with CHO cells engineered to express CD32 (CHO-CD32 cells) in the presence or absence of anti-CD 137 antibodies mAbl, mAb8, mAb4 or mAb5, isotype control.
  • FIG. 30 provides graphs showing NFK and SRF signaling in CCL-119 cells transduced with luciferase reporters for NFK or SRF in the presence of mAbl, mAB8, mAb4 or mAb5 at varying concentrations.
  • FIG. 31 provides graphs showing induction of IL-6, TNFoc, or IL-27 by bone marrow- derived mouse macrophages stimulated with TLR9 agonist CpG in the presence of anti-CD 137 antibodies mAbl, 3H3 or LOB 12.3, or isotype control.
  • FIG. 32 provides a graph showing induction of TNFoc by human monocyte derived macrophages stimulated with LPS in the presence of anti-CD 137 antibodies mAbl, mAb4 or mAb5, or isotype control.
  • FIG. 33 provides a graph showing effect of anti-CD 137 antibodies on macrophage differentiation as determined by CD64 expression of THP1 monocytes cultured with PM A in the presence of anti-CDl37 antibodies mAbl, mAb4 or mAb5, or isotype control.
  • FIGs. 34A-34C provides graphs showing percentage of hCD45+, hCD8+ or hCD4+ from immunocompetent mice that received human PBMCs and anti-CDl37 antibodies mAbl, mAb4 or mAb5, or isotype control.
  • FIG. 35A shows CT26 cells, a colon cancer cell line, stained with a fluorescent antibody against beta-2 microglobulin (B2M) or isotype control, and analyzed using flow cytometry.
  • B2M beta-2 microglobulin
  • FIG. 35B shows CT26 cells stained with a fluorescent antibody against major histocompatibility complex I (MHC I) or isotype control, analyzed using flow cytometry.
  • MHC I major histocompatibility complex I
  • FIG. 36A shows B 16-F10 cells, a skin melanoma cell line, stained with a fluorescent antibody against beta-2 microglobulin (B2M) or isotype control, and analyzed using flow cytometry.
  • B2M beta-2 microglobulin
  • FIG. 36B shows B 16-F10 cells stained with a fluorescent antibody against major histocompatibility complex I (MHC I) or isotype control, analyzed using flow cytometry.
  • MHC I major histocompatibility complex I
  • FIG. 37 shows CT26 cells, EMT6 cells, A20 cells, MC38 cells, RENCA cells, MB49 cells, 4T1 cells, ID-8 cells, B 16-F10 cells, PANC02 cells, HEPA1-6 cells, and Rl.l cells stained with a fluorescent antibody against major histocompatibility complex I (MHC I) or isotype control and analyzed using flow cytometry.
  • MHC I major histocompatibility complex I
  • FIG. 38A shows CT26 cells stained with a fluorescent antibody against major histocompatibility complex I (MHC I) as compared to isotype control which were analyzed using flow cytometry; and graphs of tumor volume in mice following treatment with either 25 pg weekly of mAb8 or vehicle.
  • FIG. 38B shows EMT6 cells stained with a fluorescent antibody against MHC I or isotype control which were analyzed using flow cytometry; and graphs of tumor volume in mice following treatment with either 5pg weekly of mAb8 or vehicle.
  • MHC I major histocompatibility complex I
  • FIG. 38C shows A20 cells stained with a fluorescent antibody against MHC I as compared to isotype control which were analyzed using flow cytometry; and graphs of tumor volume in mice following treatment with either 200 pg weekly of mAb8 or control antibodies.
  • FIG. 38D shows MC38 cells stained with a fluorescent antibody against MHC I or isotype control which were analyzed using flow cytometry; and graphs of tumor volume in mice following treatment with either 12.5 pg weekly of mAb8 or control antibodies. Histogram curves for MHC I and isotype control is indicated in blue and red, respectively.
  • FIG. 39A shows B 16-F10 cells stained with a fluorescent antibody against major histocompatibility complex I (MHC I) or isotype control which were analyzed using flow cytometry; and graphs of tumor volume in mice following treatment with either 150 pg of mAb8 or isotype control antibodies on days 0, 3, 6, and 9.
  • MHC I major histocompatibility complex I
  • FIG. 39B shows PANC02 cells stained with a fluorescent antibody against MHC I or isotype control which were analyzed using flow cytometry; and graphs of tumor volume in mice following treatment with either 150 pg of mAb8 or isotype control antibodies.
  • FIG. 39C shows 4T1 cells stained with a fluorescent antibody against MHC I or isotype control which were analyzed using flow cytometry; and graphs of tumor volume in mice following treatment with either 50pg of mAbl or isotype control antibodies on days 0, 3, 6, and 9. Units for middle panel of FIG. 39C is the same as the lower panel of FIG. 39C.
  • FIG. 40 depicts expression levels of MHC I, B2M, and PDL1, in CT26 parental cells (parental CTN) and CT26- 2M KO cells (CT26 B2M KO 873 CNT and CT26 B2M KO 882 CNT).
  • FIG. 41A graphically depicts tumor volume in mice injected with CT26 parental cells (CT26 Par CNT) or CT26- 2M KO cells (CT26 B2M KO 882 or CT26 B2M KO 883 ) following treatment with mAb8 or control (x-axis: days post-commencement of treatment, y-axis: tumor volume in mm 3 ).
  • FIG. 41B graphically depicts tumor volume in mice injected with CT26 parental cells (CT26 Par CNT) or OT26-b2M KO cells (CT26 B2M KO 882 or CT26 B2M KO 883 ) following treatment with mAb8 or control.
  • FIG. 41C graphically depicts percent survival in mice injected with CT26 parental cells (CT26 Par CNT) or CT26-p2M KO cells (CT26 B2M KO 882 or CT26 B2M KO 883 ) following treatment with mAh 8 or control.
  • the present disclosure relates to methods and composition for treating cancer in a subject.
  • the disclosure is based, at least in part, on the surprising discovery that treatment of cancer cells expressing major histocompatibility complex I (MHC I) is enhanced when treatment includes the anti-CD 137 agonist antibodies, and antigen-binding fragments thereof, disclosed herein.
  • MHC I major histocompatibility complex I
  • the present discovery can be utilized to identify a cancer patient population that would be particularly amenable to anti-cancer therapy using the anti-CD 137 agonist antibodies disclosed herein.
  • a patient population wherein the patient or subject has cancer cells that express MHC I.
  • the disclosure provides methods for the treatment of cancer in a subject with the isolated monoclonal antibodies of the disclosure, or antigen binding portions thereof, that specifically bind to an epitope of human CD 137 and agonize human CD 137.
  • the subject has cancer cells that express MHC I.
  • the antibody or antigen binding portion thereof competes with mAbl for binding to the epitope of human CD137.
  • the anti-CDl37 agonist antibodies of the disclosure induce cytokine production and expansion of CD8+ T cells in the tumor microenvironment, and protective anti-tumor immunity in vivo with a concomitant reduction in the potential for toxicity- related events, as compared to the anti-mouse CD137 3H3 antibody (Melero et al. (1997) Nature Medicine 3(6):682-685; Uno et al. (2006) Nature Medicine l2(6):693-696) and to at least two anti-human CD137 antibodies in clinical development (BMS-6635l3/Urelumab, Bristol-Meyers Squibb, and PF-05082566/Utomilumab, Pfizer).
  • the term "agonist” refers to any molecule that partially or fully promotes, induces, increases, and/or activates a biological activity of a native polypeptide disclosed herein (e.g., CD137).
  • Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc.
  • activation in the presence of the agonist is observed in a dose-dependent manner.
  • the measured signal e.g., biological activity
  • the measured signal is at least about 5%, at least about 10%, at least about 15%, at least about
  • binding assays such as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio ⁇ systems, and radioimmunoassay (RIA).
  • ELISA enzyme-linked immuno-absorbent assay
  • RIA radioimmunoassay
  • Efficacy of an agonist can also be determined using functional assays, such as the ability of an agonist to activate or promote the function of the polypeptide.
  • a functional assay may comprise contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.
  • the potency of an agonist is usually defined by its EC50 value (concentration required to activate 50% of the agonist response). The lower the EC50 value the greater the potency of the agonist and the lower the concentration that is required to activate the maximum biological response.
  • alanine scanning refers to a technique used to determine the contribution of a specific wild-type residue to the stability or function(s) (e.g., binding affinity) of a given protein or polypeptide.
  • the technique involves the substitution of an alanine residue for a wild-type residue in a polypeptide, followed by an assessment of the stability or function(s) (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparison to the wild-type polypeptide.
  • Techniques to substitute alanine for a wild-type residue in a polypeptide are known in the art.
  • ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phospho serine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • a“polar amino acid” refers to an amino acid comprising a side chain that prefers to reside in an aqueous environment.
  • a polar amino acid is selected from the group consisting of: arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, theronine and tyrosine.
  • Polar amino acids can be positive, negatively or neutrally charged.
  • a“non-polar amino acid” refers to an amino acid selected from the group consisting of: alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine.
  • amino acid substitution refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different “replacement” amino acid residue.
  • amino acid insertion refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger “peptide insertions,” can also be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non- naturally occurring as disclosed above.
  • amino acid deletion refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
  • antagonist refers to any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein.
  • Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc.
  • inhibition in the presence of the antagonist is observed in a dose-dependent manner.
  • the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured with a negative control under comparable conditions. Also disclosed herein, are methods of identifying antagonists suitable for use in the methods of the disclosure.
  • these methods include, but are not limited to, binding assays such as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio ⁇ systems, and radioimmunoassay (RIA).
  • binding assays such as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio ⁇ systems, and radioimmunoassay (RIA).
  • ELISA enzyme-linked immuno-absorbent assay
  • RIA radioimmunoassay
  • the term“amount” or“level” refers to a detectable quantity, level or abundance of a substance (e.g., a protein or a biomarker).
  • a substance e.g., a protein or a biomarker.
  • the terms“level of expression” or“expression level” in general are used interchangeably and generally refer to a detectable amount of a polypeptide or biomarker in a biological sample (e.g., on the surface of a cell).
  • a detectable amount or detectable level of a biomarker is associated with a likelihood of a response to an agent, such as those described herein.
  • an anti-CDl37 agonist antibody refers to an antibody that specifically binds to CD137 and partially or fully promotes, induces, increases, and/or activates CD137 biological activity, response, and/or downstream pathway(s) mediated by CD137 signaling or other CDl37-mediated function.
  • an anti-CD 137 agonist antibody binds to CD 137 and allows binding of CD137L.
  • an anti-CD 137 agonist antibody binds to CD 137 and induces multimerization of CD137.
  • an anti-CDl37 agonist antibody binds to CD137 and induces the dimerization of CD137 trimers.
  • an anti-CDl37 agonist antibody binds to CD 137 and induces the multimerization of CD 137 trimers.
  • anti-CD 137 agonist antibodies are provided herein. Methods for detecting formation of a trimentrimer complex are known to those of skill in the art. For example, electron microscopy has been shown to detect such complexes, see, e.g., Won, E. The Journal of Biological Chemistry, Vol. 285 (12): 9202-9210 (2010)
  • anti-CDl37 mAbl refers to an exemplary anti-CD 137 agonist antibody that comprises the variable heavy chain (V H ) amino acid sequence:
  • variable light chain (V L ) amino acid sequence (V L ) amino acid sequence:
  • anti-CD 137 mAb8 refers to an exemplary anti-CD 137 agonist antibody that comprises the variable heavy chain ((V H ) amino acid sequence:
  • variable light chain (V L ) amino acid sequence (V L ) amino acid sequence:
  • anti-CDl37 mAblO refers to an exemplary anti-CDl37 agonist antibody that comprises the variable heavy chain ((V H ) amino acid sequence:
  • variable light chain (V L ) amino acid sequence (V L ) amino acid sequence:
  • the term“antibody” refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies.
  • the term“antibody” includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody.
  • the antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.
  • the antibody can be a purified or a recombinant antibody.
  • the terms“antibody fragment,”“antibody portion”, “antigen -binding fragment,”“antigen binding portion” or similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen (e.g., CD137) and inhibit the activity of the target antigen.
  • a target antigen e.g., CD137
  • Such fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, a Fab fragment, a Fab’ fragment, or an F(ab’) 2 fragment.
  • An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived.
  • intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, e.g., Todorovska et al., (2001) J. Immunol. Methods 248(l):47-66; Hudson and Kortt, (1999) J. Immunol. Methods 231(1): 177-189; Poljak, (1994) Structure 2(12): 1121-1123; Rondon and Marasco, (1997) Annu. Rev. Microbiol. 51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety.
  • the term“antibody fragment” also includes, e.g., single domain antibodies such as camelized single domain antibodies. See, e.g., Muyldermans et al., (2001) Trends Biochem. Sci. 26:230-235; Nuttall et al., (2000) Curr. Pharm. Biotech. 1:253-263; Reichmann et al., (1999) J. Immunol. Meth. 231:25-38; PCT application publication nos. WO 94/04678 and WO 94/25591; and U.S. patent no. 6,005,079, all of which are incorporated herein by reference in their entireties.
  • the disclosure provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.
  • an antigen-binding fragment includes the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide. In some embodiments, an antigen-binding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptide of an antibody.
  • antibody- antigen complex or“immune complex” or“complex” refers to a molecular structure formed upon the specific binding of an antibody to an antigen (e.g., a biomarker).
  • the bound antibody and antigen act as a unitary object.
  • a first antibody is bound to an antigen, thereby forming a complex.
  • the complex is detected by the binding of a second antibody to the first antibody, thereby indirectly detecting the antigen.
  • APC antigen presenting cell
  • T cells recognize this complex using T cell receptor (TCR).
  • APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMC), monocytes (such as THP-l), B lymphoblastoid cells (such as C1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs).
  • DCs dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes such as THP-l
  • B lymphoblastoid cells such as C1R.A2, 1518 B-LCL
  • DCs monocyte-derived dendritic cells
  • antigen presentation refers to the process by which APCs capture antigens and enables their recognition by T cells, e.g., as a component of an MHC-I and/or MHC-II conjugate.
  • apoptosis refers to the process of programmed cell death that occurs in multicellular organisms (e.g. humans).
  • the highly-regulated biochemical and molecular events that result in apoptosis can lead to observable and characteristic morphological changes to a cell, including membrane blebbing, cell volume shrinkage, chromosomal DNA condensation and fragmentation, and mRNA decay.
  • a common method to identify cells, including T cells, undergoing apoptosis is to expose cells to a fluorophore-conjugated protein (Annexin V). Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine on the outer leaflet of the plasma membrane, which is an early indicator that the cell is undergoing the process of apoptosis.
  • MHC molecules refers to two types of molecules, MHC class I and MHC class II.
  • MHC class I molecules present antigen to specific CD8+ T cells and MHC class II molecules present antigen to specific CD4+ T cells.
  • Antigens delivered exogenously to APCs are processed primarily for association with MHC class II.
  • antigens delivered endogenously to APCs are processed primarily for association with MHC class I.
  • the term“MHC I” as used herein includes the MHC I complex as well as one or more of the subunits that make up MHC I (e.g., the class I alpha chain having 3 alpha subdomains (ocl, oc2, oc3) and beta2-microglobulin).
  • the alpha chains of the MHC I molecule are encoded by highly polymorphic HLA-A, HLA-B, and HLA-C genes.
  • “MHC I” includes the MHC class I molecules encoded by the HLA-A, HLA- B, and HLA-C genes, their fragments, as well as their polymorphs.
  • “detecting MHC I” can be performed by detecting the presence of beta2-microglobulin.
  • a method that“detects MHC I” includes the detection of any one or more of the subunits of MHC I, or MHC I in its entirety (e.g., by use of an antibody that recognizes the entire complex).
  • marker refers to MHC I itself, or any of the subunit(s) and/or component(s) that indicate the presence of MHC I (e.g., any one of more of the 3 alpha subdomains of the class I alpha chains, and polymorphs thereof, and beta2-micro globulin).
  • the term“binds to immobilized CD137,” refers to the ability of a human antibody of the disclosure to bind to CD137, for example, expressed on the surface of a cell or which is attached to a solid support.
  • biomarker refers to a characteristic (e.g., biological marker) that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions.
  • a biomarker is a molecular biomarker.
  • a molecular biomarker is a protein biomarker.
  • a molecular biomarker is a nucleic acid biomarker.
  • a biomarker is a histologic biomarker.
  • a biomarker is a radiographic biomarker.
  • a biomarker is a physiologic characteristic.
  • bispecific or“bifunctional antibody” refers to an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315- 321; Kostelny et al., (1992) J. Immunol. 148: 1547-1553.
  • bispecific antibodies are based on the co expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chain/light-chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305:537- 539).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion of the heavy chain variable region is preferably with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, CH2, and CH3 regions.
  • Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148(5): 1547-1553.
  • the leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers may be reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • scFv single-chain Fv
  • the antibodies can be“linear antibodies” as described in, e.g., Zapata et al. (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • Antibodies with more than two valencies are contemplated and described in, e.g., Tutt et al. (1991) J Immunol 147:60.
  • the disclosure also embraces variant forms of multi- specific antibodies such as the dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297.
  • DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain.
  • the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc region.
  • VH heavy chain variable domains
  • Methods for making DVD-Ig molecules from two parent antibodies are further described in, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715.
  • the bispecific antibody is a Fabs-in- Tandem immunoglobulin, in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody.
  • Such antibodies are described in, e.g., International Patent Application Publication No. WO 2015/103072.
  • cancer antigen refers to (i) tumor- specific antigens, (ii) tumor- associated antigens, (iii) cells that express tumor- specific antigens, (iv) cells that express tumor- associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor- specific membrane antigens, (viii) tumor- associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • the anti-CDl37 antibodies described herein can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • an assay e.g., a competitive binding assay; a cross-blocking assay
  • a test antigen binding protein e.g., a test antibody
  • inhibits e.g., reduces or blocks
  • a reference antigen-binding protein e.g., a reference
  • the antibodies described herein cross compete with mAbl (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 4 and 6, respectively), mab8 (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 101 and 6, respectively) or mAblO (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 26 and 6, respectively).
  • mAbl i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 4 and 6, respectively
  • mab8 i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 101 and 6, respectively
  • mAblO i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 26 and 6, respectively.
  • a polypeptide or amino acid sequence "derived from” a designated polypeptide or protein refers to the origin of the polypeptide.
  • the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence.
  • Polypeptides derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions.
  • a polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule.
  • a polypeptide consists of, consists essentially of, or comprises an amino acid sequence selected from a sequence set forth in any one of Tables 5-8.
  • a polypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from a sequence set forth in any one of Tables 5-8.
  • a polypeptide includes a contiguous amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous amino acid sequence selected from a sequence set forth in any one of Tables 5-8.
  • a polypeptide includes an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous amino acids of an amino acid sequence selected from a sequence set forth in any one of Tables 5-8.
  • the antibodies of the disclosure are encoded by a nucleotide sequence.
  • Nucleotide sequences of the invention can be useful for a number of applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like.
  • the nucleotide sequence of the invention comprises, consists of, or consists essentially of, a nucleotide sequence selected from a sequence set forth in any one of Tables 5-8.
  • a nucleotide sequence includes a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from a sequence set forth in any one of Tables 5-8.
  • a nucleotide sequence includes a contiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotide sequence selected from a sequence set forth in any one of Tables 5-8.
  • a nucleotide sequence includes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous nucleotides of a nucleotide sequence selected from a sequence set forth in any one of Tables 5-8.
  • antibodies suitable for use in the methods disclosed herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
  • nucleotide or amino acid substitutions leading to conservative substitutions or changes at "non-essential" amino acid residues may be made.
  • Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • the antibodies suitable for use in the methods disclosed herein may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • a nonessential amino acid residue in a binding polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • mutations may be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into binding polypeptides of the invention and screened for their ability to bind to the desired target.
  • antigen“cross-presentation” refers to presentation of exogenous protein antigens to T cells via MHC class I and class II molecules on APCs.
  • cross -reacts refers to the ability of an antibody of the disclosure to bind to CD137 from a different species.
  • an antibody of the present disclosure which binds human CD137 may also bind another species of CD137.
  • cross reactivity is measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA) or binding to, or otherwise functionally interacting with, cells physiologically expressing CD137.
  • Methods for determining cross-reactivity include standard binding assays as described herein, for example, by BiacoreTM surface plasmon resonance (SPR) analysis using a BiacoreTM 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.
  • SPR surface plasmon resonance
  • BiacoreTM 2000 SPR instrument Biacore AB, Uppsala, Sweden
  • flow cytometric techniques As used herein, the term“cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8 + T cells.
  • diagnosis or “diagnosis” or “diagnosing” refer to distinguishing or identifying a disease, syndrome or condition or distinguishing or identifying a person having a particular disease, syndrome, or condition.
  • diagnostic antibody denotes an antibody used for the detection and visualization of its target antigen (e.g., a biomarker).
  • diagnostic antibody is used e.g. in assay systems (e.g. ELISA), or for imaging techniques (e.g., IHC).
  • a diagnostic antibody may e.g., be a labeled therapeutic antibody.
  • diagnosis biomarker refers to a biomarker used to detect or confirm presence of a disease or condition of interest or to identify individuals with a subtype of the disease.
  • dimerization refers to the formation of a macromolecular complex by two, usually non-covalently bound, macromolecules, such as proteins or multimers of proteins.
  • Homodimerization refers to the process of dimerization when the macromolecules (e.g., proteins) are identical in nature.
  • Heterodimerization refers to the process of dimerization when the macromolecules (e.g., proteins) are non-identical in nature. Methods for determining dimerization are known to those of skill in the art.
  • such methods include, but are not limited to, yeast two-hybrid assay, fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), protein mass spectrometry, evanescent wave methods, size exclusion chromatography, analytical ultracentrifugation, scattering techniques, NMR spectroscopy, isothermal titration calorimetry, fluorescence anisotropy, fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), proximity imaging (PRIM) and bimolecular fluorescence complementation (BiFC) (see e.g., Gell D.A., Grant R.P., Mackay J.P.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • protein mass spectrometry evanescent wave methods
  • size exclusion chromatography size exclusion chromatography
  • analytical ultracentrifugation scattering techniques
  • NMR spectroscopy isothermal titration calorimetry
  • the terms "dimerization of CD 137" refers to the dimerization of two CD 137 trimers.
  • the anti-CDl37 agonist antibodies described herein induce or enhance dimerization of CD 137.
  • the anti-CD 137 agonist antibodies described herein induce or enhance dimerization of CD 137 relative to the amount of dimerization in the absence of an anti-CD 137 agonist antibody.
  • the anti-CD 137 agonist antibodies described herein induce or enhance dimerization of CD 137 relative to the amount of dimerization in the presence of a reference anti-CDl37 agonist antibody.
  • dimerization is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • EC50 refers to the concentration of an antibody or an antigen binding portion thereof, which induces a response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.
  • the term“effective dose” or“effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect.
  • therapeutically effective dose is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient’s own immune system.
  • the term“epitope” or“antigenic determinant” refers to a determinant or site on an antigen (e.g., CD137) to which an antigen-binding protein (e.g., an immunoglobulin, antibody, or antigen-binding fragment) specifically binds.
  • an antigen-binding protein e.g., an immunoglobulin, antibody, or antigen-binding fragment
  • the epitopes of protein antigens can be demarcated into“linear epitopes” and“conformational epitopes”.
  • the term“linear epitope” refers to an epitope formed from a contiguous, linear sequence of linked amino acids.
  • Linear epitopes of protein antigens are typically retained upon exposure to chemical denaturants (e.g., acids, bases, solvents, cross-linking reagents, chaotropic agents, disulfide bond reducing agents) or physical denaturants (e.g. thermal heat, radioactivity, or mechanical shear or stress).
  • an epitope is non-linear, also referred to as an interrupted epitope.
  • the term“conformational epitope” or“non-linear epitope” refers to an epitope formed from noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide. Conformational epitopes are typically lost upon treatment with denaturants.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. In some embodiments, an epitope includes fewer than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in a unique spatial conformation. Generally, an antibody, or antigen-binding fragment thereof, specific for a particular target molecule will preferentially recognize and bind to a specific epitope on the target molecule within a complex mixture of proteins and/or macromolecules. In some embodiments, an epitope does not include all amino acids of the extracellular domain of human CD 137.
  • antibodies that bind to an epitope on CD 137 which comprises all or a portion of an epitope recognized by the particular antibodies described herein (e.g ., the same or an overlapping region or a region between or spanning the region).
  • epitope mapping refers to a process or method of identifying the binding site, or epitope, of an antibody, or antigen binding fragment thereof, on its target protein antigen. Epitope mapping methods and techniques are provided herein.
  • CD 137 refers to a specific member of the tumor necrosis factor receptor (TNFR) family of transmembrane proteins.
  • Alternative names and acronyms for CD 137 in the art include“tumor necrosis factor receptor superfamily member 9” (TNFRSF9), 4-1BB and “induced by lymphocyte activation” (IFA) (Alderson et al., (1994) Eur J Immunol 24(9):22l9- 2227; Schwarz et al., (1993) Gene 134(2):295-298).
  • An exemplary amino acid sequence of full- length human CD137, including leader, transmembrane, and cytoplasmic domains is set forth in Table 7 (SEQ ID NO: 3) and here:
  • CD137F refers to a member of the tumor necrosis factor (TNF) family of transmembrane proteins.
  • Alternative names and acronyms for CD137F in the art include“tumor necrosis factor superfamily member 9” (TNFSF9) and 4-1BB ligand (4-1BBF) (Alderson et al., (1994) Eur J Immunol 24(9) :2219-2227).
  • An exemplary amino acid sequence of full-length CD137L is set forth in Table 7 (SEQ ID NO: 97).
  • “expression” generally refers to the process by which information contained within a gene is converted into the structures (e.g., a protein biomarker) present and operating in the cell. Therefore, as used herein,“expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide).
  • Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis.
  • “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).
  • “Elevated expression,”“elevated expression levels,” or“elevated levels” refers to an increased expression or increased levels of a substance within a sample relative to a control sample, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control.
  • the elevated expression of a substance in a sample refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., IHC).
  • “Reduced expression,”“reduced expression levels,” or“reduced levels” refers to a decrease expression or decreased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control.
  • reduced expression is little or no expression.
  • the reduced expression of a substance e.g., a protein or a biomarker
  • the reduced expression of a substance in a sample refers to a decrease in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., IHC).
  • the term“extent of binding” refers to the level of binding of an antibody (e.g., a diagnostic antibody) to an antigen (e.g., a biomarker) present in a sample.
  • the extent of antibody binding to an antigen can be determined by any of the methods known in the art for determining binding levels of antibodies such as ELISA, Western Blotting, or FACS.
  • the extent of binding may be determined using any detection system such as secondary immunoglobulins or fragments thereof linked to a detectable marker.
  • Exemplary detectable markers include, without limitation, radioactive groups, fluorescent or chromogenic molecules, an enzyme capable of catalyzing a reaction yielding a detectable product (such as a color reaction), and a biotin group capable of being detected by avidin.
  • the terms“Fc-mediated effector functions” or“Fc effector functions” refer to the biological activities of an antibody other than the antibody’s primary function and purpose.
  • the effector functions of a therapeutic agnostic antibody are the biological activities other than the activation of the target protein or pathway.
  • antibody effect functions include Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody- dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); lack of activation of platelets that express Fc receptor; and B cell activation. Many effector functions begin with Fc binding to an Fey receptor.
  • the term“Fc receptor” refers to a polypeptide found on the surface of immune effector cells, which is bound by the Fc region of an antibody.
  • the Fc receptor is an Fey receptor.
  • FeyRI CD64
  • FcyRII CD32
  • FycRIII CD16
  • IgG isotypes IgGl, IgG2, IgG3 and IgG4
  • FcyRIIB is an inhibitory receptor, and therefore antibody binding to this receptor does not activate complement and cellular responses.
  • FcyRI is a high affinity receptor that binds to IgG in monomeric form
  • FcyRIIA and FcyRIIA are low affinity receptors that bind IgG only in multimeric form and have slightly lower affinity.
  • the binding of an antibody to an Fc receptor and/or Clq is governed by specific residues or domains within the Fc regions. Binding also depends on residues located within the hinge region and within the CH2 portion of the antibody.
  • the agonistic and/or therapeutic activity of the antibodies described herein is dependent on binding of the Fc region to the Fc receptor (e.g., FcyR).
  • the agonistic and/or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to the Fc receptor (e.g., FcyR).
  • glycosylation pattern is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein.
  • a glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the nonhuman transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the nonhuman transgenic animal than to the species from which the CH genes of the transgene were derived.
  • hematological cancer includes a lymphoma, leukemia, myeloma or a lymphoid malignancy, as well as a cancer of the spleen and lymph nodes.
  • exemplary lymphomas include both B cell lymphomas (a B-cell hematological cancer) and T cell lymphomas.
  • B-cell lymphomas include both Hodgkin's lymphomas and most non-Hodgkin's lymphomas.
  • Non limiting examples of B cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis.
  • T cell lymphomas include extranodal T cell lymphoma, cutaneous T cell lymphomas, anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma.
  • Hematological malignancies also include leukemia, such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia.
  • Hematological malignancies further include myelomas, such as, but not limited to, multiple myeloma and smoldering multiple myeloma.
  • Other hematological and/or B cell- or T-cell- associated cancers are encompassed by the term hematological malignancy.
  • human antibody includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences.
  • Human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g ., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo ) (See, e.g., Lonberg et ah, (1994) Nature 368(6474): 856-859); Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13:65-93, and Harding & Lonberg, (1995) Ann. N.Y.
  • human antibody does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e . humanized antibodies).
  • heterologous antibody is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
  • the term“indicates” or“indicating” refers to a relationship between the presence of a substance or substances (e.g., biomarkers) and one or more events (e.g., response to treatment). In some embodiments, indicates refers to a positive relationship or positive correlation in which as one increases, the other increases as well. In some embodiments, indicates refers to a negative relationship or negative correlation in which as one increases, the other decreases.
  • biomarkers the levels of which indicate or predict a likely outcome, such as presence, amount or level of a biomarker (e.g., MHC I) in a patient sample, and the likelihood a patient will respond to treatment with an agent.
  • the presence, amount or level of a biomarker can indicate a likelihood of a positive clinical outcome for the patient, such as an increased likelihood of long-term survival without recurrence and/or a positive response to treatment.
  • a positive indication may be demonstrated statistically in various ways, e.g., by a low hazard ratio.
  • the presence, amount or level of a biomarker may negatively indicate a likelihood of good clinical outcome for the patient. In this case, for example, the patient may have a decreased likelihood of a positive response to treatment.
  • Such a negative indication may be demonstrated statistically in various ways, e.g., by a high hazard ratio.
  • inducing an immune response and“enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen.
  • inducing CDC or ADCC refer to the stimulation of particular direct cell killing mechanisms.
  • a subject“in need of prevention,”“in need of treatment,” or“in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising an anti-CD 137 antibody).
  • an appropriate medical practitioner e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals
  • in vivo refers to processes that occur in a living organism.
  • the term“isolated antibody” is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human CD 137 is substantially free of antibodies that specifically bind antigens other than CD137).
  • An isolated antibody that specifically binds to an epitope may, however, have cross-reactivity to other CD137 proteins from different species. However, the antibody continues to display specific binding to human CD137 in a specific binding assay as described herein.
  • an isolated antibody is typically substantially free of other cellular material and/or chemicals.
  • a combination of“isolated” antibodies having different CD 137 specificities is combined in a well-defined composition.
  • isolated nucleic acid molecule refers to nucleic acids encoding antibodies or antibody portions (e.g ., VH, VL, CDR3) that bind to CD137, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than CD137, which other sequences may naturally flank the nucleic acid in human genomic DNA.
  • a sequence selected from a sequence set forth in any one of Tables 5- 8 corresponds to the nucleotide sequences comprising the heavy chain (VH) and light chain (VL) variable regions of anti-CD 137 antibody monoclonal antibodies described herein.
  • a human monoclonal antibody of the disclosure is of the IgGl isotype.
  • a human monoclonal antibody of the disclosure is of the IgGl isotype and comprises a mutation.
  • a human monoclonal antibody of the disclosure is of the IgG2 isotype.
  • a human monoclonal antibody of the disclosure is of the IgG3 isotype.
  • a human monoclonal antibody of the disclosure is of the IgG4 isotype.
  • a human monoclonal antibody of the disclosure is of the IgG4 isotype and comprises a mutation.
  • the mutation is a substitution at Ser228.
  • the substitution at Ser228 is S228P.
  • isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
  • KD or“K D ” refers to the equilibrium dissociation constant of a binding reaction between an antibody and an antigen.
  • the value of K D is a numeric representation of the ratio of the antibody off-rate constant (kd) to the antibody on-rate constant (ka).
  • the value of K D is inversely related to the binding affinity of an antibody to an antigen. The smaller the K D value the greater the affinity of the antibody for its antigen. Affinity is the strength of binding of a single molecule to its ligand and is typically measured and reported by the equilibrium dissociation constant (K D ), which is used to evaluate and rank order strengths of bimolecular interactions.
  • the term“kd” or“k d ” is intended to refer to the off-rate constant for the dissociation of an antibody from an antibody/antigen complex.
  • the value of kd is a numeric representation of the fraction of complexes that decay or dissociate per second, and is expressed in units sec 1 .
  • ka is intended to refer to the on-rate constant for the association of an antibody with an antigen.
  • the value of ka is a numeric representation of the number of antibody/antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen, and is expressed in units M ⁇ sec 1 .
  • the term“likelihood” generally refers to an increase in the probability of an event (e.g., a patient will respond to treatment) and may be expressed as a probability value, fraction, or percentage.
  • the term“likelihood” may include the concept of“probability” as used in mathematics and by persons of ordinary skill in the art of statistics.
  • the term“likelihood” when used in reference to a patient’s response to treatment the term contemplates an increase in the probability that the patient responds positively to treatment.
  • the term“likelihood” when used in reference to a tumor’ s response to treatment contemplates a change in the amount of one or more biomarkers present in the tumor or tumor sample that may evidence a progression in treating the tumor.
  • the terms "linked,” “fused”, or “fusion”, are used interchangeably. These terms refer to the joining together of two more elements or components or domains, by whatever means including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.
  • “local administration” or“local delivery,” refers to delivery that does not rely upon transport of the composition or agent to its intended target tissue or site via the vascular system.
  • the composition may be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent.
  • the composition or agent, or one or more components thereof may diffuse to the intended target tissue or site.
  • the term“monitor,” carries its common usage and can refer to the surveillance or continual observation of a process or event (e.g., a response to treatment).
  • a process or event e.g., a response to treatment
  • the term“monitoring a patient’s response to treatment” contemplates, for example, observation or surveillance of disease progression.
  • the monitoring can be performed, for example, by following or determine the expression of biomarkers over time.
  • monitoring biomarker refers to a biomarker measured serially for assessing status of a disease or medical conditions or for evidence of exposure to (or effect of) a medical product or agent.
  • the term“monoclonal antibody” refers to an antibody which displays a single binding specificity and affinity for a particular epitope. Accordingly, the term“human monoclonal antibody” refers to an antibody which displays a single binding specificity, and which has variable and optional constant regions derived from human germline immunoglobulin sequences.
  • human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • the term“multimerization” refers to the formation of a macromolecular complex comprising more than two macromolecules such as proteins, typically bound by non- covalent interactions. Methods for determining multimerization are known to those of skill in the art and are described supra for dimerization.
  • the anti-CD 137 agonist antibodies described herein induce or enhance multimerization of CD 137.
  • the anti-CD 137 agonist antibodies described herein induce or enhance multimerization of CD 137 relative to the amount of multimerization in the absence of an anti-CD 137 agonist antibody.
  • the anti-CDl37 agonist antibodies described herein induce or enhance multimerization of CD 137 relative to the amount of multimerization in the presence of a reference anti-CDl37 agonist antibody.
  • multimerization is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • the term“naturally-occurring” as applied to an object refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • the term“nonswitched isotype” refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching.
  • Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene.
  • Non-classical isotype switching may occur by, for example, homologous recombination between human qm and human ⁇ m (d-associated deletion).
  • Alternative non-classical switching mechanisms such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al, 1992; Rossolini et al, Mol. Cell. Probes 8:91-98, 1994).
  • arginine and leucine modifications at the second base can also be conservative.
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • nucleic acid probe refers to a fragment of DNA or RNA that is labeled with a tag.
  • the nucleic acid probe is added to a DNA or RNA sample to detect and/or recover nucleotide sequences that are complementary to the sequence in the probe.
  • the probe hybridizes to a single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe- target base pairing due to complementarity between the nucleic acid probe and target.
  • the term encompasses nucleic acid probes labeled with tags including radioactive labels, fluorophores, enzymes, or nucleotides modified with digoxygenin or biotin.
  • Polynucleotides used herein can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that can be single- stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
  • polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • a nucleic acid is“operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • operably linked indicates that the sequences are capable of effecting switch recombination.
  • the term“panel of biomarkers” includes a group of markers, the quantity or activity of each member of which indicates responsiveness to treatment with an agent as described herein. The determination of a panel of biomarkers can provide more sensitive and specific information needed to evaluate a tumor or a patient.
  • the panel of biomarkers include one, two, three, four, five, six, seven, eight, or nine or more biomarkers.
  • the panel comprises MHC I, beta-2-microglobulin chain (b2M), and MHC class I alpha chain.
  • the panel comprises MHC I and b2M.
  • the panel comprises MHC I and MHC class I alpha chain.
  • the panel comprises b2M and MHC class I alpha chain.
  • paratope also“antigen-binding site” refers to a portion of an antibody, or antigen-binding fragment thereof, which recognizes and binds to an epitope on an antigen, comprising the set of complementarity determining regions (CDRs) located within variable heavy and light chains.
  • CDRs complementarity determining regions
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.
  • the term“patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. “Patient” is sometimes used interchangeably with“subject”.
  • percent identity in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
  • sequence comparison algorithms e.g., BLASTP and BLASTN or other algorithms available to persons of skill
  • the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et ah, infra).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et ah, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.
  • “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • a“pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66: 1-19).
  • polypeptide As used herein, the terms “polypeptide,” “peptide”, and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • the term“preventing” when used in relation to a condition refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.
  • the term“purified” or“isolated” as applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryote expressing the proteins.
  • a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample.
  • the term“rearranged” refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete V H or V L domain, respectively.
  • a rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.
  • the term“receptor clustering” refers to a cellular process that results in grouping or local accumulation of a set of receptors at a particular cellular location, often to induce or amplify a signaling response.
  • Many protein receptors bind cognate ligands and cluster, i.e., form dimers, trimers, oligomers or multimers, upon binding their cognate ligands.
  • the PDGF receptor and TNF receptor superfamily members form dimers and trimers upon ligand binding, respectively.
  • Cognate ligand-induced clustering e.g., dimerization, multimerization
  • the antibodies, or antigen-binding fragments thereof, of the present disclosure can activate a receptor by binding to more than one receptor and induce or stabilize dimerization, trimerization, and/or multimerization with or without cognate ligand binding.
  • 4-1BB CD137
  • CD40 CD40
  • GITR CD27
  • DR3, DR5 CD27
  • Fas are some of the TNFSF receptors known to require clustering in order to trigger downstream signaling.
  • Experimental evidence that the 4-1BB receptor must be cross-linked to signal comes from Rabu et al.
  • an anti-CDl37 agonist antibody induces the multimerization of 2 or more trimers of CD 137.
  • recombinant host cell (or simply“host cell”) is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term“host cell” as used herein.
  • the term“recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • variable and constant regions that utilize particular human germline immunoglobulin sequences are encoded by the germline genes, but include subsequent rearrangements and mutations which occur, for example, during antibody maturation.
  • the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen.
  • the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen.
  • the constant region will change in further response to an antigen (i.e ., isotype switch).
  • the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar (i.e., have at least 80% identity).
  • the term“reference antibody” (used interchangeably with“reference mAb”) or“reference antigen-binding protein” refers to an antibody, or an antigen-binding fragment thereof, that binds to a specific epitope on human CD 137 and is used to establish a relationship between itself and one or more distinct antibodies. In some embodiments, the relationship is the binding of the reference antibody and the one or more distinct antibodies to the same epitope on CD 137.
  • variable heavy (V H ) and light chain (V L ) amino acid sequences of an exemplary reference antibody (mAbl) are provided in Table 7 (V H l, SEQ ID NO. 4; V H 2, SEQ ID NO. 6).
  • the term connotes an anti- CD 137 antibody that is useful in a test or assay, as a comparator, wherein the assay is useful for distinguishing characteristics of the antibodies (e.g., hepatotoxicity, anti-tumor efficacy).
  • the reference antibody is urelumab. In some embodiments, the reference antibody is utomilumab.
  • sample refers to a composition that is obtained or derived from a patient, subject or individual of interest that contains or may contain a cellular or molecular entity, signature or substance (e.g., a biomarker) that is to be detected, quantified, identified, or otherwise characterized, for example based on physical, biochemical, chemical, or physiological characteristics, or a combination thereof.
  • A“tumor sample” refers to a sample taken from a subject having a tumor (e.g., a cancer patient), wherein the sample comprising tumor cells.
  • a tumor sample comprises malignant (tumor) and non-malignant cells, optionally, extracellular components.
  • A“reference sample” refers to a sample, cell, tissue, standard, or level that is used for comparison purposes.
  • a reference sample is a sample known to contain one or more biomarkers of interest. The amount or level of a biomarker in a reference sample is referred to as a“reference amount.”
  • a reference sample is a sample obtained from a healthy and/or non-diseased part of the body of the same patient, subject or individual. For example, healthy and/or non-diseased cells or tissues adjacent to the diseased cells or tissues (e.g., cells or tissues adjacent to a tumor).
  • a reference sample is obtained from an untreated tissue and/or cell of the body of the same patient, subject or individual.
  • a reference sample is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the patient or subject.
  • a reference sample is obtained from an untreated tissue and/or cell of the body of an individual who is not the patient or subject.
  • a sample may be obtained by any technique (e.g., biopsy) known to a person skilled in the art.
  • samples may comprise tissue (referred to as a “tissue sample”) or cells (referred to as a“cell sample”).
  • the terms“specific binding,”“selective binding,”“selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen.
  • the antibody binds with an equilibrium dissociation constant (K D ) of approximately less than 10 6 M, such as approximately less than 10 7 , 10 8 M, 10 9 M or 10 10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument using recombinant human CD 137 as the analyte and the antibody as the ligand and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g ., BSA, casein) other than the predetermined antigen or a closely- related antigen.
  • K D equilibrium dissociation constant
  • SPR surface plasmon resonance
  • the term“switch sequence” refers to those DNA sequences responsible for switch recombination.
  • A“switch donor” sequence typically a m switch region, will be 5' (i.e., upstream) of the construct region to be deleted during the switch recombination.
  • The“switch acceptor” region will be between the construct region to be deleted and the replacement constant region (e.g., g, e, etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.
  • the term“subject” includes any human or non-human animal.
  • the methods and compositions of the present invention can be used to treat a subject with an immune disorder.
  • the term“non-human animal” includes all vertebrates, e.g., mammals and non mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. “Subject” is sometimes used interchangeably with“patient”.
  • nucleic acids the term“substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.
  • the nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(l7):3389-3402.
  • the default parameters of the respective programs e.g ., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is“isolated” or“rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, L. Ausubel, et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
  • nucleic acid compositions of the present disclosure while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof may be mutated, in accordance with standard techniques to provide gene sequences. Lor coding sequences, these mutations, may affect amino acid sequence as desired.
  • DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).
  • tumor microenvironment refers to the cellular environment or milieu in which the tumor or neoplasm exists, including surrounding blood vessels as well as non-cancerous cells including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME.
  • the tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of tumor cells.
  • T cell refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface.
  • T helper cells a.k.a.
  • T H cells or CD4 + T cells and subtypes, including T H I , T H 2, T H 3, T H 17, T H 9, and T FH cells, cytotoxic T cells (i.e Tc cells, CD8 + T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a.
  • T reg cells or suppressor T cells and subtypes, including CD4 + FOXP3 + T reg cells, CD4 + FOXP3 T reg cells, Trl cells, Th3 cells, and T reg l7 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (gd T cells), including Vy9/ V 52 T cells.
  • NKT cells natural killer T cells
  • MAITs mucosal associated invariant T cells
  • gd T cells gamma delta T cells
  • Vy9/ V 52 T cells gamma delta T cells
  • T cell activation 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
  • These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.
  • 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 terms“therapeutically effective amount” or“therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., an anti- CD 137 antibody or an antigen-binding fragment thereof) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a cancer).
  • an agent e.g., an anti- CD 137 antibody or an antigen-binding fragment thereof
  • the terms“treat,”“treating,” and“treatment,” as used herein, refer to therapeutic or preventative measures described herein.
  • the methods of“treatment” employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • V segment configuration refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a“plasmid” refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • a viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g ., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the anti-CD 137 agonist antibody, or antigen-binding fragment thereof, described herein demonstrate anti-tumor efficacy in cancer cells that express MHC I.
  • the present discovery can be applied to identify a cancer patient population that would be amenable to anti-cancer therapy using the anti-CD 137 agonist antibodies disclosed herein, thereby enhancing therapeutic efficacy of the anti-CDl37 agonist antibodies.
  • the present disclosure provides methods for detecting the presence (or absence) of MHC I in a biological sample, e.g., a biopsy obtained from a subject having or suspected of having any one or more types of cancer disclosed herein, thereby allowing the identification of a subject amenable to anti-cancer therapy using the anti-CD 137 agonist antibodies disclosed herein.
  • cancer that is“amenable” to agonist anti-CD 137 antibody therapy includes those cancers that, upon being administered a therapeutically effective amount of an agonist anti- CD 137 antibody, can benefit from the therapy so as to be“treated”, as used herein.
  • the present disclosure also provides methods for determining whether a subject having cancer would be amenable to agonist CD 137 antibody, or antigen binding fragment thereof, therapy.
  • the method comprises contacting a biological sample, e.g., a biopsy obtained from a subject having or suspected of having any one or more types of cancer disclosed herein, from the subject with an agent directed to MHC I, and detecting the agent bound to MHC I according to known methods, and as described herein, wherein the presence of MHC I indicates that the cancer is amendable to treatment with an agonist CD 137 antibody, or an antigen binding fragment thereof.
  • the method further comprises determining that the cancer is amenable to treatment with an agonist CD 137 antibody, or antigen-binding fragment thereof (by the presence of MHC I), and administering to the subject a therapeutically effective amount of an agonist anti-CD 137 antibody, or antigen-binding fragment thereof.
  • detecting MHC I expression is performed by detecting the presence of any one or more markers selected from the group consisting of: MHC I protein, MHC I mRNA, beta-2-microglobulin chain (b2M) protein, b2M chain mRNA, MHC class I alpha chain domain protein (e.g., ocl, oc2, or oc3), and MHC class I alpha chain domain mRNA.
  • the alpha chains are encoded by any one of human leukocyte antigen (HLA) -A, -B, and -C genes, which are three of the major types of human MHC class I cell surface receptors.
  • HLA human leukocyte antigen
  • MHC I expression is detected with an agent directed to any one or more of the markers.
  • the agent is an antibody or a nucleic acid probe.
  • the antibody or probe comprises a detectable label.
  • Those skilled in the art can readily determine the appropriate agent, e.g., antibody or probe, depending on the particular MHC I (e.g., HLA-A, HLA-B, or HLA-C) being detected. While the HLA-A, -B, and -C genes encoding the alpha chains of the MHC I complex are known to be polymorphic, those skilled in the art can readily determine their sequences, and select and/or design a suitable detecting agent directed to the alpha chain and/or the b2M chain.
  • SEQ ID NO: 134 An exemplary amino acid sequence of MHC I (HLA-A) is shown in SEQ ID NO: 134 (its nucleotide sequence is shown in SEQ ID NO: 135); the amino acid sequence of b2M is shown in SEQ ID NO: 136 (its nucleotide sequence is shown in SEQ ID NO: 137).
  • SEQ ID NO: 134 an exemplary amino acid sequence of MHC I (HLA-A) is shown in SEQ ID NO: 135); the amino acid sequence of b2M is shown in SEQ ID NO: 136 (its nucleotide sequence is shown in SEQ ID NO: 137).
  • the present methods contemplate the use of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 134.
  • the present methods contemplate the use of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 135.
  • the present methods contemplate the use of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 136.
  • the present methods contemplate the use of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 137.
  • MHC I is detected in a sample or tumor using an agent that binds MHC I protein or MHC I mRNA.
  • the agent that binds MHC I protein is an antibody.
  • the agent that binds MHC I mRNA is a nucleic acid probe.
  • MHC I is detected in a sample or a tumor using an agent that binds beta-2-microglobulin (b2M protein) or b2M mRNA.
  • the agent that binds b2M protein is an antibody.
  • the agent that binds b2M mRNA is a nucleic acid probe.
  • MHC I is detected in a sample or a tumor using an agent that binds MHC class I alpha chain protein or MHC class I alpha chain mRNA.
  • the agent that binds MHC class I alpha chain protein is an antibody.
  • the agent that binds MHC class I alpha chain mRNA is a nucleic acid probe.
  • the presence of MHC I can be determined by any of the methods known in the art, and include, for example, detecting MHC I in its entirety, or detecting any one or more of its subunits and variants, as described herein. Further, various agents can be used to detect the presence of the markers (e.g., MHC I itself, or any of the subunit(s) and/or component(s) that indicate the presence of MHC I (e.g., any one of more of the 3 alpha subdomains of the class I alpha chains, and polymorphs thereof, and beta2-microglobulin), depending on the assay and/or detection method used.
  • markers e.g., MHC I itself, or any of the subunit(s) and/or component(s) that indicate the presence of MHC I (e.g., any one of more of the 3 alpha subdomains of the class I alpha chains, and polymorphs thereof, and beta2-microglobulin
  • an agent that binds to the marker are nucleic acid probes, or protein
  • an antibody against any one or more of the protein markers can be used if the detection method to be used is, e.g., ELISA.
  • the antibody is an anti- MHC I antibody, such as MHC Class I (H-2Kd/H-2Dd) antibody from ebioscience (#12-5998- 82) or ebioscience (#(14-5998-82).
  • the antibody is an anti ⁇ 2M antibody, such as anti-mouse b2M antibody from Biolegend (#154506) or anti-human b2M antibody from Biolegend (#316302).
  • the antibody is an anti-MHC class I alpha chain antibody, such as Human HLA Class I antibody from R& D Systems (#MAB7098).
  • agents such as nucleic acid probes against any one or more of the mRNA markers can be used if the detection method to be used is, e.g., RT-PCR.
  • the agent can include a detectable label commonly known in the art.
  • the presence of a marker can be determined by detecting the level of marker mRNA, marker protein, or combinations thereof (e.g., MHC I protein, MHC I mRNA, beta-2-microglobulin chain (b2M) protein, b2M chain mRNA, MHC class I alpha chain protein, and MHC class I alpha chain mRNA).
  • marker mRNA e.g., MHC I protein, MHC I mRNA, beta-2-microglobulin chain (b2M) protein, b2M chain mRNA, MHC class I alpha chain protein, and MHC class I alpha chain mRNA.
  • Suitable biological assays for detecting the marker include, but are not limited to, PCR based methods, hybridization-based methods, sequencing methods, protein detection methods, or combinations thereof.
  • suitable PCR based methods comprise any form of reverse transcriptase polymerase chain reaction (RT-PCR), any form of quantitative reverse transcriptase polymerase chain reaction (QRT-PCR), competitive RT-PCR, and real-time RT-PCR.
  • suitable hybridization methods comprise nuclease protection assay, Northern blot analysis, in situ hybridization, and microarray-based analysis based on any suitable arrays such as Affymetrix GENECHIPTM, ILLUMINATM DASLTM arrays, printed cDNA arrays, and the likes.
  • Suitable sequencing methods comprise next generation sequencing (NGS) technologies.
  • Suitable protein detecting methods comprises a Western blot analysis, an enzyme-linked immunosorbent assay (ELISA), immunohistochemistry analysis, an immunoprecipitation followed by an SDS-PAGE analysis, a proteomics analysis, such as a quantitative proteomics analysis.
  • Other assays include e.g., RNase protection assay (RPA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, complement fixation assay, fluorescence-activated cell sorting (FACS), mass spectrometry, magnetic bead-antibody immunoprecipitation, protein chip, or any combination thereof.
  • the marker (e.g., protein or nucleic acid) level need not be quantified; instead, the presence of the marker at any detectable level indicates that the cancer cell is amenable to anti-cancer therapy using the anti-CD 137 agonist antibodies disclosed herein. In some embodiments, the level of the marker is quantified.
  • a negative control is any suitable control appropriate for use in a given assay.
  • a negative control is an isotype control antibody in an assay that uses an antibody to a marker (e.g., in flow cytometry).
  • the patient is administered a therapeutically effective amount of an agonist anti-CD 137 antibody, or antigen -binding fragment thereof, as disclosed herein, according to the methods disclosed herein.
  • the disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agonist monoclonal antibody, or antigen-binding fragment thereof, that specifically binds human CD137, wherein the subject has cancer cells that express major histocompatibility complex I (MHC I).
  • MHC I major histocompatibility complex I
  • the present disclosure provides antibodies that specifically bind to and agonize CD 137 in a population of cancer cells that express MHC I.
  • the disclosure provides anti- CD137 agonist antibodies that are useful for the treatment of cancer in subjects with cancer cells that express MHC I.
  • the anti-CDl37 agonist antibodies induce cytokine production.
  • the anti-CD 137 agonist antibodies increase the number of CD8+ T cells in the tumor microenvironment.
  • the anti-CDl37 agonist antibodies induce protective anti-tumor immunity.
  • the disclosure also provides anti-CD 137 agonist antibodies that, upon administration in vivo , do not substantially increase intrasplenic or intrahepatic CD4+ and/or CD8+ T cell populations.
  • Human CD137 is a 255 amino acid transmembrane polypeptide (SEQ ID NO: 3; Accession No. NM_001561; NP_00l552) and a member of the phylogenetically-conserved tumor necrosis factor receptor (TNFR) superfamily.
  • CD137 (alternatively 4-1BB, TNFR superfamily 9) and its ligand (CD137L) are involved in the regulation of a wide range of immune activities.
  • CD137 ligand cross-links its receptor, CD137, which is expressed on activated T cells, and co-stimulates T cell activities.
  • CD137 is an activation-induced co- stimulatory molecule.
  • CD 137 ligand is a transmembrane protein on the cell surface and transmit signals into the cells on which it is expressed, a phenomenon referred to as“reverse signaling” or“back signaling”). CD 137 ligand expression is found on most types of leukocytes and on some nonimmune cells. In monocytic cells (monocytes, macrophages, and DCs), CD137 ligand signaling induces activation, migration, survival, and differentiation.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to and agonizes CD 137 and allows or promotes CD137L binding.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to and agonizes CD137.
  • the anti-CDl37 antibodies provided by the disclosure bind to and agonize CD137 and co-stimulate activation of T cells.
  • an isolated anti-CDl37 agonist antibody, or antigen-binding fragment thereof, described herein has one or more of the following properties or characteristics: a) specifically binds to human CD 137;
  • an anti-CD 137 agonist antibody, or antigen-binding fragment thereof, described herein binds to CD137 and co-stimulates T cell activities. In some embodiments, an anti-CD 137 agonist antibody, or antigen-binding fragment thereof, described herein, binds to CD 137 and induces or enhances T cell activation, a cytotoxic T lymphocyte (CTL) response, T cell proliferation, cytokine production, or a combination thereof.
  • CTL cytotoxic T lymphocyte
  • an anti-CD 137 agonist antibody, or antigen-binding fragment thereof, described herein binds to CD 137 and induces or enhances T cell activation, a cytotoxic T lymphocyte (CTL) response, T cell proliferation, cytokine production, or a combination thereof, in a tumor microenvironment.
  • CTL cytotoxic T lymphocyte
  • an anti-CDl37 antibody, or antigen-binding fragment thereof, described herein does not significantly induce or enhance intrahepatic and/or intrasplenic T cell activation and/or T cell proliferation.
  • an anti-CD 137 antibody, described herein binds to CD137 and induces the production of IFN Y .
  • the antibodies provided by the disclosure bind to CD 137 and induce the production of IL-2, TNF- Oi , IL-13, or a combination thereof.
  • the anti-CD 137 antibodies described herein specifically bind to and agonize CD137.
  • agonism of CD137 is measured by determining the concentration of cytokines produced by immune cells. Methods for analyzing cytokine production are known in the art and utilized in the Examples.
  • an increase in cytokine production by immune cells indicates CD 137 agonism.
  • agonism of CD 137 is measured by analyzing T cell proliferation.
  • an increase in T cell proliferation indicates CD137 agonism.
  • agonism of CD137 is measured by measuring the level of cell signaling either through quantitation of phosphorylation of relevant molecules or expression of a gene reporter after a relevant promoter.
  • an increase in cell signaling indicates CD 137 agonism.
  • agonism of CD 137 is measured by measuring the volume of a tumor. In some embodiments, a decrease in the volume of a tumor indicates CD 137 agonism.
  • the anti-CD 137 antibodies described herein induce, increase or stabilize oligomerization, multimerization, or other higher order clustering of CD 137.
  • the clustering of CD 137 on the cell surface is observed via fluorescence microscopy.
  • the antibodies or antigen binding fragments thereof that bind to and agonize CD 137.
  • the antibodies or antigen binding fragments thereof (i) bind human CD137 with an affinity (KD) of about 40-l00nM (e.g., between about 40 nM and about 100 nM); (ii) bind an epitope on human CD137 described herein; and/or (iii) comprise a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).
  • the antibodies or antigen binding fragments thereof (i) bind human CD137 with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM); (ii) bind an epitope on human CD137 described herein; and/or (iii) comprise a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds human CD137 with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM or between about 40 nM and about 100 nM).
  • affinity of the anti-CDl37 antibody to human CD137 is at least two (e.g., at least three, four, five, six, seven, eight, nine, or 10) fold higher than the affinity of mAblO for mouse CD 137.
  • the affinity of the anti-CD 137 antibody is no greater than 500, 450, 400, 350, 300, 250, 200, 250, 200, 175, 150, 125, 110, or 100 nM. In some embodiments, the affinity of the anti-CD 137 antibody to human CD 137 is at least two (e.g., at least three, four, five, six, seven, eight, nine, or 10) fold higher than the affinity of mAblO for mouse CD137, but no greater than 500, 450, 400, 350, 300, 250, 200, 250, 200, 175, 150, 125, 110, or 100 nM.
  • the affinity of the antibody is the strength of binding to a single CD 137 polypeptide. In some embodiments, affinity is indicated by the equilibrium dissociation constant (KD). The value of KD is inversely related to the binding affinity of an antibody to an antigen. Accordingly, the smaller the KD value, the greater the affinity of the antibody for its antigen.
  • An exemplary method for determining binding affinity employs surface plasmon resonance.
  • Surface plasmon resonance is an optical phenomenon that allows for the analysis of realtime biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
  • BIAcore Phharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.
  • the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM). In some embodiments, the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 40-100 nM (e.g., between about 40 nM and about 100 nM).
  • the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 30- 40 nM, 40-50 nM, 50-60 nM, 60-70 nM, 70-80 nM, 80-90 nM, 90-100 nM, 45-55 nM, 55-65 nM, 75-85 nM, 85-95 nM, 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM.
  • KD affinity
  • the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 60-80 nM. In some embodiments, the anti- CD137 antibodies described herein bind human CD137 with an affinity (KD) of about 60-75 nM. Iln some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 60-90 nM. In some embodiments, the anti-CD 137 antibodies described herein bind human CD137 with an affinity (KD) of about 50-80 nM. In some embodiments, the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 40-70 nM.
  • the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 55-75 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 65-75 nM. In some embodiments, the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 60-80 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 55-85 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 50-90 nM.
  • the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 45-95 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 85-95 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 75-85 nM. In some embodiments, the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 75-85 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 55-65 nM.
  • the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 45-55 nM. In some embodiments, the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 80-90 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 70-80 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 60-70 nM. In some embodiments, the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 50-60 nM.
  • the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 40-50 nM. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 30-40 nM.
  • the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 30 nM, about 31 nM, about 32 nM, about 33 nM, about 34 nM, about 35 nM, about 36 nM, about 37 nM, about 38 nM, about 39 nM, about 40 nM, about 41 nM, about 42 nM, about 43 nM, about 44 nM, about 45 nM, about 46 nM, about 47 nM, about 48 nM, about 49 nM, about 50 nM, about 51 nM, about 52 nM, about 53 nM, about 54 nM, about 55 nM, about 56 nM, about
  • KD affinity
  • the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of at least 30 nM but less than about 110 nM, at least 31 nM but less than about 109 nM, at least 32 nM but less than about 108 nM, at least 33 nM but less than about 107 nM, at least 34 nM but less than about 106 nM, at least 35 nM but less than about 105 nM, at least 36 nM but less than about 104 nM, at least 37 nM but less than about 103 nM at least 38 nM but less than about 102 nM, at least 39 nM but less than about 101 nM, at least 40 nM but less than about 100 nM; at least 41 nM but less than about 99 nM; least 42 nM but less than about 98 nM; least 43 nM but less than about 97 nM; at least 44 nM but less than about
  • KD
  • the anti-CDl37 antibodies described herein cross-react with CD137 polypeptides from more than one species. In some embodiments, the anti-CDl37 antibodies described herein bind cynomolgus CD137 and human CD137. In some embodiments, the anti- CD 137 antibodies described herein bind mouse CD 137 and human CD 137. In some embodiments, the anti-CDl37 antibodies described herein bind human CD137, mouse CD137 and cynomolgus CD137.
  • the isolated monoclonal antibody, or antigen binding portion thereof, that specifically binds to human CD137 binds to an epitope on human CD137 comprising one or more (e.g., one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all 25) of amino acids 111-132 of SEQ ID NO:3.
  • the isolated monoclonal antibody, or antigen binding portion thereof, that specifically binds to human CD137 binds to an epitope within amino acids 111-132 of SEQ ID NO:3.
  • the disclosure provides an isolated monoclonal antibody, or antigen binding portion thereof, that specifically binds to human CD137, binds to all or a portion of amino acids 111-132 of SEQ ID NO:3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD 137 comprising residue Kl 14 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD137 comprising residues El 11, T113 and K114 of SEQ ID NO: 3.
  • an isolated anti-CD 137 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD137 comprising residues El l l, T113, K114, N126 and 1132 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD 137 comprising El l l, Tl l3, K114, N126, 1132 and P135 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD137 comprising one or more residues El l l, T113, K114, N126, 1132 and P135 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD 137 comprising a sequence of one or more amino acid residues corresponding to amino acid positions 100 to 135, 101 to 135, 102 to 135, 103 to 135, 104 to 135, 105 to 135, 106 to 135, 107 to 135, 108 to 135, 109 to 135, 110 to 135, or 111 to 135 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD 137 comprising a sequence of one or more amino acid residues corresponding to amino acid positions 111 to 135 of SEQ ID NO: 3.
  • the epitope comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues corresponding to amino acid positions 111 to 135 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD 137 within amino acid positions 100 to 135, 101 to 135, 102 to 135, 103 to 135, 104 to 135, 105 to 135, 106 to 135, 107 to 135, 108 to 135, 109 to 135, 110 to 135, or 111 to 135 of SEQ ID NO: 3.
  • an isolated anti-CD 137 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD137 within amino acid positions 111 to 135 of SEQ ID NO: 3.
  • the epitope comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues corresponding to amino acid positions 111 to 135 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD 137 comprising ELTK (corresponding to amino acid residues 111-114 of SEQ ID NO: 3).
  • amino acid residue Ll 12 can be another amino acid residue.
  • the epitope is a non-linear epitope.
  • mutation of amino acid residue K114 abrogates bindings of an isolated anti-CDl37 agonist antibody, or antigen binding fragment thereof, described herein, to human CD 137.
  • isolated anti-CD 137 agonist antibody, or antigen binding fragment thereof, described herein binds to an epitope of human CD 137 comprising a sequence of one or more amino acid residues corresponding to amino acid positions 111 to 135 of SEQ ID NO: 3, wherein the epitope comprises at least amino acid K114, and wherein the antibody or antigen binding portion thereof binds mouse CD 137 and does not bind rat CD 137.
  • the epitope is a non-linear epitope.
  • the antibody or antigen binding portion thereof binds mouse CD 137 and cynomolgus CD 137 and does not bind rat CD 137.
  • binding of an isolated anti-CD 137 agonist antibody, or antigen binding fragment thereof, described herein, to human, mouse, rat and cynomolgus CD 137 is determined by surface plasmon resonance (SPR).
  • the antibody or antigen binding portion thereof binds to mouse, cynomolgus or human CD137 with an affinity that is at least 10, 20, 30, 40, 50, 100, 200, 500 or 1000 times greater than the antibody’s affinity for rat CD137.
  • the antibody or antigen binding portion thereof binds to mouse, cynomolgus or human CD 137 with an affinity that is at least 10, 20, 30, 40, 50, 100, 200, 500 or 1000 times greater than the antibody’s affinity for a CD137 polypeptide that does not comprise a lysine at position 114 relative to human CD137 of SEQ ID NO: 3.
  • an isolated anti-CDl37 agonist antibody, or antigen-binding fragment thereof, described herein binds to an epitope of human CD 137 and competes with mAbl for binding to the epitope of human CD137.
  • an isolated anti-CDl37 agonist antibody, or antigen-binding fragment thereof, described herein binds to and agonizes CD 137.
  • the anti-CD 137 antibodies provided by the disclosure bind to and agonize CD137 and co-stimulate activation of T cells.
  • the present disclosure provides antibodies that compete for binding to an epitope on CD 137 which comprises all or a portion of an epitope recognized by one or more particular reference antibodies described herein (e.g., mAbl).
  • the anti-CDl37 antibodies bind to an epitope of human CD 137 and compete with a reference antibody (e.g., mAbl) for binding to the epitope of human CD 137 and wherein the antibody, or antigen binding fragment thereof, binds human CD137 with an equilibrium dissociation constant KD of 1 X 10 6 or less.
  • the anti-CDl37 antibodies bind to an epitope on CD137, wherein one or more mutations to the epitope inhibit, reduce, or block binding to both the antibodies and a reference antibody (e.g., mAbl).
  • a reference antibody e.g., mAbl
  • the reference antibody is the mAbl antibody, described herein.
  • the reference antibody is any one antibody provided in any one of Tables 5-8.
  • the anti-CDl37 antibodies provided by the disclosure may be assessed through x-ray crystallographic analysis of a crystal structure comprising an antibody bound to CD137, or a fragment or portion thereof.
  • the epitopes that bound by the antibodies provided by the disclosure are identified by determining the residues on the human CD 137 antigen that reside or are located within 4 angstroms (A) of an antibody paratope residue, e.g., mAbl.
  • the epitope bound by the anti-CDl37 antibodies described herein is at least 3 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is at least 4 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is at least 5 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is at least 6 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is at least 7 amino acid residues. In some embodiments, the epitope bound by the anti- CD137 antibodies described herein is at least 8 amino acid residues.
  • the epitope bound by the anti-CDl37 antibodies described herein is at least 9 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is at least 10 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is at least 12 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is at least 3 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is at least 13 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is at least 14 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is at least 15 amino acid residues.
  • the epitope bound by the anti-CDl37 antibodies described herein is fewer than 25 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 24 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is fewer than 23 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 22 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 21 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is fewer than 20 amino acid residues.
  • the epitope bound by the anti-CDl37 antibodies described herein is fewer than 19 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 18 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is fewer than 17 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 16 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 15 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is fewer than 14 amino acid residues.
  • the epitope bound by the anti-CDl37 antibodies described herein is fewer than 13 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 12 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is fewer than 11 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 10 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 9 amino acid residues. In some embodiments, the epitope bound by the anti-CD 137 antibodies described herein is fewer than 8 amino acid residues.
  • the epitope bound by the anti-CD 137 antibodies described herein is fewer than 7 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 6 amino acid residues. In some embodiments, the epitope bound by the anti-CDl37 antibodies described herein is fewer than 5 amino acid residues.
  • the anti-CD 137 antibodies described herein bind to an epitope of fewer than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids and comprises amino acid residue Kl 14 of SEQ ID NO: 3.
  • isolated monoclonal antibodies or antigen binding fragments thereof comprising heavy and light chain variable sequences as set forth in any one of Tables 5-8.
  • the anti-CDl37 antibodies described herein comprise heavy and light chain CDRs selected from the group consisting of:
  • the anti-CDl37 antibodies described herein comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 101 and 103; and wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 105.
  • the anti-CDl37 antibodies described herein comprise heavy and light chain CDRs, wherein heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68.
  • the anti-CDl37 antibodies described herein comprise heavy and light chain variable regions comprising amino acid sequences selected from the group consisting of:
  • the anti-CDl37 antibodies described herein comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 101 and 103; and wherein the light chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 105.
  • the anti-CDl37 antibodies described herein comprise heavy and light chain variable regions comprising amino acid sequences at least 90% identical to the amino acid sequences selected from the group consisting of:
  • antibodies that specifically bind human CD 137 comprising heavy chain and light chain variable regions encoded by nucleotide sequences selected from the group consisting of:
  • antibodies that specifically bind human CD 137 comprising heavy chain and light chain variable regions encoded by nucleotide sequences having at least 90% identity to the nucleotide sequences selected from the group consisting of:
  • the anti-CDl37 antibodies described herein comprise heavy and light chain variable regions, wherein the heavy chain variable region is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 102 and 104; and wherein the light chain variable region is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 106.
  • the anti-CDl37 antibodies described herein comprise heavy and light chain variable regions, wherein the heavy chain variable region is encoded by a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 102 and 104; and wherein the light chain variable region is encoded by a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 106.
  • anti-CD 137 antibodies that specifically bind to human CD 137 and comprise a heavy chain CDR3 having the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid. In some embodiments, X is any amino acid except for alanine. In some embodiments, mutation of residues D95, L100, Y 100E, Y 100G, and/or Y 100H of SEQ ID NO: 126, results in loss of binding to human CD137.
  • anti-CD 137 antibodies that specifically bind to human CD 137 and comprise a heavy chain CDR3 having the amino acid sequence DXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X is any amino acid.
  • mutation of residues F98, D100A, Y100D, and/or Y100F, and/or Y100H of SEQ ID NO: 126, to alanine results in loss of binding to human CD137.
  • mutation of residues F98, D100A, Y 100D, and/or Y 100F, and/or Y 100H of SEQ ID NO: 126, to any residue except for alanine results in an increase in binding to human CD 137.
  • anti-CD 137 antibodies that specifically bind to human CD 137 and comprise a heavy chain CDR3 having the amino acid sequence DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X 2 is a non-polar amino acid, wherein X 3 is a non-polar amino acid, wherein X 4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X 7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid.
  • X 2 is proline, wherein X 3 is phenylalanine or tryptophan, wherein X5 is aspartic acid or glutamic acid, wherein X 8 is tyrosine, and wherein X9 is tyrosine.
  • alanine scanning is a technique used to determine the contribution of a specific wild-type residue to the stability or function(s) (e.g., binding affinity) of given protein or polypeptide.
  • the technique involves the substitution of an alanine residue for a wild-type residue in a polypeptide, followed by an assessment of the stability or function(s) (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparison to the wild-type polypeptide.
  • the residues identified as not critical are further evaluated to modulate the binding of the antibody to the antigen (e.g., increase or decrease binding).
  • a non-limiting example of such analysis is deep mutational scanning. This method allows for the evaluation of large numbers of mutations.
  • each amino acid residue within the heavy chain CDR3 is mutated to every amino acid residue (except for alanine), and binding is assessed.
  • Other methods for analyzing the effect of amino acid residue mutations are known in the art. In some embodiments, these methods are utilized to assess the role of residues in all of the heavy chain and light chain CDRs in binding to human CD 137.
  • the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM). In some embodiments, the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of about 40-100 nM (e.g., between about 40 nM and about 100 nM). In some embodiments, the anti-CD 137 antibodies described herein bind an epitope on human CD 137 described supra (e.g., comprising K114).
  • the anti-CDl37 antibodies described herein comprise a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126). In some embodiments, the anti-CDl37 antibodies described herein bind human CD137 with an affinity (KD) of 30-100 nM (e.g., between about 30 nM and about 100 nM) and bind an epitope on human CD137 described supra (e.g., comprising K114).
  • KD affinity
  • the anti-CD 137 antibodies described herein bind human CD 137 with an affinity (KD) of 30-100 nM (e.g., between about 30 nM and about 100 nM) and comprise a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).
  • the anti-CDl37 antibodies described herein bind an epitope on human CD137 described supra (e.g., comprising Kl 14) and comprise a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).
  • the anti- CD137 antibodies described herein bind human CD137 with an affinity (KD) of 30-l00nM (e.g., between about 30 nM and about 100 nM), bind an epitope on human CD137 described supra (e.g., comprising K114), and comprise a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).
  • KD affinity
  • the anti-CD 137 antibodies described herein bind human CD137 with an affinity (KD) of 30-l00nM (e.g., between about 30 nM and about 100 nM), bind an epitope on human CD137 described supra (e.g., comprising K114), and comprise a heavy chain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).
  • the anti-CD 137 antibodies bind human CD137 with an affinity (KD) of 30-l00nM (e.
  • DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X 2 is a non-polar amino acid, wherein X 3 is a non-polar amino acid, wherein X 4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X 7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X2 is a non-polar amino acid, wherein X3 is a non-polar amino acid, wherein X4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid.; or
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X 2 is a non-polar amino acid, wherein X 3 is a non-polar amino acid, wherein X 4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X 7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X 2 is a non-polar amino acid, wherein X 3 is a non-polar amino acid, wherein X 4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X 7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid; or
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X2 is a non-polar amino acid, wherein X3 is a non-polar amino acid, wherein X4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X 2 is a non-polar amino acid, wherein X 3 is a non-polar amino acid, wherein X 4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X 7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid; or
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • the anti-CD 137 antibodies are anti-CD 137 antibodies.
  • DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128), wherein Xi is any amino acid, wherein X 2 is a non-polar amino acid, wherein X 3 is a non-polar amino acid, wherein X 4 is any amino acid, wherein X5 is a polar amino acid, wherein X 6 is any amino acid, wherein X 7 is any amino acid, wherein X 8 is a polar amino acid, wherein X9 is a polar amino acid, and wherein X10 is any amino acid.
  • the anti-CDl37 antibodies described supra comprise heavy and light chain CDRs selected from the group consisting of:
  • the anti-CDl37 antibodies described supra comprise heavy and light chain CDRs selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 101; and wherein the light chain variable region comprises an amino acid sequence of SEQ ID NO: 6.
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions comprising amino acid sequences selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions comprising amino acid sequences selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions encoded by nucleotide sequences selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions encoded by nucleotide sequences selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 101; and wherein the light chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 6.
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 26 and 101 ; and wherein the light chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 6.
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions, wherein the heavy chain variable region is encoded by a nucleotide sequence which is least 90% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 5 and 102; and wherein the light chain variable region is encoded by a nucleotide sequence which is at least 90% identical to the nucleotide sequence of SEQ ID NO: 7.
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions, wherein the heavy chain variable region is encoded by a nucleotide sequence which is least 90% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 27 and 102; and wherein the light chain variable region is encoded by a nucleotide sequence which is at least 90% identical to the nucleotide sequence of SEQ ID NO: 7.
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions comprising amino acid sequences at least 90% identical to the amino acid sequences selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions comprising amino acid sequences at least 90% identical to the amino acid sequences selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions encoded by nucleotide sequences at least 90% identical to the nucleotide sequences selected from the group consisting of:
  • the anti-CD 137 antibodies comprise heavy and light chain variable regions encoded by nucleotide sequences at least 90% identical to the nucleotide sequences selected from the group consisting of: (a) SEQ ID NOs: 5 and 7, respectively;
  • the anti-CDl37 antibodies described herein have at least the functional properties of mAbl (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 4 and 6, respectively), mab8 (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 101 and 6, respectively) or mAblO (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 26 and 6, respectively).
  • mAbl i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 4 and 6, respectively
  • mab8 i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 101 and 6, respectively
  • mAblO i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 26 and 6, respectively.
  • the functional properties of an antibody described herein include but are not limited to: induction or enhancement of dimerization of CD137; induction or enhancement of multimerization of CD137; induction or enhancement of CDl37-mediated T cell activation; induction or enhancement of CDl37-mediated cytotoxic T cell response; induction or enhancement of CDl37-mediated T cell proliferation; induction or enhancement of CD137- mediated cytokine production; lack of induction or enhancement of intrahepatic and/or intrasplenic T cell activation and/or T cell proliferation; and reduction or inhibition of tumor growth.
  • the anti-CDl37 antibodies described herein bind human CD137 with an equilibrium dissociation constant KD of 1 X 10 6 or less. In some embodiments, the anti- CD137 antibodies described herein bind human CD137 with an equilibrium dissociation constant KD at least equivalent to that of mAbl (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 4 and 6, respectively), mab8 (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 101 and 6, respectively) or mAblO (i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 26 and 6, respectively).
  • mAbl i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 4 and 6, respectively
  • mab8 i.e., an antibody comprising the heavy and light chain variable sequences of SEQ ID NOs: 101 and 6, respectively
  • mAblO i.e., an antibody
  • the anti-CDl37 antibodies described herein comprise a human IgGl heavy chain constant region or a human IgG4 heavy chain constant region. In some embodiments, the anti-CD 137 antibodies described herein comprise a human wild-type IgGl heavy chain constant region or a human wild-type IgG4 heavy chain constant region. In some embodiments, the anti-CD 137 antibodies described herein comprise a human wild-type IgGl heavy chain constant region as set forth in SEQ ID NO: 1. In some embodiments, the anti-CDl37 antibodies described herein comprise a human wild-type IgG4 heavy chain constant region as set forth in SEQ ID NO: 138.
  • the anti-CDl37 antibodies described herein comprise a mutant IgGl heavy chain constant region an IgGl heavy chain constant region comprising an amino acid mutation relative to the human wild-type IgGl) or a mutant IgG4 heavy chain constant region ( i.e an IgG4 heavy chain constant region comprising an amino acid mutation relative to the human wild-type IgG4).
  • the anti-CD 137 antibodies described herein comprise a mutant IgG4 heavy chain constant region, wherein the mutant IgG4 heavy chain constant region comprises an amino acid substitution at residue Ser228 according to EU numbering. In some embodiments, the amino acid substitution at residue Ser228 is S228P.
  • the anti-CD 137 antibodies described herein comprise an IgG4 heavy chain constant region, wherein the c-terminal lysine residue is removed. In some embodiments, the anti- CD 137 antibodies described herein comprise an IgG4 heavy chain constant region wherein the c- terminal lysine residue is removed and comprises the S228P amino acid substitution. In some embodiments, the anti-CD 137 antibodies described herein comprise an IgG4 heavy chain constant region as set forth in SEQ ID NO: 2.
  • the anti-CDl37 antibodies described herein comprise heavy and light chains comprising the amino acid sequences set forth in SEQ ID NOs: 129 and 133, respectively. In some embodiments, the anti-CD 137 antibodies described herein comprise heavy and light chains comprising the amino acid sequences set forth in SEQ ID NOs: 130 and 133, respectively. In some embodiments, the anti-CD 137 antibodies described herein comprise heavy and light chains comprising the amino acid sequences set forth in SEQ ID NOs: 131 and 133, respectively. In some embodiments, the anti-CD 137 antibodies described herein comprise heavy and light chains comprising the amino acid sequences set forth in SEQ ID NOs: 132 and 133, respectively.
  • the anti-CDl37 antibodies described herein comprise heavy and light chains comprising amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least, 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 129 and 133, respectively. In some embodiments, the anti-CDl37 antibodies described herein comprise heavy and light chains comprising amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least, 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 130 and 133, respectively.
  • the anti-CDl37 antibodies described herein comprise heavy and light chains comprising amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least, 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 131 and 133, respectively. In some embodiments, the anti-CDl37 antibodies described herein comprise heavy and light chains comprising amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least, 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 132 and 133, respectively.
  • 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.
  • CDRs are defined according to Kabat, 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.
  • 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), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-32 (CDR1), 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain.
  • CDR1 residues 24-34
  • CDR2 50-56
  • CDR3 89-97
  • CDR3 26-32
  • CDR1, 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain.
  • 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
  • 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 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3.
  • the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
  • the CDRs recited herein comprise about residues 24-34 (CDR1), 50-56 (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 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: 4 and/or 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: 6.
  • 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%
  • 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, 2 amino acids or less, or 1 amino acid from SEQ ID NO: 4.
  • 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 CDRs of the antibody or antigen-binding portion thereof comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain of SEQ ID NO: 6, and 27-35 (CDR1), 49-60 (CDR2) and 93-102 (CDR3) in the heavy chain variable domain of SEQ ID NO: 4, when numbered according to Chothia numbering.
  • CDR2 in the light chain variable domain SEQ ID NO: 6 can comprise amino acids 49-56, when numbered according to Chothia numbering.
  • the disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that comprises heavy chain CDRs of the heavy chain variable region of SEQ ID NO: 4, and light chain CDRs of the light chain variable region of SEQ ID NO: 6, wherein the heavy and light chain CDR residues are numbered according to Rabat.
  • the disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that comprises heavy chain CDRs of the heavy chain variable region of SEQ ID NO: 4, and light chain CDRs of the light chain variable region of SEQ ID NO: 6, wherein the heavy and light chain CDR residues are numbered according to Chothia.
  • the disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that comprises heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 4, and light chain CDRs of the light chain variable region of SEQ ID NO: 6, wherein the heavy and light chain CDR residues are numbered according to MacCallum.
  • the disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that comprises heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 4, and light chain CDRs of the light chain variable region of SEQ ID NO: 6, wherein the heavy and light chain CDR residues are numbered according to AbM.
  • the disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that comprises heavy chain CDRs of the heavy chain variable regions of SEQ ID NO: 4, and light chain CDRs of the light chain variable region of SEQ ID NO: 6, wherein the heavy and light chain CDR residues are numbered according to IMGT.
  • an anti-CDl37 antibody described herein binds human CD137 with an affinity (KD) of about 40-100 nM (e.g., between about 40 nM and about 100 nM) as determined by an antigen-binding assay. In some embodiments, an anti-CDl37 antibody described herein binds human CD137 with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM) as determined by an antigen-binding assay.
  • an anti-CDl37 antibody described herein binds human CD137 with an affinity (KD) of about 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM as determined by an antigen-binding assay.
  • KD affinity
  • the antigen-binding assay determines a binding affinity of the anti- CD 137 antibody for a CD 137 polypeptide.
  • the antigen binding assay is surface plasmon resonance.
  • an anti-CD 137 antibody described herein binds human CD137 with an affinity (KD) of about 40-100 nM (e.g., between about 40 nM and about 100 nM) as determined using surface plasmon resonance.
  • an anti-CDl37 antibody described herein binds human CD137 with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM) as determined using surface plasmon resonance.
  • an anti-CDl37 antibody described herein binds human CD137 with an affinity (KD) of about 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM, 55- 75 nM, 40-70 nM, 50-80 nM, or 60-90 nM as determined using surface plasmon resonance.
  • KD affinity
  • surface plasmon resonance includes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).
  • BIAcore Pharmaacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ.
  • the antigen binding assay is biolayer interferometry (BLI).
  • BBI biolayer interferometry
  • an anti-CD 137 antibody described herein binds human CD137 with an affinity (KD) of about 40-100 nM (e.g., between about 40 nM and about 100 nM) as determined using biolayer interferometry.
  • an anti-CD 137 antibody described herein binds human CD137 with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM) as determined using biolayer interferometry.
  • an anti-CDl37 antibody described herein binds human CD137 with an affinity (KD) of about 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM as determined using biolayer interferometry.
  • KD affinity
  • biolayer interferometry includes an optical phenomenon that allows for the measurement of sub-nanometer changes in the thickness of its optical layer detection surface.
  • biomolecules bind at a sensor surface and change the optical layer thickness.
  • the magnitude of the optical layer thickness change is proportional to the mass or molecular weight of the binding molecule.
  • CD137 is immobilized to the sensor surface to measure binding by an antibody, wherein binding creates a change in the molecular weight to produce a corresponding change in the optical layer thickness.
  • BLI is performed with an OCTET system (ForteBio).
  • an anti-CDl37 antibody described herein induces or enhances cytokine production by an immune cell as determined by a cytokine assay.
  • the cytokine assay determines an amount of at least one cytokine secreted from an immune cell contacted with the anti-CD 137 antibody, wherein an increase in the amount of the at least one cytokine indicates induction or enhancement of cytokine production by the anti-CD 137 antibody.
  • an increase in cytokine production is at least 1 fold, 2 fold, 3 fold, 4 fold or 5 fold more compared to a control antibody (e.g., an antibody that does not bind to CD 137 and does not induce cytokine production).
  • an anti-CDl37 antibody described herein induces or enhances cytokine production by an immune cell as determined by a cytokine assay, wherein the cytokine assay comprises the following steps:
  • an anti-CDl37 antibody described herein induces or enhances cytokine production by an immune cell as determined by a cytokine assay, wherein the cytokine assay comprises the following steps:
  • the reference immune cell is contacted with a control antibody, and wherein an increase in the amount of the at least one cytokine produced from the immune cell relative to the reference immune cell indicates induction or enhancement of human CDl37-mediated cytokine production.
  • an anti-CDl37 antibody described herein induces or enhances cytokine production by an immune cell as determined by a cytokine assay, wherein the cytokine assay comprises the following steps:
  • the reference immune cell is not contacted with the anti-CD 137 antibody, and wherein an increase in the amount of the at least one cytokine produced from the immune cell relative to the reference immune cell indicates induction or enhancement of human CD137- mediated cytokine production by the immune cell.
  • the at least one cytokine is selected from a group consisting of IL- 2, IFNy, TNFa, IL-13, and combinations thereof.
  • the cytokine is IL-2.
  • the cytokine is IFNy.
  • the cytokine is TNFa.
  • the cytokine is IL-13.
  • an anti-CDl37 antibody induces or enhances IL-2 production.
  • an anti-CDl37 antibody induces or enhances TNFa production.
  • an anti-CD 137 antibody induces or enhances IL-13 production.
  • the cytokine produced is IL-2.
  • the cytokine produced is TNFa. In some embodiments, the cytokine produced is IL-13. In some embodiments, the cytokine produced is IFNy. In some embodiments, the cytokine produced is IL- 2 and TNFa. In some embodiments, the cytokine produced is IL-2 and IL-13. In some embodiments, the cytokine produced is IL-2 and IFNy. In some embodiments, the cytokine produced is TNFa and IL-13. In some embodiments, the cytokine produced is TNFa and IFNy. In some embodiments, the cytokine produced is IL-13 and IFNy.
  • the cytokine produced is IL-2, TNFa and IL-13. In some embodiments, the cytokine produced is IL- 2, TNFa and IFNy. In some embodiments, the cytokine produced is IFNy, TNFa and IL-13.
  • the immune cell is a T cell. In some embodiments, the reference immune cell is a T cell. In some embodiments the T cell is a CD8+ T cell.
  • the cytokine assay is a cytokine bead array assay.
  • a cytokine bead array assay is a bead-based immunoassay that allows for multianalyte flow cytometric determination of multiple cytokines in a sample.
  • the use of microspheres of different size or color is the basis of a cytokine bead array assay, wherein each microsphere (or“bead”) is coated with an antibody that specifically binds to an antigen (e.g., a cytokine).
  • Antibody-coated beads are then introduced to a sample in combination with detector antibodies.
  • the bead: antigen: detector antibody complexes are then analyzed by flow cytometry.
  • cytokine bead array assays include, but are not limited to, BDTM Cytometric Bead Array Systems (BD Biosciences) and LUMINEX® Assays (R&D Systems).
  • induction or enhancement of human CDl37-mediated cytokine production is determined by a cytokine bead array assay.
  • induction or enhancement of human CDl37-mediated cytokine production is determined by a LUMINEX® Assay.
  • the cytokine assay is a Meso Scale Discovery (MSD) assay (Meso Scale Diagnostics; Rockville, MD).
  • MSD assay is a commercially available assay based on detection of electrochemiluminescent-labeled antibodies that specifically bind to an antigen (e.g., a cytokine) of interest.
  • An MSD assay comprises high binding carbon electrodes in the bottom of microplate wells that allow for attachment of biological reagents (e.g., capture antibodies specific for a cytokine).
  • MSD assays use electrochemiluminescent labels that are conjugated to detection antibodies.
  • a sample is added to the microplate wells and electricity is applied to the plate electrodes by an MSD instrument leading to light emission by the electrochemiluminescent labels. Light intensity is measured to quantify analytes (e.g., cytokines) in the sample.
  • analytes e.g., cytokines
  • induction or enhancement of human CDl37-mediated cytokine production is determined by a Meso Scale Discovery (MSD) assay.
  • MSD Meso Scale Discovery
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay.
  • the T cell activation assay determines an amount of at least one cytokine secreted from T cells contacted with an anti-CD 137 antibody described herein, wherein an increase in the amount of the at least one cytokine indicates induction or enhancement of T cell activation.
  • an increase in cytokine production is at least 1 fold, 2 fold, 3 fold, 4 fold or 5 fold more compared to a control antibody (e.g., an antibody that does not bind to CD137 and does not induce cytokine production).
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises the following steps:
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises the following steps:
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises the following steps:
  • the reference T cells are contacted with a control antibody, and wherein an increase in the amount of the at least one cytokine produced from the T cells relative to the reference T cells indicates the anti-CD 137 antibody induces or enhances T cell activation.
  • the T cell activation assay comprises determining the level of at least one cytokine secreted by the T cells after contact with an anti-CD 137 antibody described herein, wherein the at least one cytokine is selected from the group consisting of IL-2, IFNy, TNFa and IL-13.
  • the cytokine is IL-2.
  • the cytokine is IFNy.
  • the cytokine is TNFa.
  • the cytokine is IL-13.
  • the T cell activation assay comprises a cytokine assay, such as those described herein, to determine the amount of the at least one cytokine.
  • the cytokine produced is IL-2. In some embodiments, the cytokine produced is TNFa. In some embodiments, the cytokine produced is IL-13. In some embodiments, the cytokine produced is IFNy. In some embodiments, the cytokine produced is IL-2 and TNFy. In some embodiments, the cytokine produced is IL-2 and IL-13. In some embodiments, the cytokine produced is IL-2 and IFNa. In some embodiments, the cytokine produced is TNFa and IL-13. In some embodiments, the cytokine produced is TNFa and IFNy. In some embodiments, the cytokine produced is IL-13 and IFNy.
  • the cytokine produced is IL-2, TNFa and IL-13. In some embodiments, the cytokine produced is IL-2, TNFa and IFNy. In some embodiments, the cytokine produced is IFNy, TNFa and IL-13.
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises detecting surface expression of at least one activation marker on T cells, and wherein an increase in the expression level of the at least one activation marker indicates induction or enhancement of T cell activation.
  • “increase in surface expression” refers to at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% increase in surface expression relative to surface expression in the presence of a control antibody or in the absence of an antibody.
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay in vitro , wherein the T cell activation assay comprises the following steps:
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises the following steps:
  • the reference T cells are not contacted with the anti-CD 137 antibody, and wherein an increase in surface expression of at least one activation marker on the T cells relative to the reference T cells indicates the anti-CD 137 antibody induces or enhances T cell activation.
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises the following steps:
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay in vivo , wherein the T cell activation assay comprises the following steps:
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises the following steps:
  • the reference T cells are isolated from a subject not administered the anti-CD 137 antibody, and wherein an increase in surface expression of the at least one activation marker on the T cells relative to the reference T cells indicates the anti-CD 137 antibody induces or enhances T cell activation.
  • an anti-CD 137 antibody described herein induces or enhances T cell activation as determined by a T cell activation assay, wherein the T cell activation assay comprises the following steps:
  • the reference T cells are isolated from a subject contacted with a control antibody, and wherein an increase in surface expression of the at least one activation marker on the T cells relative to surface expression of the at least one activation marker on the reference T cells indicates the anti-CD 137 antibody induces or enhances T cell activation.
  • an anti-CD 137 antibody described herein does not induce or enhance intrahepatic T cell activation as determined by a T cell activation assay in vivo , wherein the T cell activation assay comprises the following steps:
  • the reference T cells are isolated from a subject not administered the anti-CD 137 antibody, optionally, wherein the reference T cells are isolated from a subject administered a control antibody, and wherein an absence of an increase in surface expression of the at least one activation marker on the T cells relative to surface expression of the at least one activation marker on the reference T cells indicates the anti-CD 137 antibody induces or enhances T cell activation.
  • an anti-CD 137 antibody described herein does not induce or enhance intrasplenic T cell activation as determined by a T cell activation assay in vivo, wherein the T cell activation assay comprises the following steps:
  • the reference T cells are isolated from a subject not administered the anti-CD 137 antibody, optionally, wherein the reference T cells are isolated from a subject administered a control antibody, and wherein an absence in an increase in surface expression of the at least one activation marker on the T cells relative to surface expression of the at least one activation marker on the reference T cells indicates the anti-CD 137 antibody induces or enhances T cell activation.
  • “does not induce or enhance” is intended to refer to the absence of an activity (e.g., T cell activation) or a lack of increase of an activity relative to an increase by a reference antibody.
  • a surface expression of a T cell activation marker is equivalent to the surface expression in the absence of an antibody. In some embodiments a surface expression of a T cell activation marker is less than the surface expression in the presence of a reference antibody that induces or enhance surface expression at least 1 fold, 5 fold, 10 fold, 50 fold, or 100 fold higher compared to surface expression in the absence of an antibody.
  • the at least one activation marker is selected from the group consisting of CD25, CD69 and CD40L. In some embodiments, the one or more activation markers is CD25.
  • T cells are isolated from a subject having a tumor. In some embodiments, the T cells are isolated from the tumor. In some embodiments, the control antibody is an isotype control antibody.
  • an anti-CDl37 antibody described herein induces or enhances infiltration of one or more immune cells into a tumor microenvironment as determined by an immune cell infiltration assay. In some embodiments, an anti-CD 137 antibody described herein decreases infiltration of one or more immune cells into a tumor microenvironment as determined by an immune cell infiltration assay.
  • the immune cell infiltration assay determines a quantity of immune cells expressing one or more immune cell markers in a tumor.
  • the one or more immune cell markers is labeled with an antibody.
  • the one or more immune cell markers is selected from the group consisting of CD45, CD25, FOXP3, CD4, CD8, F4/80, CDl lb, TIGIT and PD-l.
  • the quantity of immune cells expressing the one or more immune cell markers in a tumor is determined by flow cytometry. Methods of quantifying immune cells expressing one or more immune cell markers by flow cytometry are known in the art.
  • the anti-CD 137 antibody induces or enhances infiltration of one or more immune cells into a tumor microenvironment relative to a reference antibody, as determined by an immune cell infiltration assay.
  • the reference antibody is an antibody comprising the same isotype as the anti-CD 137 antibody and does not specifically bind to CD 137.
  • the reference antibody is an antibody comprising the same isotype as the anti-CD 137 antibody and specifically binds to CD 137.
  • the reference antibody is an antibody comprising the different isotype as the anti-CD 137 antibody and does not specifically bind to CD137.
  • the reference antibody is an antibody comprising a different isotype as the anti-CD 137 antibody and specifically binds to CD 137.
  • an anti-CDl37 antibody described herein increases infiltration of immune cells expressing CD45 into a tumor microenvironment in a subject as determined by an immune cell infiltration assay, wherein the assay comprises the following steps:
  • an increase in the quantity of fluorescently-immune cells expressing CD45 in the tumor indicates the anti-CD 137 antibody induces or enhances infiltration of immune cells into the tumor microenvironment.
  • an increase in the quantity of immune cells expressing CD45 is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of total cells in the tumor microenvironment.
  • an anti-CDl37 antibody described herein reduces or inhibits infiltration of one or more immune cells into a tumor microenvironment as determined by an immune cell infiltration assay.
  • the anti-CDl37 antibody decreases infiltration of one or more immune cells into a tumor microenvironment relative to a reference antibody, as determined by an immune cell infiltration assay.
  • the reference antibody is an antibody comprising the same isotype as the anti-CD 137 antibody and does not specifically bind to CD137.
  • the reference antibody is an antibody comprising the same isotype as the anti-CD 137 antibody and specifically binds to CD 137.
  • the reference antibody is an antibody comprising the different isotype as the anti- CD 137 antibody and does not specifically bind to CD 137. In some embodiments, the reference antibody is an antibody comprising a different isotype as the anti-CD 137 antibody and specifically binds to CD137. In some embodiments, a decrease in immune cells is less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of total cells in a tumor microenvironment.
  • an anti-CDl37 antibody described herein decreases infiltration of tumor associated macrophages into a tumor microenvironment in a subject as determined by an immune cell infiltration assay, wherein the assay comprises the following steps:
  • tumor-associated macrophages are less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of immune cells in the tumor microenvironment.
  • tumor-associated macrophages express F4/80, CDl lb and CD45.
  • an anti-CDl37 antibody described herein decreases infiltration of T regulatory cells (Tregs) into a tumor microenvironment in a subject as determined by an immune cell infiltration assay, wherein the assay comprises the following steps:
  • Treg cells are less than 35%, 30%, 25%, 20%, 15%, 10%, or 5% of CD4+ T cells in the tumor microenvironment.
  • Treg cells express CD4, FOXP-3 and CD25.
  • an anti-CD 137 antibody described herein protects T cells from T cell exhaustion and/or reverses T cell exhaustion as determined by a T cell exhaustion assay. Exhausted T cells can be distinguished from other T cell dysfunctions such as anergy and senescence based on their underlying molecular mechanisms (Crespo et ah, (2013) Curr Opin Immunol 25(2):24l-22l).
  • T cells arise from T cells which initially gained and provided T cell effector function, but that exhibit a gradual deterioration of T cell effector function due to continuous T cell receptor (TCR) stimulation from persistent antigen and inflammatory mediators, both of which commonly occur in tumors (Wherry & Kurachi (2015) Nat Rev Immunol l5(8):486-99).
  • TCR T cell receptor
  • Hallmarks of T cell exhaustion include, but are not limited to, continuous deterioration of in vivo and/or ex vivo T cell function, an increased expression of multiple inhibitory receptors (IRs) (e.g., PD-l, CTLA-4, LAG-3, TIM-3, CD244, CD160, TIGIT), progressive loss or decrease of effector cytokine secretion (e.g., IL-2, interferon gamma (IFNy), tumor necrosis factor alpha (TNFa)), loss or decrease of CC chemokine (b-chemokine) production, poor responsiveness to IL-7 and IL-15, loss or decrease of proliferative capacity, loss or decrease of in vivo and/or ex vivo cytolytic activity, altered cell metabolism and a different transcriptional profile relative to non-exhausted T cells. Severely exhausted T cells can succumb to deletion (Yi et al., (2010) Immunology l29(4):474- 481).
  • IRs inhibitory
  • an anti-CD 137 antibody described herein protects T cells from T cell exhaustion and/or reverses T cell exhaustion as determined by a T cell exhaustion assay wherein the T cell exhaustion assay determines an amount or level of one or more effector cytokines secreted from T cells treated with an anti-CD 137 antibody described herein, wherein the amount or level of the one or more effector cytokines indicates protection from or reversion of T cell exhaustion.
  • the T cell exhaustion assay comprises the following steps:
  • the one or more effector cytokines is selected from IL-2, IFNy, and TNFa.
  • the amount or level of the one or more effector cytokines is determined by ELISA. ELIS As suitable for the determination of the amount or level of the one or more effector cytokines are known in the art.
  • the amount or level of the one or more effector cytokines is determined by Meso Scale Discovery.
  • the amount or level of the one or more effector cytokines is determined by any one of the cytokine production assays described herein.
  • an anti-CD 137 antibody described herein protects T cells from T cell exhaustion and/or reverses T cell exhaustion as determined by a T cell exhaustion assay wherein the T cell exhaustion assay determines an expression level of one or more inhibitory receptors on T cells treated with an anti-CD 137 antibody described herein, wherein the expression level of the one or more inhibitory receptors indicates protection from or reversion of T cell exhaustion.
  • the T cell exhaustion assay comprises the following steps:
  • the one or more inhibitory receptors is selected from TIGIT and PD-l
  • the expression level of the one or more inhibitory receptors is determined by flow cytometry. Methods to determine expression levels of inhibitory receptors on immune cells (e.g. T cells) by flow cytometry are known in the art.
  • the amount of exhausted T cells is less than 20%, 15%, 10% or 5% of total CD8+ or CD4+ T cells in a tumor microenvironment.
  • assays described herein refer to‘isolating T cells from a subject’; it is to be understood that the assay may suitably be performed on T cells previously isolated from a subject.
  • the assays described herein refer to (i) administering the anti-CD 137 antibody to a subject and (ii) isolating T cells from the subject; it is to be understood that the assay may suitably be performed on T cells previously isolated from a subject to whom the anti-CD 137 antibody has been administered.
  • the assays described herein refer to‘obtaining a sample of the tumor’; it is to be understood that the assay may suitably be performed on a sample of a tumor previously isolated from a subject.
  • the assays described herein refer to (i) administering the anti-CD 137 antibody to a subject having a tumor and (ii) obtaining a sample of the tumor; it is to be understood that the assay may suitably be performed a sample of a tumor previously isolated from a subject to whom the anti-CD 137 antibody has been administered.
  • an anti-CD 137 antibody described herein binds to a non-ligand binding region of CD137, as determined by a ligand binding assay.
  • a ligand binding assay is an assay, or an analytic procedure, that provides a measure of the interactions that occur between two reactant molecules (e.g., a receptor and ligand polypeptides).
  • the LBA provides a measure of the degree of affinity between the two reactant molecules (e.g., a receptor and ligand polypeptides).
  • a ligand binding assay is used to determine the presence, rate, extent of binding, or combinations thereof, of a ligand molecule (e.g., CD137L) to a receptor (e.g., CD 137).
  • a ligand binding assay comprises detecting the formation of a ligand:receptor complex.
  • a ligand binding assay comprises determining the dissociation of a ligand:receptor complex.
  • the formation and/or dissociation of a ligand:receptor complex is determined by detection of a fluorescently-labeled ligand in complex with a receptor. In some embodiments, the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of fluorescently-labeled receptor in complex with a ligand. In some embodiments, the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of a fluorescently-labeled antibody that specifically binds to the ligand:receptor complex. Methods of detecting and quantifying fluorescence are known in the art and include, but are not limited to, fluorescence polarization (FP) and fluorescence anisotropy (FA).
  • FP fluorescence polarization
  • FA fluorescence anisotropy
  • the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of a radioactively-labeled ligand in complex with a receptor. In some embodiments, the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of radioactively-labeled receptor in complex with a ligand. In some embodiments, the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of a radioactively-labeled antibody that specifically binds to the ligand:receptor complex. Methods of detecting and quantifying radioactivity are known in the art and include, but are not limited to, quantitative autoradiography and scintillation counting.
  • the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of a bioluminescently-labeled ligand in complex with a receptor. In some embodiments, the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of bioluminescently-labeled receptor in complex with a ligand. In some embodiments, the formation and/or dissociation of a ligand:receptor complex is determined by detection and/or quantification of an amount of a bioluminescently-labeled antibody that specifically binds to the ligand:receptor complex. Methods of detecting and quantifying bioluminescence are known in the art and include, but are not limited to, luminometry.
  • formation and/or dissociation of the ligand:receptor complex is determined by surface plasmon resonance (SPR) as described supra.
  • SPR surface plasmon resonance
  • a ligand binding assay determines if an antibody that specifically binds to a receptor (e.g., an anti-CDl37 antibody) affects the formation of a ligand:receptor complex by determining the presence, rate and/or extent of ligand binding to the receptor in the presence of the antibody.
  • an antibody e.g., an anti-CD 137 antibody
  • a receptor e.g., CD137

Abstract

La présente invention concerne des procédés de traitement ou d'amélioration d'un ou plusieurs symptômes du cancer, à l'aide d'anticorps agonistes, et de fragments de liaison à l'Antigène de ceux-ci, qui se lient à CD137, les cellules cancéreuses exprimant le complexe majeur d'histocompatibilité I (CMH I).
PCT/US2019/026675 2018-04-09 2019-04-09 Anticorps agonistes contre le cd137 humain dans le cancer exprimant le cmh i WO2019199896A1 (fr)

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