Anti-CD137 molecules and their uses

Abstract

Provided are antibodies or antigen-binding fragments thereof that bind to human CD137, nucleic acids encoding said antibodies or antigen-binding fragments thereof, therapeutic compositions of said antibodies or antigen-binding fragments thereof, and their uses for enhancing T cell function by upregulating cell-mediated immune responses and for treating T cell dysfunction disorders, such as tumor immunity, and for treating cancer.

Description

[0001] This application is a divisional application of the invention patent application filed on August 21, 2018, with application number 201880063358.5 and invention title: "Anti-CD137 molecule and its use".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to International Patent Application Serial No. PCT / CN2017 / 098332, filed on August 21, 2017, which is incorporated herein by reference in its entirety.

[0004] References to sequence lists

[0005] The following submission on an ASCII text file is incorporated herein by reference in its entirety: Sequence List in Computer-Readable Form (CRF) (filename: 695402000341seqlist.txt, record date: August 20, 2018, size: 542KB). Technical Field

[0006] This disclosure relates to antibodies or antigen-binding fragments thereof that bind to human CD137, nucleic acids encoding said antibodies or antigen-binding fragments thereof, therapeutic compositions thereof, and their antitumor uses. Background Technology

[0007] CD137 (also known as CD137 receptor, 4-1BB, TNFRSF9, etc.) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). Current understanding of CD137 indicates that its expression is generally activation-dependent and present in a broad subset of immune cells, including activated NK and NKT cells, regulatory T cells, dendritic cells (DCs), stimulated mast cells, differentiating bone marrow cells, monocytes, neutrophils, and eosinophils (Wang, 2009, Immunological Reviews 229:192-215). CD137 expression has also been confirmed on tumor angiography (Broll, 2001, Amer. J. Clin. Pathol. 115(4): 543-549; Seaman, 2007, Cancer Cell 11: 539-554) and on inflamed or atherosclerotic endothelial sites (Drenkard, 2007 FASEB J. 21: 456-463; Olofsson, 2008, Circulation 117: 1292-1301). The ligand stimulating CD137, CD137 ligand (CD137L), is expressed on activated antigen-presenting cells (APCs), bone marrow progenitor cells, and hematopoietic stem cells.

[0008] Human CD137 is a protein of 255 amino acids (GenBank accession number NM). _ 001561; NP _ 001552; SEQ ID NO.: 1). The protein comprises a signal sequence (amino acid residues 1-17), followed by an extracellular domain (169 amino acids), a transmembrane region (27 amino acids), and an intracellular domain (42 amino acids) (Cheuk ATC et al. 2004 Cancer Gene Therapy 11:215-226). The receptor is expressed on the cell surface in monomeric and dimeric forms and may trimerize with CD137 ligands for signal transduction.

[0009] Numerous studies on mouse and human T cells have demonstrated that CD137 promotes enhanced cell proliferation, survival, and cytokine production (Croft, 2009, Nat Rev Immunol 9:271-285). Studies have shown that some CD137 agonist mAbs increase the expression of co-stimulatory molecules and significantly enhance cytolytic T lymphocyte responses, thereby leading to antitumor efficacy in various models. CD137 agonist mAbs have demonstrated efficacy in both prophylactic and therapeutic settings. Furthermore, durable antitumor protective T-cell memory responses have been established in tumor models using CD137 monotherapy and combination therapy (Lynch, 2008, Immunol Rev. 22:277-286). CD137 agonists have also been shown to suppress autoimmune responses in several well-established autoimmune models (Vinay, 2006, J Mol Med 84:726-736). This dual activity of CD137 offers the potential to provide antitumor activity while reducing the autoimmune side effects associated with immunotherapies that disrupt immune tolerance.

[0010] There is a long-standing unmet need for antibodies that bind to human CD137, enhance CD137-mediated responses, and thus provide potential therapeutic agents for treating a variety of diseases and conditions, including cancer and autoimmune diseases. Furthermore, there is a need for anti-CD137 antibodies that cross-react between different species (such as humans and laboratory animals (mice, monkeys, dogs, etc.)) to enable animal model studies while simultaneously providing therapeutic candidates. Summary of the Invention

[0011] One object of this disclosure is to provide a separate binding molecule, such as an antibody or a binding fragment thereof, or a derivative thereof, that binds to human CD137. Another object of this disclosure is to provide a composition comprising a binding molecule that binds to CD137. Yet another object of this disclosure is to provide a method for treating diseases and / or symptoms related to or mediated by CD137 signaling, which is achieved by using one or more binding molecules of this disclosure. These and other objects of this disclosure are described more fully herein.

[0012] Therefore, in one aspect, this document provides one or more antibodies (e.g., isolation antibodies) or one or more antigen-binding fragments thereof, said antibodies or antigen-binding fragments thereof binding to the extracellular domain of human CD137, and comprising one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all 10) of the following functional features: (a) binding to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO:1; (b) not binding to one or more amino acid residues within amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO:1; (c) at 100 nM or less K D (d) binds to human CD137; (e) has agonist activity on human CD137; (f) does not bind to human OX40, CD40, GITR and / or CD27 receptors at concentrations up to 1000 nM; (g) cross-reacts with CD137 in monkeys, mice, rats and / or dogs; (h) does not induce ADCC effects; (i) inhibits tumor cell growth; (j) has therapeutic effects on cancer; and / or (j) blocks the binding between CD137 and CD137L.

[0013] Therefore, in one aspect, this document provides an antibody (e.g., an isolation antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (I): X1TFX2X3YX4IHWV (SEQ ID NO:2), wherein X1 is F or Y, X2 is S or T, X3 is G, N, or S, and X4 is A, G, or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO:3), wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T; and Formula (III): FSLSTX1GVX2VX3WI (SEQ ID NO:3). NO:4), wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T; wherein HVR-H2 contains an amino acid sequence selected from the group consisting of: Formula (IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO:5), wherein X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y, and X4 is P or T; Formula (V): IGX1IYHSGX2TYYX3PSLKSRV (SEQ ID NO:6), wherein X1 is D or E, X2 is N or S, and X3 is N or S; and Formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO:6). NO:7), wherein X1 is A, G, S, V or Y, X2 is A, D, S or Y, X3 is D, G or S, and X4 is S or T; and wherein HVR-H3 contains an amino acid sequence according to the following formula: Formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO:8), wherein X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T or V, X5 is A, I, L, T or V, and X6 is A, D or G.

[0014] In some embodiments, this document provides an antibody (e.g., an isolation antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (XII): X1TFSX2YWIHWV (SEQ ID NO: 853), wherein X1 is F or Y, and X2 is N or S; Formula (XIII): YSIX1SGX2X3WX4WI (SEQ ID NO: 854), wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, or S; and Formula (XIV): FSLSTX1GVX2VX3WI (SEQ ID NO: 854). NO:855), wherein X1 is G or S, X2 is A or G, and X3 is A, G, or S; wherein HVR-H2 contains an amino acid sequence according to the group selected from the following: Formula (IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO:5), wherein X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y, and X4 is P or T; and Formula (XV): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO:856), wherein X1 is G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T; and wherein HVR-H3 contains an amino acid sequence according to the following: Formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO:855). NO:8), where X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T or V, X5 is A, I, L, T or V, and X6 is A, D or G.

[0015] On the other hand, this document provides an antibody (e.g., an isolation antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises an amino acid sequence according to the following formula: Formula (VIII): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO: 9), wherein X1 is Q or R, X2 is D, G, or S, X3 is I or V, X4 is G, R, S, or T, X5 is P, R, S, or T, X6 is A, D, F, S, V, or Y, X7 is L or V, and X8 is A, G, or N; wherein HVR-L2 comprises an amino acid sequence according to the following formula: Formula (IX): X1ASX2X3X4X5GX6 (SEQ ID NO: 9). NO:10), wherein X1 is A or D, X2 is N, S or T, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and wherein HVR-L3 contains an amino acid sequence according to the group selected from the following: Formula (X): YCQQX1YX2X3X4T (SEQ ID NO:11), wherein X1 is A, G, S or Y, X2 is Q, S or Y, X3 is I, L, T or Y, and X4 is I, S, V or W; and Formula (XI): YCX1QX2X3X4X5PX6T (SEQ ID NO:12), wherein X1 is E or Q, X2 is P, S or Y, X3 is D, L, S, T or Y, X4 is D, E, H, S or T, X5 is D, LT or W, and X6 is L, P, R or V.

[0016] In some embodiments, this document provides an antibody (e.g., an isolation antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises an amino acid sequence according to the following formula: Formula (XVI): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO: 857), wherein X1 is Q or R, X2 is D, G, or S, X3 is I or V, X4 is G, R, S, or T, X5 is P, R, S, or T, X6 is A, F, S, V, or Y, X7 is L or V, and X8 is A or G; wherein HVR-L2 comprises an amino acid sequence according to the following formula: Formula (XVII): X1ASX2X3X4X5GX6 (SEQ ID NO: 857). NO:858), wherein X1 is A or D, X2 is N or S, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and wherein HVR-L3 contains an amino acid sequence according to the following formula: Formula (XVIII): YCQQX1YX2X3WT (SEQ ID NO:859), wherein X1 is A or G, X2 is S or Y, and X3 is I, L or T.

[0017] On the other hand, this document provides an antibody (e.g., an isolation antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3 of the following: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, ... VH24, VH25, VH26, VH27, VH28, VH29, VH30, VH31, VH32, VH33, VH34, VH35, VH36, VH37, VH38, VH39, VH40, VH 41. VH42, VH43, VH44, VH45, VH46, VH47, VH48, VH49, VH50, VH51, VH52, VH53, VH54, VH55, VH56, VH57, VH58 VH59 or VH60; and / or the light chain variable region contains HVR-L1, HVR-L2 and HVR-L3 of the following: VL1, VL2, VH3, VL4, VH5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL22, VL23, VL24, VL25, VL26, VL27 VL28, VL29, VL30, VL31, VL32, VL33, VL34, VL35, VL36, VL37, VL38, VL39, VL40, VL41, VL42, VL43, VL44, VL45, VL46, VL47, VL48, VL49, VL50, VL51, VL52, VL53, VL54, VL55, VL56, VL57, VL58, VL59 or VL60 (as shown in Table 1c).In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the following: VH1 and VL1, VH2 and VL2, VH3 and VL3, VH4 and VL4, VH5 and VL5, VH6 and VL6, VH7 and VL7, VH8 and VL8, VH9 and VL9, VH10, and VL10, VH11 and VL11, VH12 and VL12, VH13 and VL13, VH14 and VL14, VH15 and VL15, VH16 and VL16, VH17 and VL17, VH18 and VL18, VH19 and VL19, VH20 and VL20, VH21 and VL21, VH22 and VL22, VH23 and VL23, VH24 and VL24, VH25 and VL25, VH26 and VL26, VH27 and V L27, VH28 and VL28, VH29 and VL29, VH30 and VL30, VH31 and VL31, VH32 and VL32, VH33 and VL33, VH34 and VL34, VH35 and VL35, VH36 and VL36, VH37 and VL37, VH38 and VL38, VH39 and VL39, VH40 and VL40, VH41 and VL41, VH42 and VL42, VH43 and VL43, VH44 and VL 44, VH45 and VL45, VH46 and VL46, VH47 and VL47, VH48 and VL48, VH49 and VL49, VH50 and VL50, VH51 and VL51, VH52 and VL52, VH53 and VL53, VH54 and VL54, VH55 and VL55, VH56 and VL56, VH57 and VL57, VH58 and VL58, VH59 and VL59, or VH60 and VL60 (as shown in Table 1c).

[0018] On the other hand, this document provides an antibody (e.g., an isolating antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain variable regions of the following: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24, VH2 5. VH26, VH27, VH28, VH29, VH30, VH31, VH32, VH33, VH34, VH35, VH36, VH37, VH38, VH39, VH40, VH41, V H42, VH43, VH44, VH45, VH46, VH47, VH48, VH49, VH50, VH51, VH52, VH53, VH54, VH55, VH56, VH57, VH58 VH59 or VH60; and / or the light chain variable region includes the following light chain variable regions: VL1, VL2, VH3, VL4, VH5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL22, VL23, VL24, VL25, VL26, VL27, VL28, V L29, VL30, VL31, VL32, VL33, VL34, VL35, VL36, VL37, VL38, VL39, VL40, VL41, VL42, VL43, VL44, VL45, VL46, VL47, VL48, VL49, VL50, VL51, VL52, VL53, VL54, VL55, VL56, VL57, VL58, VL59 or VL60 (as shown in Table 1c).In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region include the heavy chain variable region and the light chain variable region of the following: VH1 and VL1, VH2 and VL2, VH3 and VL3, VH4 and VL4, VH5 and VL5, VH6 and VL6, VH7 and VL7, VH8 and VL8, VH9 and VL9, VH10 and VL10, VH11 and VL11, VH12 and VL 12, VH13 and VL13, VH14 and VL14, VH15 and VL15, VH16 and VL16, VH17 and VL17, VH18 and VL18, VH19 and VL19, VH20 and VL20, VH21 and VL21, VH22 and VL22, VH23 and VL23, VH24 and VL24, VH25 and VL25, VH26 and VL26, VH27 and VL27, VH28 and VL28, V H29 and VL29, VH30 and VL30, VH31 and VL31, VH32 and VL32, VH33 and VL33, VH34 and VL34, VH35 and VL35, VH36 and VL36, VH37 and VL37, VH38 and VL38, VH39 and VL39, VH40 and VL40, VH41 and VL41, VH42 and VL42, VH43 and VL43, VH44 and VL44, VH45 and VL45, VH46 and VL46, VH47 and VL47, VH48 and VL48, VH49 and VL49, VH50 and VL50, VH51 and VL51, VH52 and VL52, VH53 and VL53, VH54 and VL54, VH55 and VL55, VH56 and VL56, VH57 and VL57, VH58 and VL58, VH59 and VL59, or VH60 and VL60 (as shown in Table 1c).

[0019] In another embodiment, this document provides an antibody (e.g., a separating antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137. In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues from amino acid residues 34-108 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues from amino acid residues 34-93 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more of amino acid residues 34-36, one or more of amino acid residues 53-55, and one or more of amino acid residues 92-93 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment does not bind to one or more amino acid residues selected from the group consisting of amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment does not bind to amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from cynomolgus monkeys, mice, rats, and / or dogs. In some embodiments, the antibody or antigen-binding fragment binds to cynomolgus monkey CD137.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1 containing the amino acid sequence SEQ ID NO:711, HVR-H2 containing the amino acid sequence SEQ ID NO:735, and HVR-H3 containing the amino acid sequence SEQ ID NO:759; and / or wherein the light chain variable region comprises HVR-L1 containing the amino acid sequence SEQ ID NO:783, HVR-L2 containing the amino acid sequence SEQ ID NO:807, and HVR-L3 containing the amino acid sequence SEQ ID NO:831. In some embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO:41, and / or the light chain variable region comprises the amino acid sequence SEQ ID NO:42. In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:617, and / or the light chain comprises the amino acid sequence SEQ ID NO:618.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1 containing the amino acid sequence SEQ ID NO:712, HVR-H2 containing the amino acid sequence SEQ ID NO:736, and HVR-H3 containing the amino acid sequence SEQ ID NO:760; and / or wherein the light chain variable region comprises HVR-L1 containing the amino acid sequence SEQ ID NO:784, HVR-L2 containing the amino acid sequence SEQ ID NO:808, and HVR-L3 containing the amino acid sequence SEQ ID NO:832. In some embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO:61, and / or the light chain variable region comprises the amino acid sequence SEQ ID NO:62. In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:619, and / or the light chain comprises the amino acid sequence SEQ ID NO:620.

[0022] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1 containing the amino acid sequence SEQ ID NO:731, HVR-H2 containing the amino acid sequence SEQ ID NO:755, and HVR-H3 containing the amino acid sequence SEQ ID NO:779; and / or wherein the light chain variable region comprises HVR-L1 containing the amino acid sequence SEQ ID NO:803, HVR-L2 containing the amino acid sequence SEQ ID NO:827, and HVR-L3 containing the amino acid sequence SEQ ID NO:851. In some embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO:71, and / or the light chain variable region comprises the amino acid sequence SEQ ID NO:72. In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:657, and / or the light chain comprises the amino acid sequence SEQ ID NO:658.

[0023] On the other hand, this article provides an antibody (e.g., an isolation antibody) that binds to the extracellular domain of human CD137, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to the formula selected from the group consisting of: formula (I), formula (II), and formula (III); HVR-H2 comprises an amino acid sequence according to the formula selected from the group consisting of: formula (IV), formula (V), and formula (VI); and HVR-H3 comprises an amino acid sequence according to formula (VII); and / or the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises an amino acid sequence according to formula (VIII); HVR-L2 comprises an amino acid sequence according to formula (IX); and HVR-L3 comprises an amino acid sequence according to the formula selected from the group consisting of: formula (X) and formula (XI). In some embodiments, this document provides an antibody (e.g., an isolation antibody) that binds to the extracellular domain of human CD137, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to formulas (XIII) and (XVI) selected from the group consisting of; HVR-H2 comprises an amino acid sequence according to formulas (IV) and (XV); and HVR-H3 comprises an amino acid sequence according to formula (VII); and / or the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises an amino acid sequence according to formula (XVI); HVR-L2 comprises an amino acid sequence according to formula (XVII); and HVR-L3 comprises an amino acid sequence according to formula (XVIII).

[0024] In some embodiments, HVR-H1 contains an amino acid sequence selected from the group consisting of SEQ ID NO:253-312, HVR-H2 contains an amino acid sequence selected from the group consisting of SEQ ID NO:313-372, HVR-H3 contains an amino acid sequence selected from the group consisting of SEQ ID NO:373-432, HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NO:433-492, HVR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NO:493-552, and / or HVR-L3 contains an amino acid sequence selected from the group consisting of SEQ ID NO:553-612. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, and 131, and / or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, ... The amino acid sequences of NO:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, and 132.

[0025] In some embodiments, HVR-H1 comprises an amino acid sequence according to the group consisting of formulas (XII), (XIII), and (XIV); HVR-H2 comprises an amino acid sequence according to formula (IV) or (XV); and HVR-H3 comprises an amino acid sequence according to formula (VII); and / or wherein HVR-L1 comprises an amino acid sequence according to formula (XVI); HVR-L2 comprises an amino acid sequence according to formula (XVII); and HVR-L3 comprises an amino acid sequence according to formula (XVIII). In some embodiments, HVR-H1 contains an amino acid sequence selected from the group consisting of SEQ ID NO:709-732, HVR-H2 contains an amino acid sequence selected from the group consisting of SEQ ID NO:733-756, HVR-H3 contains an amino acid sequence selected from the group consisting of SEQ ID NO:757-780, HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NO:781-804, HVR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NO:805-828, and HVR-L3 contains an amino acid sequence selected from the group consisting of SEQ ID NO:829-852. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 17, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 61, 63, 65, 67, 71, 73, 75, 79, 83, 85, and 87, and / or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 18, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 62, 64, 66, 68, 72, 74, 76, 80, 84, 86, and 88. In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, and 659, and / or the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, and 660.

[0026] In some embodiments, HVR-H1 comprises the amino acid sequence SEQ ID NO: 711 or 731; HVR-H2 comprises the amino acid sequence SEQ ID NO: 735 or 755; HVR-H3 comprises the amino acid sequence SEQ ID NO: 759 or 779; HVR-L1 comprises the amino acid sequence SEQ ID NO: 783 or 803; HVR-L2 comprises the amino acid sequence SEQ ID NO: 807 or 827; and HVR-L3 comprises the amino acid sequence SEQ ID NO: 831 or 851. In some embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO: 41 or 71, and the light chain variable region comprises the amino acid sequence SEQ ID NO: 42 or 72. In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO: 617 or 657, and the light chain comprises the amino acid sequence SEQ ID NO: 618 or 658.

[0027] In some embodiments, HVR-H1 comprises the amino acid sequence SEQ ID NO:712; HVR-H2 comprises the amino acid sequence SEQ ID NO:736; HVR-H3 comprises the amino acid sequence SEQ ID NO:760; HVR-L1 comprises the amino acid sequence SEQ ID NO:784; HVR-L2 comprises the amino acid sequence SEQ ID NO:808; and HVR-L3 comprises the amino acid sequence SEQ ID NO:832. In some embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence SEQ ID NO:62. In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:619, and the light chain comprises the amino acid sequence SEQ ID NO:620.

[0028] In some embodiments that can be combined with any of the foregoing embodiments, the antibody or antigen-binding fragment is at a Kc of 100 nM or less. D Binding to human CD137 (e.g., as measured by surface plasmon resonance). In some embodiments, the antibody or antigen-binding fragment is at 50 nM or less K. D Combined with human CD137 (e.g., as measured by surface plasmon resonance).

[0029] In some embodiments that can be combined with any of the foregoing embodiments, the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from cynomolgus monkeys (e.g., GenBank Gene ID 102127961), mice (e.g., GenBank Gene ID 21942), rats (e.g., GenBank Gene ID 500590), and / or dogs (e.g., GenBank Gene ID 608274). In some embodiments, the antibody or antigen-binding fragment binds to cynomolgus monkey CD137.

[0030] In some embodiments that can be combined with any of the foregoing embodiments, the activity of human CD137 (e.g., when expressed on cells such as human cells) is reduced when it comes into contact with an antibody or antigen-binding fragment.

[0031] In some embodiments that can be combined with any of the foregoing embodiments, the antibody or antigen-binding fragment has a half-maximum inhibitory concentration (IC50) of about 100 nM or less for blocking the in vitro binding of human CD137 to human CD137L. 50 In some embodiments, when the antibody or antigen-binding fragment is provided at a concentration of about 1 μM or greater, the antibody or antigen-binding fragment completely blocks the in vitro binding of human CD137 to human CD137L. In some embodiments that can be combined with any of the foregoing embodiments, the activity of human CD137 (e.g., when expressed on cells such as human cells) increases upon contact with the antibody or antigen-binding fragment. In some embodiments, contacting CD137 (e.g., expressed on human cells) with the antibody or antigen-binding fragment results in an increase in NF-κB-dependent transcription. In some embodiments of any of the above embodiments, the antibody or antigen-binding fragment blocks one or more aspects of CD137 signaling stimulated by CD137L in cells expressing CD137, such as CD137L-stimulated NF-κB-dependent transcription.

[0032] In some embodiments that can be combined with any of the foregoing embodiments, the antibody comprises a human IgG2 Fc region. In some embodiments that can be combined with any of the foregoing embodiments, the antibody comprises a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises an S241P mutation, wherein the numbering is based on Kabat. In some embodiments, the antibody or antigen-binding fragment does not induce an ADCC effect.

[0033] On the other hand, this document provides a method comprising a subset of SEQ ID NO: 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 1 The antibody heavy chain variable region encoded by polynucleotides of sequences 99, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and 251, and / or by sequences containing sequences selected from SEQ ID. NO:134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 16 4, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196 The polynucleotide sequences 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, and 252 encode the variable region of the antibody light chain. In some embodiments, this document provides antibody heavy chains encoded by polynucleotides comprising sequences selected from SEQ ID NO:661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, and 707, and / or antibody light chains encoded by polynucleotides comprising sequences selected from SEQ ID NO:662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, and 708.

[0034] In some embodiments, HVR is performed according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising HVR-H1 containing the sequence GFSLSTSGVGVG (SEQ ID NO: 866), HVR-H2 containing the sequence LIDWDDDKYYSPSLKS (SEQ ID NO: 867), and HVR-H3 containing the sequence GGSDTVLGDWFAY (SEQ ID NO: 868); and / or a light chain variable (VL) domain comprising HVR-L1 containing the sequence RASQSVSPYLA (SEQ ID NO: 869), HVR-L2 containing the sequence DASSLES (SEQ ID NO: 870), and HVR-L3 containing the sequence QQGYSLWT (SEQ ID NO: 871).

[0035] In some embodiments, HVR is performed according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising HVR-H1 containing the sequence GYSITSGHYWA (SEQ ID NO: 872), HVR-H2 containing the sequence SISGYGSTTYYADSVKG (SEQ ID NO: 873), and HVR-H3 containing the sequence GGSDAVLGDWFAY (SEQ ID NO: 874); and / or a light chain variable (VL) domain comprising HVR-L1 containing the sequence RASQGIGSFLA (SEQ ID NO: 875), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 876), and HVR-L3 containing the sequence QQGYYLWT (SEQ ID NO: 877).

[0036] In some embodiments, HVR is performed according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising HVR-H1 containing the sequence GFSLSTGGVGVG (SEQ ID NO: 878), HVR-H2 containing the sequence LIDWADDKYYSPSLKS (SEQ ID NO: 879), and HVR-H3 containing the sequence GGSDTVIGDWFAY (SEQ ID NO: 880); and / or a light chain variable (VL) domain comprising HVR-L1 containing the sequence RASQSIGSYLA (SEQ ID NO: 881), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 882), and HVR-L3 containing the sequence QQGYYLWT (SEQ ID NO: 883).

[0037] In some embodiments of any of the above embodiments, the antibody disclosed herein is a multimeric antibody (e.g., a bispecific antibody). In some embodiments of any of the above embodiments, the antibody disclosed herein is an IgM antibody, for example, comprising an IgM Fc region (e.g., a human IgM Fc region).

[0038] On the other hand, this document provides a polynucleotide that encodes any of the antibody or antigen-binding fragments described herein. In some embodiments, this document provides a polynucleotide comprising a sequence selected from SEQ ID NO:133-252.

[0039] On the other hand, this document provides a vector comprising any of the polynucleotides described above. In some embodiments, the vector is an expression vector.

[0040] On the other hand, this document provides a host cell (e.g., bacterial cells, yeast cells, insect cells, mammalian cells (such as CHO cells or 293T cells), etc.) containing any of the polynucleotides or vectors described herein. In some embodiments, this document provides a method for preparing an antibody or antigen-binding fragment, comprising culturing a host cell under conditions suitable for producing the antibody or antigen-binding fragment. In some embodiments, the method further includes recovering the antibody or antigen-binding fragment produced by the host cell.

[0041] On the other hand, this article provides a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments (or any derivatives thereof) described herein, and a pharmaceutically acceptable carrier.

[0042] On the other hand, this document provides methods for treating abnormal cell growth (e.g., cancer) in a subject in need, comprising administering to the subject a therapeutically effective amount of any one of the antibodies, antigen-binding fragments, and / or pharmaceutical compositions described herein. In some embodiments, this document provides methods for reducing tumor cell metastasis in a subject, comprising administering to the subject a therapeutically effective amount of any one of the antibodies, antigen-binding fragments, and / or pharmaceutical compositions described herein. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of: viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiotherapy, and chemotherapy. In some embodiments, at least one additional therapeutic agent is selected from the group consisting of: pomalyst, lenalidomide, lenalidomide, thalidomide, DNA-alkylated platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiotherapy, single-dose radiation, fractionated radiation, focal radiation. Radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptors, adoptive T cells, anticancer vaccines, and oncolytic viruses. This document also provides for the use of any of the pharmaceutical compositions, antibodies, and / or antigen-binding fragments (or any derivatives thereof) described herein for the treatment of abnormal cell growth (e.g., cancer) and / or reduction of tumor cell metastasis in subjects in need. This document also provides for the use of any of the antibodies or antigen-binding fragments (or any derivatives thereof) described herein for the manufacture of a medicament for the treatment of abnormal cell growth (e.g., cancer) and / or reduction of tumor cell metastasis in subjects in need.

[0043] It should be understood that one, some, or all of the features of the various embodiments described above and herein can be combined to form other embodiments of this disclosure. These and other aspects of this disclosure will become apparent to those skilled in the art. These and other embodiments of this disclosure are further described in the following detailed description.

[0044] More specifically, this application provides the following:

[0045] 1. An isolated antibody or antigen-binding fragment thereof that binds to the extracellular domain of human CD137, wherein the antibody or antigen-binding fragment thereof binds to one or more amino acid residues from amino acid residues 34-108 of SEQ ID NO:1.

[0046] 2. The antibody or antigen-binding fragment as described in item 1, wherein the antibody or antigen-binding fragment binds to one or more amino acid residues within amino acid residues 34-93 of SEQ ID NO:1.

[0047] 3. The antibody or antigen-binding fragment as described in item 1 or 2, wherein the antibody or antigen-binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO:1.

[0048] 4. The antibody or antigen-binding fragment as described in item 3, wherein the antibody or antigen-binding fragment is bound to one or more of amino acid residues 34-36, one or more of amino acid residues 53-55, and one or more of amino acid residues 92-93 of SEQ ID NO:1.

[0049] 5. The antibody or antigen-binding fragment as described in any one of items 1-4, wherein the antibody or antigen-binding fragment does not bind to one or more amino acid residues selected from the group consisting of amino acid residues 109-112, 125, 126, 135-138, 150 and 151 of SEQ ID NO:1.

[0050] 6. The antibody or antigen-binding fragment as described in item 5, wherein the antibody or antigen-binding fragment does not bind to amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO:1.

[0051] 7. An antibody or antigen-binding fragment as described in any one of items 1-6, wherein the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, rats and / or dogs.

[0052] 8. The antibody or antigen-binding fragment as described in item 7, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey CD137.

[0053] 9. The antibody or antigen-binding fragment as described in any one of items 1-8, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region.

[0054] The heavy chain variable region comprises HVR-H1 containing the amino acid sequence SEQ ID NO:711, HVR-H2 containing the amino acid sequence SEQ ID NO:735, and HVR-H3 containing the amino acid sequence SEQ ID NO:759; and / or

[0055] The light chain variable region includes HVR-L1 containing the amino acid sequence SEQ ID NO:783, HVR-L2 containing the amino acid sequence SEQ ID NO:807, and HVR-L3 containing the amino acid sequence SEQ ID NO:831.

[0056] 10. The antibody or antigen-binding fragment as described in item 9, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID NO:41, and / or the light chain variable region comprises the amino acid sequence SEQ ID NO:42.

[0057] 11. The antibody or antigen-binding fragment as described in item 10, wherein the antibody comprises a heavy chain and a light chain, and wherein the heavy chain comprises the amino acid sequence SEQ ID NO:617, and / or the light chain comprises the amino acid sequence SEQ ID NO:618.

[0058] 12. The antibody or antigen-binding fragment as described in any one of items 1-8, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region.

[0059] The heavy chain variable region comprises HVR-H1 containing the amino acid sequence SEQ ID NO:712, HVR-H2 containing the amino acid sequence SEQ ID NO:736, and HVR-H3 containing the amino acid sequence SEQ ID NO:760; and / or

[0060] The light chain variable region includes HVR-L1 containing the amino acid sequence SEQ ID NO:784, HVR-L2 containing the amino acid sequence SEQ ID NO:808, and HVR-L3 containing the amino acid sequence SEQ ID NO:832.

[0061] 13. The antibody or antigen-binding fragment as described in item 12, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID NO:61, and / or the light chain variable region comprises the amino acid sequence SEQ ID NO:62.

[0062] 14. The antibody or antigen-binding fragment as described in item 13, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:619, and / or the light chain comprises the amino acid sequence SEQ ID NO:620.

[0063] 15. The antibody or antigen-binding fragment as described in any one of items 1-8, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region.

[0064] The heavy chain variable region comprises HVR-H1 containing the amino acid sequence SEQ ID NO:731, HVR-H2 containing the amino acid sequence SEQ ID NO:755, and HVR-H3 containing the amino acid sequence SEQ ID NO:779; and / or

[0065] The light chain variable region includes HVR-L1 containing the amino acid sequence SEQ ID NO:803, HVR-L2 containing the amino acid sequence SEQ ID NO:827, and HVR-L3 containing the amino acid sequence SEQ ID NO:851.

[0066] 16. The antibody or antigen-binding fragment as described in item 15, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID NO:71, and / or the light chain variable region comprises the amino acid sequence SEQ ID NO:72.

[0067] 17. The antibody or antigen-binding fragment as described in item 16, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:657, and / or the light chain comprises the amino acid sequence SEQ ID NO:658.

[0068] 18. An isolated antibody that binds to the extracellular domain of human CD137, or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region.

[0069] a) The heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3.

[0070] The HVR-H1 contains an amino acid sequence selected from the group consisting of:

[0071] Formula (I): X1TFX2X3YX4IHWV (SEQ ID NO:2), where X1 is F or Y, X2 is S or T, X3 is G, N or S, and X4 is A, G or W;

[0072] Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO:3), where X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T; and

[0073] Formula (III): FSLSTX1GVX2VX3WI (SEQ ID NO:4), where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T;

[0074] The HVR-H2 contains an amino acid sequence selected from the group consisting of:

[0075] Formula (IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO:5), where X1 is A, D or Y, X2 is D or G, X3 is R, S or Y, and X4 is P or T;

[0076] Formula (V): IGX1IYHSGX2TYYX3PSLKSRV (SEQ ID NO:6), where X1 is D or E, X2 is N or S, and X3 is N or S; and

[0077] Formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO:7), where X1 is A, G, S, V or Y, X2 is A, D, S or Y, X3 is D, G or S, and X4 is S or T; and

[0078] The HVR-H3 contains an amino acid sequence according to the following formula: Formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO:8), where X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T or V, X5 is A, I, L, T or V, and X6 is A, D or G; and / or

[0079] b) wherein the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3,

[0080] The HVR-L1 contains an amino acid sequence according to the following formula: Formula (VIII): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO:9), wherein X1 is Q or R, X2 is D, G or S, X3 is I or V, X4 is G, R, S or T, X5 is P, R, S or T, X6 is A, D, F, S, V or Y, X7 is L or V, and X8 is A, G or N;

[0081] The HVR-L2 described therein comprises an amino acid sequence according to the following formula: Formula (IX): X1ASX2X3X4X5GX6 (SEQ ID NO: 10), where X1 is A or D, X2 is N, S or T, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and

[0082] The HVR-L3 contains an amino acid sequence selected from the group consisting of:

[0083] Formula (X): YCQQX1YX2X3X4T (SEQ ID NO:11), where X1 is A, G, S or Y, X2 is Q, S or Y, X3 is I, L, T or Y, and X4 is I, S, V or W; and

[0084] Formula (XI): YCX1QX2X3X4X5PX6T (SEQ ID NO:12), where X1 is E or Q, X2 is P, S or Y, X3 is D, L, S, T or Y, X4 is D, E, H, S or T, X5 is D, LT or W, and X6 is L, P, R or V.

[0085] 19. The antibody or antigen-binding fragment of claim 18, wherein HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:253-312, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:313-372, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:373-432, HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:433-492, HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:493-552, and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:553-612.

[0086] 20. An antibody or antigen-binding fragment as described in item 18 or 19, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, and 131, and / or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, and 131, and / or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 1 ... The amino acid sequences of NO:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, and 132.

[0087] 21. The antibody or antigen-binding fragment as described in item 18, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of:

[0088] Formula (XII): X1TFSX2YWIHWV (SEQ ID NO:853), where X1 is F or Y, and X2 is N or S;

[0089] Formula (XIII): YSIX1SGX2X3WX4WI (SEQ ID NO: 854), where X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, or S; and

[0090] Formula (XIV): FSLSTX1GVX2VX3WI (SEQ ID NO:855), where X1 is G or S, X2 is A or G, and X3 is A, G, or S;

[0091] The HVR-H2 contains an amino acid sequence selected from the group consisting of:

[0092] Formula (IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO:5), where X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y, and X4 is P or T; and

[0093] Formula (XV): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO:856), where X1 is G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T; and

[0094] The HVR-H3 contains an amino acid sequence according to the following formula: Formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO:8), where X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T or V, X5 is A, I, L, T or V, and X6 is A, D or G; and / or

[0095] The HVR-L1 contains an amino acid sequence according to the following formula: Formula (XVI): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO:857), wherein X1 is Q or R, X2 is D, G or S, X3 is I or V, X4 is G, R, S or T, X5 is P, R, S or T, X6 is A, F, S, V or Y, X7 is L or V, and X8 is A or G;

[0096] The HVR-L2 described therein comprises an amino acid sequence according to the following formula: Formula (XVII): X1ASX2X3X4X5GX6 (SEQ ID NO: 858), where X1 is A or D, X2 is N or S, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and

[0097] The HVR-L3 contains an amino acid sequence according to the following formula: Formula (XVIII): YCQQX1YX2X3WT (SEQ ID NO: 859), where X1 is A or G, X2 is S or Y, and X3 is I, L, or T.

[0098] 22. The antibody or antigen-binding fragment of claim 21, wherein HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:709-732, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:733-756, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:757-780, HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:781-804, HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:805-828, and HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:829-852.

[0099] 23. The antibody or antigen-binding fragment as described in item 21 or 22, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 17, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 61, 63, 65, 67, 71, 73, 75, 79, 83, 85, and 87, and / or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 18, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 62, 64, 66, 68, 72, 74, 76, 80, 84, 86, and 88.

[0100] 24. An antibody or antigen-binding fragment as described in any one of items 21-23, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, and 659, and / or the light ...57, and 659, and / or the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 613, 615, 617, 619, 621, 623, 625, 627, 629, 651, 653, 655, 657, and 659, and / or The amino acid sequences consisting of NO:614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658 and 660.

[0101] 25. The antibody or antigen-binding fragment as described in item 21, wherein HVR-H1 comprises the amino acid sequence SEQ ID NO: 711 or 731; HVR-H2 comprises the amino acid sequence SEQ ID NO: 735 or 755; HVR-H3 comprises the amino acid sequence SEQ ID NO: 759 or 779; HVR-L1 comprises the amino acid sequence SEQ ID NO: 783 or 803; HVR-L2 comprises the amino acid sequence SEQ ID NO: 807 or 827; and HVR-L3 comprises the amino acid sequence SEQ ID NO: 831 or 851.

[0102] 26. The antibody or antigen-binding fragment as described in item 25, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID NO:41 or 71, and the light chain variable region comprises the amino acid sequence SEQ ID NO:42 or 72.

[0103] 27. The antibody or antigen-binding fragment of claim 26, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO: 617 or 657, and the light chain comprises the amino acid sequence SEQ ID NO: 618 or 658.

[0104] 28. The antibody or antigen-binding fragment as described in item 21, wherein HVR-H1 comprises the amino acid sequence SEQ ID NO:712; HVR-H2 comprises the amino acid sequence SEQ ID NO:736; HVR-H3 comprises the amino acid sequence SEQ ID NO:760; HVR-L1 comprises the amino acid sequence SEQ ID NO:784; HVR-L2 comprises the amino acid sequence SEQ ID NO:808; and HVR-L3 comprises the amino acid sequence SEQ ID NO:832.

[0105] 29. The antibody or antigen-binding fragment as described in item 28, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence SEQ ID NO:62.

[0106] 30. The antibody or antigen-binding fragment of claim 29, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:619, and the light chain comprises the amino acid sequence SEQ ID NO:620.

[0107] 31. An antibody or antigen-binding fragment as described in any one of items 1-30, wherein, as measured by surface plasmon resonance, the antibody or antigen-binding fragment has a Kc concentration of 100 nM or less. DCombined with human CD137.

[0108] 32. The antibody or antigen-binding fragment as described in item 31, wherein, as measured by surface plasmon resonance, the antibody or antigen-binding fragment has a Kc of 50 nM or less. D Combined with human CD137.

[0109] 33. An antibody or antigen-binding fragment as described in any one of items 18-32, wherein the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, rats and / or dogs.

[0110] 34. The antibody or antigen-binding fragment as described in item 33, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey CD137.

[0111] 35. The antibody or antigen-binding fragment as described in any one of items 1-34, wherein the activity of human CD137 expressed on human cells is reduced upon contact with said antibody or antigen-binding fragment.

[0112] 36. The antibody or antigen-binding fragment as described in any one of items 1-34, wherein, for blocking the in vitro binding of human CD137 to human CD137L, said antibody or antigen-binding fragment has a half-maximum inhibitory concentration (IC50) of about 100 nM or less. 50 ).

[0113] 37. The antibody or antigen-binding fragment of any one of items 1-34, wherein when the antibody or antigen-binding fragment is provided at a concentration of about 1 μM or greater, the antibody or antigen-binding fragment completely blocks the in vitro binding of human CD137 to human CD137L.

[0114] 38. An antibody or antigen-binding fragment as described in any one of items 1-34, wherein the antibody or antigen-binding fragment blocks one or more aspects of CD137 signaling stimulated by CD137L in cells expressing CD137.

[0115] 39. The antibody or antigen-binding fragment as described in any one of items 1-34, wherein the activity of human CD137 expressed on human cells increases upon contact with said antibody or antigen-binding fragment.

[0116] 40. An antibody or antigen-binding fragment as described in item 39, wherein contacting CD137-expressing human cells with said antibody or antigen-binding fragment results in increased NF-κB-dependent transcription.

[0117] 41. The antibody or antigen-binding fragment of any one of items 1-40, wherein the antibody comprises a human IgG2 Fc region.

[0118] 42. The antibody or antigen-binding fragment of any one of items 1-41, wherein the antibody comprises the human IgG4 Fc region.

[0119] 43. The antibody or antigen-binding fragment as described in item 42, wherein the human IgG4 Fc region contains the S241P mutation, wherein the numbering is based on Kabat.

[0120] 44. An antibody or antigen-binding fragment as described in any one of items 18-20, wherein the heavy chain variable region comprises a subset selected from SEQ ID NO: 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, The polynucleotide encoding a sequence of the group consisting of 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and 251, and / or the light chain variable region is composed of sequences selected from SEQ ID. NO:134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 1 64, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 1 The polynucleotide codes of the sequence group consisting of 96, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, and 252.

[0121] 45. The antibody or antigen-binding fragment as described in item 24, wherein the heavy chain is encoded by a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, and 707, and / or the light chain is encoded by a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, and 708.

[0122] 46. ​​A polynucleotide encoding an antibody or antigen-binding fragment as described in any one of items 1-45.

[0123] 47. A polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:133-252.

[0124] 48. A vector comprising a polynucleotide as described in any one of items 46-47.

[0125] 49. The carrier as described in item 48, wherein the carrier is an expression carrier.

[0126] 50. A host cell comprising a vector as described in item 48 or 49.

[0127] 51. A method for preparing an antibody or antigen-binding fragment, comprising culturing a host cell as described in item 50 under conditions suitable for producing said antibody or antigen-binding fragment.

[0128] 52. The method of claim 51, further comprising recovering the antibody or antigen-binding fragment produced by the cells.

[0129] 53. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of items 1-45, and a pharmaceutically acceptable carrier.

[0130] 54. A method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment as described in any one of items 1-45.

[0131] 55. The method of claim 54, further comprising administering to the subject a therapeutically effective amount of at least one additional therapeutic agent.

[0132] 56. The method of claim 55, wherein the at least one additional therapeutic agent is selected from the group consisting of: viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiotherapy, and chemotherapy.

[0133] 57. The method of claim 55 or 56, wherein the at least one additional therapeutic agent is selected from the group consisting of: pomalidomide, lenalidomide, lenalidomide, pomalidomide, thalidomide, DNA-alkylated platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiotherapy, single-dose radiation, fractionated radiation, lesion radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptive T cells, anticancer vaccines, and oncolytic viruses. Attached Figure Description

[0134] Figure 1A The diagram illustrates the high variable region (HVR) definition as defined in the Kabat CDR for the exemplary heavy chain variable region (VH) (SEQ ID NO:13) and the exemplary light chain variable region (VL) (SEQ ID NO:14).

[0135] Figure 1B The selection of Fab hits that cross-react with mouse CD137 is shown.

[0136] Figure 2 The ELISA assays of exemplary antibodies binding to CD137 in humans, monkeys, and mice are shown. Each small figure represents a different antibody as indicated at the top of the small figure.

[0137] Figure 3A The diagram illustrates FACS-based binding assays of exemplary antibodies against CD137 in humans, monkeys, mice, and rats. Each small figure represents a different antigen as indicated at the top of the small figure.

[0138] Figure 3B A comparison of species cross-reactivity between exemplary and reference antibodies is shown.

[0139] Figure 4A Exemplary antibodies bind to activated human and monkey T cells, but do not bind to primary T cells. Human T cells.

[0140] Figure 4BThe binding of AG10131 to activated human, monkey, mouse, and rat T cells is shown.

[0141] Figure 5 The example antibody demonstrates binding specificity to CD137, but not to other members of the TNFR family.

[0142] Figure 6A and Figure 6B This illustrates an exemplary antibody that blocks the binding of CD137 to its homologous ligand CD137L, which is demonstrated by ELISA (...). Figure 6A ) and flow cytometry assay ( Figure 6B )get.

[0143] Figure 7A The results of epitope mapping obtained by flow cytometry are shown.

[0144] Figure 7B This paper shows multiple sequence alignments of a portion of human (SEQ ID NO:1), cynomolgus monkey (SEQ ID NO:860), and mouse (SEQ ID NO:861) CD137 with target CD137 sequences / regions (annotated) identified from epitope mapping experiments.

[0145] Figure 8 The agonist activity of an exemplary antibody is shown in an NFκB reporter gene assay.

[0146] Figure 9 The agonist activity of exemplary antibodies in CD8+ T cell proliferation (top inset) and INF-γ secretion (bottom inset) is shown.

[0147] Figure 10 This demonstrates the antitumor efficacy of exemplary antibodies in an H22 mouse liver cancer model, as well as CD4. + and CD8 + T cell infiltration in tumors.

[0148] Figure 11 This demonstrates the antitumor efficacy of an exemplary antibody in a CT26 mouse colon cancer model.

[0149] Figure 12 This demonstrates the antitumor efficacy of an exemplary antibody in an EMT6 mouse breast cancer model.

[0150] Figure 13 The study shows that CT26 mice treated with the exemplary antibody remained tumor-free after re-excitation with the same tumor cells.

[0151] Figure 14 This demonstrates tumor cell killing achieved using spleen cells from tumor-rejected re-excited mice.

[0152] Figure 15The AG10131 does not show an ADCC effect.

[0153] Figure 16 The example antibody shows minimal aggregation at high concentrations.

[0154] Figure 17 This demonstrates the stability of an exemplary antibody under accelerated stress conditions.

[0155] Figure 18 This demonstrates thermal stability.

[0156] Figure 19 AG10131 was shown to have no hematologic toxicity in normal mice at up to 100 mg / kg (BIW) x 2 every two weeks.

[0157] Figure 20 The results showed that AG10131 did not cause histological liver abnormalities in normal mice at up to 100 mg / kg (BIW) x 2 every two weeks.

[0158] Figure 21 The results showed that AG10131 had no hematologic toxicity in cynomolgus monkeys at 10 mg / kg / week x 4.

[0159] Figure 22 The results showed that AG10131 had no hepatotoxicity in monkeys at 10 mg / kg / week x 4.

[0160] Figure 23 The pharmacokinetic characteristics of AG10131 in monkeys are shown.

[0161] Figure 24 The pharmacokinetic characteristics of AG10131 in rats are shown.

[0162] Figure 25 The pharmacokinetic characteristics of various antibodies in mice are shown.

[0163] Figure 26 The crystal structure of the human CD137-CD137L complex with the indicated CD137 cysteine ​​enriched domain (CRD) is shown.

[0164] Figure 27 Epitopes of the indicated anti-CD137 antibody, obtained by flow cytometry, are plotted to bind to the indicated human CD137 CRD.

[0165] Figure 28This demonstrates how the anti-CD137 antibody AG10131 blocks CD137L-stimulated CD137 signaling. Results were obtained from a cellular NFκB luciferase reporter gene assay, in which 293T cells stably expressing the NFκB luciferase reporter gene, transfected with a DNA construct expressing human CD137, were co-cultured with human B-cell lymphoma cells Daudi (top row) or Raji (bottom row) at an indicated ratio, and then incubated overnight with serial dilutions of isotype control (left column) or ligand-blocking anti-CD137 antibody (right column) as indicated, followed by luciferase activity measurement.

[0166] Figure 29 The diagram illustrates the activation of human CD137-mediated NFκB signaling by anti-CD137 antibodies AG10131, AC1121, or AC1097 in the presence or absence of cross-linked antibodies. The EC50 (nM) for each antibody targeting NFκB signaling activation (in the presence or absence of cross-linking) is indicated.

[0167] Figure 30 The results showed that AG10131 and its human IgG4 isotype control antibody lacked the ability to bind human complement C1q components within the tested concentration range, while the human IgG1 isotype control antibody was able to bind C1q.

[0168] Figure 31 This section illustrates the enhancement of tumor-infiltrating T lymphocytes achieved by AG10131 treatment in various tumor models. Top left: Representative IHC staining images of mouse CD4+ (top inset) and CD8+ (bottom inset) T cells in H22 tumors after treatment. Top right: Representative IHC staining images of mouse CD4+ (top inset) and CD8+ (bottom inset) T cells in EMT6 tumors after treatment. Bottom center: Representative IHC staining images of mouse CD4+ (top inset) and CD8+ (bottom inset) T cells in CT26 tumors after treatment. CD4+ or CD8+ T cells are stained black with hematoxylin against a background of nuclear counterstaining. CD4+ and CD8+ T cells are indicated by black arrows.

[0169] Figure 32 For those from Figure 31 The experiment shown quantified the number of tumor-infiltrating T lymphocytes. The percentage of CD4+ in tumors from H22, EMT6, and CT26 tumor samples was compared between the mediator group and the AG10131 treatment group. + T cells (apical row) and %CD8 + T cells (bottom row). **, p<0.01; ***, p<0.001.

[0170] Figure 33This diagram illustrates the antitumor effects of AG10131 and anti-PD-1 antibodies, alone or in combination, in an established mouse CT26 colon cancer syngeneic model. Top inset: Graph of mean tumor growth for each treatment group. Bottom inset: Spider plot of individual tumor growth for each group. Detailed Implementation

[0171] A. Definition

[0172] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure will have meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature used in conjunction with the antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein, as well as the techniques used in antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, are those well known and commonly used in the art.

[0173] As used in this article, the following terms have the meanings associated with them in this section.

[0174] The article “a / kind (“a” and “an”)” refers to the grammatical object of the article, which is one / kind or more / kinds (i.e., at least one / kind). By way of example, “element” means one element or more elements.

[0175] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, but whose C-terminal carboxyl group, N-terminal amino group, or side chain functional group has been chemically modified to another functional group. The term "amino acid mimic" refers to a chemical compound that has a structure different from the general chemical structure of an amino acid but functions similarly to a naturally occurring amino acid.

[0176] The term “antibody” is used in the broadest sense herein and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., single-chain variable fragments or scFvs) as long as they exhibit the desired biological activity.

[0177] The term "antibody" is a recognized term in the art and can refer to an antigen-binding protein (i.e., an immunoglobulin) having a basic tetrapeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to the H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the homotype of the H chains. Each heavy chain has a variable region (abbreviated herein as V) at its N-terminus. H Next is the constant region. The heavy chain constant region contains three structural domains: C H1 C H2 and C H3 Each light chain has a variable region at the N-terminus (abbreviated as V in this paper). I ), followed by the constant region at its other end. The constant region of the light chain contains a structural domain, C L V L With V H Compare and put C L Alignment with the first constant structural domain (CH1) of the heavy chain. V H and V L The two molecules pair together to form a single antigen-binding site. IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, thus containing 10 antigen-binding sites; while secretory IgA antibodies can polymerize to form multivalent assemblies containing 2-5 basic tetrameric units and the J chain.

[0178] V H and V L The region can be further subdivided into highly variable regions, called high-variability regions (HVRs), based on structural and sequence analysis. HVRs are scattered with more conserved regions, called frame regions (FWs). For comparison, the Kabat CDR definition from Yvonne Chen et al. (Selection and Analysis of an Optimized Anti-VEGF Antibody: Crystal Structure of an Affinity-matured Fab in Complex with Antigen, J. Mol. Biol. (1999) 293, 865-881) is listed below (see also Figure 1a). Each V H and V L It consists of three heavy chain HVRs and four light chain FWs, arranged in the following order from the amino terminus to the carboxyl terminus: FW1, HVR1, FW2, HVR2, FW3, HVR3, FW4. Throughout this disclosure, the three heavy chain HVRs are referred to as HVR_H1, HVR_H2, and HVR_H3. Similarly, the three light chain HVRs are referred to as HVR_L1, HVR_L2, and HVR_L3.

[0179] The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Within both the light and heavy chains, the variable and constant regions are linked by a “J” region having about 12 or more amino acids, with the heavy chain also including a “D” region having about 10 or more amino acids. See generally Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)).

[0180] The L-chain from any vertebrate species can be designated as one of two distinct types (called κ and λ) based on the amino acid sequence of its constant domain. Antibodies can be designated as different species or isotypes depending on the amino acid sequence of their constant domain (CH). Five classes of antibodies exist: IgA, IgD, IgE, IgG, and IgM, which have heavy chains named α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. IgG antibodies can be further classified into four subclasses—IgG1, IgG2, IgG3, and IgG4—based on the γ heavy chains Y1–Y4.

[0181] The term "antibody derivative" or "derivative of an antibody" refers to a molecule capable of binding to the same antigen (e.g., CD137) bound by the antibody and comprising the amino acid sequence of the antibody linked to another molecular entity. The amino acid sequence of the antibody contained in the antibody derivative can be the full-length heavy chain, the full-length light chain, any one or more portions of the full-length heavy chain, any one or more portions of the full-length light chain, any other one or more fragments of the antibody, or a complementary antibody. The other molecular entity can be a chemical or biological molecule. Examples of the other molecular entity include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies), and chemical compounds. The other molecular entity can have any utility, such as being used as a detection agent, label, biomarker, pharmaceutical agent, or therapeutic agent. The amino acid sequence of the antibody can be attached to or linked to the other molecular entity by chemical conjugation, gene fusion, non-covalent association, or other means. The term "antibody derivative" also encompasses chimeric antibodies, humanized antibodies, and molecules derived from modifications of the amino acid sequence of the CD137 antibody, such as conserved amino acid substitutions, additions, and insertions.

[0182] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of the antibody that retain the ability to bind to the antigen (e.g., CD137) to which the antibody is bound. Examples of "antigen-binding fragments" of antibodies include (i) Fab fragments, which are composed of V... L VH C L and C H1 (ii) a monovalent segment composed of structural domains; (iii) a F(ab′)2 segment, which is a divalent segment containing two Fab segments connected by disulfide bonds in the hinge region; (iv) a segment composed of V H and C H1 (iv) Fd fragments composed of structural domains; V-shaped segments formed by the single arm of the antibody. L and V H The Fv segment is composed of structural domains; (v) is composed of V H dAb fragments composed of structural domains (Ward et al., Nature 341:544-546 (1989)); and (vi) separated complementarity-determining regions (CDRs).

[0183] The term “binding molecule” encompasses (1) an antibody, (2) an antigen-binding fragment of an antibody, and (3) an antibody derivative, each as defined herein.

[0184] The terms “binding CD137” or “binding to CD137” or “binding to CD137” refer to the binding of a molecule to CD137 with an affinity (K) of 100 nM or less in in vitro assays, as defined herein. D The binding of CD137 to human CD137, the in vitro assay being such as the Biacore assay described in Example 4.

[0185] The terms “CD137” and “CD137 receptor” are used interchangeably in this application and include the human CD137 receptor, as well as its variants, isotypes, and species homologs. Therefore, binding molecules as defined and disclosed herein may also bind CD137 from species other than humans. In other cases, binding molecules may be completely specific to human CD137 and may not exhibit species- or other types of cross-reactivity.

[0186] The term "CD137 antibody" refers to an antibody as defined herein that can bind to the human CD137 receptor.

[0187] The term "chimeric antibody" refers to antibodies that contain amino acid sequences derived from different animal species, such as those with variable regions derived from human antibodies and constant regions of mouse immunoglobulins.

[0188] The term "competitive binding" refers to the interaction between two antibodies in their binding to a target. If, in the presence of a second antibody, the binding of the first antibody to its homologous epitope is detectably reduced compared to the binding of the first antibody in the absence of the second antibody, then the first antibody competes with the second antibody for binding. Alternative scenarios where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody are possible, but not necessarily. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, if each antibody detectably inhibits the binding of the other antibody to its homologous epitope, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to one or more of their respective epitopes.

[0189] The term "epitope" refers to a portion of an antigen that binds to an antibody (or its antigen-binding fragment). Epitopes can be formed from consecutive or discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes can comprise different numbers of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, 2D nuclear magnetic resonance, deuterium exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with computer modeling of the antigen and its complex structure containing the bound antibody and its variants. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, edited by GEMorris (1996). Once the desired epitope of an antigen is determined, an antibody against said epitope can be generated, for example, using the techniques described herein. The generation and characterization of the antibody can also elucidate information about the desired epitope. This information allows for competitive screening of antibodies that bind to the same epitope. This is achieved by conducting cross-competition studies to identify antibodies that compete to bind to each other—that is, antibodies that compete to bind to the antigen. A high-throughput method based on this cross-competitive "binning" of antibodies is described in PCT Publication No. WO 03 / 48731.

[0190] The term "germline" refers to the nucleotide sequence of antibody genes and gene segments that are passed from parent to offspring via germ cells. Germline sequences differ from the nucleotide sequences encoding antibodies in mature B cells, which have been altered during B cell maturation through recombination and hypermutation events.

[0191] The term "glycosylation site" refers to an amino acid residue recognized by eukaryotic cells as a location for attaching sugar residues. The amino acids that attach carbohydrates (such as oligosaccharides) are typically asparagine (N-bond), serine (O-bond), and threonine (O-bond) residues. Specific attachment sites are usually identified by an amino acid sequence referred to herein as the "glycosylation site sequence." The glycosylation site sequence for N-linked glycosylation is -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid except proline. The terms "N-linked" and "O-linked" refer to the chemical groups that serve as attachment sites between the sugar molecule and the amino acid residues. N-linked sugars are attached via an amino group; O-linked sugars are attached via a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety attached to the glycosylation site (i.e., the glycan site is occupied). In the presence of at least two potential glycosylation sites on a polypeptide, none of the sites (occupied by 0-glycan sites), one (occupied by 1-glycan sites), or both (occupied by 2-glycan sites) can be occupied by the carbohydrate moiety.

[0192] The term "host cell" refers to a cellular system that can be engineered to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells included within transgenic animals or cultured tissues. This term covers not only the specific subject cell but also its progeny. Because certain modifications may occur in successive generations due to mutations or environmental influences, the progeny may not be identical to the parent cells but are still included within the scope of the term "host cell."

[0193] The term "human antibody" refers to an antibody in which the entire amino acid sequence of both the light and heavy chains is derived from the human immunoglobulin gene. If produced in mice, mouse cells, or hybridomas derived from mouse cells, human antibodies may contain mouse carbohydrate chains. Human antibodies can be prepared using a variety of methods known in the art.

[0194] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDRs or HVRs derived from non-human animals or synthetic antibodies, while the frame and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0195] The term "exemplary antibody" refers to any of the antibodies described in this disclosure and named as those listed in Tables 1a and 1b. These antibodies may belong to any class (e.g., IgA, IgD, IgE, IgG, and IgM). Therefore, the antibodies identified above cover those for V L and V H Antibodies in all five classes that share the same amino acid sequence are included. Furthermore, antibodies in the IgG class can belong to any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Therefore, the antibodies identified above in the IgG subclass cover the V... L and V H Antibodies in all four subclasses have the same amino acid sequence. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes and four IgG subclasses are known in the art. The full-length heavy and light chain amino acid sequences of each of the IgG4 subclass of exemplary antibodies shown in Table 1b are provided in this disclosure.

[0196] The terms “isolated antibody” or “isolated binding molecule” refer to an antibody or binding molecule as defined herein that: (1) is not associated with a naturally associated component adjacent to it in its native state; (2) does not contain other proteins from the same species; (3) is expressed by cells from a different species; or (4) does not exist in nature. Examples of isolated antibodies include CD137 antibodies that have been purified using CD137 affinity, CD137 antibodies that have been generated in vitro via hybridoma or other cell lines, and CD137 antibodies derived from transgenic animals.

[0197] The term "isolated nucleic acid" refers to a genomic, cDNA, or synthetically derived nucleic acid molecule, or a combination thereof, that is separate from other nucleic acid molecules present in natural sources of nucleic acids. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that are separate from chromosomes naturally associated with the genomic DNA. Preferably, the "isolated" nucleic acid does not contain sequences naturally side-attached to the nucleic acid (i.e., sequences located at the 5' and 3' ends of the target nucleic acid).

[0198] The term "k" a "" refers to the association rate constant of a specific antibody-antigen interaction, while the term "k" refers to the rate of association of that interaction. d "" refers to the dissociation rate constant of a specific antibody-antigen interaction.

[0199] The term "K" D "" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. It is determined by k d With k a The ratio (i.e., k) d / k a The concentration obtained is expressed as molar concentration (M). K DUsed as a measure of the binding affinity between an antibody and its binding partner. K D The smaller the K value, the tighter the antibody binding, or the higher the affinity between the antibody and the antigen. For example, antibodies with a nanomolar (nM) dissociation constant bind more tightly to a specific antigen than antibodies with a micromolar (μM) dissociation constant. The K value of an antibody... D The value can be determined using well-established methods in the art. Determining antibody K D One approach is to use surface plasmon resonance, typically employing biosensor systems such as System. Using BIACORE TM The measurement procedure performed by the system (BIAcore measurement) is described in the Embodiments section of this disclosure.

[0200] The term "mammal" refers to any animal species of mammal species. Examples of mammals include: humans; laboratory animals such as rats, mice, apes, and guinea pigs; domesticated animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0201] The term "preventing" in relation to the symptoms of a disease in mammals refers to preventing or delaying the onset of a disease, or preventing the appearance of its clinical or subclinical symptoms.

[0202] As used herein, “sequence identity” between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. The amino acid sequence identity of a polypeptide can be routinely determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence has, for example, 95% identity with a reference amino acid sequence, parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and allows for a maximum interval of 5% for the total number of amino acid residues in the reference sequence that are homologous. The above-described method for determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants disclosed herein.

[0203] When referring to the interaction between a binding molecule (e.g., an antibody) and its binding partner (e.g., an antigen) as defined herein, the terms "specifically binds" or "specifically binds to" refer to the ability of a binding molecule, under a given set of conditions, to distinguish between a target antigen from one animal species and an antigen orthologue from a different animal species. As determined in in vitro assays, a CD137 binding molecule is said to specifically bind to human CD137 if it binds to human CD137 at less than 50% of the EC50 of rat or mouse CD137. The binding specificity of an antibody can be determined using methods known in the art. Examples of such methods include FACS using PHA-stimulated primary cells, Western blots, ELISA, RIA, ECL, IRMA assays, and peptide scans.

[0204] When referring to the interaction between a binding molecule (e.g., an antibody) and its binding partner (e.g., an antigen) as defined herein, the terms "selectively binds" or "selectively binds to" refer to the ability of a binding molecule, under a given set of conditions, to distinguish between a target antigen (such as human CD137) from one animal species and a different antigen (such as human CD40) from the same animal species. As determined in an in vitro assay, a CD137 binding molecule is said to selectively bind to human CD137 if it binds to less than 10% of the EC50 of human CD40 or human CD134.

[0205] The term "treatment" (or "treat") in relation to a disease symptom in mammals refers to an effect that results in a desired or beneficial outcome in a mammal suffering from that disease symptom. A desired or beneficial outcome may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells, etc.), or cessation or inhibition of further development of the disease, symptom, or condition. In the context of treating cancer in mammals, a desired or beneficial outcome may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or increased survival rate in the mammal. The effect may be subjective or objective. For example, if the mammal is a human, then an improvement in energy or vitality or a reduction in pain may be recorded as an improved subjective symptom or response to therapy. Alternatively, a clinician may notice a reduction in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or imaging findings. For a response to treatment, clinicians can observe several laboratory signs, including standardized tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, clinicians may observe a reduction in detectable tumor markers. Alternatively, other tests can be used to assess objective improvement, such as sonography, MRI, and positron emission tomography (PET).

[0206] The term "vector" refers to a nucleic acid molecule capable of transporting foreign nucleic acid molecules. Foreign nucleic acid molecules are attached to vector nucleic acid molecules via recombination techniques such as ligation or recombination. This allows for the multiplication, selection, further manipulation, or expression of foreign nucleic acid molecules in a host cell or organism. Vectors can be plasmids, bacteriophages, transposons, granules, chromosomes, viruses, or virions. One class of vectors integrates into the host cell's genome after introduction and thus replicates along with the host genome (e.g., non-attached mammalian vectors). Another class of vectors is capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attached mammalian vectors). Another specific type of vector capable of directing the expression of an expressible foreign nucleic acid that it is operatively linked to is often called an "expression vector." Expression vectors typically have control sequences that drive the expression of the expressible foreign nucleic acid. Simpler vectors known as "transcription vectors" can only transcribe, but not translate: they can replicate in target cells, but do not express. The term "vector" encompasses all types of vectors, regardless of their function. The vector that can guide the expression of the expressible nucleic acid that it is operatively linked to is usually called an "expression vector".

[0207] Unless otherwise indicated, the methods and techniques disclosed herein are generally performed according to methods well known in the art and described in various general and more specific references cited and discussed throughout this specification. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly practiced in the art or as described herein. The nomenclature used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein, as well as the laboratory procedures and techniques of said analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0208] As used in this article, the 20 common amino acids and their abbreviations follow their usual usage. See Immunology—ASynthesis (2nd edition, edited by ESGolob and DRGren, Sinauer Associates, Sunderland, Mass. (1991)).

[0209] B. The binding molecule that binds to human CD137

[0210] This disclosure provides isolated binding molecules that bind to human CD137, including CD137 antibodies, antigen-binding fragments of CD137 antibodies, and derivatives of CD137 antibodies. In some embodiments, the binding molecule is any antibody described herein, including antibodies described with respect to epitope binding and antibodies described with respect to specific amino acid sequences of HVR, variable regions (VL, VH), and the light and heavy chains of IgG (e.g., IgG4). In some embodiments, this disclosure relates to a binding molecule that binds to human CD137 and has at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, eight, or all nine) of the following functional properties: (a) binding to human CD137 at a KD of 500 nM or less; (b) having agonist activity against human CD137; (c) not binding to human OX40, CD40, GITR, and / or CD27 receptors at concentrations up to 1000 nM; (d) cross-reacting with CD137 in monkeys, mice, rats, or dogs; (e) not causing ADCC effects; (f) being able to inhibit tumor cell growth; (g) having a therapeutic effect on cancer; (h) blocking the binding between CD137 and CD137L; and (i) blocking CD137 signaling stimulated by CD137L (e.g., CD137L-stimulated NF-κB-dependent transcription) in cells expressing CD137. In some embodiments, the antibodies disclosed herein can also block, for example, completely block, the binding between CD137 and its ligand CD137L. This document also provides one or more anti-CD137 antibody or antigen-binding fragments that cross-competitively bind to human CD137 with one or more of the antibody or antigen-binding fragments described herein.

[0211] In some embodiments, the antibody or its antigen-binding fragment binds to one or more amino acid residues from amino acid residues 34-108 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues from amino acid residues 34-93 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more of amino acid residues 34-36, one or more of amino acid residues 53-55, and one or more of amino acid residues 92-93 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment does not bind to one or more amino acid residues selected from the group consisting of amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment does not bind to amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO:1. The ability of the antibody or antigen-binding fragment to bind to the target antigen can be measured using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filtration binding assay, EMSA, etc. In some embodiments, the ability of the antibody or antigen-binding fragment to bind to the target antigen is measured by surface plasmon resonance (see, for example, Example 1 below).

[0212] In some embodiments, the antibody or antigen-binding fragment binds to human CD137 at a KD of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). In some embodiments, the antibody or antigen-binding fragment binds to human CD137 at a KD of about 100 nM or less. In some embodiments, the antibody or antigen-binding fragment binds to human CD137 with a KD of about 50 nM or less. The KD of the antibody or antigen-binding fragment can be measured using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filtration binding assay, EMSA, etc. In some embodiments, KD is measured by surface plasmon resonance (see, for example, Example 1 below).

[0213] Anti-CD137 antibodies must be cross-linked to become agonistic. For example, cross-linking is achieved in vivo via Fcγ receptors, while polyclonal anti-Fc antibodies are typically used in vitro in cell-based experiments. In some embodiments, the antibody or antigen-binding fragment described herein has agonistic activity against human CD137. In some embodiments, when cells expressing human CD137 (e.g., human cells) are exposed to the antibody or antigen-binding fragment, the antibody or antigen-binding fragment induces one or more (e.g., one or more, two or more, three or more, etc.) activities of human CD137. Various CD137 activities are known in the art and may include, but are not limited to, induction of NF-κB-dependent transcription, induction of T cell proliferation, prolongation of T cell survival, co-stimulation of activated T cells, induction of cytokine secretion (such as IL-2), and induction of monocyte activation. In some embodiments, one or more CD137 activities are not the binding of CD137 to its ligand. Methods for measuring CD137 activity (e.g., induction of NF-κB-dependent transcription and / or T cell proliferation, etc.) are known in the art, including, for example, the methods described in Examples 8 and 9 below. In some embodiments, the antibody or antigen-binding fragment increases NF-κB-dependent transcription in cells expressing human CD137 (e.g., human cells). In some embodiments, relative to corresponding cells not contacted with the antibody or antigen-binding fragment (e.g., corresponding cells not contacted with the antibody, or contacted with an isotype control antibody), in CD137-expressing cells (e.g., human cells) contacted with the antibody or antigen-binding fragment, NF-κB-dependent transcription is increased by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 99% or more. In some implementations, compared to corresponding cells that are not in contact with the antibody or antigen-binding fragment (e.g., corresponding cells that are not in contact with the antibody or are in contact with an isotype control antibody), in CD137-expressing cells (e.g., human cells) that are in contact with the antibody or antigen-binding fragment, NF-κB-dependent transcription is increased by approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, or more.

[0214] In some embodiments, the antibody or antigen-binding fragment cross-reacts with CD137 in monkeys (e.g., cynomolgus monkeys), mice, rats, and / or dogs. In some embodiments, the antibody or antigen-binding fragment cross-reacts with monkey CD137. In some embodiments, the antibody or antigen-binding fragment cross-reacts with mouse CD137. In some embodiments, the antibody or antigen-binding fragment cross-reacts with rat CD137. In some embodiments, the antibody or antigen-binding fragment cross-reacts with dog CD137. In some embodiments, the antibody or antigen-binding fragment cross-reacts with CD137 in monkeys and mice; CD137 in monkeys and rats; CD137 in monkeys and dogs; CD137 in mice and rats; CD137 in mice and dogs; CD137 in rats and dogs; CD137 in monkeys, mice, and rats; CD137 in monkeys, mice, and dogs; CD137 in monkeys, rats, and dogs; or CD137 in monkeys, mice, rats, and dogs. In some embodiments, the antibody or antigen-binding fragment cross-reacts at about 100 nM (e.g., at about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, and about 100 nM). Methods for measuring antibody cross-reactivity are known in the art, including but not limited to surface plasmon resonance, ELISA, isothermal titration calorimetry, filtration binding assay, EMSA, etc. In some embodiments, cross-reactivity is measured by ELISA (see, for example, Example 2 below).

[0215] In some embodiments, the antibody does not induce an ADCC effect. Methods for measuring ADCC effects (e.g., in vivo methods) are known in the art, including but not limited to those described in Example 11 below. In some embodiments, the antibody does not induce an ADCC effect greater than about 10% relative to a control (not induced an ADCC effect greater than about 10%, greater than about 5%, greater than about 1%, greater than about 0.1%, or greater than about 0.01%).

[0216] In some embodiments, the antibody or antigen-binding fragment is capable of inhibiting tumor cell growth / proliferation. In some embodiments, when the antibody or antigen-binding fragment is contacted, tumor cell growth / proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%), relative to corresponding tumor cells that are not in contact with the antibody or antigen-binding fragment. In some embodiments, when the antibody or antigen-binding fragment is administered to a subject, the antibody or antigen-binding fragment is capable of reducing the tumor volume of the subject. In some embodiments, the antibody or antigen-binding fragment is capable of reducing the subject's tumor volume by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the subject's initial tumor volume (e.g., before administration of the antibody or antigen-binding fragment). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor suppression are known in the art, including, for example, the methods described in Example 10 below.

[0217] In some implementations, the antibody or antigen-binding fragment has a therapeutic effect on cancer. In some implementations, the antibody or antigen-binding fragment reduces one or more signs or symptoms of cancer. In some implementations, when the antibody or antigen-binding fragment is administered, a subject with cancer enters partial or complete remission.

[0218] In another aspect, this disclosure provides isolated antibodies that compete or cross-compete with any of the exemplary antibodies of this disclosure, such as AG10058, AG10059, and / or AG10131, for binding to human CD137. In a particular embodiment, this disclosure provides isolated antibodies that compete or cross-compete with any of the exemplary antibodies of this disclosure for binding to the same epitope on human CD137. The ability of an antibody to compete or cross-compete with another antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, the exemplary antibody of this disclosure can be allowed to bind to human CD137 under saturation conditions, and then the ability of the test antibody to bind to CD137 can be measured. If the test antibody can bind to CD137 simultaneously with the exemplary antibody, then the test antibody binds to a different epitope than the exemplary antibody. However, if the test antibody cannot bind to CD137 simultaneously, then the test antibody binds to the same epitope, overlapping epitope, or closely adjacent epitope of the epitope bound by the exemplary antibody. This experiment can be performed using various methods, such as ELISA, RIA, FACS, or surface plasmon resonance.

[0219] In some embodiments, the antibody or antigen-binding fragment blocks the binding between CD137 and its ligand (e.g., human CD137 and human CD137L). In some embodiments, the antibody or antigen-binding fragment blocks the binding between CD137 and its ligand in vitro. In some embodiments, the antibody or antigen-binding fragment has a half-maximum inhibitory concentration (IC50) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for blocking the binding of CD137 and its ligand. In some embodiments, the antibody or antigen-binding fragment has a half-maximum inhibitory concentration (IC50) of about 100 nM or less for blocking the binding of CD137 and its ligand. In some embodiments, when provided at concentrations of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.), the antibody or antigen-binding fragment completely blocks the binding of human CD137 to its ligand. As used herein, the term "complete blocking" or "completely blocks" refers to the ability of the antibody or antigen-binding fragment to reduce the binding between the first protein and the second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods for measuring the ability of an antibody or antigen-binding fragment to block the binding of the first protein (e.g., CD137) to the second protein (e.g., CD137L) are known in the art, including but not limited to BIAcore analysis, ELISA assays, and flow cytometry (see, for example, Example 6 below).

[0220] B-1.CD137 antibody

[0221] In some aspects, this disclosure provides an isolation antibody that binds to human CD137 at an epitope within amino acid residues 34-108 or 34-93 of SEQ ID NO.:1. In some embodiments, the antibody is expressed at 50 nM or less K D The antibody binds to human CD137, as measured by surface plasmon resonance. In some embodiments, the antibody may cross-react with at least one non-human species selected from a list consisting of cynomolgus monkeys, mice, rats, and dogs.

[0222] In one aspect, this disclosure provides an isolation antibody comprising a heavy chain variable region and a light chain variable region, a) wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a group selected from the following: Formula (I): X1TFX2X3YX4IHWV (SEQ ID NO:2), wherein X1 is F or Y, X2 is S or T, X3 is G, N, or S, and X4 is A, G, or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO:3), wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T; and Formula (III): FSLSTX1GVX2VX3WI (SEQ ID NO:3). NO:4), wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T; wherein HVR-H2 contains an amino acid sequence selected from the group consisting of: Formula (IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO:5), wherein X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y, and X4 is P or T; Formula (V): IGX1IYHSGX2TYYX3PSLKSRV (SEQ ID NO:6), wherein X1 is D or E, X2 is N or S, and X3 is N or S; and Formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO:6). ID NO:7), wherein X1 is A, G, S, V or Y, X2 is A, D, S or Y, X3 is D, G or S, and X4 is S or T; and wherein HVR-H3 contains an amino acid sequence according to the following formula: Formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO:8), wherein X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T or V, X5 is A, I, L, T or V, and X6 is A, D or G; and / or b) wherein the light chain variable region contains HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 contains an amino acid sequence according to the following formula: Formula (VIII): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO:8) NO:9), wherein X1 is Q or R, X2 is D, G or S, X3 is I or V, X4 is G, R, S or T, X5 is P, R, S or T, X6 is A, D, F, S, V or Y, X7 is L or V, and X8 is A, G or N; wherein HVR-L2 contains an amino acid sequence according to the following formula: Formula (IX): X1ASX2X3X4X5GX6 (SEQ ID NO:10), wherein X1 is A or D, X2 is N, S or T, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V;Furthermore, HVR-L3 contains amino acid sequences selected from the group consisting of: Formula (X): YCQQX1YX2X3X4T (SEQ ID NO:11), where X1 is A, G, S, or Y; X2 is Q, S, or Y; X3 is I, L, T, or Y; and X4 is I, S, V, or W; and Formula (XI): YCX1QX2X3X4X5PX6T (SEQ ID NO:12), where X1 is E or Q; X2 is P, S, or Y; X3 is D, L, S, T, or Y; X4 is D, E, H, S, or T; X5 is D, LT, or W; and X6 is L, P, R, or V.

[0223] In some embodiments, the antibody may comprise HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NO:253-312, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NO:313-372, HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NO:373-432, HVR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NO:433-492, HVR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NO:493-552, and / or HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO:553-612.

[0224] In some embodiments, the antibody may comprise a VL and / or a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:13-132, which may preferably be encoded by a DNA sequence selected from the group consisting of SEQ ID NO:133-252, respectively.

[0225] In some embodiments, the antibody may comprise HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NO:709-732, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NO:733-756, HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NO:757-780, HVR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NO:781-804, HVR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NO:805-828, and / or HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO:829-852.

[0226] In some embodiments, the antibody may comprise a light chain and / or a heavy chain (e.g., those of IgG such as IgG4) having an amino acid sequence selected from the group consisting of SEQ ID NO:613-660, which may preferably be encoded by a DNA sequence selected from the group consisting of SEQ ID NO:661-708, respectively.

[0227] In some embodiments, HVR is performed according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising HVR-H1 containing the sequence GFSLSTSGVGVG (SEQ ID NO: 866), HVR-H2 containing the sequence LIDWDDDKYYSPSLKS (SEQ ID NO: 867), and HVR-H3 containing the sequence GGSDTVLGDWFAY (SEQ ID NO: 868); and / or a light chain variable (VL) domain comprising HVR-L1 containing the sequence RASQSVSPYLA (SEQ ID NO: 869), HVR-L2 containing the sequence DASSLES (SEQ ID NO: 870), and HVR-L3 containing the sequence QQGYSLWT (SEQ ID NO: 871).

[0228] In some embodiments, HVR is performed according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising HVR-H1 containing the sequence GYSITSGHYWA (SEQ ID NO: 872), HVR-H2 containing the sequence SISGYGSTTYYADSVKG (SEQ ID NO: 873), and HVR-H3 containing the sequence GGSDAVLGDWFAY (SEQ ID NO: 874); and / or a light chain variable (VL) domain comprising HVR-L1 containing the sequence RASQGIGSFLA (SEQ ID NO: 875), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 876), and HVR-L3 containing the sequence QQGYYLWT (SEQ ID NO: 877).

[0229] In some embodiments, HVR is performed according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising HVR-H1 containing the sequence GFSLSTGGVGVG (SEQ ID NO: 878), HVR-H2 containing the sequence LIDWADDKYYSPSLKS (SEQ ID NO: 879), and HVR-H3 containing the sequence GGSDTVIGDWFAY (SEQ ID NO: 880); and / or a light chain variable (VL) domain comprising HVR-L1 containing the sequence RASQSIGSYLA (SEQ ID NO: 881), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 882), and HVR-L3 containing the sequence QQGYYLWT (SEQ ID NO: 883).

[0230] The CD137 antibody described herein can belong to any class, such as IgG, IgM, IgE, IgA, or IgD. Preferably, the CD137 antibody belongs to the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The CD137 antibody can be converted from one class or subclass to another using methods known in the art. An exemplary method for generating an antibody belonging to the desired class or subclass includes the steps of: separating the nucleic acid of the heavy chain encoding the CD137 antibody and the nucleic acid of the light chain encoding the CD137 antibody; separating the nucleic acid of the V... H The sequence of regions; V H Sequence linking with sequences encoding the heavy chain constant region of the desired species or subclass; expression of light chain genes and heavy chain constructs in cells; and collection of CD137 antibodies.

[0231] Furthermore, the antibodies provided in this disclosure can be monoclonal or polyclonal, but are preferably monoclonal.

[0232] Examples of specific isolated antibodies provided in this disclosure include those listed in Tables 1a and 1b. The nucleotide and amino acid sequences of the heavy chain variable regions, full-length heavy chains, light chain variable regions, and full-length light chains of these antibodies are also provided below.

[0233] The antibodies disclosed herein can be produced using techniques known in the art, including conventional monoclonal antibody methods, such as standard somatic cell hybridization (see, for example, Kohler and Milstein, Nature 256:495 (1975), viral or oncogenic transformation of B lymphocytes, or recombinant antibody techniques, as described in detail below.

[0234] Hybridoma generation is a well-established procedure. The mouse system is a common animal system used for hybridoma preparation. Immunoassay protocols and techniques for isolating spleen cells for fusion are known in the art. Fusion couples (e.g., mouse myeloma cells) and fusion procedures are also known. A well-known method for preparing the human CD137 antibody disclosed herein involves XenoMouse. TM The use of animal systems. XenoMouse TMThe mice are engineered strains containing large fragments of human immunoglobulin heavy and light chain loci and defective antibody production in mice. See, for example, Green et al., Nature Genetics 7:13-21 (1994) and WO2003 / 040170. Animals are immunized with the CD137 antigen. The CD137 antigen is isolated and / or purified CD137, preferably CD137. It can be a fragment of CD137, such as the extracellular domain of CD137, particularly a fragment containing amino acid residues 34-108 or 34-93 of SEQ ID NO:1. Immunization of the animals can be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle, and horses are well known in the art. See, for example, Harlow and Lane, ibid., and U.S. Patent No. 5,994,619. The CD137 antigen may be administered with an adjuvant to stimulate an immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramicyl dipeptide), or ISCOM (immunostimulatory complex). After immunization of an animal with the CD137 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. Following immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized. Methods for immortalizing cells include, but are not limited to, transfecting the cells with an oncogene, infecting the cells with an oncogene, culturing the cells under conditions selected for immortalized cells, subjecting the cells to oncogenes or mutagenic compounds, fusing the cells with immortalized cells (e.g., myeloma cells), and inactivating tumor suppressor genes. See, for example, Harlow and Lane, ibid. If a fusion containing myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin peptides (non-secreting cell line). Immortalized cells are screened using CD137, or cells that partially or fully express CD137. Cells that produce CD137 antibodies, such as hybridomas, are selected, cloned, and further screened for desired characteristics, including robust growth, high antibody yield, and desired antibody signatures, as discussed further below. Hybridomas can be expanded in vivo in syngeneic animals, in animals lacking an immune system (e.g., nude mice), or in vitro in cell cultures. Methods for selecting, cloning, and expanding hybridomas are well known to those skilled in the art.

[0235] The antibodies disclosed herein can also be prepared using phage display or yeast display methods. Such display methods for isolating human antibodies have been established in the art, such as Achim Knappik et al., “Fully Synthetic Human Combinatorial Antibody Libraries (HuCAL) Based on Modular Consensus Frameworks and CDRs Randomized with Trinucleotides.” J. Mol. Biol. (2000) 296, 57-86; and Michael J. Feldhaus et al., “Flow-cytometric isolation of human antibodies from anon-immune Saccharomyces cerevisiae surface display library” Nat Biotechnol (2003) 21: 163-170.

[0236] B-2. Antigen-binding fragment

[0237] In some other respects, this disclosure provides antigen-binding fragments of any CD137 antibody provided herein.

[0238] The antigen-binding fragment may contain any sequence of the antibody. In some embodiments, the antigen-binding fragment contains the following amino acid sequence: (1) a light chain of the CD137 antibody; (2) a heavy chain of the CD137 antibody; (3) a variable region from the light chain of the CD137 antibody; (4) a variable region from the heavy chain of the CD137 antibody; (5) one or more HVRs (two, three, four, five or six HVRs) of the CD137 antibody; or (6) three HVRs from the light chain of the CD137 antibody and three HVRs from the heavy chain of the CD137 antibody.

[0239] In some specific embodiments, this disclosure provides antigen-binding fragments of antibodies selected from those listed in Tables 1a and 1b.

[0240] In some other specific embodiments, the antigen-binding fragment of the CD137 antibody includes: (i) a Fab fragment, which is composed of V L V H C L and C H (ii) a monovalent segment composed of a structural domain; (iii) a F(ab′)2 segment, which is a divalent segment containing two Fab segments connected by disulfide bonds in the hinge region; H and C H(iv) The Fd fragment composed of a single-arm antibody; L and V H The Fv segment is composed of structural domains; (v) is composed of V H The dAb fragment composed of the structural domain (Ward et al., (1989) Nature 341:544-546); (vi) the isolated CDR, and (vii) the single-chain antibody (scFv), which is a V containing the antibody. H V of the region-linked antibody L Polypeptides in the region. Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0241] In some specific implementations, the antigen-binding fragment is a Fab fragment selected from those listed in Table 1a.

[0242] B-3. ​​Antibody derivatives

[0243] In some other aspects, this disclosure provides derivatives of any CD137 antibody provided herein.

[0244] In one aspect, the antibody derivative is derived from modifications of the amino acid sequence of the exemplary antibody (“parent antibody”) of this disclosure, while retaining the overall molecular structure of the parent antibody’s amino acid sequence. The amino acid sequence of any region of the parent antibody chain may be modified, such as a frame region, HVR region, or constant region. Types of modification include substitution, insertion, deletion, or combinations thereof of one or more amino acids of the parent antibody.

[0245] In some implementations, the antibody derivative contains V L or V HThe antibody derivative comprises an HVR_H1 amino acid sequence region that has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 13-132. In some embodiments, the antibody derivative comprises an HVR_H2 amino acid sequence region that has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 253-312. In some embodiments, the antibody derivative comprises an HVR_H2 amino acid sequence region that has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 313-372. In some embodiments, the antibody derivative comprises the HVR_H3 amino acid sequence region having at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequences shown in any of SEQ ID NO:373-432. In some embodiments, the antibody derivative comprises the HVR_L1 amino acid sequence of all Fab hits shown in Table 1a, which is available in SEQ ID NO:433-492. In some embodiments, the antibody derivative comprises the HVR_L2 amino acid sequence of all Fab hits shown in Table 1a, which is available in SEQ ID NO:493-552.

[0246] In some embodiments, the antibody derivative comprises the HVR_L3 amino acid sequence of all Fab hits shown in Table 1a, which is available in SEQ ID NO:553-612. In some specific embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved or non-conserved substitutions of the amino acid sequence shown in any of SEQ ID NO:13-132 and 253-612, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions.

[0247] In some embodiments, the antibody derivative comprises a light or heavy chain having at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence as shown in any of SEQ ID NO:613-660.

[0248] In some embodiments, the antibody derivative comprises the HVR_H1 amino acid sequence region, which has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO:709-732. In some embodiments, the antibody derivative comprises the HVR_H2 amino acid sequence region, which has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO:733-756. In some embodiments, the antibody derivative comprises the HVR_H3 amino acid sequence region, which has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO:757-780. In some embodiments, the antibody derivative comprises the HVR_L1 amino acid sequence region, which has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO:781-804. In some embodiments, the antibody derivative comprises the HVR_L2 amino acid sequence region, which has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO:805-828. In some embodiments, the antibody derivative comprises the HVR_L3 amino acid sequence region, which has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO:829-852. In some embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conserved or non-conserved substitutions for the amino acid sequence shown in any of SEQ ID NO: 613-660 and 709-852, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additions and / or deletions.

[0249] Amino acid substitution encompasses both conserved and non-conserved substitutions. The term "conserved amino acid substitution" refers to the replacement of one amino acid with another, where the two amino acids share similar physicochemical properties, such as the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, substitution can typically occur within each of the following groups: (a) nonpolar (hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids, such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids, such as aspartic acid and glutamic acid.

[0250] Modifications can be made at any position in the amino acid sequence of the antibody, including HVR, frame regions, or constant regions. In one embodiment, this disclosure provides an antibody derivative comprising the V of an exemplary antibody of this disclosure. H and V L The HVR sequence also includes frame sequences that differ from those of the exemplary antibody. Such frame sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database or the “VBase” human germline sequence database (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson, IM et al., J. Mol. Biol. 227:776-798 (1992); and Cox, JPL et al., Eur. J. Immunol. 24:827-836 (1994)). Frame sequences that can be used to construct antibody derivatives include those structurally similar to those used in the exemplary antibody of this disclosure, such as those similar to the V... H 3-23 Frame Sequence and / or V LThose of the λ3 or λ1-13 frame sequences. For example, the HVR_H1, HVR_H2, and HVR_H3 sequences of exemplary antibodies, as well as the HVR_L1, HVR_L2, and HVR_L3 sequences, can be transplanted into frame regions having the same sequences found in the germline immunoglobulin genes from which the frame sequences originate, or the HVR sequences can be transplanted into frame regions containing one or more mutations compared to the germline sequences.

[0251] In one particular embodiment, the antibody derivative is a chimeric antibody comprising the amino acid sequence of an exemplary antibody of this disclosure. In one example, one or more HVRs derived from one or more exemplary human antibodies are combined with HVRs derived from an antibody from a non-human animal (such as a mouse or rat). In another example, all HVRs of the chimeric antibody are derived from one or more exemplary antibodies. In some particular embodiments, the chimeric antibody comprises one, two, or three HVRs derived from the heavy chain variable region or light chain variable region of an exemplary antibody. Chimeric antibodies can be generated using conventional methods known in the art.

[0252] Another type of modification is to make V H and / or V L Amino acid residue mutations are performed within the HVR region of the chain. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effects on antibody binding or other functional properties of interest can be assessed in in vitro or in vivo assays known in the art. Typically, conserved substitutions are introduced. Mutations can be amino acid additions and / or deletions. Furthermore, typically no more than one, two, three, four, or five residues within the HVR region are altered. In some embodiments, the antibody derivative contains 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVR and / or light chain HVR. In another embodiment, the amino acid substitution is the replacement of one or more cysteine ​​residues in the antibody with another residue, such as, but not limited to, alanine or serine. The cysteine ​​can be classical or non-classical cysteine. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conserved amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of the exemplary antibody.

[0253] It can also be used for V H and / or V L The framework residues within the region are modified. Typically, such framework variants are prepared to reduce the immunogenicity of the antibody. One approach is to "revert" one or more framework residues to their corresponding germline sequences. Antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody originated. These residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originated. To restore the framework region sequence to its germline structure, somatic mutations can be "reverted" to germline sequences using, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0254] Furthermore, modifications can be made to the Fc region of exemplary antibodies, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. In one example, the hinge region of CH1 is modified to change the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing them. This method is further described in U.S. Patent No. 5,677,425. Changing the number of cysteine ​​residues in the hinge region of CH1 can, for example, favor the assembly of the light and heavy chains, or increase or decrease the stability of the antibody. In another case, the Fc hinge region of the antibody is mutated to reduce the antibody's biological half-life.

[0255] Furthermore, the antibodies of this disclosure can be modified according to conventional experiments known in the art to alter their potential glycosylation sites or patterns. In another aspect, this disclosure provides derivatives of the CD137 antibody of this disclosure containing at least one mutation in a variable region of the light or heavy chain, said mutation altering the glycosylation pattern in the variable region. Such antibody derivatives may have increased affinity and / or altered specificity for antigen binding. Mutations may add new glycosylation sites in the V region, alter the position of one or more V region glycosylation sites, or remove previously existing V region glycosylation sites. In one embodiment, this disclosure provides a derivative of a CD137 antibody having a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein one of the potential N-linked glycosylation sites in the heavy chain variable region is removed. In another embodiment, this disclosure provides a derivative of a CD137 antibody having a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein both potential N-linked glycosylation sites in the heavy chain variable regions are removed. Methods for altering the glycosylation pattern of antibodies are known in the art, such as those described in U.S. Patent No. 6,933,368, the disclosure of which is incorporated herein by reference.

[0256] In another aspect, this disclosure provides an antibody derivative comprising a CD137 antibody, as described herein, linked to another molecular entity, or an antigen-binding fragment thereof. Examples of the other molecular entity include pharmaceuticals, peptides or proteins, detection agents or labels, and antibodies.

[0257] In some embodiments, the antibody derivative comprises an antibody of the present disclosure linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, as well as radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthraquinone, mitoxantrone, photomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologs. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil azithromycin), alkylating agents (e.g., dichloromethyldiethylamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly known as actinomycin), bleomycin, photomycin and atrazomycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated with antibodies for diagnostic or therapeutic purposes include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 Helu 177Methods for linking antibodies to drugs are known in the art, such as the use of various linker techniques. Examples of linker types include linkers containing hydrazones, thioethers, esters, disulfides, and peptides. For further discussion of the connectors and methods used to link therapeutic agents to antibodies, see also Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan, I. and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter, PD and Springer, CJ (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0258] In one particular embodiment, the antibody derivative is a CD137 antibody multimer, which is a multimeric form of the CD137 antibody, such as an antibody dimer, trimer, or higher-order multimer of a monomeric antibody. The monomers within the antibody multimer can be the same or different. Furthermore, the monomers within the multimer can have the same or different binding specificities. Antibody multimerization can be achieved through the natural aggregation of antibodies. For example, a certain percentage of purified antibody formulations (e.g., purified IgG4 molecules) spontaneously form protein aggregates comprising antibody homodimers and other higher-order antibody multimers. Alternatively, antibody homodimers can be formed using chemical linking techniques known in the art, such as by using cross-linking agents. Suitable crosslinking agents include those that are heterobifunctional, have two distinct reactive groups separated by suitable spacer groups (such as m-maleimide benzoyl-N-hydroxysuccinimide, 4-(maleimide methyl)cyclohexane-1-carboxylic acid succinimide, and S-acetylthio-acetic acid N-succinimide), or are homobifunctional (such as bis(succinimide) octanoate). Such linkers are commercially available, for example, from Pierce Chemical Company, Rockford, IL. Antibody polymerization can also be achieved using recombinant DNA techniques known in the art.

[0259] In some embodiments, the antibody disclosed herein is a multimeric antibody (e.g., a bispecific antibody). In some embodiments, the antibody disclosed herein is an IgM antibody, for example, comprising an IgM Fc region (e.g., a human IgM Fc region).

[0260] Other examples of antibody derivatives provided in this disclosure include single-chain antibodies, biantibodies, domain antibodies, nanobodies, and unibodies. A "single-chain antibody" (scFv) consists of antibodies containing V... H V connected by structural domains L The domain is composed of a single polypeptide chain, in which V L domain and V H Domain pairing forms a monovalent molecule. Single-chain antibodies can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). “Biantibodies” consist of two chains, each containing a heavy chain variable region linked to a light chain variable region on the same polypeptide chain via a short peptide linker, wherein the two regions on the same chain do not pair with each other, but pair with complementary domains on the other chain to form a bispecific molecule. Methods for preparing biantibodies are known in the art (see, for example, Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 and Poljak RJ et al., (1994) Structure2:1121-1123). Domain-bound antibodies (dAbs) are small functional binding units of antibodies that correspond to the variable regions of the antibody's heavy or light chain. Domain-bound antibodies are well expressed in bacterial, yeast, and mammalian cell systems. Further details regarding domain-bound antibodies and methods of their production are known in the art (see, for example, U.S. Patents 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patents 0368684 & 0616640; WO05 / 035572, WO04 / 101790, WO04 / 081026, WO04 / 058821, WO04 / 003019, and WO03 / 002609). Nanobodies are derived from the heavy chain of antibodies. Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding capacity of the original antibody. Nanobodies can be prepared using methods known in the art (see, for example, U.S. Patent Nos. 6,765,087, 6,838,254, and WO 06 / 079372). Mini-antibodies consist of one light chain and one heavy chain of an IgG4 antibody. Mini-antibodies can be prepared by removing the hinge region of the IgG4 antibody. Further details of mini-antibodies and their preparation methods can be found in WO2007 / 059782.

[0261] C. Nucleic acids, vectors, host cells, and recombinant methods for producing CD137 antibodies

[0262] Another aspect of this disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of the binding molecule provided herein. The amino acid sequence encoded by said nucleotide sequence can be any portion of an antibody, such as an HVR, a sequence containing one, two, or three HVRs, a variable region of the heavy chain, a variable region of the light chain, or can be a full-length heavy chain or a full-length light chain. The nucleic acid of this disclosure can be, for example, DNA or RNA, and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0263] In some embodiments, this disclosure provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: (1) the amino acid sequence of HVR_H3 or HVR_L3 of an exemplary antibody; (2) a variable region of the heavy chain or a variable region of the light chain of an exemplary antibody; or (3) the full-length heavy chain or the full-length light chain of an exemplary antibody.

[0264] In other embodiments, the nucleic acid molecule comprises or is composed of a nucleotide sequence that encodes an amino acid sequence as shown in any one of SEQ ID NO: 13-132, 253-612, 613-660, and 709-852.

[0265] In other embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from or composed of the group consisting of SEQ ID NO:133-252 and 661-708.

[0266] The nucleic acids disclosed herein can be obtained using any suitable molecular biology technique. For antibodies expressed via hybridoma, the cDNA encoding the light and heavy chains of the antibody prepared via hybridoma can be obtained by PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding said antibodies can be recovered from the library.

[0267] By encoding V H The DNA is operationally linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3) to encode V. HThe isolated DNA from the region is converted into a full-length heavy chain gene. The sequences of human heavy chain constant regions are known in the art (see, for example, Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are most preferably IgG4 or IgG2 constant regions that do not exhibit ADCC effects. The IgG4 constant region sequence can be any of the various alleles or allotypes known to exist in different individuals. These allotypes represent naturally occurring amino acid substitutions in the IgG4 constant region. For Fab fragment heavy chain genes, the sequence encoding V... H The DNA is operatively linked to another DNA molecule that encodes only the CH1 constant region of the heavy chain.

[0268] By encoding V L The DNA is operatively linked to another DNA molecule encoding the light chain constant region CL, thereby encoding V. L The isolated DNA from the region is converted into full-length light chain genes (and Fab light chain genes). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions.

[0269] In order to produce the scFv gene, the gene encoding V will be... H and V L The DNA fragment is operatively linked to another fragment encoding a flexible adapter, such as another fragment encoding the amino acid sequence (Gly4-Ser)3, so that V H and V L The sequence can be expressed as having V connected by a flexible joint. L and V H The region consists of continuous single-chain proteins (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990)).

[0270] This disclosure also provides a vector comprising a nucleic acid molecule provided herein. The nucleic acid molecule may encode an amino acid sequence of a portion of a light chain or heavy chain (such as CDR or HVR), a full-length light chain or heavy chain, a polypeptide comprising a portion of a heavy chain or light chain or of full length, or an antibody derivative or antigen-binding fragment. In some embodiments, the vector is an expression vector that can be used to express a binding molecule such as an antibody or an antigen-binding fragment thereof. In some embodiments, vectors are provided herein wherein a first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein, and a second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises polynucleotides encoding both a heavy chain variable region as described herein and a light chain variable region as described herein.

[0271] To express the binding molecules of this disclosure, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector, such that the DNA molecule is operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" means that the antibody gene is linked into the vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the DNA molecule. The chosen expression vector and expression control sequences should be compatible with the host cells used for expression. The antibody light chain gene and antibody heavy chain gene may be inserted into separate vectors, or more commonly, both genes may be inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (e.g., linking the antibody gene fragment to a complementary restriction site on the vector, or DNA ligation based on homologous recombination). The variable regions of the light and heavy chains of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype and subclass by inserting them into expression vectors that already encode the desired isotype and subclass heavy chain constant regions and light chain constant regions, such that V H The segment is operatively connected to one or more Cs within the carrier. H Section and V L The segment is operatively connected to the C within the carrier. L This can be achieved through segmentation. Alternatively, recombinant expression vectors can encode signal peptides that facilitate the secretion of antibody chains by host cells. The antibody chain gene can be cloned into the vector, with the signal peptide linked in frame to the N-terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0272] In addition to the antibody chain gene, the expression vectors disclosed herein typically carry regulatory sequences that control the expression of the antibody chain gene in host cells. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Those skilled in the art will recognize that the design of expression vectors, including the selection of regulatory sequences, can depend on factors such as the choice of host cells to be transformed and the desired protein expression level. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitous protein promoters or β-globulin promoters, may be used. In addition, regulatory elements contain sequences from various sources, such as the SR promoter system, which contains sequences from the SV40 early promoter and long terminal repeat sequences from human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8: 466-472).

[0273] In addition to antibody chain genes and regulatory sequences, expression vectors may carry other sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes aid in the selection of host cells in which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017, all filed by Axel et al.). For instance, selection marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells in which the vector has been introduced. Selection marker genes include the dihydrofolate reductase (DHFR) gene (for selecting / amplifying dhfr-host cells using methotrexate) and the neo gene (for G418 selection).

[0274] To express the light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into host cells using any suitable technique. The term "transfection" in various forms is intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. Although the antibodies disclosed herein can be expressed in prokaryotic or eukaryotic host cells, they are most commonly expressed in eukaryotic cells and, more commonly, in mammalian host cells.

[0275] This disclosure also provides a host cell comprising the nucleic acid molecule provided herein. The host cell can be virtually any cell to which the expression vector is applicable. The cell can be, for example, a higher eukaryotic host cell, such as a mammalian cell; a lower eukaryotic host cell, such as a yeast cell; and can be a prokaryotic cell, such as a bacterial cell. The introduction of the recombinant nucleic acid construct into the host cell can be achieved via calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

[0276] Suitable eukaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0277] Mammalian host cells used to express the binding molecules of this disclosure include, for example, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980), which are used with DHFR selective markers, for example, as described in Kaufman and Sharp, J. Mol. Biol. 159:601-621 (1982)), NSO myeloma cells, COS cells, and Sp2 cells. Specifically, another expression system for use with NSO myeloma or CHO cells is the GS (glutamine synthase) gene expression system disclosed in WO87 / 04462, WO 89 / 01036, and EP338,841. When an expression vector encoding an antibody gene is introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period sufficient to allow the antibodies to be expressed in the host cells or secreted into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using any suitable protein purification method.

[0278] D. Composition

[0279] In other aspects, this disclosure provides a composition comprising the binding molecule provided herein. In one aspect, the composition is a pharmaceutical composition comprising the binding molecule and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0280] In some embodiments, this disclosure provides a composition comprising an antibody or antigen-binding fragment thereof provided herein, and a pharmaceutically acceptable carrier, wherein the antibody comprises a variable domain comprising the HVR amino acid sequence disclosed herein, and wherein the composition comprises no more than about 11%, 10%, 8%, 5%, 3%, or 2% of the antibody or antigen-binding moiety thereof glycosylated at asparagine in the amino acid sequence compared to the total amount of the antibody or antigen-binding moiety present in the composition. In another embodiment, the composition comprises at least about 2% of the antibody or antigen-binding moiety thereof glycosylated at asparagine in the amino acid sequence compared to the total amount of the antibody or antigen-binding moiety present in the composition.

[0281] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering bound molecules. Carriers can be anti-adhesives, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delayers, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0282] The composition can be in any suitable form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusionable solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A specific form suitable for delivering the binding molecule is a sterile liquid, such as a solution, suspension, or dispersion, for injection or infusion. Sterile solutions can be prepared by incorporating the desired amount of antibody into a suitable carrier followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the antibody into a sterile medium containing a basic dispersion medium and other carriers. For sterile powders used to prepare sterile liquids, preparation methods include vacuum drying and freeze-drying (lyophilization) to produce a powder of the active ingredient plus any additional desired components from its previously sterile filtered solution. Various dosage forms of the composition can be prepared using conventional techniques known in the art.

[0283] The relative amount of bound molecules contained in a composition will vary depending on a number of factors, such as the specific bound molecules and carriers used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of bound molecules in a single dosage form will typically be the amount that produces a therapeutic effect, but can also be a smaller amount. Generally, this amount will range from about 0.01% to about 99%, from about 0.1% to about 70%, or from about 1% to about 30% relative to the total weight of the dosage form.

[0284] In addition to the binding molecule, one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described below. The appropriate amount of the additional therapeutic agent to be included in the composition can be readily selected by those skilled in the art and will vary depending on a number of factors, such as the specific pharmaceutical agent and carrier used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of additional therapeutic agent included in a single dosage form will generally be the amount of the pharmaceutical agent that produces the therapeutic effect, but may also be a smaller amount.

[0285] E. Use of combined molecules and pharmaceutical compositions

[0286] The conjugate molecules and pharmaceutical compositions disclosed herein can be used for therapeutic, diagnostic, or other purposes, such as modulating immune responses, treating cancer, enhancing the efficacy of other cancer therapies, enhancing vaccine efficacy, or treating autoimmune diseases. Therefore, in other aspects, this disclosure provides methods of using the conjugate molecules or pharmaceutical compositions. In one aspect, this disclosure provides a method of treating a condition in a mammal, comprising administering a therapeutically effective amount of the conjugate molecules provided herein to the mammal in need of treatment. The conjugate molecules may be CD137 agonists or antagonists. In some embodiments, the conjugate molecules are CD137 agonists. In some embodiments, the mammal is a human.

[0287] In some implementations, the condition is cancer. CD137 is involved in a variety of cancers, whether malignant or benign, and whether primary or secondary, which can be treated or prevented using the methods provided in this disclosure. Examples of such cancers include lung cancers such as bronchial carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyosarcoma; bone cancers such as osteochondroma, condromas, chondroblastoma, chondromycinoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; and brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, and pituitary gland. Somatic adenomas, pineal tumors, osteomas, hemangioblastomas, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermoid cysts, and hemangiomas; cancers of the digestive system, such as leiomyomas, epidermoid carcinomas, adenocarcinomas, leiomyosarcomas, gastric adenocarcinomas, intestinal lipomas, intestinal neurofibromas, intestinal fibromas, polyps in the large intestine, and colorectal cancer; liver cancers, such as hepatocellular adenomas, hemangiomas, hepatocellular carcinomas, fibrolamellar carcinomas, bile duct carcinomas, hepatoblastomas, and angiosarcomas; kidney cancers, such as renal adenocarcinomas, renal cell carcinomas, adrenoid adenomas, and transitional cell carcinomas of the renal pelvis; bladder cancer; blood cancers, such as acute lymphoblastic leukemia, acute myeloid leukemia (myeloid, myeloblastic, myelomonocytic), and chronic lymphocytic leukemia (e.g., Sezary syndrome). Hodgkin's syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloblastic, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocytosis, and chronic myeloid leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancers; eye-related cancers such as retinoblastoma and intraocular melanoma; and male reproductive system cancers, etc. Examples of cancers include benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system, such as uterine cancer (endometrial cancer), cervical cancer (cervical cancer), ovarian cancer (ovarian cancer), vulvar cancer, vaginal cancer, fallopian tube cancer, and vesicular birth defects; thyroid cancer (including papillary carcinoma, follicular carcinoma, degenerative carcinoma, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid gland; pancreatic cancer; and blood cancers, such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

[0288] In some other implementations, the condition is an autoimmune disease. Examples of autoimmune diseases for which conjugate molecules can be used for treatment include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. Conjugate molecules can also be used to treat inflammation such as allergic asthma and chronic graft-versus-host disease.

[0289] In another aspect, this disclosure provides a method for enhancing an immune response in mammals, comprising administering a therapeutically effective amount of the binding molecule provided in this disclosure to the mammal. In some embodiments, the binding molecule is a CD137 antibody or an antigen-binding fragment thereof and the mammal is a human. In another embodiment, the binding molecule is a CD137 agonist antibody or an antigen-binding fragment thereof. The term “enhanced immune response” or its grammatical variations refer to any response that stimulates, arouses, increases, elevates, or strengthens the immune system of a mammal. An immune response can be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., an antibody-mediated response), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+ helper T cell activity and the production of cytolytic T cells. Enhancement of an immune response can be assessed using a variety of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, the methods of this disclosure enhance the immune response of mammals when compared to the immune response of untreated mammals or mammals not treated using the claimed method. In one embodiment, the binding molecule is used to enhance a human immune response to a microbial pathogen, such as a virus. In another embodiment, the binding molecule is used to enhance a human immune response to a vaccine. The binding molecule may be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In one embodiment, the method enhances a cellular immune response, particularly a cytotoxic T cell response. In another embodiment, the cellular immune response is a T helper cell response. In another embodiment, the immune response is cytokine production, particularly IL-2 production. The binding molecule can be used to enhance a human immune response to a microbial pathogen, such as a virus, or to a vaccine. The binding molecule may be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist.

[0290] In practice, the binding molecule can be administered alone as a monotherapy or in combination with one or more other therapeutic agents or therapies. Therefore, in another aspect, this disclosure provides a combination therapy comprising a binding molecule in combination with one or more other therapeutic agents or therapies for separate, sequential, or simultaneous administration. The term "other therapy" refers to a therapy that does not employ the binding molecule provided in this disclosure as a therapeutic agent. The term "other therapeutic agent" refers to any therapeutic agent other than the binding molecule provided in this disclosure. In one particular aspect, this disclosure provides a combination therapy for treating cancer in mammals, comprising administering to the mammal a therapeutically effective amount of the binding molecule provided in this disclosure in combination with one or more other therapeutic agents. In another embodiment, the mammal is a human.

[0291] A wide variety of cancer therapeutic agents can be used in combination with the binding molecules provided in this disclosure. Those skilled in the art will recognize the existence and development of other cancer therapies that can be used in combination with the methods and binding molecules of this disclosure, and will not be limited to those forms of the therapies described herein. Examples of other classes of therapeutic agents that can be used in combination therapies for treating cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents.

[0292] The term "chemotherapy agent" refers to a chemical or biological substance that can cause cancer cell death or interfere with the growth, division, repair, and / or function of cancer cells. Examples of chemotherapy agents include those disclosed in WO 2006 / 129163 and US 20060153808, the disclosures of which are incorporated herein by reference. Examples of specific chemotherapeutic agents include: (1) alkylating agents such as chlorambucil (LEUKERAN), mcyclophosphamide (CYTOXAN), ifosfamide (IFEX), nitrogen mustard hydrochloride (MUSTARGEN), thioplex (THIOPLEX), streptozotocin (ZANOSAR), carmustine (BICNU, GLIADELWAFER), lomustine (CEENU), and dacarbazine (DTIC-DOME); (2) alkaloids or vinca alkaloids, including cytotoxic antibiotics such as doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), nemorubicin, and idarubicin (IDAMYCIN). PFS, ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE), cytarabine (actinomycin D, COSMEGEN), procainamide (MITHRACIN), mitomycin (MUTAMYCIN) and bleomycin (BLENOXANE), vinorelbine tartrate (NAVELBINE), vinblastine (VELBAN), vincristine (ONCOVIN) and vindesine (ELDISINE); (3) antimetabolites, such as capecitabine (XELODA), cytarabine (CYTOSAR-U), fludarabine (FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate (FOLEX, MEXATE, TREXALL), nerabine (ARRANON), trimethoprim. (3) NEUTREXIN and PEMRTE (ALIMTA); (4) Pyrimidine antagonists, such as 5-fluorouracil (5-FU), capecitabine (XELODA), raltitrexed (TOMUDEX), tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5) Taxanes, such as docetaxel (TAXOTERE) and paclitaxel (TAXOL); (6) Platinum-based drugs, such as cisplatin (PLATINOL), carboplatin (PARAPLATIN), and oxaliplatin (ELOXATIN); (7) Topoisomerase inhibitors, such as irinotecan (CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), and teniposide (VUMON);(8) Podophyllotoxin (podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID, TOPOSAR); (9) Folic acid derivatives, such as folinic acid (WELLCOVORIN); (10) Nitrosoureas, such as carmustine (BiCNU) and lomustine (CeeNU); (11) Receptor tyrosine kinases, including inhibitors of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA), bortezomib (VELCADE), imatinib mesylate (GLEEVEC), gefitinib (genefitinib), lapatinib, sorafenib, thalidomide, and sunitinib (SUTE). (12) Antibodies against insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596, including NT, axitinib, rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN), ranibizumab (LUCENTIS), lym-1 (ONCOLYM), and sulphurin (ANCOLYM). (13) Antibodies against insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596; (14) Angiogenesis inhibitors, such as bevacizumab (AVASTIN), suramin (GERMANIN), angiostatin, SU5416, thalidomide, and matrix metalloproteinase inhibitors (such as batimastat and marimastat), and those disclosed in WO2002055106; and (15) proteasome inhibitors, such as bortezomib (VELCADE).

[0293] The term "immunotherapy agent" refers to a chemical or biological substance that enhances the immune response in mammals. Examples of immunotherapy agents include: BCG; cytokines such as interferon; vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX, and NeuVax; and antibodies such as alemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX), gemtuzumab, and oxazolidin. ozogamicin (MYLOTARG), ibritumomabtiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), tosimoumab (BEXXAR), ipilimumab (YERVOY), tremelimumab, CAT-3888, agonist antibodies against the OX40 receptor (such as those disclosed in WO2009 / 079335), agonist antibodies against the CD40 receptor (such as those disclosed in WO2003 / 040170), and TLR-9 agonists (such as those disclosed in WO2003 / 015711, WO2004 / 016805 and WO2009 / 022215).

[0294] The term "hormone therapy agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones, or inhibits or counteracts the effects of hormones on the growth and / or survival of cancer cells. Examples of such agents suitable for the methods described herein include those disclosed in US20070117809. Specific examples of hormone therapy agents include tamoxifen (NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole (ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The binding molecules of this disclosure may also be used in combination with non-pharmacological hormone therapies such as (1) surgical methods that remove all or part of organs or glands involved in hormone production, such as the ovaries, testes, adrenal glands, and pituitary gland, and (2) radiation therapy, wherein the patient's organ or gland is subjected to a dose of radiation sufficient to inhibit or eliminate the production of the target hormone.

[0295] Combination therapies for cancer treatment also encompass the combination of binding molecules with surgical procedures to remove tumors. Binding molecules can be administered to mammals before, during, or after surgery.

[0296] Combination therapies for cancer treatment also encompass combinations of binding molecules with radiotherapy such as ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) and particle beam radiotherapy (e.g., high linear energy radiation). The radiation source can be external or internal for mammals. Binding molecules can be administered to mammals before, during, or after radiotherapy.

[0297] The binding molecules and compositions disclosed herein can be administered via any suitable enteric or parenteral route of administration. The term "enteric route of administration" means administration via any part of the gastrointestinal tract. Examples of enteric routes include oral, mucosal, buccal, and rectal routes, or intragastric routes. "Paratrical route of administration" refers to a route of administration other than the enteric route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrasheathal, intracystic, intraorbital, intracardiac, intratracheal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or local administration. The antibodies and compositions disclosed herein can be administered using any suitable method, such as oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The appropriate route and method of administration can vary depending on a variety of factors, such as the specific antibody used, the desired absorption rate, the specific formulation or dosage form used, the type or severity of the disease being treated, the specific site of action, and the patient's condition, and can be readily selected by those skilled in the art.

[0298] The term "therapeutic effective amount" for a molecule refers to an amount effective for the intended therapeutic purpose. For example, in the context of enhancing an immune response, a "therapeutic effective amount" is any amount effective in stimulating, arousing, increasing, enhancing, or strengthening any response of the mammalian immune system. In the context of treating a disease, a "therapeutic effective amount" is any amount sufficient to cause any desired or beneficial effect in the treated mammal. Specifically, in the treatment of cancer, examples of desired or beneficial effects include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or increased mammalian survival. Therapeutic effective amounts of CD137 antibodies typically range from about 0.001 to about 500 mg / kg, and more typically from about 0.01 to about 100 mg / kg of mammalian body weight. For example, the amount may be about 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, or 100 mg / kg of mammalian body weight. In some embodiments, the therapeutically effective dose of the CD137 antibody is in the range of about 0.01-30 mg / kg of mammalian body weight. In other embodiments, the therapeutically effective dose of the CD137 antibody is in the range of about 0.05-15 mg / kg of mammalian body weight. The precise dose level to be administered can be readily determined by those skilled in the art and will depend on a variety of factors, such as the type and severity of the condition to be treated, the specific binding molecule used, the route of administration, the time of administration, the duration of treatment, the specific additional therapy used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0299] The conjugate molecule or composition is typically administered in multiple doses. The intervals between single doses can be, for example, once weekly, once monthly, once every 3 months, or once annually. Exemplary treatment regimens include administration once weekly, once every 2 weeks, once every 3 weeks, once every 4 weeks, once monthly, once every 3 months, or once every 3 to 6 months. Typical dosing regimens for CD137 antibodies include intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight using one of the following dosing regimens: (i) 6 doses every 4 weeks, followed by every 3 months; (ii) every 3 weeks; (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight every 3 weeks.

[0300] This disclosure will be more fully understood by referring to the following embodiments. However, the embodiments should not be construed as limiting the scope of this disclosure. It should be understood that the embodiments and implementations described herein are for illustrative purposes only and will suggest various modifications or variations to be made by those skilled in the art, and these will be included within the spirit and scope of this application and the appended claims. All drawings and all references, patents and published patent applications cited throughout this disclosure are expressly incorporated herein by reference in their entirety.

[0301] Example

[0302] Example 1

[0303] Generation of primary Fab that specifically binds to human CD137

[0304] Human CD137 antigen was panned using proprietary plasmid libraries (see the concurrently filed PCT international application entitled "Dynamic Human Antibody Light Chain Libraries" under Agent's File No. 69540-2000140, which is incorporated herein by reference in its entirety; also see the concurrently filed PCT international application entitled "Dynamic Human Heavy Chain Antibody Libraries" under Agent's File No. 69540-2000240, which is incorporated herein by reference in its entirety). A total of three or four rounds of panning were performed. After the final round of panning, single-colony supernatant ELISA was performed to identify primary hits that specifically recognize human CD137. Primary hits were defined as those whose ELISA signal was at least twice the background signal. They were sequenced, and unique clones were expressed and purified by ForteBio and Biacore for affinity measurements. The list was finely adjusted to 124 from the Fab containing both ELISA-positive hits and unique sequences. According to K... D The response signal R > 0.1, R 2 >0.9 and affinity K D The <100 nM standard further refined the list to 60 hits (Table 1a). Twenty-four of these were then converted to IgG (Table 1b) for detailed biophysical and functional characterization.

[0305] The Fabs corresponding to the unique hits were expressed and purified in *E. coli*. Their affinity for human CD137 was measured using the ForteBio OctetRED96 system. Briefly, CD137-hisFc fusion protein (Sino Biological catalog number 10041-H03H) was captured using an AHC sensor (Anti-Human IgG FcCapture Dip and Read Biosensors) and dipped into wells containing purified Fab diluted to 5–10 μg / mL with kinetic buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v / v surfactant P20, pH 7.4). The obtained ForteBio data were processed using Data Acquisition software 7.1, and the kinetic data were fitted to a 1:1 Langmuir binding model. Affinity and kinetic parameters (background removed) are listed in Table 1a. The affinity of their corresponding IgG for human CD137 was measured by Biacore and is shown in Table 1b.

[0306] Table 1a: Affinity of the selected Fab to human CD137 and corresponding amino acid sequences (in SEQ ID NO.)

[0307]

[0308]

[0309]

[0310] The corresponding DNA sequences encoding the amino acid sequences SEQ ID NO:13-132 are found in SEQ ID NO:133-252. The HVR_H1 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NO:253-312. The HVR_H2 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NO:313-372. The HVR_H3 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NO:373-432. The HVR_L1 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NO:433-492. The HVR_L2 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NO:493-552. The HVR_L3 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NO:553-612 (see also Table 1c).

[0311] Table 1b: Affinity of Fab and corresponding IgG to human CD137

[0312]

[0313]

[0314] The corresponding DNA sequences encoding the amino acid sequences SEQ ID NO:613-660 are found in SEQ ID NO:661-708, respectively. The HVR_H1 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NO:709-732, respectively. The HVR_H2 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NO:733-756, respectively. The HVR_H3 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NO:757-780, respectively. The HVR_L1 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NO:781-804, respectively. The HVR_L2 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NO:805-828, respectively. The HVR_L3 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NO:829-852, respectively.

[0315] Table 1c: CDR sequences of Fab

[0316]

[0317]

[0318]

[0319] Example 2

[0320] Selection of Fab hits that cross-react with mouse CD137

[0321] Species cross-reactivity of Fab-targeted antibodies was determined using ELISA. Briefly, 200 μL of 5 μg / mL anti-human IgG (Fab-specific) (Sigma#I5260) was spread onto Maxisorp microplates (Thermo Scientific 446469) and incubated overnight at 4°C. After blocking, 100 μL of Fab 5310 (5 μg / mL), 5351 (2.8 μg / mL), and 5365 (5 μg / mL) were added and incubated for 1 hr. After three washes, serial dilutions of human or mouse CD137 antigen fused with the human FC fragment were added and incubated for 1 hr. After washing, HRP-labeled goat anti-human FC was diluted 1:2000 with PBS and added to each well, incubated for 1 hr. The plate was washed three times and incubated with TMB substrate at room temperature for 20 min. After the reaction was stopped, absorbance was measured at 450 nm. The results are shown in Figure 1b. The lower inset shows that Fab 5310 and 5365 bind to both human and mouse CD137, while Fab 5351 binds to human CD137 but not to mouse CD137.

[0322] Example 3

[0323] IgG conversion and expression: AG10058, AG10059 and AG10131

[0324] The heavy and light chains of Fab 5310, 5351, and 5365 were individually cloned into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific) of the IgG4 isotype with the S241P mutation. Additionally, the heavy and light chains of two reference antibodies were cloned into pCDNA3.3 of the IgG4 and IgG2 isotypes, respectively.

[0325] The heavy chain variable region used in reference antibody AC1097 contains the sequence EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSS (SEQ ID NO:862), and the light chain variable region of reference antibody AC1097 contains the sequence SYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVL (SEQ ID NO:863). The heavy chain variable region used in reference antibody AC1121 contains the sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS (SEQ ID NO: 864), and the light chain variable region of reference antibody AC1121 contains the sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK (SEQ ID NO: 865). The IgG used in this paper is shown in Table 2.

[0326] Table 2: List of IgG

[0327]

[0328] Following the manufacturer's instructions, the plasmid pairs were transiently transfected into HEK293F cells. The supernatant was harvested, clarified by centrifugation and filtration, and IgG was purified using standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The protein was eluted and neutralized, and the buffer was exchanged into PB buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). Protein concentration was determined by UV-spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions.

[0329] Example 4

[0330] Binding affinity to CD137 in humans, monkeys, and mice

[0331] The binding affinity of IgG to CD137 in humans, monkeys, and mice was measured using BIAcore, ELISA, and flow cytometry. The results are summarized in Table 3.

[0332] Table 3: Binding affinity of antibodies to human, monkey, and mouse CD137

[0333]

[0334] NC: No cross-reaction

[0335] 4a. Combining affinity and kinetics through SPR measurements

[0336] Use Biacore TMUsing a T200 instrument (Biacore AB, Uppsala, Sweden), the binding affinity and kinetics of antibodies to human, monkey, and mouse CD137 proteins were examined by surface plasmon resonance (SPR) analysis, following the manufacturer's instructions. Anti-human IgG (Fc) antibodies from the Human Antibody Capture Kit (GE BR-1008-39) were immobilized on a CM5 chip by amino-coupling them to the carboxylated surface of the sensor chip, according to the instructions for the amine conjugation kit (GE Biacore#BR-1000-50). AG10058, AG10059, AG10131, AC1121, and AC1097 were captured using the immobilized anti-human IgG (Fc) antibodies. Finally, six concentrations (3.13, 6.25, 12.5, 25, 50, 100) (nM) of human CD137-His6 (Sino Biological #10041-H08H) (diluted in running buffer) were injected at a flow rate of 30 μL / min for 300 seconds, with a dissociation time of 300 seconds. The running buffer used was 1×HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v / v surfactant P20, pH 7.4, 25°C). Blank flow cells without fixed proteins were used as corresponding controls for each condition to subtract “background”. Assignment and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines. As shown in Table 3, all antibodies bound to human CD137. AG10058 and AG10059 showed higher affinity than the two reference antibodies. All antibodies, except for the AC1121 reference mAb, bound monkey CD137. Only AG10058 and AG10131 bound mouse and rat CD137. AG10058 had a higher affinity (15.2 nM) than AG10131 (64.5 nM).

[0337] 4b. Measure the binding affinity to soluble CD137 using ELISA.

[0338] Serial dilutions of human, monkey, or mouse CD137 fused with the human FC fragment were prepared and used to coat ELISA plates, incubated at 37°C for 1 hour. After blocking, 100 μL of IgG (5 μg / mL) was added and incubated at 37°C for 1 hour. The plates were washed three times and then incubated at 37°C for 1 hour with HRP-conjugated protein L (1:2000 dilution). The plates were washed three times again and incubated at room temperature for 20 minutes with TMB substrate. After the reaction was stopped, the absorbance was measured at 450 nm. Data were analyzed using nonlinear fitting with Graphpad Prism 6. Figure 2 As shown, all antibodies bound human CD137 (FC fusion protein) with similar sub-nM affinity. Except for the AC1121 reference mAb, all antibodies bound monkey CD137 with similar sub-nM affinity. Consistent with results from Biacore, only AG10058 and AG10131 bound mouse CD137. AG10058 had a higher affinity (0.3 nM) than AG10131 (23.9 nM).

[0339] 4c. Measure the binding affinity to CD137 overexpressed on the cell surface by flow cytometry.

[0340] The affinity of the antibody for human, monkey, and mouse CD137 transiently expressed on the surface of HEK293F cells was also evaluated. Briefly, HEK293F cells were transfected with plasmids expressing full-length human, monkey, or mouse CD137 from bicistronic IRES vectors, and transfected cells were identified using EGFP. After 48 hours, transfected cells were harvested and then washed once with cold FACS buffer (PBS supplemented with 1% BSA). Cells were then incubated on ice for 1 hour with various IgGs (100 nM each), washed twice with pre-cooled FACS buffer, and compared with Alexa... The 647-conjugated mouse anti-human FC antibody was incubated together on ice for 30 min. The cells were washed once and then subjected to flow cytometry (…). Analysis was performed using CytoFlex. As shown in Figure 3a, all antibodies bound to human CD137 expressed on the cell surface with low nM affinity. AG10058, AG10059, and AG10131 were slightly superior to the two reference antibodies. Except for the AC1121 reference mAb, all antibodies bound to monkey CD137 with low nM affinity, with AG10058, AG10059, and AG10131 being slightly superior to the AC1097 reference antibody. Consistent with results from Biacore and ELISA, only AG10058 and AG10131 bound to mouse and rat CD137. AG10058 had a higher affinity for mouse CD137 than AG10131. In addition, AG10058 and AG10131 (each at 100 nM) also bound to rat and canine CD137 overexpressed on the surface of HEK293F cells (Figure 3b).

[0341] 4d. IgG binds to activated human, monkey, mouse, and rat T cells.

[0342] Species cross-reactivity of the exemplary antibodies was further confirmed using PMA and iomycin-stimulated PBMCs or T cells from humans, monkeys, mice, and rats. Human and cynomolgus monkey PBMCs were isolated by Ficoll-density gradient centrifugation. Briefly, fresh whole blood from healthy donors or cynomolgus monkeys was diluted with an equal volume of PBS and carefully loaded onto the top of a Histopaque 1077 (14 ml in a 50 ml centrifuge tube). Centrifugation was performed at 1,200 × g for 30 min at room temperature using brake-off. After centrifugation, the supernatant was carefully aspirated to a 0.5 cm opaque interface containing monocytes using a pipette. The supernatant was discarded. The opaque interface (approximately 3–5 ml) was carefully transferred to a clean 50 ml conical centrifuge tube using a pipette. Cells were washed with 20 ml of PBS, collected by centrifugation at 400 × g for 5 min, and resuspended in 20 ml of PBS. Cells were counted using a hemocytometer and collected again by centrifugation at 400 × g for 5 min. Mouse or rat spleen cells were isolated as follows: Spleens were passed through a 45 μm cell sieve attached to a 50-mL conical tube to obtain a single-cell suspension, and the cells were washed through the sieve with PBS. The cells were centrifuged at 1600 rpm for 5 min and the supernatant was discarded. The cell pellet was resuspended in 2 mL of erythrocyte lysis solution and centrifuged for 2 min. Cells were collected by adding 10-fold excess PBS and centrifuging at 1600 rpm for 5 min. The supernatant was discarded, and the spleen cells were resuspended in RPMI 1640 / 10% FBS. Pan-T cells were enriched from PBMCs (human / monkey) or spleen cells (mouse / rat) by negative selection using magnetic beads from a commercially available kit (Stemcell Technologies) that is specific to humans, monkeys, mice, and rats. Activation of human / monkey PBMCs or mouse / rat spleen cells was performed by incubating the cells overnight at 37°C and 5% CO2 with 50 ng / mL PMA + 1 μM iomycin.

[0343] Activate cells (~2x10) 5 Cells / tube were washed in pre-cooled staining buffer (PBS supplemented with 2% FBS) and incubated on ice for 1 hour with 100 nM test antibody. Cells were then washed twice with 1 mL staining buffer and resuspended in Alexa-contaminated water. Cells were incubated in 100 μL of staining buffer with 647-conjugated mouse anti-human FC antibody and species-specific T cell marker antibody. The T cell marker antibody used was CD3, CD4, or CD8. After incubation in the dark for 30 min, cells were washed twice with staining buffer. Finally, cells were resuspended in 300 μL of staining buffer and analyzed using Beckman CytoFlex. Data analysis was performed using Flowjo 10 software. As shown in Figure 4a, all tested antibodies bound activated human and monkey T cells, but not naïve human T cells. The binding affinity of AG10131 to activated mouse and rat T cells was further evaluated (Figure 4b). AG10131 binds to activated mouse and rat T cells.

[0344] In summary, the AG10058 and AG10131 antibodies showed higher affinity for human and monkey CD137. They exhibited broad species cross-reactivity, including humans, cynomolgus monkeys, mice, rats, and dogs for AG10131, but including humans, cynomolgus monkeys, mice, and dogs for AG10058, thus allowing for rapid evaluation of in vivo efficacy in mouse syngeneic models.

[0345] Example 5

[0346] Antibody binding selectivity to CD137

[0347] The selectivity of the antibody for CD137 was evaluated using flow cytometry analysis of its binding affinity to members of the TNFR superfamily. TNFRSF receptors, including CD137, OX40, CD40, GITR, and CD27, were transiently overexpressed on the surface of HEK293F cells. Transfected cells were washed in pre-cooled staining buffer (PBS supplemented with 2% FBS) and incubated on ice for 1 hour with 100 nM of the test antibody. Cells were washed twice with staining buffer and Alexa was added. 647-conjugated mouse anti-human FC antibody was incubated on ice for 30 min. The sample was washed once with staining buffer and then analyzed by flow cytometry. Figure 5 As shown, AG10058, AG10059, and AG10131 specifically bind to CD137, but do not specifically bind to any other family members tested or parental cells transfected with an empty vector.

[0348] Example 6

[0349] Ligand competition was tested using ELISA and flow cytometry.

[0350] The antibodies were tested by ELISA and flow cytometry to determine their ability to block the binding of CD137 to its homologous ligand CD137L. As shown in Figures 6a and 6b, all tested antibodies blocked the binding of CD137 and CD137L.

[0351] 6a. Ligand competitive binding was tested using ELISA.

[0352] Recombinant human CD137 (fused with human Fc and His tags) was diluted to 1 μg / mL in PBS and plated onto Maxisorp plates, incubated overnight at 4°C. The plates were blocked with PBS supplemented with 3% skim milk at 37°C for 1 hour. After washing, 50 μL of biotinylated CD137L (4 μg / mL) and a total volume of 100 μL of various concentrations of test antibody (eight 1:2 serial dilutions ranging from 500 μg / mL to 2 μg / mL) were added to each well and incubated at 37°C for 1 hour. The plates were washed three times and 100 μL of HRP-conjugated neutralavidin (1:1000) was added to each well and incubated at 37°C for 1 hour. The plates were washed as previously described, 50 μL of TMB substrate solution was added and incubated at room temperature for 20 minutes, then the reaction was stopped by 50 μL of H2SO4. As shown in Figure 6a, all tested antibodies AG10058, AG10059, and AG10131 blocked the binding of CD137 to CD137L. AG10131 exhibited the strongest or complete blocking ability in the approximately μM range, followed by AG10058 with significant blocking > μM; and AG10059 with effective blocking in the μM range. These data indicate that, under the tested conditions and using the reagents used, the broadly species-cross-reactive antibodies AG10131 and AG10058 are highly effective inhibitors of the interaction between CD137 and its ligand CD137L, while AG10059 only showed moderately effective blocking of the interaction between CD137 and its ligand CD137L. It should be noted that the reference antibody AC1097 cross-reacts with human and monkey CD137, while AC1121, which reacts only with human CD137, showed almost no blocking.

[0353] 6b. Detection of ligand competitive binding by flow cytometry

[0354] The plasmid encoding full-length human CD137 was transiently expressed in HEK293F cells. Cells were washed with staining buffer (PBS supplemented with 1% BSA) and resuspended in staining buffer containing 100 nM of the test antibody. After incubation on ice for 30 min, 33 nM of biotinylated CD137L was added to each well and the cells were incubated on ice for another 1 hr. Cells were washed twice with staining buffer, 50 μL of staining buffer containing Alexa fluor 647-conjugated streptavidin was added, and the cells were incubated on ice for 30 min. Cells were washed once and analyzed by CytoFlex flow cytometry. As shown in Figure 6b, all three test antibodies blocked the binding between CD137 and CD137L in a concentration-dependent manner. AG10131 showed the strongest blocking ability, followed by AG10058 with significant blocking ability, and AG10059 with less effective blocking ability. These data indicate that the broadly species-cross-reactive antibodies AG10131 and AG10058 highly effectively block the interaction between CD137 and its ligand CD137L, while AG10059 shows partial blockade of this interaction. In contrast, the AC1097 reference antibody, which cross-reacts with human and monkey CD137, shows only partial blockade, while the AC1121 reference antibody, which reacts only with human CD137, shows no blockade.

[0355] Example 7

[0356] Epitope plotting

[0357] To determine the binding region of the test antibody at the amino acid residue level, a series of mutations were performed on the extracellular domain of human CD137 (Table 5). HEK293F cells were transfected with these CD137 mutant plasmids. Binding of the antibody to the human CD137 mutant was assessed by flow cytometry analysis, as previously described in Example 5 and in [the following text is incomplete and requires further context to translate accurately]. Figure 7A The results are shown in Table 5. Together with the cross-reactivity of these antibodies with CD137 in human, monkey, mouse, and rat samples at the differentiation of interest, they indicate fine epitopes of the hits derived from the Adagene library. AG10131 bound to all four species, while AG10058 bound to all three CD137 species but not to rat CD137. AG10058, AG10059, and AG10131 lost their binding affinity to the GFT34AAA, FSS53AAA, and FH92AA mutations, indicating that their binding epitopes are located in these regions, such as amino acid residues 34-93 or 34-108 of SEQ ID NO.:1 (see also...). Figure 7BAG10058 and AG10131 can combine with the same or highly similar epitopes, while AG10059 can combine with epitopes that are different from AG10058 and AG10131.

[0358] The mutant constructs were designed to distinguish the epitopes bound by AG10058, AG10059, and AG10131 from those of the reference antibodies AC1121 and AC1097. Clearly, all three antibodies, AG10058, AG10059, and AG10131, target epitopes that are very different from those of AC1121 and AC1097. AG10058, AG10059, and AG10131 differ from AC1121 in the regions defined by the mutants Hu_FH92AA and Hu_FSS53AAA, and possibly Hu_GTF34AAA, while AG10058, AG10059, and AG10131 differ from AC1097 in the regions defined by most of the mutants used, except for Hu_FH92AA and their species cross-reactivity with monkeys, but differing in other species cross-reactivity such as mouse, rat, and dog CD137. In some embodiments, AG10058, AG10059, and AG10131, or other antibodies disclosed herein, do not bind to epitopes located within amino acid residues 115-156 of SEQ ID NO.:1. Figure 7A Table 5 also shows that the binding patterns of human CD137 ligands to wild-type human CD137 relative to mutant human CD137 are well matched with the binding patterns of the tested antibodies, which is consistent with the observation that these antibodies block the binding of CD137 ligands to their receptors.

[0359] Table 5: Plotting of Table Positions

[0360]

[0361]

[0362] Example 8

[0363] Agonist activity of antibodies in NFκB luciferase reporter gene assay

[0364] The agonist activity of the antibody was assessed using an NFκB reporter gene assay. 293T cells were transfected with a plasmid expressing human, monkey, or mouse CD137 and an NFκB luciferase reporter gene plasmid. Four hours later, the antibody was injected with 0.4 x 10⁻⁶ cells / mL. 650 μL of cells were plated into each well of a 96-well plate at a density of / mL. A total volume of 50 μL antibody mixture containing the test antibody and a 3:1 ratio of cross-linked antibody (Fab's goat anti-human IgG FC) was added and incubated for 18 h. After removing the culture medium, 50 μL of Passive lysis buffer (Promega E1980) was added and incubated at 37 °C for 30 min. 20 μL of the lysate was transferred to a white plate and luciferase substrate was added. The luminescence signals of fireflies and renin (renina) were measured, and their ratios were used for data analysis using GraphPad Prism 6.0 software. Figure 8 As shown, compared to the isotype control antibody, all tested antibodies activated NFκB reporter gene expression when human and monkey CD137 was expressed. When mouse CD137 was expressed, AG10058 and AG10131, but not AG10059, activated NFκB reporter gene expression. This is consistent with previous observations that AG10058 and AG10131 bound mouse CD137, while AG10059 did not.

[0365] Example 9

[0366] Antibody agonist activity in T cell activation assay

[0367] The agonist activity of the antibody was further confirmed in a T-cell activation assay. 50 μL of anti-CD3 antibody (2 μg / mL) was spread onto 96-well cell culture plates, either alone or together with 50 μL of the test antibody (60 μg / mL, 20 μg / mL, 6 μg / mL, 2 μg / mL, and 0 μg / mL) in 1×PBS, and incubated overnight at 4°C. CD8+ T cells were isolated using the manufacturer's instructions. The assay was performed at 1×10⁻⁶ cells / mL. 7 Cells were prepared at a density of 10 cells / mL in RPMI 1640 medium supplemented with 10% FBS. 200 μL of cells were plated into each well and incubated at 37°C, 5% CO2 for 4 days. Cell proliferation was examined daily under a microscope. After 96 hours of incubation, 100 μL of supernatant was transferred to a new 96-well plate for IFN-γ assay. T cell proliferation was measured using the Cell Titer Glow Kit (Promega). Figure 9 As shown, compared to the isotype control antibody, all tested antibodies induced CD8+ T cell proliferation and IFN-γ secretion in a dose-dependent manner.

[0368] Example 10

[0369] Antitumor activity in mouse syngeneic models

[0370] Species cross-reactivity with mouse CD137 allows for rapid in vivo functional assessment. AG10058 and AG10131 have been tested in multiple mouse syngeneic models. 2 × 10⁻⁶ cells were subcutaneously transplanted into BALB / c mice (n = 8 / group). 6 One H22 hepatocellular carcinoma cell (Xiao et al., Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo. Clin Cancer Res. 2007; 13(6):1823-30.), 5×10 5 One CT26 colon cancer cell, or 5 × 10 5 EMT6 breast cancer cells. When the tumor is established (>50mm) 3 Treatment began with intraperitoneal injections of an allotype control antibody, AG10058, or AG10131 twice weekly for up to 3 weeks. Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. Figure 10-13 As shown, compared with the isotype control antibody, AG10058 and AG10131 exhibited effective in vivo antitumor activity in these different syngeneic mouse tumor models.

[0371] 10a.CD137 agonist antibody showed antitumor efficacy in an H22 mouse hepatocellular carcinoma model.

[0372] First, AG10058 or AG10131 were administered twice weekly at a dose of 50 mg / kg for 3 weeks. Both molecules showed almost 100% TGI (tumor growth inhibition). Figure 10 (Figure a). Immunohistochemical staining for CD4 and CD8 markers showed that AG10131 significantly increased the infiltration of CD4+ and CD8+ T cells in the H22 tumor microenvironment (Xiao et al., Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo. Clin Cancer Res. 2007; 13(6):1823-30). Figure 10 (Figure b). Further titration at a dose reduced to 3 mg / kg still showed ~100% TGI, indicating that both molecules possess potent antitumor activity. Figure 10(See figures c and d). Further dose titrations of AG10131 reduced to 1 and 0.1 mg / kg at 0.1 mg / kg and 1 mg / kg showed a TGI greater than 50% ( ). Figure 10 (small picture e).

[0373] 10b.CD137 agonist antibody showed antitumor efficacy in a CT26 mouse model of colon cancer.

[0374] like Figure 10 As shown, AG10058 and AG10131 exhibited nearly 100% TGI (tumor growth inhibition) at a dose of 50 mg / kg in the CT26 mouse colon cancer model. Figure 11 (Figure a)(Martinez-Forero et al., T cell costimulation with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes. J Immunol. 2013; 190(12):6694-706). Further dose titration of AG10131 ( Figure 11 Figure b) shows nearly 100% TGI at doses of 5 mg / kg and 1 mg / kg. At a dose of 0.1 mg / kg, approximately 40% TGI was achieved, indicating dose-dependent antitumor activity.

[0375] 10c.EMT6 breast cancer model

[0376] Further evaluation of antitumor activity in an EMT6 mouse breast cancer syngeneic model (Shi and Siemann, Augmented antitumor effects of radiation therapy by 4-1BB antibody (BMS-469492) treatment. Anticancer Res. 2006; 26:3445-53) Figure 12 Both AG10058 and AG10131 showed almost 100% inhibition of tumor growth.

[0377] 10 days. Mice that fully responded to CD137 agonist antibody treatment remained tumor-free after rechallenge with new tumor cells.

[0378] In the CT26 tumor model, mice treated with AG10058 or AG10131 for 3 weeks showed complete tumor regression and remained untreated for over a month. Then, on day 62, mice maintaining a complete response were administered 5 × 10⁻⁶ oz. ...5 Subcutaneous re-challenge was performed using CT26 tumor cells, and tumor growth was monitored. A re-challenge control group was established using naïve mice inoculated with the same number of CT26 tumor cells. Figure 13 As shown, treatment with AG10131 (at 1 and 5 mg / kg, see [reference]). Figure 13 (Top and bottom insets, respectively) demonstrated potent antitumor activity in the CT26 tumor model, with 5 / 8 of the AG10131 (1 mg / kg group) and 6 / 8 of the AG10131 (5 mg / kg group) showing complete response within 60 days prior to re-excitation with CT26 tumor cells. Furthermore, these mice remained tumor-free after re-excitation with the same tumor cells, indicating the development of specific antitumor memory in these mice.

[0379] To test this hypothesis, spleen cells were collected from these tumor-rejection re-challenged mice and control mice and co-cultured in vitro for 7 days with mitomycin C-arrested CT26 tumor cells to expand tumor-specific memory T cells. These spleen cells were then recovered and mixed with fluorescently labeled live CT26 tumor cells at different E / T ratios for 4 hours, and tumor cell killing was detected by live / dead staining and FACS analysis. Figure 14 As shown, significantly increased tumor cell killing was observed using spleen cells from tumor-rejected re-excited mice previously treated with AG10058 and AG10131.

[0380] Example 11

[0381] AG10131-IgG4 does not induce ADCC effect

[0382] Using EasySep CD8 + T-cell enrichment kit (StemCell Technologies) isolates human CD8 from peripheral blood from healthy donors. + T cells were then stimulated in vitro for 18 hours with PMA (50 ng / ml) + iomycin (1 uM). These activated CD8 cells were then labeled with calcein-AM. + T cells were used as target cells. NK cells from different healthy donors were isolated using a human NK cell isolation kit (StemCell Technologies) and used as effector cells. For antibody-dependent cytotoxicity (ADCC) assays, effector cells (NK) and target cells (activated CD8+) were mixed in the absence and with serially diluted antibodies. +T cells were mixed in 96-well plates at a 5:1 ratio and incubated for 4 hours. Supernatant from each well was then collected, and fluorescence was detected using a SpectraMax i3x reader (Ex 488nm, Em 520nm). Allotype hIgG4 mAb was used as a negative control, while humanized OKT3 (anti-CD3 hIgG1 from Novoprotein) was used as a positive control. % lysis was then calculated using the following formula: % lysis = [(experimental release) – Ave(target + NK)] / [Ave(target Max) – Ave(target only)] × 100% Figure 15 ).

[0383] Example 12

[0384] Developmental characteristics of antibodies

[0385] For developmental evaluation, purified AG10058, AG10059, AG10131, and AC1097 were exchanged for PB buffer (20 mM PB, 150 mM NaCl, pH 7.0). All experiments, including filtration, centrifugation, and accelerated stress tests, were performed in PB buffer. For all SEC-HPLC analyses, a TSKgel column (Tosoh Bioscience G3000SWxl) was used.

[0386] 12a. Solubility

[0387] All three antibodies could be concentrated to above 100 mg / ml in PB buffer without significant precipitation (Table 6). The antibodies were then adjusted to 20 mg / ml in PB buffer. The samples (10 μg each) were then analyzed by SEC-HPLC to detect high molecular weight (HMW) aggregates. See the chromatograms (…). Figure 16 As shown in the figure, no increase in HMW aggregates was observed at high concentrations (20 mg / ml) for all tested antibodies.

[0388] Table 6: Antibody Solubility

[0389]

[0390] 12b. Antibody solubility under accelerated stress conditions

[0391] Antibody solubility was also examined under accelerated stress conditions, and the results are summarized in Table 7. All antibodies remained stable after six freeze (-80°C) and thaw (room temperature) cycles. Figure 17 After seven days at 50°C, the changes in HMW aggregates or LMW fragments were minimal. Figure 17In experiments with longer timeframes (up to 28 days at 40°C), all antibodies remained stable, and there was no significant increase in HMW aggregates or LMW fragments. Figure 17 ).

[0392] Table 7: Changes in HMW under acceleration conditions

[0393]

[0394] Furthermore, thermal stability measurements using differential scanning calorimetry (DSC) showed that AG10131 and AG10058 were stable at temperatures up to at least approximately 59 °C. The transition midpoint Tm (the characteristic temperature at which almost all protein domains undergo unfolding transitions) is... Figure 18 And as shown in Table 8 below.

[0395] Table 8: Thermal stability measured by DSC

[0396] Tm starting point (°C) Tm1 (°C) Tm2 (°C) AG10058 61.5 67.3 76.9 AG10131 59.3 67.6 81.5

[0397] Furthermore, the highest achievable concentrations of AG10131 and AG10058 after centrifugation exceeded 180 mg / mL and 220 mg / mL, respectively.

[0398] Example 13

[0399] Safety characteristics in relevant species: mice and cynomolgus monkeys

[0400] Repeated-dose toxicity study of 13a.AG10131 in normal C57BL / 6 mice.

[0401] Repeat-dose toxicity assessment of AG10131 was conducted in normal C57BL / 6 mice. The vehicle, AG10131 (100 mg / kg), was administered intraperitoneally (ip) on days 1, 4, 8, and 11. Each group included five female mice (7–8 weeks old). Mice were monitored daily for abnormal behavior and symptoms, and food intake and body weight were measured daily. On day 14, animals were euthanized for necropsy and other analyses. Blood was collected from each animal, with two blood samples from each group used for hematological analysis (RBC, platelets, WBC, WBC differential) and three additional blood samples from each group used for blood biochemical analysis (AL, AST, ALB, GLB, A / G, TBIL, ALP, GGT, and LDH). The following organs from each mouse were collected and preserved in FFPE: heart, lung, thymus, liver, spleen, and kidney. FFPE blocks and sections of liver tissue were prepared and stained with H&E for histopathological analysis.

[0402] No abnormal behavior or unplanned animal deaths were observed throughout the study's in-life period. AG10131 did not affect food intake or body weight compared to vector treatment. No significant lesions were observed upon necropsy in mice treated with AG10131. Hematological analyses showed no significant changes, as did blood biochemical parameters tested in mice treated with AG10131. Figure 19 No obvious abnormalities were found in the histopathological sections of the livers of all these mice. Figure 20 Overall, AG10131 was well tolerated in this study and no significant toxicity was observed in mice.

[0403] 13b. Repeated-dose study of AG10131 in cynomolgus monkeys

[0404] A repeated-dose study of AG10131 was conducted in normal cynomolgus monkeys. Human IgG4 isotype control (10 mg / kg) and AG10131 (0.5 and 10 mg / kg) were administered intravenously (iv) (1 mL / kg) on ​​days 0, 7, 14, and 22. Each group included one male and one female cynomolgus monkey (3–5 years old). Abnormal behavior and clinical signs were monitored daily, and food intake was measured daily. Body weight was measured on days (-15) and (-5) before administration and on days 6, 13, 18, and 26 after administration. Hematological and blood chemistry parameters were measured on days -12 and -5 before administration and on days 7, 14, 19, and 27 after administration (10 mg / kg group only). Urine analysis was performed on days -12 and -5 before administration and on days 6, 13, and 18 after administration. Animals in the 10 mg / kg group were euthanized on day 27 for necropsy and other analyses. Major organs were removed and weighed. FFPE liver tissue blocks were prepared, sections were prepared, and H&E staining was performed for histopathological analysis.

[0405] No abnormal behavior or unplanned animal deaths were observed in any group throughout the study's in-life period. Treatment with 10 mg / kg AG10131 did not affect food intake or body weight compared to vector treatment. No clinical signs, including injection site reactions, were observed. Autopsy examination of all organs in cynomolgus monkeys treated with 10 mg / kg AG10131 revealed no significant lesions or weight abnormalities. Hematological, blood chemistry, and urinary parameters were also within normal ranges in all treatment groups. Figure 21 Following repeated administration of AG10131 at 10 mg / kg, histopathological analysis of the liver showed no significant abnormalities, including lymphocytic infiltration. Figure 22Overall, AG10131 was well tolerated in cynomolgus monkeys at doses up to 10 mg / kg per week, and no significant toxicity was detected.

[0406] Example 14

[0407] Pharmacokinetics of AG10131 in cynomolgus monkeys

[0408] Pharmacokinetics of 14a.AG10131 in cynomolgus monkeys

[0409] Pharmacokinetic studies of AG10131 were conducted in cynomolgus monkeys. Three groups of monkeys were administered AG10131 intravenously at three dose levels (10 mg / kg, 30 mg / kg, and 100 mg / kg). Each group contained three males and three females. Serum samples were collected before administration and at 0.083, 0.25, 0.5, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, 336, 408, 504, 672, and 840 hours after administration. Serum concentrations of AG10131 were determined by ELISA.

[0410] Of the 16 animals, AG10131 was rapidly cleared on day 14 (336 hours), i.e., all animals from the low- and medium-dose groups, and 2 of the 6 animals from the high-dose group. On day 21, two more animals from the high-dose group showed rapid clearance. Serum concentrations in these 14 animals were low or below the quantification limit. This is consistent with the observation of the development of anti-drug antibodies in these animals. Data from two animals from the high-dose group with potentially unaffected pharmacokinetics were fitted to predict pharmacokinetic parameters (…). Figure 23 The half-life of AG10131 is in the range of 7.3 to 8.8 days.

[0411] Pharmacokinetics of 14b.AG10131 in rats

[0412] Pharmacokinetic studies of AG10131 were conducted in naïve SD rats. Three groups of animals were administered AG10131 intravenously at three dose levels (10 mg / kg, 30 mg / kg, and 100 mg / kg). Each group contained 15 males and 15 females. Serum samples were collected from all three animals at the following time points: before administration, and 0.083, 0.25, 0.5, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, 336, 408, 504, 672, and 840 hours after administration. Serum concentrations of AG10131 were determined by ELISA, and data were analyzed using Phoenix Professional V6.3.

[0413] Results: PK parameters from low, medium, and high doses were similar. Figure 24 The clearance rate of AG10131 is approximately 0.004 ml / kg / min. The half-life of AG10131 ranges from 11.5 to 14.6 days.

[0414] Pharmacokinetics of 14c.AG10131 in mice

[0415] Pharmacokinetic studies of AG10131 were conducted in approximately 8-week-old BALB / c mice. Three female BALB / c mice in each administration group were intravenously injected with a test antibody containing AG10131 via tail vein at a dose of 1 mg / kg. Blood samples (approximately 100 μL per sample) were collected at 1 h, 8, 48, 168, and 336 hours post-administration. Blank control blood was collected from three primiparous female mice that did not receive the antibody. Serum concentrations of each test antibody containing AG10131 were determined by ELISA, with anti-human IgG (Fc-specific) antibody used for capture and HRP-labeled anti-human IgG (Fab-specific) antibody used for detection.

[0416] All tested antibodies, including the isotype control (AG10154), two benchmark antibodies (AC1020 and AC1021), and three Adagene antibodies (AG10131, AG10058, and AG10059), exhibited comparable pharmacokinetic activity in mice. Figure 25 ).

[0417] Example 15

[0418] Further epitope mapping

[0419] To determine the binding epitopes of the Adagene antibody presented in this paper, as well as other reference antibodies, we adopted a systematic approach to dissect the epitopes at three resolution levels: domains, motifs, and residues. Extracellular domains of CD137 containing four CRD motifs and CD137 from four different species, such as human, monkey, mouse, and rat CD137, were used (Table 9). A series of human CD137 CRD motifs (cysteine-enriched domains) and their constructs containing one, two, and three units of the human CRD motif are shown (Table 9). Human CD137 CRD was expressed in *Saccharomyces cerevisiae* under the control of an inducible GAL1-10 promoter using a low copy number CEN / ARS-based vector (Boder and Wittrup (1997) Nat Biotechnol 15(6):553-7). Antibody binding to human CD137 CRD was evaluated by flow cytometry and other techniques, as previously described in Example 5 and in [the following text is missing from the original] Figure 27 As shown in the image.

[0420] The results are summarized in Table 9. These antibodies selectively bind only to the CD137 target, not to any of the non-CD137 targets listed in the table; however, the species-specific cross-reactivity of these antibodies with CD137 from human, monkey, mouse, and rat species is significant, highlighting the fine epitope coverage of the different hits screened from the Adagene Dynamic Precision library. For example, AG10131 binds to CD137 from human, monkey, mouse, rat, and dog (not shown); AG10058 binds to human, monkey, and mouse CD137, but not rat CD137; while AG10059 binds to human and monkey CD137. In contrast, the reference antibody AC1121 from transgenic mice binds only to human CD137, while another reference antibody obtained from the morphosys phage library binds to both human and monkey CD137. For comparison purposes, it should be noted that the human ligand CD137L interacts only with the human CD137 receptor and not with the mouse CD137 receptor, and the mouse CD137L interacts only with the mouse CD137 receptor and not with the human CD137 receptor (see Table 9).

[0421] Table 9

[0422]

[0423] To further dissect the binding motifs of these antibodies to the human CD137 target summarized in Table 9, and comparing the dissected CD137 CRD motifs with their combined binding CD137 ligands, the identified binding sites of Adagene and other reference antibodies were well separated and noted: AG10058, AG10059, and AG10131 antibodies and human CD137 ligands do not bind to a single CRD or both CRD units (CRD2-CRD3) of human CD137. Similar to human CD137 ligands, AG10058, AG10059, and AG10131 antibodies can bind to all three CRD units (CRD1–CRD2-CRD3) of human CD137. Although the reference antibody AC1121 can also bind to all three CRD units (CRD1–CRD2-CRD3) of human CD137, it binds to only two specific CRD units (CRD1-CRD2) of human CD137. In contrast, the three CRD units (CRD1-CRD2-CRD3) of human CD137 are required for the binding of AG10058, AG10059, AG10131, and human CD137 ligands. Reference antibody AC1097 can bind two CDR units (CRD3-CRD4), encompassing all three CDR units of human CD137. These indicate that the Adagene AG10058, AG10059, and AG10131 antibodies bind to epitopes covered by the three CDR units (CRD1–CRD2-CRD3) of human CD137, similar to CD137 ligands, but they differ significantly from reference antibodies AC1121 (binding two CDR units (CRD1–CRD2)) and AC1097 (binding two CDR units (CRD3–CRD4)) of human CD137. In summary, the CD137 binding epitopes of Adagene's antibodies differ from those bound by the two reference antibodies (AC1121 and CRD1-CRD2; and AC1097 and CRD3-CRD4), as shown by the differences in the specific CDRs used and the number of CDR units required (see Table 9B), and are highly similar (if not overlapping) to the CD137 epitopes bound by the human CD137L ligand; the CD137-CD137L epitopes are confirmed by recently reported crystal structure complexes, such as... Figure 26 Shown (Gilbreth, RN, Oganesyan, VY, Amdouni, H., Novarra, S., Grinberg, L., Barnes, A., Baca, M. (2018) J. Biol. Chem. 293:9880-9891).

[0424] Table 9B.

[0425]

[0426]

[0427] To determine the binding epitopes of Adagene's antibody and reference antibody at the amino acid residue level, a series of mutations were performed on the extracellular domain of human CD137 (Table 5). HEK293F cells were transfected with these CD137 mutant plasmids. Binding of the antibody to the human CD137 mutant was assessed by flow cytometry analysis, as previously described in Example 5 and in [the following text is incomplete and requires further context]. Figure 7A The results are summarized in Table 5, along with the cross-reactivity of these antibodies with human, monkey, mouse, and rat CD137 at the differences of interest, indicating fine epitopes from the hits derived from the Adagene library. AG10131 binds human, monkey, mouse, and rat CD137, while AG10058 binds human, monkey, and mouse CD137 but not rat CD137. The binding epitopes of CD137 bound by AG10058, AG10059, and AG10131 map to the CRD1-CRD2-CRD3 units of CD137, and they lose their binding ability to GFT34AAA, FSS53AAA, and FH92AA mutations, indicating that their binding epitopes are located in these regions, such as amino acid residues 34-93 or 34-108 of SEQ ID NO.:1 (see also...). Figure 7B AG10058 and AG10131 can bind to the same or highly similar epitopes, while AG10059 can bind to epitopes different from those of AG10058 and AG10131. Single mutants such as T35A, F36A, F53A, R66A, F72A, N83A, and F92A showed loss of binding of Adagene antibodies AG10058, AG10059, and AG10131 to human CD137, as well as loss of binding to CD137L, with the exception of R66A, which still maintains binding of CD137 to its ligand. However, single mutants P32A and P49A lost the binding between CDL137L and CD137, but their effect on the interaction between the antibody and CD137 was altered. F125A showed that AC1097 does not bind to CD137 at all, but has no effect on the binding of other antibodies (including human CD137L). In summary, the overall binding pattern of mutants throughout CD137 does indeed reveal significant information: the Adagene antibody and its reference antibody differ at their binding sites.

[0428] The mutant constructs were designed to distinguish the epitopes bound by AG10058, AG10059, and AG10131 from those of the reference antibodies AC1121 and AC1097. These antibodies, AG10058, AG10059, and AG10131, target epitopes very different from those of AC1121 and AC1097. AG10058, AG10059, and AG10131 differ from AC1121 in the regions defined by the mutants Hu_FH92AA and Hu_FSS53AAA, and possibly Hu_GTF34AAA, while AG10058, AG10059, and AG10131 differ from AC1097 in the regions defined by most of the mutants used, except for Hu_FH92AA and their species cross-reactivity with monkeys, but differing in other species cross-reactivity such as mouse, rat, and dog CD137. In some embodiments, AG10058, AG10059, and AG10131, or other antibodies disclosed herein, do not bind to epitopes located within amino acid residues 115-156 of SEQ ID NO.:1. Figure 7A Table 5 also shows that the binding patterns of human CD137 ligands to wild-type human CD137 relative to mutant human CD137 are well matched with the binding patterns of the tested antibodies, which is consistent with the observation that these antibodies block the binding of CD137 ligands to their receptors.

[0429]

[0430]

[0431]

[0432] Example 16

[0433] Blocking natural CD137L signal transduction via AG10131

[0434] In vitro binding assays performed by ELISA demonstrated that AG10131 can block the interaction between recombinant CD137 and its ligand. To further validate this ligand-blocking activity of AG10131 functionally, a cellular NFκB luciferase reporter gene assay was performed. Briefly, 293T cells stably expressing the NFκB luciferase reporter gene were transfected with a DNA construct expressing human CD137, and the cells were co-cultured with human B-cell lymphoma cells Daudi or Raji at different ratios. The cell mixture was incubated overnight with serial dilutions of an allotype control or a ligand-blocking anti-CD137 antibody, and luciferase activity was measured using the Promega luciferase assay kit according to the manufacturer's instructions. Relative luciferase units (RLUs) were calculated relative to the level of luciferase expressed in 293T cells in the absence of antibody treatment.

[0435] like Figure 28 As shown, Daudi (top row) and Raji (bottom row) cells express functional CD137 ligands to activate the NFκB luciferase reporter gene in 293T cells. Compared with the isotype control antibody (left column), the addition of AG10131 (right column) to the co-culture system significantly inhibited NFκB signaling stimulated by both cell types, indicating that the AG10131 antibody can functionally block CD137 signaling stimulated by CD137 ligands expressed on Daudi and Raji B lymphoma cells.

[0436] Example 17

[0437] Anti-CD137 antibody crosslinking in NFκB reporter gene assay

[0438] Three anti-CD137 antibodies (AG10131, AC1121, and AC1097) were tested using a functional cellular NFκB reporter gene assay. Figure 29 As shown, upon cross-linking, all three anti-CD137 antibodies were able to stimulate human CD137 receptor signaling at comparable levels in a dose-dependent manner. The EC50 values ​​of antibody-induced NFκB signaling activation responses for all three anti-CD137 antibodies were in similar ranges. However, AC1121 exhibited unique properties distinct from AG10131 and AC1097. AC1121 was able to significantly activate human CD137 receptor signaling in the absence of cross-linking, while AG10131 and AC1097 could not. The EC50 values ​​of AC1121 were found to be at similar levels in the presence or absence of cross-linking during stimulation of CD137 receptor signaling.

[0439] Example 18

[0440] AG10131 does not cause CDC

[0441] The CDC activity of AG10131 was determined by direct binding of AG10131 to purified C1q fractions of human complement using ELISA. Figure 30 As shown, AG10131 and its human IgG4 isotype control antibody lacked the ability to bind human complement C1q components within the tested concentration range, while the human IgG1 isotype control antibody was able to bind C1q. This result suggests that AG10131 is also unlikely to cause complement-dependent cytotoxicity, consistent with its IgG4 isotype framework.

[0442] Example 19

[0443] Anti-CD137 antibody AG10131 enhances tumor-infiltrating T lymphocytes

[0444] The in vivo antitumor efficacy studies in the syngeneic mouse H22 hepatocellular carcinoma, EMT6 breast cancer, and CT26 colon cancer models shown in Example 10 demonstrated that AG10131 treatment strongly inhibited tumor growth. AG10131 is an agonist antibody that activates T cells, and therefore it is expected that AG10131 treatment will stimulate tumor-infiltrating T cells into the tumor microenvironment, thereby mediating the intracellular antitumor effect. To evaluate the effect of AG10131 treatment on tumor-infiltrating lymphocytes, tumors from the in vivo antitumor efficacy studies of AG10131 in the mouse H22, EMT6, and CT26 cancer models were collected at the end of the study.

[0445] Figure 31 Representative IHC staining images of mouse CD4+ and CD8+ T cells in H22 tumor (top left), EMT6 tumor (top right), and CT26 tumor (bottom middle) are shown. Figure 32 As shown, a small number of T lymphocytes (CD4+ or CD8+ T cells) were present in H22, EMT6, and CT26 tumors treated with the control drug, while AG10131 treatment significantly stimulated CD4+ and CD8+ T cells (in Figure 31 (Indicated by black arrows) Infiltrates into the tumor. These data are consistent with the function of AG10131 as an immune agonist, mediating antitumor effects by stimulating T cell proliferation, activation, and infiltration into the tumor microenvironment.

[0446] Example 20

[0447] Enhanced antitumor efficacy in a CT26 colon cancer model by combining anti-CD137 antibody AG10131 and anti-PD1 antibody.

[0448] Next, the combined effect of anti-CD137 antibody AG10131 and anti-PD1 antibody was tested in a CT26 colon cancer model. CT26 tumor cells (3 × 10⁻⁶) were subcutaneously injected into the right lower ventral region of each female BALB / c mouse. 5 This allows for tumor development. When the average tumor volume reaches 98 mm... 3 Ten mice were assigned to each experimental group. These groups received intraperitoneal injections twice weekly of a mediator (PBS), 5 or 10 mg / kg of AG10131, 10 mg / kg of anti-PD-1, or a combination of 5 or 10 mg / kg of AG10131 and 10 mg / kg of anti-PD-1 mAb, for 3 weeks. Tumor volume was measured, and tumors reaching 2000 mm² were counted. 3 Each mouse was euthanized either at the endpoint volume or on the last day (day 42) (whichever comes first).

[0449] like Figure 33As shown, AG10131 (5 mg / kg or 20 mg / kg) and anti-PD1 (10 mg / kg) delayed tumor progression, but AG10131 delayed tumor progression by a slightly longer number of days and, in rare cases, caused tumor shrinkage. However, almost all mice treated with either AG10131 or anti-PD1 eventually died from tumor progression. Importantly, when AG10131 (5 mg / kg or 20 mg / kg) was administered in combination with anti-PD1 (10 mg / kg), the tumors in most mice were essentially cured, with only 2 (out of 10) or 1 (out of 10) mice escaping tumor suppression in the combinations of AG10131 (5 mg / kg) with anti-PD1 (10 mg / kg) or AG10131 (20 mg / kg) with anti-PD1 (10 mg / kg), respectively. These results demonstrate the strong synergistic enhancement effect of AG10131 and anti-PD1, indicating that the combination of AG10131 and anti-PD1 can be effective in anti-PD1 resistant tumors. sequence list <110> Adagene Inc. <120> Anti-CD137 molecule and its uses <130> 695402000341 <140> Not yet allocated <141> at the same time <150> PCT / CN2017 / 098332 <151> 2017-08-21 <160> 883 <170> PatentIn version 3.5 <210> 1 <211> 255 <212> PRT <213> Homo sapiens <400> 1 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> variants <222> (1)..(1) <223> X1: F or Y <220> <221> variants <222> (4)..(4) <223> X2: S or T <220> <221> variants <222> (5)..(5) <223> X3: G or N or S <220> <221> variants <222> (7)..(7) <223> X4: A, G, or W <400> 2 Xaa Thr Phe Xaa Xaa Tyr Xaa Ile His Trp Val 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Variants <222> (4)..(4) <223> X1: S or T <220> <221> Variants <222> (7)..(7) <223> X2: H or Y <220> <221> Variants <222> (8) <223> X3: H or Y <220> <221> Variants <222> (10)..(10) <223> X4: A or D or G or N or S or T <400> 3 Tyr Ser Ile Xaa Ser Gly Xaa Xaa Trp Xaa Trp Ile 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Variants <222> (6) <223> X1: G or S <220> <221> Variants <222> (9)..(9) <223> X2: A or G <220> <221> Variants <222> (11)..(11) <223> X3: A or G or S or T <400> 4 Phe Ser Leu Ser Thr Xaa Gly Val Xaa Val Xaa Trp Ile 1 5 10 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Variants <222> (7)..(7) <223> X1: A, D, or Y <220> <221> Variants <222> (8) <223> X2: D or G <220> <221> Variants <222> (11)..(11) <223> X3: R or S or Y <220> <221> Variants <222> (14)..(14) <223> X4: P or T <400> 5 Leu Ala Leu Ile Asp Trp Xaa Xaa Asp Lys Xaa Tyr Ser Xaa Ser Leu 1 5 10 15 Lys Ser Arg Leu 20 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> variants <222> (3) <223> X1: D or E <220> <221> variants <222> (9)..(9) <223> X2: N or S <220> <221> variants <222> (13)..(13) <223> X3: N or S <400> 6 Ile Gly Xaa Ile Tyr His Ser Gly Xaa Thr Tyr Tyr Xaa Pro Ser Leu 1 5 10 15 Lys Ser Arg Val 20 <210> 7 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> variants <222> (3) <223> X1: A or G or S or V or Y <220> <221> variants <222> (7)..(7) <223> X2: A or D or S or Y <220> <221> variants <222> (9)..(9) <223> X3: D or G or S <220> <221> variants <222> (10)..(10) <223> X4: S or T <400> 7 Val Ser Xaa Ile Ser Gly Xaa Gly Xaa Xaa Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg Phe 20 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> variants <222> (3) <223> X1: E or G <220> <221> variants <222> (5)..(5) <223> X2: E or S <220> <221> variants <222> (6)..(6) <223> X3: D or T <220> <221> variants <222> (7)..(7) <223> X4: A or T or V <220> <221> variants <222> (9)..(9) <223> X5: A or I or L or T or V <220> <221> variants <222> (14)..(14) <223> X6: A, D, or G <400> 8 Ala Arg Xaa Gly Xaa Xaa Xaa Val Xaa Gly Asp Trp Phe Xaa Tyr 1 5 10 15 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Variants <222> (1)..(1) <223> X1: Q or R <220> <221> Variants <222> (5)..(5) <223> X2: D or G or S <220> <221> Variants <222> (6) <223> X3: I or V <220> <221> Variants <222> (7)..(7) <223> X4: G or R or S or T <220> <221> Variants <222> (8) <223> X5: P or R or S or T <220> <221> Variants <222> (9)..(9) <223> X6: A or D or F or S or V or Y <220> <221> Variants <222> (10)..(10) <223> X7: L or V <220> <221> Variants <222> (11)..(11) <223> X8: A, G, or N <400> 9 Xaa Ala Ser Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Variants <222> (1)..(1) <223> X1: A or D <220> <221> Variants <222> (4)..(4) <223> X2: N or S or T <220> <221> Variants <222> (5)..(5) <223> X3: L or R <220> <221> Variants <222> (6) <223> X4: A, E, or Q <220> <221> Variants <222> (7)..(7) <223> X5: S or T <220> <221> Variants <222> (9)..(9) <223> X6: I or V <400> 10 Xaa Ala Ser Xaa Xaa Xaa Xaa Gly Xaa 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> Variants <222> (5)..(5) <223> X1: A, G, S, or Y <220> <221> variants <222> (7)..(7) <223> X2: Q or S or Y <220> <221> variants <222> (8)..(8) <223> X3: I or L or T or Y <220> <221> variants <222> (9)..(9) <223> X4: I or S or V or W <400> 11 Tyr Cys Gln Gln Xaa Tyr Xaa Xaa Xaa Thr 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> variants <222> (3) <223> X1: E or Q <220> <221> variants <222> (5)..(5) <223> X2: P or S or Y <220> <221> variants <222> (6)..(6) <223> X3: D or L or S or T or Y <220> <221> variants <222> (7)..(7) <223> X4: D or E or H or S or T <220> <221> variants <222> (8)..(8) <223> X5: D or L or T or W <220> <221> Variants <222> (10)..(10) <223> X6: L or P or R or V <400> 12 Tyr Cys Xaa Gln Xaa Xaa Xaa Xaa Pro Xaa Thr 1 5 10 <210> 13 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ala Gly Asp Thr Thr Tyr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Asp Tyr Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 14 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 14 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Ser His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 15 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Ala Val Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ile Ile Asn Pro Asn Phe Gly Asp Thr Asn Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Glu Tyr Tyr Gly Gly Ser Tyr Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 16 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 17 <211> 121[[ID=​​​​​​​​​​Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Gly Ser Gly Gly Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Asp Leu Tyr Ser Trp Tyr Phe Asp Val Trp Gly [[ID=,,26]]100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 18 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80<00016३6>Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <21०> 19 <211> 120 <212> PRT <213> Artificial Sequence <22०> <223> Synthetic Sequence <40०> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly '1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 It should be noted that there seems to be a typo in the original text where "<00016३6>" should probably be " ", and "<21०>" should probably be "<210>". The translation has been done based on the provided text with these potential typos.Tyr His Trp Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ala Ile Ser Gly Ala Gly Gly Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Gly Tyr Gly Gly Ser Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence[[ID=3)]] <400> 20 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Thr Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 21 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Thr Asp Gly Tyr Thr Thr Glu Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Gly Gly His Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 22 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Glu Gln Pro Leu Glu Leu Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 23 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 His His Trp Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Ser Gly Tyr Gly Asp Thr Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Pro Tyr Ser Ser Gly Ser Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 24 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Tyr Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 25 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Thr Tyr Ser Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 26 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Ile Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 27 <211> 117 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 28 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 28 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Thr Ser Thr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Trp Pro 85 90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 29 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr His Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ser Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Pro Asn Tyr Tyr Ser Ser Gly Ser Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 30 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Phe 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 31 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Asp Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ser Ile Ser Gly Asp Gly Asp Thr Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Tyr Tyr Gly Tyr Gly Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 108 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 32 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 33 <211> 119 <212> PRT <213> Artificial Sequence [[ID=!16]]<220> <223> Synthetic Sequence <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 His Tyr Trp Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Ala Leu Ile Asp Trp Tyr Gly Asp Lys Tyr Tyr Ser Thr Ser Leu 50 55 60 Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Tyr Gly Ser His Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 34 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 34 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Phe Asp 20 25 30 Gly Phe Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp 85 90 95 Thr Leu Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 35 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly Asp Gly Asp Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ser Thr Thr Val Ala Gly Asp Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 36 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Phe 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Leu Val Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg <210> 37 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Ser Asp Ala Val Leu Gly Asp Trp Phe Gly 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 38 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 39 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Asp Ala Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 40 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 40 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 41 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Thr Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 42 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 42 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Pro Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 43 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Ala Gly Asp Lys Ser Tyr Ser Thr Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Thr Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 44 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 44 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Pro Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Thr Trp Thr 0]85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 45 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr His Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ser Ile Ser Gly Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Asp Ala Val Thr Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 46 <211> 107<​​​​​​<223> Synthetic Sequence <400> 46 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Gly Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg[[ID=...]] 100 105 <210> 47 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Thr Ala Val Ala Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 48 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Arg Thr Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro[[ID=!2]] 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 49 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Asp Val Val Thr Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 50 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 50 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Val 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 51 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 [[ID=ID=33]]Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 His His Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ala Ile Ser Gly Asp Gly Ser Thr Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Thr Ala Val Thr Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 52 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 52 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val Gly Tyr Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Thr Trp Thr Phe 85 90 95 Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 53 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Ala Gly Asp Lys Ser Tyr Ser Thr Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Glu Asp Ala Val Thr Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 54 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Arg Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 55 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Gly Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp<​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> Synthetic Sequence <400> 56 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 57 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Gly Ile Ser Gly Asp Gly Gly Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Glu Asp Tyr Gly Pro His Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 58 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 58 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Thr Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Tyr Ser Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 59 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 His Tyr Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ala Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Thr Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 60 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 60 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 61 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 His Tyr Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ser Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Ala Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 62 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 62 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 63 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 63 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Asp Gly Asp Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Arg Gly Leu Val Leu Asp Ala Phe Asp Tyr Trp Gly Gln 100 105 110 [[ID=*]]Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 64 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 64 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Gly Arg 20 25 30 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 四 45 Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Tyr Trp Pro 85 90 95 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 65 <211> 123 <212> PRT <213> Artificial Sequence <220> It should be noted that there is an unclear "四" in line 27 which might be an error in the original text. If this is a specific code or term that has a correct value, it needs to be adjusted accordingly. <223> Synthetic sequence <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Ala Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 66 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 66 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 67 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 His Tyr Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Ser Gly Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Ala Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 68 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 68 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 69 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 69 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 His Tyr Trp Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ala Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Ala Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 70 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 70 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Gly Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gln Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 71 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Ser Asp Thr Val Ile Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 72 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 72 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 73 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Gly Ile Ser Gly Ala Gly Asp Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Ser Asp Thr Val Leu Gly Asp Trp Phe Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 74 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 74 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 75 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Ala Val Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Arg Tyr Ser Thr Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Thr Thr Val Val Gly Asp Trp Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 76 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 76 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 77 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 77 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Ser Asp Val Val Ala Gly Asp Trp Phe Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 78 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 78 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Val 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gln Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 79 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr His Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Tyr Tyr Tyr Gly Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 80 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthetic sequence <400> 80 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val Gly Ser Val 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr His Asp Pro Val Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 81 <211> 124 <212> PRT[[ID= / / ID=17]] <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Ala[[ID= / / ID=27]] 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Gly Tyr Gly Ser Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Ser Asp Ala Val Leu Gly Asp Trp Phe Gly 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 82 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 82 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 83 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 83 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr His Trp Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ser Ile Ser Gly Tyr Gly Asp Thr Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Asp Thr Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 84 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 84 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Arg Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ile Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 85 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 85 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 His His Trp Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Asp Thr Val Leu Gly Asp Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 86 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 86 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 87 <211> 123 <212> PRT <213> Artificial Sequence<00..3140><220> <223> Synthetic sequence <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Ala Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Leu Ile Asp Trp Asp Gly Asp Lys Ser Tyr Ser Thr Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Thr Ala Val Val Gly Asp Trp Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 88 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 88 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 89 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 89 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Gly Ala Gly Gly Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Gly Ser Thr Ala Val Thr Gly Asp Trp Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 90 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 90 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Pro Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 91 <211> 124[[ID=I5]] <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 91 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Gly Ser Gly Asp Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Ser Asp Thr Val Val Gly Asp Trp Phe Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 92 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 92 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 93 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 93 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Gly Asp Gly Asp Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Ser Asp Thr Val Ala Gly Asp Trp Phe Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 94 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 94 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Thr Thr Pro Leu<00,03309>85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 95 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 95 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Gly Tyr Gly Gly Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly Ser Asp Thr Val Leu Gly Asp Trp Phe Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser[[ID=三十二]] [[ID=三十三]]115 120[[ID=三十四]] [[ID=三十五]]<210> 96[[ID=三十六]] [[ID=三十七]]<211> 108[[ID=三十八]] [[ID=三十九]]<212> PRT[[ID=四十]] [[ID=四十一]]<213> Artificial Sequence[[ID=四十二]] [[ID=四十三]]<2​​​​​Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 97 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 97 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Gly Ala Gly Gly Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gl...

Claims

1. An isolated antibody that binds to the extracellular domain of human CD137, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises HVR-H1 consisting of the amino acid sequence of SEQ ID NO:878, HVR-H2 consisting of the amino acid sequence of SEQ ID NO:879, and HVR-H3 consisting of the amino acid sequence of SEQ ID NO:880; and (ii) the light chain variable region comprises HVR-L1 consisting of the amino acid sequence of SEQ ID NO:881, HVR-L2 consisting of the amino acid sequence of SEQ ID NO:882, and HVR-L3 consisting of the amino acid sequence of SEQ ID NO:883, And wherein the HVRs are numbered according to Kabat.

2. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

72.

3. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 657, and the light chain comprises the amino acid sequence of SEQ ID NO:

658.

4. A vector comprising a polynucleotide encoding the antibody or antigen-binding fragment according to any one of claims 1 to 3.

5. The vector of claim 4, wherein the vector is an expression vector. A host cell comprising the vector according to claim 4 or 5.

7. A method for preparing an antibody or an antigen-binding fragment, comprising culturing the host cell according to claim 6 under conditions suitable for producing the antibody or the antigen-binding fragment.

8. The method of claim 7, further comprising recovering the antibody or antigen-binding fragment produced by the cell.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

10. Use of the antibody or antigen-binding fragment of any one of claims 1 to 3 in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer is breast cancer, lung cancer, colon cancer, liver cancer, endometrial cancer, skin cancer, bile duct cancer, nasopharyngeal carcinoma (NPC), head and neck cancer, B-cell lymphoma or non-Hodgkin's lymphoma.

11. The use according to claim 10, wherein the medicament further comprises at least one additional therapeutic agent, wherein: (i) the at least one additional therapeutic agent is selected from the group consisting of: viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, and chemotherapy; and / or (ii) the at least one additional therapeutic agent is selected from the group consisting of pomalidomide, lenalidomide, lenalidomide, pomalidomide, thalidomide, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, IL-12, IFNα, GM-CSF, adoptively transferred T cells, anti-cancer vaccines and oncolytic viruses.

12. The use according to claim 11, wherein the radiation therapy is single dose radiation, fractionated radiation, focal radiation or whole organ radiation.

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