Anti-CD137 molecules and their uses

Abstract

The present disclosure provides antibodies or antigen-binding fragments thereof that bind to human CD137, nucleic acids encoding the antibodies or antigen-binding fragments thereof, therapeutic compositions of the antibodies or antigen-binding fragments thereof, and their uses for enhancing T cell function to upregulate cell-mediated immune response and for treating T cell dysfunction disorders, such as tumor immunity, and for treating cancer.

Description

[0001] Reference to a sequence listing

[0002] The following submission on ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 695402000340seqlist.txt, Record Date: August 3, 2017, Size: 537 KB). Technical Field

[0003] The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to human CD137, nucleic acids encoding the antibodies or antigen-binding fragments thereof, therapeutic compositions of the antibodies or antigen-binding fragments thereof, and their anti-tumor uses. Background Art

[0004] 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 shows that its expression is usually activation-dependent and is present in a wide subset of immune cells, including activated NK and NKT cells, regulatory T cells, dendritic cells (DC), stimulated mast cells, differentiating myeloid cells, monocytes, neutrophils and eosinophils (Wang, 2009, Immunological Reviews 229: 192-215). CD137 expression has also been demonstrated on tumor blood vessels (Broll, 2001, Amer. J. Clin. Pathol. 115(4): 543-549; Seaman, 2007, Cancer Cell 11: 539-554) and on endothelial sites of inflammation or atherosclerosis (Drenkard, 2007 FASEB J. 21: 456-463; Olofsson, 2008, Circulation 117: 1292-1301). The ligand that stimulates CD137, CD137 ligand (CD137L), is expressed on activated antigen-presenting cells (APCs), bone marrow progenitor cells, and hematopoietic stem cells.

[0005] Human CD137 is a 255 amino acid protein (GenBank Accession Nos. NM_001561; NP_001552; SEQ ID NO.: 1). The protein contains 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 the CD137 ligand for signal transduction.

[0006] Many studies on mouse and human T cells have shown 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, resulting in anti-tumor efficacy in various models. CD137 agonist mAbs have demonstrated efficacy in both preventive and therapeutic settings. In addition, CD137 monotherapy and combination therapy tumor models have established long-lasting anti-tumor protective T cell memory responses (Lynch, 2008, Immunol Rev. 22: 277-286). CD137 agonists have also been shown to inhibit autoimmune reactions in a variety of art-recognized autoimmune models (Vinay, 2006, J Mol Med 84: 726-736). This dual activity of CD137 provides the potential to provide anti-tumor activity while reducing autoimmune side effects that can be associated with immunotherapy methods that break immune tolerance.

[0007] There is a long-standing unmet need for antibodies that bind to human CD137, increase CD137-mediated responses, and thereby provide potential therapeutic agents for treating various diseases and conditions, including cancer and autoimmune diseases. In addition, there is a need for anti-CD137 antibodies that cross-react between different species, such as humans and experimental animals (mice, monkeys, dogs, etc.), to enable animal model studies and simultaneously provide therapeutic agent candidates. Summary of the Invention

[0008] An object of the present disclosure is to provide an isolated binding molecule, such as an antibody or its binding fragment, or a derivative thereof, that binds to human CD137. Another object of the present disclosure is to provide a composition comprising a binding molecule that binds to CD137. Yet another object of the present disclosure is to provide a method for treating diseases and / or conditions associated with or mediated by CD137 signaling, which is achieved by using one or more binding molecules of the present disclosure. These and other objects of the present disclosure are described more fully herein.

[0009] Thus, in one aspect, provided herein are one or more antibodies (e.g., isolated antibodies) or one or more antigen-binding fragments thereof that bind to the extracellular domain of human CD137 and comprise 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 characteristics: (a) binds to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO: 1; (b) does not bind 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) binds to the extracellular domain of human CD137 with a K of 100 nM or less. D (d) binds to human CD137; (e) does not bind to human OX40, CD40, GITR and / or CD27 receptors at concentrations up to 1000 nM; (f) cross-reacts with monkey, mouse, rat and / or dog CD137; (g) does not induce ADCC effect; (h) can inhibit tumor cell growth; (i) has therapeutic effects on cancer; and / or (j) blocks the binding between CD137 and CD137L.

[0010] Thus, in one aspect, provided herein is an antibody (e.g., an isolated 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: NO: 4), wherein X1 is G or S, X2 is A or G, and X3 is A, G, S or T; wherein HVR-H2 comprises an amino acid sequence according to a formula 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: 7). 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 comprises an amino acid sequence according to 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.

[0011] In some embodiments, provided herein is an antibody (e.g., an isolated antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137, the antibody or antigen-binding fragment thereof 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: NO: 855), wherein X1 is G or S, X2 is A or G, and X3 is A, G or S; wherein HVR-H2 comprises an amino acid sequence according to a formula 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; 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 comprises an amino acid sequence according to 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.

[0012] In another aspect, provided herein is an antibody (e.g., an isolated 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), 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. 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 comprises an amino acid sequence according to a formula selected from the group consisting of: 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.

[0013] In some embodiments, provided herein is an antibody (e.g., an isolated antibody) or an antigen-binding fragment thereof that binds to the extracellular domain of human CD137, the antibody or antigen-binding fragment thereof 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: 858). 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 comprises an amino acid sequence according to 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.

[0014] On the other hand, provided herein is an antibody (e.g., an isolated 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 comprises 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: 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 VL28 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).

[0015] On the other hand, provided herein is an antibody (e.g., an isolated 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 a heavy chain variable region 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, VH41, VH42, VH43, VH44, VH45, VH46, VH47, VH48, VH49, VH50, VH51, VH52, VH53, VH54, VH55, VH56, VH57, VH58, VH59, VH6 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 , 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, VL60, VL61, VL62, VL63, VL64, VL65, VL66, VL67, VL68, VL69, VL70, VL71, VL72, VL73, VL74, VL75 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: 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 VH40 and VL40, VH41 and VL41, VH42 and VL42, VH43 and VL43, VH44 and VL44, VH45 and VL45, VH46 and VL47, VH48 and VL49, VH50 and VL51, VH52 and VL53, VH54 and VL55, VH56 and VL57, VH58 and VL59, VH59 and VL60, VH61 and VL61, VH62 and VL62, VH63 and VL64, VH65 and VL66 and VL59, or VH60 and VL60 (as shown in Table 1c).

[0016] On the other hand, provided herein is an antibody (e.g., an isolated 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 within 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 within 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 amino acid residues 34-36, one or more amino acid residues 53-55, and one or more 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 of the 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 monkey, mouse, rat, and / or dog. In some embodiments, the antibody or antigen-binding fragment binds to cynomolgus monkey CD137.

[0017] 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 comprising the amino acid sequence of SEQ ID NO: 711, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 735, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 759; and / or wherein the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 783, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 807, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 831. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody comprises a heavy chain and a light chain, and wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 617, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 618.

[0018] 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 comprising the amino acid sequence of SEQ ID NO: 712, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 736, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 760; and / or wherein the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 784, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 808, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 832. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and / or the light chain variable region comprises the amino acid sequence of 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 of SEQ ID NO: 619, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 620.

[0019] 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 comprising the amino acid sequence of SEQ ID NO: 731, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 755, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 779; and / or wherein the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 803, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 827, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 851. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, and / or the light chain variable region comprises the amino acid sequence of 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 of SEQ ID NO: 657, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 658.

[0020] In another aspect, provided herein is an antibody (e.g., an isolated 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 a formula selected from the group consisting of: Formula (I), Formula (II), and Formula (III); HVR-H2 comprises an amino acid sequence according to a 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 a formula selected from the group consisting of: Formula (X) and Formula (XI). In some embodiments, provided herein is an antibody (e.g., an isolated 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 a formula selected from the group consisting of: Formula (XIII) and Formula (XVI); HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (IV) and Formula (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).

[0021] In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 253-312, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 313-372, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 373-432, HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 433-492, HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 493-552, and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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: The amino acid sequence of the group consisting 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.

[0022] In some embodiments, HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (XII), Formula (XIII) and Formula (XIV); HVR-H2 comprises an amino acid sequence according to Formula (IV) or Formula (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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 709-732, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 733-756, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 757-780, HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 781-804, HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 805-828, and HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 829-852. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 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.

[0023] In some embodiments, HVR-H1 comprises the amino acid sequence of SEQ ID NO: 711 or 731; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 735 or 755; HVR-H3 comprises the amino acid sequence of SEQ ID NO: 759 or 779; HVR-L1 comprises the amino acid sequence of SEQ ID NO: 783 or 803; HVR-L2 comprises the amino acid sequence of SEQ ID NO: 807 or 827; and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 831 or 851. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41 or 71, and the light chain variable region comprises the amino acid sequence of 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 of SEQ ID NO: 617 or 657, and the light chain comprises the amino acid sequence of SEQ ID NO: 618 or 658.

[0024] In some embodiments, HVR-H1 comprises the amino acid sequence of SEQ ID NO: 712; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 736; HVR-H3 comprises the amino acid sequence of SEQ ID NO: 760; HVR-L1 comprises the amino acid sequence of SEQ ID NO: 784; HVR-L2 comprises the amino acid sequence of SEQ ID NO: 808; and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 832. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and the light chain variable region comprises the amino acid sequence of 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 of SEQ ID NO: 619, and the light chain comprises the amino acid sequence of SEQ ID NO: 620.

[0025] In some embodiments that may be combined with any of the preceding embodiments, the antibody or antigen-binding fragment has a K of 100 nM or less. D Binds to human CD137 (e.g., as measured by surface plasmon resonance). In some embodiments, the antibody or antigen-binding fragment binds to human CD137 with a K of 50 nM or less. D Binds to human CD137 (e.g., as measured by surface plasmon resonance).

[0026] In some embodiments that may be combined with any of the preceding embodiments, the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from cynomolgus monkey (e.g., GenBank Gene ID 102127961), mouse (e.g., GenBank Gene ID 21942), rat (e.g., GenBank Gene ID 500590), and / or dog (e.g., GenBank Gene ID 608274). In some embodiments, the antibody or antigen-binding fragment binds to cynomolgus monkey CD137.

[0027] In some embodiments that may be combined with any of the preceding embodiments, the activity of human CD137 (eg, when expressed on cells such as human cells) is reduced when contacted with the antibody or antigen-binding fragment.

[0028] In some embodiments that may be combined with any of the preceding embodiments, the antibody or antigen-binding fragment has a half-maximal inhibitory concentration (IC) of about 100 nM or less for blocking the binding of human CD137 to human CD137L in vitro. 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 may be combined with any of the preceding embodiments, the activity of human CD137 (e.g., when expressed on cells such as human cells) is increased when contacted 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 increased NF-κB-dependent transcription.

[0029] In some embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG2 Fc region. In some embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises an S241P mutation, wherein numbering is according to Kabat. In some embodiments, the antibody or antigen-binding fragment does not elicit an ADCC effect.

[0030] In another aspect, provided herein is a composition comprising a polypeptide selected from the group consisting 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, 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 a polynucleotide comprising a sequence 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 , 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 sequences. In some embodiments, provided herein are antibody heavy chains encoded by a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 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 a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 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.

[0031] In another aspect, provided herein is a polynucleotide encoding any one of the antibodies or antigen-binding fragments described herein. In some embodiments, provided herein is a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 133-252.

[0032] In another aspect, provided herein is a vector comprising any one of the polynucleotides described above. In some embodiments, the vector is an expression vector.

[0033] On the other hand, provided herein is a host cell (e.g., a bacterial cell, a yeast cell, an insect cell, a mammalian cell (such as a CHO cell or a 293T cell), etc.), comprising any of the polynucleotides or vectors described herein. In some embodiments, provided herein is a method for preparing an antibody or antigen-binding fragment, comprising culturing the host cell under conditions suitable for producing the antibody or antigen-binding fragment. In some embodiments, the method further comprises recovering the antibody or antigen-binding fragment produced by the host cell.

[0034] In another aspect, provided herein is a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments described herein (or any derivatives thereof) and a pharmaceutically acceptable carrier.

[0035] On the other hand, provided herein is a method for treating abnormal cell growth (for example, cancer) in a subject in need thereof, comprising administering to the subject any one of an antibody, Fab and / or pharmaceutical composition as described herein in a therapeutically effective amount. In some embodiments, provided herein is a method for reducing tumor cell metastasis in a subject, comprising administering to the subject any one of an antibody, Fab and / or pharmaceutical composition as described herein in a therapeutically effective amount. In some embodiments, the method further comprises administering to the subject at least one (for example, at least one, at least two, at least three, at least four, at least five, at least 10, etc.) other therapeutic agent of a therapeutically effective amount. In some embodiments, at least one other therapeutic agent is selected from the group consisting of: viral gene therapy, immune checkpoint inhibitors, target therapy, radiotherapy and chemotherapy. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of pomalyst, revlimid, 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, radiation therapy, single-dose radiation, fractionated radiation, focal radiation radiation), whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptors, adoptively transferred T cells, anticancer vaccines, and oncolytic viruses. Also provided herein are pharmaceutical compositions, antibodies, and / or antigen-binding fragments (or any derivatives thereof) described herein for use in treating abnormal cell growth (e.g., cancer) and / or reducing tumor cell metastasis in subjects in need. Also provided herein are uses of any of the antibodies or antigen-binding fragments (or any derivatives thereof) described herein for the manufacture of a drug for treating abnormal cell growth (e.g., cancer) and / or reducing tumor cell metastasis in subjects in need.

[0036] It will be appreciated that one, some, or all of the features of the various embodiments described above and herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A Shown are hypervariable region (HVR) definitions compared to the Kabat CDR definitions for an exemplary heavy chain variable region (VH) (SEQ ID NO: 13) and an exemplary light chain variable region (VL) (SEQ ID NO: 14).

[0038] Figure 1B Shown is a selection of Fab hits that cross-react with mouse CD137.

[0039] Figure 2 ELISA binding assays of exemplary antibodies to human, monkey and mouse CD 137 are shown. Each panel is for a different antibody as indicated at the top of the panel.

[0040] Figure 3A Shown are FACS-based binding assays of exemplary antibodies to human, monkey, mouse and rat CD 137. Each panel is directed against a different antigen as indicated at the top of the panel.

[0041] Figure 3B A comparison of species cross-reactivity between exemplary antibodies and a reference antibody is shown.

[0042] Figure 4A The exemplary antibodies are shown to bind to activated human and monkey T cells but not to naive Human T cells.

[0043] Figure 4B Binding of AG10131 to activated human, monkey, mouse and rat T cells is shown.

[0044] Figure 5 The binding specificity of exemplary antibodies is shown for CD137, but not other TNFR family members.

[0045] Figure 6A and Figure 6B Exemplary antibodies are shown to block the binding of CD137 to its cognate ligand CD137L, as determined by ELISA ( Figure 6A ) and flow cytometry ( Figure 6B )get.

[0046] Figure 7A Shown are the results of epitope mapping obtained by flow cytometry.

[0047] Figure 7B Shown are multiple sequence alignments of portions 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.

[0048] Figure 8 Agonist activity of exemplary antibodies in an NFκB reporter gene assay is shown.

[0049] Figure 9 Agonist activity of exemplary antibodies in CD8+ T cell proliferation (top panel) and INF-γ secretion (bottom panel) is shown.

[0050] Figure 10 The anti-tumor efficacy of the exemplary antibodies in the H22 mouse liver cancer model is shown, as well as the CD4 + and CD8 + T cell infiltration in tumors.

[0051] Figure 11 The anti-tumor efficacy of exemplary antibodies in the CT26 mouse colon cancer model is shown.

[0052] Figure 12 The anti-tumor efficacy of exemplary antibodies in the EMT6 mouse breast cancer model is shown.

[0053] Figure 13 It is shown that CT26 mice treated with exemplary antibodies remained tumor-free after rechallenge with the same tumor cells.

[0054] Figure 14 Tumor cell killing achieved using splenocytes from tumor-rejecting rechallenged mice is shown.

[0055] Figure 15 It was shown that AG10131 did not show ADCC effect.

[0056] Figure 16 The exemplary antibodies are shown to exhibit little aggregation at high concentrations.

[0057] Figure 17 Shown are the stability of exemplary antibodies under accelerated stress conditions.

[0058] Figure 18 Shows thermal stability.

[0059] Figure 19 AG10131 was shown to have no hematological toxicity in normal mice at doses up to 100 mg / kg every two weeks (BIW) x 2.

[0060] Figure 20 AG10131 was shown to have no histological liver abnormalities in normal mice at up to 100 mg / kg biweekly (BIW) x 2.

[0061] Figure 21 AG10131 was shown to have no hematological toxicity in cynomolgus monkeys at 10 mg / kg / week x 4.

[0062] Figure 22 AG10131 was shown to be non-hepatotoxic in monkeys at 10 mg / kg / week x 4.

[0063] Figure 23 Shown are the pharmacokinetic characteristics of AG10131 in monkeys.

[0064] Figure 24 Shown are the pharmacokinetic characteristics of AG10131 in rats.

[0065] Figure 25 Shown are the pharmacokinetic characteristics of various antibodies in mice. DETAILED DESCRIPTION

[0066] A. Definition

[0067] Unless otherwise defined herein, the scientific and technical terms used in connection with the present disclosure will have the meaning commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural, and plural terms should include singular. In general, the nomenclature used in conjunction with antibody engineering as described herein, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and the technology of antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry are well known in the art and commonly used those.

[0068] As used herein, each of the following terms has the meaning associated with it in this section.

[0069] The articles “a” and “an” refer to one or to a plurality of (i.e., to at least one) the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0070] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, 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 the C-terminal carboxyl group, the N-terminal amino group, or a side chain functional group has been chemically modified to another functional group. The term "amino acid mimetics" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid.

[0071] The term "antibody" is used herein in the broadest sense 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), so long as they exhibit the desired biological activity.

[0072] The term "antibody" is a recognized term in the art and may refer to an antigen binding protein (i.e., an immunoglobulin) having a basic four polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain has a variable region (abbreviated herein as V) at the N-terminus. H , ), followed by the constant region. The heavy chain constant region consists of three domains: C H1 、C H2 and C H3 Each light chain has a variable region (abbreviated herein as V I ), followed by a constant region at its other end. The light chain constant region consists of one domain, C L . V L With V H Compare and C L Aligned with the first constant domain (CH1) of the heavy chain. H and V L IgM antibodies are composed of five basic heterotetrameric units and another polypeptide called the J chain, thus containing 10 antigen-binding sites; whereas secretory IgA antibodies can polymerize to form multivalent assemblies containing 2-5 basic four-chain units and the J chain.

[0073] V H and V LThe regions can also be further subdivided based on structural and sequence analysis into regions of high variability, termed hypervariable regions (HVRs). HVRs are interspersed with more conserved regions, termed framework regions (FWs). For comparison, the Kabat CDR definitions of 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) are listed below (see also Figure 1a). Each V H and V L It consists of three HVRs and four FWs, arranged in the following order from amino terminus to carboxyl terminus: FW1, HVR1, FW2, HVR2, FW3, HVR3, FW4. Throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR_H1, HVR_H2, and HVR_H3. Similarly, the three HVRs of the light chain are referred to as HVR_L1, HVR_L2, and HVR_L3.

[0074] The variable region of heavy chain and light chain contains the binding domain that interacts with antigen.The constant region of antibody can mediate the combination of immunoglobulin and host tissue or factor, including various cells (for example, effector cells) of immune system and the first component (C1q) of classical complement system.In light chain and heavy chain, variable region and constant region are connected by having about 12 or more amino acid " J " district, wherein heavy chain also comprises having about 10 or more amino acid " D " district.Generally referring to Fundamental Immunology the 7th chapter (Paul, W. compiles, 2nd edition, Raven Press, NY (1989)).

[0075] 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. Depending on the amino acid sequence of the constant domain (CH) of its heavy chain, antibodies can be designated as different species or isotypes. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. IgG class antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, by the γ heavy chains Y1-Y4, respectively.

[0076] The term "antibody derivative" or "derivative" of an antibody refers to a molecule that can bind to the same antigen (e.g., CD137) to which the antibody is bound and comprises an amino acid sequence of the antibody connected to another molecular entity. The amino acid sequence of the antibody contained in the antibody derivative can be the full-length heavy chain, full-length light chain, any one or more parts of the full-length heavy chain, any one or more parts of the full-length light chain, any other one or more fragments of the antibody, or a complementary antibody. Another molecular entity can be a chemical or biological molecule. Examples of another molecular entity include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies), and chemical compounds. Another molecular entity can have any practicality, such as being used as a detection agent, label, marker, medicament, or therapeutic agent. The amino acid sequence of an antibody can be attached or connected to another molecular entity by chemical coupling, gene fusion, non-covalent association, or other means. The term "antibody derivative" also encompasses chimeric antibodies, humanized antibodies, and modifications of the amino acid sequence of CD137 antibodies, such as conservative amino acid substitutions, additions, and insertions derived molecules.

[0077] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retain the ability to bind to the antigen to which the antibody binds (e.g., CD137). Examples of "antigen-binding fragments" of an antibody include (i) a Fab fragment, which is a fragment consisting of V L 、V H 、C L and C H1 (ii) F(ab')2 fragment, which is a bivalent fragment consisting of two Fab fragments linked by a disulfide bond in the hinge region; (iii) H and C H1 (iv) an Fd fragment consisting of a single-arm V L and V H Fv fragment composed of structural domains; (v) composed of V H domains (Ward et al., Nature 341:544-546 (1989)); and (vi) isolated complementarity determining regions (CDRs).

[0078] The term "binding molecule" encompasses (1) antibodies, (2) antigen-binding fragments of antibodies, and (3) derivatives of antibodies, each as defined herein.

[0079] The term "binding CD137 / binds CD137" or "binding to CD137 / binds to CD137" refers to a binding molecule as defined herein that binds to CD137 with an affinity (K) of 100 nM or less in an in vitro assay.D ) to human CD137, as determined by an in vitro assay such as the Biacore assay described in Example 4.

[0080] The terms "CD137" and "CD137 receptor" are used interchangeably herein and include the human CD137 receptor, as well as variants, isoforms, and species homologs thereof. Thus, the binding molecules as defined and disclosed herein may also bind to CD137 from species other than human. In other cases, the binding molecules may be completely specific for human CD137 and may not exhibit species or other types of cross-reactivity.

[0081] The term "CD137 antibody" refers to an antibody as defined herein that is capable of binding to the human CD137 receptor.

[0082] The term "chimeric antibody" refers to antibodies comprising amino acid sequences derived from different animal species, such as those having variable regions derived from human antibodies and murine immunoglobulin constant regions.

[0083] The term "competitive binding" refers to the interaction of two antibodies in their binding to a binding target. A first antibody competes with a second antibody for binding if the binding of a first antibody to its cognate epitope is detectably reduced in the presence of a second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternative situations in which 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 so. That is, a first antibody may inhibit the binding of a second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, where each antibody detectably inhibits the binding of the other antibody to its cognate 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 corresponding epitopes.

[0084] The term "epitope" refers to the part on an antigen that is combined with an antibody (or its antigen-binding fragment). An epitope can be formed by continuous or discontinuous amino acids juxtaposed by the tertiary folding of a protein. The epitope formed by continuous amino acids is usually retained when exposed to a denaturing solvent, while the epitope formed by the tertiary folding is usually lost when treated with a denaturing solvent. An epitope can include different numbers of amino acids in a unique spatial conformation. The method for determining the spatial conformation of an epitope includes, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with antigen and computer modeling of its complex structure with its binding antibody and variants thereof. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, G.E. Morris, ed., (1996). Once the desired epitope of an antigen has been determined, antibodies against the epitope can be generated, for example, using technology as described herein. The generation and characterization of antibodies can also illustrate information about the desired epitope. From this information, one can then competitively screen for antibodies that bind to the same epitope. A method for achieving this is to perform cross-competition studies to find antibodies that compete with each other for binding, i.e., antibodies that compete for binding to the antigen. A high-throughput method for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0085] The term "germline" refers to the nucleotide sequences of antibody genes and gene segments as they are passed from parent to offspring via germ cells. Germline sequences differ from nucleotide sequences encoding antibodies in mature B cells, which have been altered by recombination and hypermutation events during B cell maturation.

[0086] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a position for attaching a sugar residue. The amino acids to which carbohydrates (such as oligosaccharides) are attached are typically asparagine (N-bond), serine (O-bond), and threonine (O-bond) residues. The specific site of attachment is typically marked by an amino acid sequence referred to herein as a "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 chemical groups that serve as attachment sites between a sugar molecule and an amino acid residue. N-linked sugars are attached via amino groups; O-linked sugars are attached via hydroxyl groups. The term "glycan occupancy" refers to the presence of a carbohydrate moiety attached to a glycosylation site (i.e., the glycan site is occupied). Where there are at least two potential glycosylation sites on a polypeptide, neither (0-glycan site occupied), one (1-glycan site occupied), or both (2-glycan site occupied) sites may be occupied by carbohydrate moieties.

[0087] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, for example, mammalian cultured cells derived from rodents (rat, mouse, guinea pig or hamster), 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 in transgenic animals or cultured tissues. The term encompasses not only specific subject cells, but also progeny of such cells. Because certain modifications may occur in succession due to mutations or environmental influences, the progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

[0088] The term "human antibody" refers to antibodies in which the entire amino acid sequence of both the light and heavy chains is derived from human immunoglobulin genes. If produced in mice, mouse cells, or hybridomas derived from mouse cells, human antibodies may contain murine carbohydrate chains. Human antibodies can be prepared by a variety of means known in the art.

[0089] The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues derived from human antibody sequences. A humanized antibody may comprise some or all CDRs or HVRs from non-human animals or synthetic antibodies, while the framework and constant regions of the antibody comprise amino acid residues derived from human antibody sequences.

[0090] The term "exemplary antibodies" refers to any of the antibodies described in this disclosure and designated as those listed in Tables 1a and 1b. These antibodies may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above encompasses antibodies for V L and V H The present invention also includes antibodies in all five classes having the same amino acid sequence in the V region. In addition, antibodies in the IgG class may belong to any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each of the above-identified antibodies in the IgG subclass encompasses antibodies for V L and V H The amino acid sequences of the heavy chain constant regions of human antibodies in all four subclasses of IgG4 and IgG5 are known in the art. The amino acid sequences of the full-length heavy and light chains of each of the IgG4 subclass of the exemplary antibodies shown in Table 1b are provided in the present disclosure.

[0091] The term "isolated antibody" or "isolated binding molecule" refers to an antibody or binding molecule as defined herein that: (1) is not associated with naturally associated components with which it is adjacent in its native state; (2) is free of other proteins from the same species; (3) is expressed by cells from a different species; or (4) does not occur in nature. Examples of isolated antibodies include CD137 antibodies that have been affinity purified using CD137, CD137 antibodies that have been produced in vitro by hybridomas or other cell lines, and CD137 antibodies derived from transgenic animals.

[0092] The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules separated from chromosomes with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the target nucleic acid).

[0093] The term "k a ” refers to the association rate constant for a specific antibody-antigen interaction, while the term “k d ” refers to the dissociation rate constant for a specific antibody-antigen interaction.

[0094] 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 ) and expressed as molar concentration (M). K DUsed as a measure of the binding affinity of an antibody to its binding partner. D The smaller the K, the tighter the antibody binds, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant will bind more tightly to a specific antigen than an antibody with a micromolar (μM) dissociation constant. D The value can be determined using well-established methods in the art. D One approach is to use surface plasmon resonance, typically with biosensor systems such as System. Using BIACORE TM The assay procedure performed by the system (BIAcore assay) is described in the Examples section of this disclosure.

[0095] The term "mammal" refers to any animal species of the mammalian species. Examples of mammals include humans; laboratory animals such as rats, mice, apes, and guinea pigs; domestic animals such as cats, dogs, rabbits, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like.

[0096] The terms "prevent" or "preventing" with respect to a disease condition in a mammal means preventing or delaying the onset of the disease, or preventing the appearance of clinical or subclinical symptoms thereof.

[0097] 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 conventionally determined using known computer programs such as Bestfit, FASTA or BLAST (see, e.g., 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 Bestfit or any other sequence alignment program is used to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are set so that the percentage of identity is calculated over the full length of the reference amino acid sequence and an interval of up to 5% homology is allowed for the total number of amino acid residues in the reference sequence. This above-described method of determining percent identity between polypeptides is applicable to all proteins, fragments or variants thereof disclosed herein.

[0098] When referring to the interaction of a binding molecule (e.g., an antibody) with its binding partner (e.g., an antigen) as defined herein, the term "specifically binds" or "specifically binds to" refers to the ability of a binding molecule to discriminate between a target antigen from one animal species and an antigen orthologue from a different animal species under a given set of conditions. A CD137 binding molecule is said to specifically bind to human CD137 if it binds to human CD137 with an EC50 less than 50% of its EC50 for binding to rat or mouse CD137 as determined in an in vitro assay. The binding specificity of an antibody can be determined using methods known in the art. Examples of such methods include FACS, Western blots, ELISA-, RIA-, ECL-, IRMA-tests, and peptide scanning using PHA-stimulated primary cells.

[0099] The term "selectively binds" or "selectively binds to" when referring to the interaction of a binding molecule (e.g., an antibody) with its binding partner (e.g., an antigen) as defined herein refers to the ability of a binding molecule to discriminate between a target antigen from one animal species (such as human CD137) and a different antigen from the same animal species (such as human CD40) under a given set of conditions. A CD137 binding molecule is said to selectively bind to human CD137 if it binds to human CD137 with an EC50 that is less than 10% of its EC50 for binding to human CD40 or human CD134, as determined in an in vitro assay.

[0100] The term "treat," "treating," or "treatment" with respect to a disease condition in a mammal refers to causing a desired or beneficial effect in a mammal suffering from the disease condition. The desired or beneficial effect 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 a cessation or inhibition of further development of the disease, condition, or illness. In the context of treating cancer in a mammal, the desired or beneficial effect 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 improvement in mammalian survival. The effect may be subjective or objective. For example, if the mammal is a human, improvement in energy or vitality or reduction in pain may be recorded as an improved symptom or response to therapy. Alternatively, a clinician may notice a decrease in tumor size or tumor burden based on a physical examination, laboratory parameters, tumor markers, or imaging findings. Some laboratory signs that clinicians can observe for response to treatment include standardized tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, clinicians can observe a decrease in detectable tumor markers. Alternatively, other tests can be used to assess objective improvement, such as sonograms, magnetic resonance imaging, and positron emission tomography.

[0101] The term "vector" refers to a nucleic acid molecule capable of transporting an exogenous nucleic acid molecule. The exogenous nucleic acid molecule is connected to a carrier nucleic acid molecule by recombinant techniques such as connection or recombinant reaction. This allows multiplication, selection, further manipulation or expression of the exogenous nucleic acid molecule in a host cell or organism. A vector can be a plasmid, phage, transposon, cosmid, chromosome, virus or virion. A type of vector can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome (for example, non-additional mammalian vectors). Another type of vector can replicate autonomously in the host cell into which it is introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Another specific type of vector capable of directing the expression of the exogenous nucleic acid to which it is operatively connected is commonly referred to as an "expression vector". An expression vector typically has a control sequence that drives the expression of the exogenous nucleic acid. The simpler vector known as a "transcription vector" can only be transcribed, but not translated: they can be replicated in target cells, but not expressed. The term "vector" encompasses all types of vectors, without regard to their function. Vectors capable of directing the expression of expressible nucleic acids to which they are operatively linked are generally referred to as "expression vectors."

[0102] Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to the methods described in various general and more specific references well known in the art and as 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 according to manufacturer specifications, as generally achieved in the art or performed as described herein. The nomenclature used in combination with analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein, and the laboratory procedures and techniques of the analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry are those well known in the art and commonly used. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and the treatment of patients.

[0103] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0104] B. Binding molecules that bind to human CD137

[0105] The present disclosure provides isolated binding molecules bound 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 for epitope binding and antibodies described for the specific amino acid sequences of HVRs, variable regions (VL, VH), and IgG (e.g., IgG4) light and heavy chains. In some embodiments, the present disclosure relates to binding molecules that bind to human CD137 and have 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, or all eight) of the following functional properties: (a) binds to human CD137 with a KD of 500 nM or less; (b) has agonist activity on human CD137; (c) does not bind to human OX40, CD40, GITR, and / or CD27 receptors at concentrations up to 1000 nM; (d) cross-reacts with monkey, mouse, rat, or dog CD137; (e) does not induce ADCC effects; (f) is capable of inhibiting tumor cell growth; (g) has a therapeutic effect on cancer; and (h) blocks the binding between CD137 and CD137L. In some embodiments, the antibodies disclosed herein can also block, for example, completely block, the binding between CD137 and its ligand CD137L. Also provided herein are one or more anti-CD137 antibodies or antigen-binding fragments that cross-compete with one or more of the antibodies or antigen-binding fragments described herein for binding to human CD137.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within 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 within 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 of the 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. Methods for measuring the ability of an antibody or antigen-binding fragment to bind to a target antigen can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc. In some embodiments, the ability of an antibody or antigen-binding fragment to bind to a target antigen is measured by surface plasmon resonance (see, e.g., Example 1 below).

[0107] In some embodiments, the antibody or antigen-binding fragment binds to human CD137 with 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 with 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. Methods for measuring the KD of an antibody or antigen-binding fragment can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filtration binding assays, EMSA, etc. In some embodiments, KD is measured by surface plasmon resonance (see, for example, Example 1 below).

[0108] Anti-CD137 antibodies must be cross-linked to become agonistic. For example, cross-linking is achieved in vivo by Fcγ receptors, and polyclonal anti-Fc antibodies are generally used in vitro for cell-based experiments. In some embodiments, antibodies or antigen-binding fragments as described herein have agonist activity to human CD137. In some embodiments, when cells expressing human CD137 (e.g., human cells) contact antibodies or antigen-binding fragments, antibodies or antigen-binding fragments induce one or more (e.g., one or more, two or more, three or more) activities of human CD137. Various CD137 activities are known in the art and may include but are not limited to the induction of NF-κB-dependent transcription, the induction of T cell proliferation, the extension of T cell survival, the costimulation of activated T cells, the induction of cytokine secretion (such as IL-2), and the induction of monocyte activation. In some embodiments, one or more CD137 activities are not the combination of CD137 and its ligand. Methods for measuring CD137 activity (e.g., NF-κB-dependent transcription and / or induction of T cell proliferation, etc.) are known in the art, including, for example, 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., not contacted with the antibody, or corresponding cells contacted with an isotype control antibody), in cells expressing CD137 (e.g., human cells) contacted with the antibody or antigen-binding fragment, NF-κB-dependent transcription increases 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 embodiments, NF-κB-dependent transcription is increased by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, or more in cells expressing CD137 (e.g., human cells) contacted with the antibody or antigen-binding fragment 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).

[0109] In some embodiments, the antibody or antigen binding fragment cross-reacts with monkey (e.g., cynomolgus monkey), mouse, rat and / or dog CD137. 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 monkey and mouse CD137; monkey and rat CD137; monkey and dog CD137; mouse and rat CD137; mouse and dog CD137; rat and dog CD137; monkey, mouse and rat CD137; monkey, mouse and dog CD137; monkey, rat and dog CD137; mouse, rat and dog CD137; or monkey, mouse, rat and dog CD137 cross-reacts. In some embodiments, the antibodies or antigen-binding fragments cross-react at about 100 nM (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about 100 nM). Methods for measuring antibody cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filtration binding assays, EMSA, and the like. In some embodiments, cross-reactivity is measured by ELISA (see, e.g., Example 2 below).

[0110] In some embodiments, the antibody does not cause an ADCC effect. Methods for measuring ADCC effects (e.g., in vivo methods) are known in the art, including but not limited to the methods described in Example 11 below. In some embodiments, the antibody does not cause an ADCC effect of more than about 10% relative to a control (does not cause an ADCC effect of more than about 10%, more than about 5%, more than about 1%, more than about 0.1%, more than about 0.01%).

[0111] In some embodiments, the antibody or antigen binding fragment can inhibit tumor cell growth / proliferation. In some embodiments, relative to corresponding tumor cells not contacted with the antibody or antigen binding fragment, when contacted with the antibody or antigen binding fragment, 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%). In some embodiments, when the antibody or antigen binding fragment is administered to a subject, the antibody or antigen binding fragment can reduce the tumor volume of the subject. In some embodiments, the antibody or antigen-binding fragment is capable of reducing the volume of a tumor in a subject 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., prior to administration of the antibody or antigen-binding fragment). Methods of monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art and include, for example, the methods described in Example 10 below.

[0112] In some embodiments, the antibody or antigen-binding fragment has a therapeutic effect on cancer. In some embodiments, the antibody or antigen-binding fragment reduces one or more signs or symptoms of cancer. In some embodiments, when the antibody or antigen-binding fragment is administered, the subject suffering from cancer enters partial or complete remission.

[0113] On the other hand, the present disclosure provides an isolated antibody that competes or cross-competes with any of the exemplary antibodies of the present disclosure, such as AG10058, AG10059 and / or AG10131 for binding to human CD137. In a specific embodiment, the present disclosure provides an isolated antibody that competes or cross-competes with any of the exemplary antibodies of the present disclosure for binding to the same epitope on human CD137. The ability of an antibody to compete or cross-compete for binding 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, an exemplary antibody of the present disclosure can be allowed to bind to human CD137 under saturating conditions, and then the ability of the test antibody to bind to CD137 is measured. If the test antibody is able to bind to CD137 simultaneously with the exemplary antibody, then the test antibody binds to an epitope different from 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 an epitope that is closely adjacent to the epitope bound by the exemplary antibody. This experiment can be performed using various methods, such as ELISA, RIA, FACS, or surface plasmon resonance.

[0114] 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, for blocking the binding of CD137 to its ligand, the antibody or antigen binding fragment has a half-maximal inhibitory concentration (IC50) of about 500nM or less (e.g., about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 100nM or less, about 50nM or less, about 25nM or less, about 10nM or less, about 1nM or less, etc.). In some embodiments, for blocking the binding of CD137 to its ligand, the antibody or antigen binding fragment has a half-maximal inhibitory concentration (IC50) of about 100nM or less. In some embodiments, when provided at a concentration 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 an antibody or antigen-binding fragment to reduce the binding between a first protein and a 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 a first protein (e.g., CD137) to a second protein (e.g., CD137L) are known in the art and include, but are not limited to, BIAcore analysis, ELISA assays, and flow cytometry (see, e.g., Example 6 below).

[0115] B-1. CD137 Antibody

[0116] In some aspects, the present disclosure provides an isolated 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 binds to human CD137 with a K of 50 nM or less. D Binds to human CD 137 as measured by surface plasmon resonance.In certain embodiments, the antibody may cross-react with at least one non-human species selected from the list consisting of cynomolgus monkey, mouse, rat, and dog.

[0117] In one aspect, the present disclosure provides an isolated 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 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: 4), wherein X1 is G or S, X2 is A or G, and X3 is A, G, S or T; wherein HVR-H2 comprises an amino acid sequence according to a formula 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: 7). 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 comprises an amino acid sequence according to 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 comprises HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises an amino acid sequence according to 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 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;and wherein HVR-L3 comprises an amino acid sequence according to a formula selected from the group consisting of: 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.;

[0118] In some embodiments, the antibody may comprise an HVR_H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 253-312, an HVR_H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 313-372, an HVR_H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 373-432, an HVR_L1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 433-492, an HVR_L2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 493-552, and / or an HVR_L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 553-612.

[0119] In certain embodiments, the antibody may comprise a VL and / or VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-132, which may preferably be encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 133-252, respectively.

[0120] In some embodiments, the antibody may comprise an HVR_H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 709-732, an HVR_H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 733-756, an HVR_H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 757-780, an HVR_L1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 781-804, an HVR_L2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 805-828, and / or an HVR_L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 829-852.

[0121] In certain embodiments, the antibody may comprise a light chain and / or a heavy chain (e.g., an IgG such as IgG4) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 613-660, which may preferably be encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 661-708, respectively.

[0122] The CD137 antibodies described herein can belong to any class, such as IgG, IgM, IgE, IgA or IgD. Preferably, the CD137 antibodies belong to the IgG class, such as IgG1, IgG2, IgG3 or IgG4 subclass. CD137 antibodies can be converted from one class or subclass to another class or subclass using methods known in the art. An exemplary method for producing an antibody belonging to a desired class or subclass comprises the following steps: isolating nucleic acid encoding the heavy chain of the CD137 antibody and nucleic acid encoding the light chain of the CD137 antibody; isolating nucleic acid encoding the V H The sequence of the region; V H The sequence is linked to a sequence encoding a heavy chain constant region of the desired class or subclass; the light chain gene and heavy chain construct are expressed in cells; and CD137 antibodies are collected.

[0123] Furthermore, the antibodies provided by the present disclosure can be monoclonal or polyclonal, but are preferably monoclonal.

[0124] Examples of specific isolated antibodies provided herein include those listed in Tables 1a and 1b. The nucleotide and amino acid sequences of the heavy chain variable regions, full-length heavy chains of IgG2 and IgG4 subclasses, light chain variable regions, and full-length light chains of these antibodies are also provided below.

[0125] Antibodies of the present disclosure can be produced by techniques known in the art, including conventional monoclonal antibody methods, e.g., standard somatic cell hybridization techniques (see, e.g., Kohler and Milstein, Nature 256:495 (1975), viral or oncogenic transformation of B lymphocytes, or recombinant antibody technology, as described in detail below.

[0126] Hybridoma production is a well-established process. A common animal system for preparing hybridomas is the mouse system. Immunization protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. A well-known method for preparing human CD137 antibodies provided herein involves the XenoMouse TM Animal system used: XenoMouse TMThe mouse is an engineered mouse strain that contains large fragments of human immunoglobulin heavy and light chain loci and is defective in mouse antibody production. See, for example, Green et al., Nature Genetics 7: 13-21 (1994) and WO2003 / 040170. The animal is immunized with a 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, in particular a fragment of the extracellular domain of CD137 comprising amino acid residues 34-108 or 34-93 of SEQ ID NO: 1. Immunization of the animal 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, supra and U.S. Patent No. 5,994,619. The CD137 antigen can be administered with an adjuvant to stimulate an immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex). After immunizing an animal with the CD137 antigen, an immortalized cell line producing antibodies is prepared by separating cells from the immune animal. After immunization, the animal is sacrificed and lymph nodes and / or spleen B cells are immortalized. The method of immortalizing cells includes but is not limited to transfecting the cells with oncogenes, infecting the cells with oncoviruses, culturing the cells under conditions selected for immortalized cells, subjecting the cells to carcinogenic or mutant compounds, fusing the cells with immortalized cells (e.g., myeloma cells), and inactivating tumor suppressor genes. See, for example, Harlow and Lane, supra. If a fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using CD137, portions thereof, or cells expressing CD137. Cells producing CD137 antibodies, such as hybridomas, are selected, cloned, and further screened for desired characteristics, including robust growth, high antibody yield, and desired antibody characteristics, 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 cell culture in vitro. Methods for selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art.

[0127] Antibodies disclosed herein can also be prepared using phage display or yeast display methods. Such display methods for isolating human antibodies are 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.

[0128] B-2. Antigen-binding fragments

[0129] In some other aspects, the present disclosure provides antigen-binding fragments of any of the CD137 antibodies provided herein.

[0130] The antigen-binding fragment may comprise any sequence of an antibody. In some embodiments, the antigen-binding fragment comprises the following amino acid sequence: (1) a light chain of a CD137 antibody; (2) a heavy chain of a CD137 antibody; (3) a variable region from the light chain of a CD137 antibody; (4) a variable region from the heavy chain of a CD137 antibody; (5) one or more HVRs (two, three, four, five, or six HVRs) of a CD137 antibody; or (6) three HVRs from the light chain of a CD137 antibody and three HVRs from the heavy chain of the CD137 antibody.

[0131] In some specific embodiments, the present disclosure provides antigen-binding fragments of antibodies selected from those listed in Tables 1a and 1b.

[0132] In some other specific embodiments, the antigen-binding fragment of the CD137 antibody comprises: (i) a Fab fragment consisting of V L 、V H 、C L and C H 1 domain; (ii) F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) H and C H1 domain; (iv) a V fragment consisting of a single arm of the antibody L and V H Fv fragment composed of V H dAb fragments composed of the V domains (Ward et al., (1989) Nature 341: 544-546); (vi) isolated CDRs, and (vii) single-chain antibodies (scFv), which are fragments containing V domains that are closely related to antibodies. H The V region of the antibody is connected L Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883.

[0133] In some specific embodiments, the antigen binding fragment is a Fab fragment selected from those listed in Table 1a.

[0134] B-3. ​​Antibody Derivatives

[0135] In some additional aspects, the present disclosure provides derivatives of any of the CD137 antibodies provided herein.

[0136] In one aspect, antibody derivatives are derived from modifications of the amino acid sequences of exemplary antibodies of the present disclosure ("parent antibodies") while retaining the overall molecular structure of the parent antibody amino acid sequences. The amino acid sequence of any region of the parent antibody chain may be modified, such as the framework region, HVR region, or constant region. The types of modifications include substitutions, insertions, deletions, or combinations thereof of one or more amino acids of the parent antibody.

[0137] In some embodiments, the antibody derivative comprises V L or V HIn some embodiments, the antibody derivative comprises an HVR_H1 amino acid sequence region that is 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% identical to the amino acid sequence as shown in any one of SEQ ID NOs: 13-132. In some embodiments, the antibody derivative comprises an HVR_H2 amino acid sequence region that is 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% identical to the amino acid sequence as shown in any one of SEQ ID NOs: 313-372. In some embodiments, the antibody derivative comprises an HVR_H3 amino acid sequence region that is 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% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 373-432. In some embodiments, the antibody derivative comprises an HVR_L1 amino acid sequence of all Fab hits shown in Table 1a, which can be found in SEQ ID NOs: 433-492. In some embodiments, the antibody derivative comprises an HVR_L2 amino acid sequence of all Fab hits shown in Table 1a, which can be found in SEQ ID NOs: 493-552.

[0138] In some embodiments, the antibody derivative comprises the HVR_L3 amino acid sequence of all Fab hits shown in Table 1a, which can be found in SEQ ID NOs: 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 conservative or non-conservative substitutions, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions to the amino acid sequence shown in any one of SEQ ID NOs: 13-132 and 253-612.

[0139] In some embodiments, the antibody derivative comprises a light chain or heavy chain that is 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% identical to the amino acid sequence as shown in any one of SEQ ID NOs: 613-660.

[0140] In some embodiments, the antibody derivative comprises an HVR_H1 amino acid sequence region that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs: 709-732. In some embodiments, the antibody derivative comprises an HVR_H2 amino acid sequence region that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs: 733-756. In some embodiments, the antibody derivative comprises an HVR_H3 amino acid sequence region that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs: 757-780. In some embodiments, the antibody derivative comprises an HVR_L1 amino acid sequence region that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs: 781-804. In some embodiments, the antibody derivative comprises an HVR_L2 amino acid sequence region that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs: 805-828. In some embodiments, the antibody derivative comprises an HVR_L3 amino acid sequence region that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs: 829-852. In some embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative or non-conservative substitutions, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additions and / or deletions to the amino acid sequence as shown in any one of SEQ ID NOs: 613-660 and 709-852.

[0141] Amino acid substitution encompasses conservative and non-conservative substitutions. The term "conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid where the two amino acids have similarities in certain physical-chemical properties, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. For example, substitutions can typically be made within each of the following groups: (a) non-polar (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.

[0142] Modifications can be made at any position of the amino acid sequence of the antibody, including HVR, framework region or constant region. In one embodiment, the present disclosure provides an antibody derivative comprising V and V sequences of an exemplary antibody of the present disclosure. H and V L HVR sequences, further comprising framework sequences different from those of the exemplary antibodies. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the 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 Edition, U.S. Department of Health and Human Services (USDepartment 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)). Framework sequences that can be used to construct antibody derivatives include framework sequences that are structurally similar to those used for the exemplary antibodies of the present disclosure, for example, V sequences similar to those used for the exemplary antibodies of the present disclosure. H 3-23 framework sequence and / or V LFor example, the HVR_H1, HVR_H2, and HVR_H3 sequences, and the HVR_L1, HVR_L2, and HVR_L3 sequences of the exemplary antibodies can be grafted onto framework regions having the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the HVR sequences can be grafted onto framework regions that contain one or more mutations compared to the germline sequences.

[0143] In a specific embodiment, antibody derivatives are chimeric antibodies comprising the amino acid sequence of an exemplary antibody of the present disclosure. In one example, one or more HVRs from one or more exemplary human antibodies are combined with HVRs from antibodies of non-human animals (such as mice or rats). In another example, all HVRs of the chimeric antibody are derived from one or more exemplary antibodies. In some specific embodiments, the chimeric antibody comprises one, two or three HVRs 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.

[0144] Another type of modification is to make V H and / or V L The amino acid residues in the HVR region of the heavy chain are mutated. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. Mutations can be amino acid additions and / or deletions. In addition, no more than one, two, three, four, or five residues in the HVR region are typically changed. In some embodiments, the antibody derivatives comprise 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 to change one or more cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. Cysteine ​​can be a classical or non-classical cysteine. In one embodiment, the antibody derivatives have 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of the exemplary antibody.

[0145] You can also V H and / or V L In some embodiments, the framework residues in the region are modified. Typically, such framework variants are prepared to reduce the immunogenicity of the antibody. One method is to "revert" one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to restore the framework region sequence to its germline structure, the somatic mutation can be "reverted" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0146] In addition, modifications may also be made in the Fc region of the 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 cellular cytotoxicity. In one example, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is altered, for example, increased or decreased. This method is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains, or to increase or decrease the stability of the antibody. In another case, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody.

[0147] In addition, the antibodies of the present invention can be modified according to routine experiments known in the art to change their potential glycosylation sites or patterns. On the other hand, the present disclosure provides derivatives of the CD137 antibodies of the present disclosure, which contain at least one mutation in the variable region of the light chain or heavy chain, and the mutation changes the glycosylation pattern in the variable region. For antigen binding, such antibody derivatives may have increased affinity and / or changed specificity. The mutation may add new glycosylation sites in the V region, change the position of one or more V region glycosylation sites, or remove pre-existing V region glycosylation sites. In one embodiment, the present disclosure provides derivatives of the CD137 antibody, which have potential N-linked glycosylation sites at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in one heavy chain variable region are removed. In another embodiment, the present disclosure provides derivatives of the CD137 antibody, which have potential N-linked glycosylation sites at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in both 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. Pat. No. 6,933,368, the disclosure of which is incorporated herein by reference.

[0148] In another aspect, the present disclosure provides an antibody derivative comprising a CD137 antibody as described herein, or an antigen-binding fragment thereof, linked to another molecular entity. Examples of another molecular entity include a pharmaceutical agent, a peptide or protein, a detection agent or label, and an antibody.

[0149] In some embodiments, the antibody derivative comprises an antibody of the present invention linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, and radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., dichloromethyl diethylamide, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 and lutetium 177Methods for linking antibodies to pharmaceutical agents are known in the art, such as using various linker technologies. Examples of linker types include those containing hydrazones, thioethers, esters, disulfides, and peptides. For further discussion of linkers and methods for attaching 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.

[0150] In a specific 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 individual monomers in the antibody multimer can be the same or different. In addition, the individual monomers in the multimer can have the same or different binding specificities. The multimerization of the antibody can be achieved by the natural aggregation of the antibody. For example, a certain percentage of purified antibody preparations (e.g., purified IgG4 molecules) spontaneously form protein aggregates comprising antibody homodimers and other higher-order antibody multimers. Alternatively, antibody homodimers can be formed by chemical connection techniques known in the art, such as by using a cross-linking agent. Suitable cross-linking agents include those that are heterobifunctional, having two different reactive groups separated by an appropriate spacer, such as m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4-(maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester, and S-acetylthio-acetic acid N-succinimidyl ester, or homobifunctional, such as disuccinimidyl suberate. Such linkers are commercially available, for example, from Pierce Chemical Company, Rockford, IL. Antibodies can also be multimerized by recombinant DNA techniques known in the art.

[0151] Examples of other antibody derivatives provided herein include single-chain antibodies, diabodies, domain antibodies, nanobodies, and unibodies. "Single-chain antibodies" (scFv) consist of a single-chain antibody comprising a V H Domain-linked V LThe single polypeptide chain of the domain, in which V L domain and V H The domains are paired to form 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). A "diabody" consists of two chains, each chain comprising a heavy chain variable region connected to a light chain variable region on the same polypeptide chain connected by 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 diabodies 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) Structure 2: 1121-1123). Domain antibodies (dAbs) are small functional binding units of antibodies, which correspond to the variable regions of the heavy or light chains of antibodies. Domain antibodies are well expressed in bacteria, yeast, and mammalian cell systems. The other details of domain antibodies and their production methods are known in the art (see, e.g., U.S. Patent Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patent Nos. 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 comprise a single variable domain and two constant domains (CH2 and CH3) and maintain the antigen binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Patent No. 6,765,087, U.S. Patent No. 6,838,254, WO 06 / 079372). Minibodies are composed of one light chain and one heavy chain of an IgG4 antibody. Minibodies can be prepared by removing the hinge region of an IgG4 antibody. Further details of minibodies and methods for their preparation can be found in WO 2007 / 059782.

[0152] C. Nucleic Acids, Vectors, Host Cells, and Recombinant Methods for Producing CD137 Antibodies

[0153] Another aspect of the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the binding molecule provided by the present disclosure. The amino acid sequence encoded by the nucleotide sequence can be any part of an antibody, such as an HVR, comprising one, two or three HVR sequences, a variable region of a heavy chain, a variable region of a light chain, or can be a full-length heavy chain or a full-length light chain. The nucleic acid of the present disclosure can be, for example, DNA or RNA, and may or may not comprise intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0154] In some embodiments, the present 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) the variable region of the heavy chain or the 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.

[0155] In other embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NOs: 13-132, 253-612, 613-660, and 709-852.

[0156] In other embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 133-252 and 661-708.

[0157] Nucleic acids disclosed herein can be obtained using any suitable molecular biology technique. For antibodies expressed by hybridomas, cDNA encoding the light and heavy chains of the antibodies prepared by hybridomas can be obtained by PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the libraries.

[0158] By encoding V H The DNA encoding the V constant region (CH1, CH2 and CH3) is operatively linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). HThe isolated DNA of the heavy chain constant region is converted into full-length heavy chain gene. The sequence of human heavy chain constant region gene is known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH publication number 91-3242) and the DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably IgG4 or IgG2 constant region without ADCC effect. The IgG4 constant region sequence can be any one of the various alleles or allotypes known to be present in different individuals. These allotypes represent naturally occurring amino acid substitutions in the IgG4 constant region. For the Fab fragment heavy chain gene, the encoding V H The DNA encoding the heavy chain CH1 constant region is operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0159] By encoding V L The DNA encoding the light chain constant region CL is operatively linked to another DNA molecule encoding the V L The isolated DNA of the light chain constant region is converted into full-length light chain genes (and Fab light chain genes). The sequence of human light chain constant region genes is known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH publication number 91-3242) and the DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0160] To generate scFv genes, the V H and V L The DNA fragment encoding the flexible linker is operably linked to another fragment encoding the amino acid sequence (Gly4-Ser)3, such that V H and V L The sequence can be expressed as a V with V connected by a flexible linker L and V H The invention relates to a continuous single-chain protein containing a cleavage region (see, e.g., 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)).

[0161] The present disclosure further provides a vector comprising a nucleic acid molecule provided by the present disclosure. The nucleic acid molecule can encode 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 the full length, or an amino acid sequence of an antibody derivative or an antigen-binding fragment. In some embodiments, the vector is an expression vector that can be used to express binding molecules such as antibodies or their antigen-binding fragments. In some embodiments, a vector is provided herein, wherein the first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein, and the second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises polynucleotides encoding a heavy chain variable region as described herein and a light chain variable region as described herein.

[0162] In order to express binding molecules of the present invention, the DNA encoding part or full-length light chain and heavy chain is inserted into the expression vector so that the DNA molecule is operatively connected to transcription and translation control sequences. In this context, the term "operatively connected" refers to that the antibody gene is connected to the vector so that the transcription and translation control sequences in the vector play the expected function of transcription and translation of its regulatory DNA molecule. The selected expression vector and expression control sequence should be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into an independent vector, or more generally, two genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (for example, connecting the complementary restriction sites on the antibody gene fragment and the vector, or connecting based on the DNA of homologous recombination). The light chain and heavy chain variable regions of antibody described herein can be used to produce the full-length antibody genes of any antibody isotype and subclass, and this is done by inserting them into the expression vector of the heavy chain constant region and the light chain constant region of the desired isotype and subclass, so that V H The segment is operably linked to one or more C H Segment and V L The segment is operatively linked to the C L The antibody chain can be expressed in a manner that allows the host cell to express the antibody chain. The antibody chain gene can be cloned into a vector such that the signal peptide is connected to the amino terminus of the antibody chain gene in the same frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0163] In addition to the antibody chain genes, the expression vectors of the present disclosure generally also carry regulatory sequences for controlling the expression of the antibody chain genes 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 genes. Such regulatory sequences are described in, for example, 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 the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. 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), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyomavirus. Alternatively, non-viral regulatory sequences may be used, such as ubiquitin promoters or β-globin promoters. Additionally, regulatory elements include sequences from various sources, such as the SR promoter system, which includes sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0164] In addition to antibody chain genes and regulatory sequences, expression vectors can also carry other sequences, such as sequences (for example, replication origin) and selective marker genes for the replication of regulatory vectors in host cells. Selective marker genes contribute to the selection of host cells into which vectors have been introduced (see, for example, U.S. Patent numbers 4,399,216, 4,634,665 and 5,179,017, all applied for by the people such as Axel). For example, conventional selective marker genes will confer resistance to drugs such as G418, hygromycin or methotrexate on host cells into which vectors have been introduced. Selective marker genes include dihydrofolate reductase (DHFR) genes (for use in dhfr- host cells selected / amplified using methotrexate) and neo genes (selected for G418).

[0165] To express the light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into host cells by any suitable technique. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the present disclosure can be expressed in prokaryotic or eukaryotic host cells, the antibodies are most commonly expressed in eukaryotic cells and typically in mammalian host cells.

[0166] The present disclosure also provides a host cell comprising a nucleic acid molecule provided herein. The host cell can be virtually any cell for which the expression vector is suitable. 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; or a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant nucleic acid construct into the host cell can be achieved by calcium phosphate transfection, DEAE- or dextran-mediated transfection, electroporation, or phage infection.

[0167] Suitable eukaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0168] Mammalian host cells for expressing the binding molecules of the present 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 a DHFR selective marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol. 159: 601-621 (1982)), NSO myeloma cells, COS cells, and Sp2 cells. In particular, for use with NSO myeloma or CHO cells, another expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When an expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or to be secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using any suitable protein purification method.

[0169] D. Composition

[0170] In other aspects, the present disclosure provides a composition comprising a binding molecule provided by the present disclosure. In one aspect, the composition is a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0171] In some embodiments, the present 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 an 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 portion thereof glycosylated at asparagine of the amino acid sequence, compared to the total amount of the antibody or antigen-binding portion thereof present in the composition. In another embodiment, the composition comprises at least about 2% of the antibody or antigen-binding portion thereof glycosylated at asparagine of the amino acid sequence, compared to the total amount of the antibody or antigen-binding portion thereof present in the composition.

[0172] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering a binding molecule. Carriers can be antiadherents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterials or antifungals), sweeteners, absorption delaying agents, wetting agents, emulsifiers, buffers, etc. The example of a suitable pharmaceutically acceptable carrier includes water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents, such as sugar, polyols, sorbitol, and sodium chloride.

[0173] The composition can be in any suitable form, such as liquid, semisolid and solid dosage form. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules and powders. The specific form of the composition 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 mixing the desired amount of antibody into a suitable carrier and then performing sterilization microfiltration. In general, dispersions are prepared by mixing the antibody into a sterile vehicle comprising a basic dispersion medium and other carriers. For sterile powders for the preparation of sterile liquids, preparation methods include vacuum drying and freeze drying (lyophilization) to produce a powder of any other desired component of the active ingredient plus a solution thereof that has previously been sterile filtered. The various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0174] The relative amount of the binding molecule included in the composition will vary depending on a variety of factors, such as the specific binding molecule and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of binding molecule in a single dosage form will generally be that which produces a therapeutic effect, but may also be a lesser amount. Generally speaking, this amount will be in the range of about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% relative to the gross weight of the dosage form.

[0175] 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 easily selected by those skilled in the art and will vary depending on a variety of factors, such as the specific agent and carrier used, dosage form, and the desired release and pharmacodynamic characteristics. The amount of the additional therapeutic agent included in a single dosage form will generally be that amount of the agent that produces the therapeutic effect, but may also be a lesser amount.

[0176] E. Uses of Binding Molecules and Pharmaceutical Compositions

[0177] The binding molecules and pharmaceutical compositions provided by the present disclosure can be used for treatment, diagnosis or other purposes, such as regulating immune response, treating cancer, enhancing the efficacy of other cancer therapies, enhancing vaccine efficacy, or treating autoimmune diseases. Therefore, in other aspects, the present disclosure provides methods of using binding molecules or pharmaceutical compositions. In one aspect, the present disclosure provides a method for treating a disease in a mammal, comprising administering a therapeutically effective amount of the binding molecules provided by the present disclosure to a mammal in need of treatment. The binding molecule can be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In some embodiments, the mammal is a human.

[0178] In some embodiments, the disorder is cancer. A variety of cancers in which CD137 is involved, whether malignant or benign and whether primary or secondary, can be treated or prevented using the methods provided herein. Examples of such cancers include lung cancers such as bronchogenic carcinomas (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, enchondromatous hamartomas (non-cancerous), and sarcomas (cancerous); heart cancers such as myxomas, fibromas, and rhabdomyomas; bone cancers such as osteochondromas, condromas, chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, chondrosarcomas, multiple myeloma, osteosarcomas, fibrosarcomas, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, astrocytomas, oligodendrogliomas, medulloblastomas, chordomas, schwannomas, ependymomas, meningiomas, epithelial cells, and gliomas. adenomas, pinealomas, 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, adrenal tumors, and transitional cell carcinoma of the renal pelvis; bladder cancer; blood cancers, such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myelocytic, myeloid, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome), and leukemia. syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloid, 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, thrombocythemia, 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; male reproductive system cancers such as Such as 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 cancer of the uterus (endometrium), cervix (cervical cancer), ovary (ovarian cancer), vulva, vagina, fallopian tube cancer and hygroma; thyroid cancer (including papillary, follicular, anaplastic or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid glands; pancreatic cancer; and blood cancers, such as leukemia, myeloma, non-Hodgkin's lymphoma and Hodgkin's lymphoma.

[0179] In some other embodiments, the condition is an autoimmune disease. Examples of autoimmune diseases that can be treated with binding molecules include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. Binding molecules can also be used to treat inflammation (such as allergic asthma) and chronic graft-versus-host disease,

[0180] On the other hand, the present disclosure provides a method for enhancing immune response in mammals, which includes administering a therapeutically effective amount of the binding molecules provided by the present disclosure to mammals. In some embodiments, the binding molecule is a CD137 antibody or its antigen-binding fragment and the mammal is a human. In another embodiment, the binding molecule is a CD137 agonist antibody or its antigen-binding fragment. The term "enhancing immune response" or its grammatical variants refers to any response that stimulates, provokes, increases, improves or strengthens the immune system of a mammal. The 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 immune response or a secondary immune response. Examples of immune response enhancement include an increase in CD4+ helper T cell activity and the generation of cytolytic T cells. The enhancement of the 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, when compared to the immune response of an untreated mammal or a mammal not treated with the claimed method, the method of the present disclosure enhances the immune response of a mammal. In one embodiment, the binding molecule is used to enhance a person's immune response to a microbial pathogen (such as a virus). In another embodiment, the binding molecule is used to enhance a person's immune response to a vaccine. The binding molecule can 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, especially 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, especially IL-2 production. The binding molecule can be used to enhance a person's immune response to a microbial pathogen (such as a virus) or to a vaccine. The binding molecule can be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist.

[0181] When practicing treatment methods, the binding molecule can be administered alone as a monotherapy, or administered in combination with one or more additional therapeutic agents or therapies. Therefore, on the other hand, the present disclosure provides a combination therapy comprising a binding molecule in combination with one or more additional therapies or therapeutic agents for separate, sequential or simultaneous administration. The term "additional therapy" refers to a therapy that does not employ a binding molecule provided by the present disclosure as a therapeutic agent. The term "additional therapeutic agent" refers to any therapeutic agent other than the binding molecule provided by the present disclosure. In a specific aspect, the present disclosure provides a combination therapy for treating cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule provided by the present disclosure in combination with one or more additional therapeutic agents. In another embodiment, the mammal is a human.

[0182] A wide variety of cancer therapeutics can be used in combination with the binding molecules provided herein. One of ordinary skill 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 disclosed herein and will not be limited to those forms of therapy described herein. Examples of classes of additional therapeutic agents that can be used in combination therapies for treating cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents.

[0183] The term "chemotherapeutic 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 chemotherapeutic 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), cyclophosphamide (CYTOXAN), ifosfamide (IFEX), nitrogen mustard hydrochloride (MUSTARGEN), thiotepa (THIOPLEX), streptozotocin (ZANOSAR), carmustine (BICNU, GLIADELWAFER), lomustine (CEENU), and dacarbazine (DTIC-DOME); (2) alkaloids or plant vinca alkaloids, including cytotoxic antibiotics such as doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN), and daunorubicin (DAMYCIN). PFS, ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE), dactinomycin (actinomycin D, COSMEGEN), plicamycin (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), nelarabine (ARRANON), trimetazidine (TREXALL), (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), paclitaxel (TAXOL); (6) platinum 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) Epipodophyllotoxins (podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID, TOPOSAR); (9) Folic acid derivatives, such as folinic acid (WELLCOVORIN); (10) Nitrosoureas, such as carmustine (BiCNU), lomustine (CeeNU); (11) Inhibitors of receptor tyrosine kinases, including 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, sunitinib (SUTE NT), axitinib, rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN) and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), antibodies to the insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596); (12) 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 (13) proteasome inhibitors such as bortezomib (VELCADE). ;

[0184] The term "immunotherapeutic agent" refers to a chemical or biological substance that can enhance the immune response of a mammal. Examples of immunotherapeutic agents include: Bacillus Calmette-Guérin (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 ozogamicin (gemtuzumab), and oxazolidinone (oxazolidinone).

[00135] The present invention relates to an anti-cancer drug that is directed against adenomatous polymorphisms (PD-1 / PD-1), ...

[0185] The term "hormonal therapeutic agent" refers to a chemical or biological substance that suppresses or eliminates the production of a hormone, or suppresses or offsets the effect of a hormone on the growth and / or survival of cancer cells. Examples of such agents suitable for use in the methods herein include those disclosed in US20070117809. Examples of specific hormone therapeutic 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 the present disclosure can also be used in combination with non-drug hormone therapies, such as (1) surgical methods to remove all or part of the organs or glands involved in hormone production, such as ovaries, testicles, adrenal glands and pituitary glands, and (2) radiotherapy, wherein the patient's organs or glands are subjected to radiation sufficient to suppress or eliminate the production of targeted hormones.

[0186] Combination therapy for treating cancer also encompasses the combination of binding molecules with surgery to remove the tumor. The binding molecules can be administered to the mammal before, during or after surgery.

[0187] Combination therapies for treating cancer also encompass combinations of binding molecules with radiation therapy, such as ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high linear energy radiation). The radiation source can be external or internal to the mammal. The binding molecule can be administered to the mammal before, during, or after radiation therapy.

[0188] The binding molecules and compositions provided by the present disclosure can be administered by any suitable enteral or parenteral routes. The "enteral route" administered by the term refers to administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal and rectal routes, or intragastric routes. The "parenteral route" administered refers to the route of administration except the enteral route. Examples of parenteral routes administered include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal, subcutaneous or topical administration. Any suitable method can be used to administer antibodies and compositions of the present disclosure, such as by oral ingestion, nasogastric tube, gastrostomy tube (gastrostomy tube), injection, infusion, implantable infusion pump, and osmotic pump. The appropriate route and method of administration may vary depending on a variety of factors, such as the particular antibody used, the desired rate of absorption, the particular formulation or dosage form used, the type or severity of the condition being treated, the particular site of action, and the condition of the patient, and can be readily selected by one skilled in the art.

[0189] The term "therapeutically effective amount" of a binding molecule refers to an amount effective for the intended treatment purpose. For example, in the context of enhancing an immune response, a "therapeutically effective amount" is any amount effective in stimulating, stimulating, increasing, improving or strengthening any response of the mammalian immune system. In the context of treating a disease, a "therapeutically 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 improvement in mammalian survival rate. The therapeutically effective amount of a CD137 antibody typically ranges 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 can 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 amount of the CD137 antibody is in the range of about 0.01-30 mg / kg of mammal body weight. In some other embodiments, the therapeutically effective amount of the CD137 antibody is in the range of about 0.05-15 mg / kg of mammal body weight. The exact dosage level to be administered can be readily determined by one 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 previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0190] Typically, the binding molecule or composition is administered in multiple situations. The interval between single doses can be, for example, once a week, once a month, once every 3 months, or once a year. Exemplary treatment regimens include administration once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 3 months, or once every 3 to 6 months. Typical dosage 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) once every 4 weeks for 6 doses, then once every 3 months; (ii) once every 3 weeks; (iii) 3 mg / kg body weight once, then 1 mg / kg body weight, once every 3 weeks.

[0191] The present disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and it is recommended that those skilled in the art make various modifications or variations thereto, and that they are included within the spirit and authority of this application and the scope of the appended claims. The contents of all figures cited throughout this disclosure and all references, patents, and published patent applications are expressly incorporated herein by reference in their entirety.

[0192] Example

[0193] Example 1

[0194] Generation of primary Fabs that specifically bind to human CD137

[0195] A proprietary plasmid library (see PCT International Application entitled "Dynamic Human Antibody Light Chain Libraries" filed concurrently under attorney docket number 69540-2000140, which is incorporated herein by reference in its entirety; see also PCT International Application entitled "Dynamic Human Heavy Chain Antibody Libraries" filed concurrently under attorney docket number 69540-2000240, which is incorporated herein by reference in its entirety) was panned against human CD137 antigen. 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 recognized human CD137. Primary hits were defined as those whose ELISA signal was at least twice that of the background. They were sequenced, and unique clones were expressed and purified for affinity measurement by ForteBio and Biacore. The list was fine-tuned to 124 with both ELISA-positive hits and unique sequences in the Fab. According to K D Response signal R>0.1, R 2 >0.9 and affinity K D Using a standard of <100 nM, the list was further refined to 60 hits (Table 1a). 24 of these were subsequently converted to IgG (Table 1b) for detailed biophysical and functional characterization.

[0196] The Fab corresponding to the unique hit was expressed in Escherichia coli and purified. Their affinity for human CD137 was measured by the ForteBio OctetRED96 system. In brief, CD137-hisFc fusion protein (Sino Biological catalog number 10041-H03H) was captured using AHC sensors (Anti-Human IgG FcCapture Dip and Read Biosensors) and immersed in wells containing purified Fab diluted to 5-10 μg / ml with kinetic buffer (kinetic buffer) (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% volume / volume surfactant P20, pH 7.4). The acquired ForteBio data were processed with Data Acquisition software 7.1, and the kinetic data were fitted to a 1:1 Langmuir binding model. Affinity and kinetic parameters (background subtracted) are listed in Table 1a. The affinity of their corresponding IgG to human CD137 was measured by Biacore and is shown in Table 1b.

[0197] Table 1a: Affinity of selected Fabs for human CD137 and corresponding amino acid sequences (in SEQ ID NO.)

[0198]

[0199]

[0200]

[0201]

[0202] The corresponding DNA sequences encoding the amino acid sequences of SEQ ID NOs: 13-132 are found in SEQ ID NOs: 133-252, respectively. The HVR_H1 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NOs: 253-312, respectively. The HVR_H2 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NOs: 313-372, respectively. The HVR_H3 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NOs: 373-432, respectively. The HVR_L1 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NOs: 433-492, respectively. The HVR_L2 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NOs: 493-552, respectively. The HVR_L3 amino acid sequences of all Fab hits shown in Table 1a are found in SEQ ID NOs: 553-612, respectively (see also Table 1c).

[0203] Table 1b: Affinity of Fab and corresponding IgG for human CD137

[0204]

[0205]

[0206] The corresponding DNA sequences encoding the amino acid sequences of SEQ ID NOs: 613-660 are found in SEQ ID NOs: 661-708, respectively. The HVR_H1 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NOs: 709-732, respectively. The HVR_H2 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NOs: 733-756, respectively. The HVR_H3 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NOs: 757-780, respectively. The HVR_L1 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NOs: 781-804, respectively. The HVR_L2 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NOs: 805-828, respectively. The HVR_L3 amino acid sequences of all IgG sequences shown in Table 1b are found in SEQ ID NOs: 829-852, respectively.

[0207] Table 1c: CDR sequences of Fab

[0208]

[0209]

[0210] Example 2

[0211] Selection of Fab hits cross-reactive with mouse CD137

[0212] The species cross-reactivity of Fab hits was determined using ELISA. Briefly, 200 μL of 5 μg / mL anti-human IgG (Fab specificity) (Sigma # I5260) was coated on a Maxisorp microplate (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 hour. After washing three times, a serial dilution of human or mouse CD137 antigen fused to human FC fragments was added and incubated for 1 hour. After washing, HRP-labeled goat anti-human FC was diluted 1: 2000 with PBS and added to each well and incubated for 1 hour. The plate was washed three times and incubated at room temperature for 20 minutes with TMB substrate. After the reaction stopped, the absorbance at 450 nm was measured. The results are presented in Figure 1 b, the lower panel shows that Fabs 5310 and 5365 bind to both human and mouse CD137, whereas Fab 5351 binds to human CD137 but not mouse CD137.

[0213] Example 3

[0214] IgG conversion and expression: AG10058, AG10059, and AG10131

[0215] The heavy and light chains of Fab 5310, 5351 and 5365 were cloned individually into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific) in the IgG4 isotype with the S241P mutation. In addition, the heavy and light chains of two reference antibodies were cloned into pCDNA3.3 in the IgG4 and IgG2 isotypes, respectively.

[0216] The heavy chain variable region used in the reference antibody AC1097 comprised the sequence EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSS (SEQ ID NO: 862), and the light chain variable region in the reference antibody AC1097 comprised the sequence SYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVL (SEQ ID NO: 863). The heavy chain variable region used in the reference antibody AC1121 comprises the sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS (SEQ ID NO: 864), and the light chain variable region in the reference antibody AC1121 comprises the sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK (SEQ ID NO: 865). The IgGs used herein are shown in Table 2.

[0217] Table 2: List of IgG

[0218] IgG Fab Isotype describe AC1097 Reference 1 IgG2 Reference Ab AC1121 Reference 2 IgG4(S241P) Reference Ab AG10058 5310 IgG4(S241P) Adagene mAb AG10059 5351 IgG4(S241P) Adagene mAb AG10131 5365 IgG4(S241P) Adagene mAb AG10154 IgG4(S241P) Isotype control

[0219] According to the manufacturer's instructions, plasmid is transiently transfected into HEK293F cells. Harvest supernatant, by centrifugation and filtration, make it clear, and utilize standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare) IgG purification. Protein is eluted and neutralized, and buffer exchanged into PB buffer (20mM sodium phosphate, 150mM NaCl, pH 7.0). Protein concentration is determined by ultraviolet spectrophotometry, and by SDS-PAGE or SEC-HPLC, under denaturation, reduction and non-reduction conditions, IgG purity is analyzed.

[0220] Example 4

[0221] Binding affinity to human, monkey, and mouse CD137

[0222] The binding affinity of IgG to human, monkey and mouse CD137 was measured by BIAcore, ELISA and flow cytometry. The results are summarized in Table 3.

[0223] Table 3: Binding affinity of antibodies to human, monkey and mouse CD137

[0224]

[0225] NC: Not cross-reactive

[0226] 4a. Measurement of binding affinity and kinetics by SPR

[0227] Using Biacore TMThe binding affinity and kinetics of antibodies to human, monkey and mouse CD137 proteins were examined by surface plasmon resonance (SPR) analysis using a T200 instrument (Biacore AB, Uppsala, Sweden) according to 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 coupling their amino groups to the carboxylated surface of the sensor chip according to the instructions of the amine coupling kit (GE Biacore #BR-1000-50). AG10058, AG10059, AG10131, AC1121 and AC1097 were captured using fixed anti-human IgG (Fc) antibodies. Finally, human CD137-His6 (Sino Biological #10041-H08H) at six concentrations (3.13, 6.25, 12.5, 25, 50, 100) (nM) (diluted in running buffer) was injected for 300 seconds at a flow rate of 30 μl / min and 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% volume / volume surfactant P20, pH 7.4, 25°C). A corresponding control was performed in each case using a blank flow cell without immobilized protein for "background" subtraction. Association 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 instructions. As shown in Table 3, all antibodies bound to human CD137. AG10058 and AG10059 showed higher affinity than both reference antibodies. Except for the AC1121 reference mAb, all antibodies bound to monkey CD137. Only AG10058 and AG10131 bound to mouse and rat CD137. AG10058 had a higher affinity (15.2 nM) than AG10131 (64.5 nM).

[0228] 4b. Measurement of binding affinity to soluble CD137 using ELISA assay

[0229] Serial dilutions of human, monkey or mouse CD137 fused to human FC fragments were prepared and used to coat ELISA plates at 37°C for 1 hour. After blocking, 100 μL IgG (5 μg / mL) was added and incubated at 37°C for 1 hour. The plates were washed three times and then incubated with HRP-conjugated protein L (1:2000 dilution) at 37°C for 1 hour. The plates were washed three times again and incubated with TMB substrate at room temperature for 20 minutes. After the reaction was stopped, the absorbance at 450 nm was measured. The data were analyzed using nonlinear fitting by Graphpad Prism 6. Figure 2 As shown, all antibodies bind to human CD137 (FC fusion protein) with similar sub nM affinity. Except for the AC1121 reference mAb, all antibodies bind to monkey CD137 with similar sub nM affinity. Consistent with the results from Biacore, only AG10058 and AG10131 bind to mouse CD137. AG10058 has a higher affinity (0.3 nM) than AG10131 (23.9 nM).

[0230] 4c. Measurement of Binding Affinity to CD137 Overexpressed on the Cell Surface by Flow Cytometry

[0231] The affinity of the antibodies to 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 a bicistronic IRES vector, and transfected cells were identified using EGFP. After 48 hours, the transfected cells were harvested and then washed once with cold FACS buffer (PBS supplemented with 1% BSA). The cells were then incubated on ice for 1 hour with various IgGs (each at 100 nM), washed twice with pre-cooled FACS buffer, and incubated with Alexa Fluor 500. The cells were incubated with mouse anti-human FC antibody conjugated with 647 on ice for 30 min. The cells were washed once and then analyzed by flow cytometry ( CytoFlex) was used for analysis. As shown in Figure 3a, all antibodies bind to human CD137 expressed on the cell surface with low nM affinity. AG10058, AG10059 and AG10131 are slightly better than the two reference antibodies. With the exception of the AC1121 reference mAb, all antibodies bind to monkey CD137 with low nM affinity, with AG10058, AG10059 and AG10131 being slightly better than the AC1097 reference antibody. Consistent with the results from Biacore and ELISA, only AG10058 and AG10131 bind to mouse and rat CD137. AG10058 has a higher affinity for mouse CD137 than AG10131. In addition, AG10058 and AG10131 (each at 100 nM) also bind to rat and dog CD137 overexpressed on the surface of HEK293F cells (Figure 3b).

[0232] 4d. Binding of IgG to activated human, monkey, mouse and rat T cells.

[0233] The species cross-reactivity of the exemplary antibodies was further confirmed using PBMC or T cells stimulated with PMA and ionomycin from humans, monkeys, mice, and rats. Human and cynomolgus monkey PBMC were separated by Ficoll-density gradient centrifugation. In short, fresh whole blood from healthy donors or cynomolgus monkeys was diluted with an equal volume of PBS and carefully loaded onto the top of Histopaque 1077 (14 ml in a 50 ml centrifuge tube). Utilizing brake-free deceleration (brake off), centrifuge at room temperature at 1,200 × g for 30 minutes. After centrifugation, the upper layer was carefully aspirated to within the opaque interface containing 0.5 cm mononuclear cells with a pipette. The upper layer was discarded. The opaque interface (about 3-5 ml) was carefully transferred to a clean 50 ml conical centrifuge tube with a pipette. The cells were washed with 20 ml of PBS, collected by centrifugation at 400 × g for 5 minutes, and resuspended in 20 ml of PBS. Cells were counted with a hemocytometer, and cells were collected again by centrifugation at 400 × g for 5 minutes. Mouse or rat splenocytes were isolated in the following manner: the spleen was 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 red blood cell lysis solution for 2 min. 10 times the volume of PBS was added in excess and the cells were collected by centrifugation at 1600 rpm for 5 min. The supernatant was discarded and the splenocytes were resuspended in RPMI 1640 / 10% FBS. Magnetic beads in a commercial kit (Stemcell Technologies) specific for humans, monkeys, mice, and rats were used to enrich pan-T cells (Pan-T cell) from PBMC (human / monkey) or splenocytes (mouse / rat) by negative selection. The activation of human / monkey PBMC or mouse / rat splenocytes was performed by incubating the cells with 50 ng / ml PMA+1 μM ionomycin at 37° C., 5% CO 2 overnight.

[0234] Activate cells (~2x10 5 Cells / tube) were washed in pre-chilled staining buffer (PBS supplemented with 2% FBS) and incubated with 100 nM test antibody on ice for 1 hr. The cells were then washed twice with 1 mL staining buffer and resuspended in Alexa Fluor 500 μL PBS containing Alexa Fluor 500 μL PBS. AG10131 was added to 100 μL of staining buffer containing 647-conjugated mouse anti-human FC antibody and species-specific T cell marker antibodies. T cell marker antibodies were used as follows: CD3, CD4 or CD8. After incubation in the dark for 30 minutes, the cells were washed twice with staining buffer. Finally, the cells were resuspended in 300 μL of staining buffer and analyzed by Beckman CytoFlex. Data analysis was performed using Flowjo 10 software. As shown in Figure 4a, all tested antibodies bound to activated human and monkey T cells, but not to naive 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.

[0235] In summary, AG10058 and AG10131 antibodies show higher affinity for human and monkey CD137. They exhibit broad species cross-reactivity, including human, cynomolgus monkey, mouse, rat, and dog for AG10131, but including human, cynomolgus monkey, mouse, and dog for AG10058, allowing rapid evaluation of in vivo efficacy in mouse syngeneic models.

[0236] Example 5

[0237] Antibody binding selectivity for CD137

[0238] The selectivity of the antibodies for CD137 was assessed using flow cytometric analysis of their 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-chilled staining buffer (PBS supplemented with 2% FBS) and incubated with 100 nM test antibodies on ice for 1 hour. Cells were washed twice with staining buffer and Alexa Fluor 500 was added. 647-conjugated mouse anti-human FC antibody and incubated on ice for 30 min. The samples were washed once with staining buffer and then analyzed by flow cytometry. Figure 5 As shown, AG10058, AG10059, and AG10131 specifically bound to CD137, but not to any other family member tested or to parental cells transfected with empty vector.

[0239] Example 6

[0240] Testing ligand competition using ELISA and flow cytometry

[0241] The antibodies were tested for their ability to block the binding of CD137 to its cognate ligand CD137L as determined by ELISA and flow cytometry. As shown in Figures 6a and 6b, all tested antibodies blocked the binding of CD137 and CD137L.

[0242] 6a. Ligand competition binding assay by ELISA

[0243] Recombinant human CD137 (fused to human Fc and His tags) was diluted to 1 μg / mL in PBS and coated on Maxisorp plates overnight at 4°C. The plates were blocked with PBS supplemented with 3% skim milk for 1 hour at 37°C. After washing, a total volume of 100 μL of a mixture of 50 μL biotinylated CD137L (4 μg / mL) and various concentrations of test antibodies (eight 1:2 serial dilutions ranging from 500 μg / mL to 2 μg / mL) was added to each well and incubated at 37°C for 1 hour. The plates were washed three times and 100 μL of HRP-conjugated neutravidin (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, and 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 showed the strongest or complete blocking ability, in the range of about uM, followed by AG10058 with significant blocking of >uM; and AG10059 with effective blocking in the uM range. These data indicate that under the conditions tested and using the reagents used, the broad 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, which cross-reacts with human and monkey CD137, and AC1121, which reacts only with human CD137, showed almost no blocking at all.

[0244] 6b. Testing ligand competitive binding by flow cytometry

[0245] The plasmid encoding full-length human CD137 was transiently expressed in HEK293F cells. The cells were washed with staining buffer (PBS supplemented with 1% BSA) and resuspended in staining buffer containing 100nM test antibody. After incubation on ice for 30min, 33nM biotinylated CD137L was added to each well and incubated on ice for 1hr. The cells were washed twice with staining buffer, 50 μL of staining buffer containing Alexa fluor 647-conjugated streptavidin was added and incubated on ice for 30min. The cells were washed once and analyzed by CytoFlex flow cytometry. As shown in Figure 6b, all three test antibodies can block the combination between CD137 and CD137L in a concentration-dependent manner. AG10131 shows the strongest blocking ability, followed by AG10058 with significant blocking, and AG10059 with less effective blocking. These data demonstrate that the broadly cross-reactive antibodies AG10131 and AG10058 highly effectively block the interaction between CD137 and its ligand CD137L, while AG10059 exhibits partial blocking of the interaction between CD137 and its ligand CD137L. In contrast, the AC1097 reference antibody, which cross-reacts with both human and monkey CD137, exhibited only partial blocking, while the AC1121 reference antibody, which reacts only with human CD137, exhibited no blocking.

[0246] Example 7

[0247] Epitope mapping

[0248] In order to determine the binding region of the test antibodies at the amino acid residue level, a series of mutations were made on the extracellular domain of human CD137 (Table 5). HEK293F cells were transfected with these CD137 mutant plasmids. The binding of the antibodies to human CD137 mutants was assessed by flow cytometry analysis, as previously described in Example 5 and in Figure 7A The results are summarized in Table 5, along with the cross-reactivity of these antibodies to human, monkey, mouse, and rat CD137 at the differentiations of interest, indicating the refined epitopes from the hits derived from the Adagene library. AG10131 bound to all four species, while AG10058 bound to all three CD137s but not to rat CD137. AG10058, AG10059, and AG10131 lost their ability to bind to the GFT34AAA, FSS53AAA, and FH92AA mutations, indicating that their binding epitopes are within these regions, such as amino acid residues 34-93 or 34-108 of SEQ ID NO.: 1 (see also Figure 7BAG10058 and AG10131 may bind to the same or a highly similar epitope, whereas AG10059 may bind to a different epitope than AG10058 and AG10131.

[0249] The mutant constructs are intended to distinguish the epitopes bound by AG10058, AG10059, and AG10131 from the reference antibodies AC1121 and AC1097. Clearly, all three antibodies, AG10058, AG10059, and AG10131, target very different epitopes from AC1121 and AC1097. AG10058, AG10059, and AG10131 differ from AC1121 in the region defined by the mutants Hu_FH92AA and Hu_FSS53AAA, and possibly Hu_GTF34AAA, while AG10058, AG10059, and AG10131 differ from AC1097 in the region defined by most of the mutants used, with the exception of Hu_FH92AA and their species cross-reactivity with monkey, but differ 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 an epitope located within amino acid residues 115-156 of SEQ ID NO.:1. Figure 7A As also shown in Table 5, the binding of human CD137 ligand to wild-type human CD137 versus mutant human CD137 matched well with the binding pattern of the tested antibodies, which is consistent with the observation that these antibodies block the binding of CD137 ligand to its receptor.

[0250] Table 5: Epitope mapping

[0251]

[0252] Example 8

[0253] Agonist activity of antibodies in NFκB luciferase reporter assay

[0254] The agonist activity of the antibodies was assessed using an NFκB reporter gene assay. 293T cells were transfected with plasmids expressing human, monkey, or mouse CD137 and a NFκB luciferase reporter gene plasmid. After 4 h, 0.4 x 10 650 μL of cells were plated into each assay well of a 96-well plate at a density of / mL. A total volume of 50 μL of an antibody mixture containing the test antibody and a cross-linking antibody (Fab' goat anti-human IgG FC) at a ratio of 3:1 was added and incubated for 18 hours. After removing the culture medium, 50 μL of Passive lysis buffer (Promega E1980) was added and incubated at 37°C for 30 minutes. 20 μL of the lysate was transferred to a white plate and luciferase substrate was added. The luminescent signals of firefly and renin (Renina) were measured and their ratio was used for data analysis by GraphPad Prism6.0 software. Figure 8 As shown, all tested antibodies activated NFκB reporter gene expression when human and monkey CD137 were expressed, compared to an isotype control antibody. 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 bind to mouse CD137, while AG10059 does not.

[0255] Example 9

[0256] Agonist activity of antibodies in T cell activation assays

[0257] The agonist activity of the antibodies was further confirmed in a T cell activation assay. 50 μL of anti-CD3 antibody (2 μg / ml) was coated on a 96-well cell culture plate alone or together with 50 μL of test antibody (60 μg / mL, 20 μg / mL, 6 μg / mL, 2 μg / mL, and 0 μg / mL) in 1×PBS overnight at 4°C. CD8+ T cells were isolated using a protocol according to the manufacturer's instructions. 1×10 7 Cells were prepared in RPMI 1640 medium supplemented with 10% FBS at a density of 10 cells / mL. 200 μL of cells were plated into each assay well and incubated in a 37°C, 5% CO2 incubator for 4 days. Cell proliferation was examined under a microscope every day. After 96 hours of incubation, 100 μL of supernatant was transferred to a new 96-well plate for IFN-γ detection. T cell proliferation was determined using the Cell Titer Glow kit (Promega). Figure 9 As shown, all tested antibodies induced CD8+ T cell proliferation and IFN-γ secretion in a dose-dependent manner compared to an isotype control antibody.

[0258] Example 10

[0259] Antitumor activity in syngeneic mouse models

[0260] Species cross-reactivity with mouse CD137 allows for rapid in vivo functional assessment. AG10058 and AG10131 have been tested in multiple syngeneic mouse models. BALB / c mice (n=8 / group) were subcutaneously implanted with 2×10 6 H22 hepatocarcinoma cells (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 CT26 colon cancer cells, or 5×10 5 EMT6 breast cancer cells. When the tumor is established (>50mm 3 ), treatment with isotype control antibody, AG10058, or AG10131 was started by intraperitoneal injection twice a week for up to 3 weeks. Tumor growth was monitored twice a week and reported as mean tumor volume ± sem over time. Figure 10-13 As shown, AG10058 and AG10131 exhibited potent in vivo antitumor activity in these different syngeneic mouse tumor models compared to an isotype control antibody.

[0261] 10a. CD137 agonist antibodies exhibit antitumor efficacy in the H22 mouse liver cancer model

[0262] First, AG10058 or AG10131 was administered twice weekly at a dose of 50 mg / kg for 3 weeks. Both molecules showed almost 100% TGI (tumor growth inhibition) ( Figure 10 , panel a). Immunohistochemical staining of 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 , panel b). Further dose titration down to 3 mg / kg still showed ∼100% TGI, indicating that both molecules have potent antitumor activity ( Figure 10, panels c and d). Further dose titration of AG10131 down to 1 and 0.1 mg / kg showed greater than 50% TGI at 0.1 mg / kg and 1 mg / kg ( Figure 10 , small picture e).

[0263] 10b. CD137 agonist antibodies exhibit antitumor efficacy in the CT26 mouse colon cancer model

[0264] like Figure 10 As shown, AG10058 and AG10131 showed almost 100% TGI (tumor growth inhibition) at a dose of 50 mg / kg in the CT26 mouse colon cancer model ( Figure 11 , panel 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 , Panel b) showed almost 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 a dose-dependent antitumor activity.

[0265] 10c.EMT6 breast cancer model

[0266] The antitumor activity was further evaluated in the 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% tumor growth inhibition.

[0267] 10d. Mice that fully responded to CD137 agonist antibody treatment remained tumor-free after rechallenge with new tumor cells

[0268] Mice with complete tumor regression after 3 weeks of treatment with AG10058 or AG10131 in the CT26 tumor model remained untreated for more than a month. Mice that maintained a complete response were then treated with 5 × 105 CT26 tumor cells were subcutaneously re-stimulated and tumor growth was monitored. At the same time, a re-stimulation control group was established using naive mice inoculated with the same number of CT26 tumor cells. Figure 13 As shown, treatment with AG10131 (at 1 and 5 mg / kg, see Figure 13 , top and bottom panels, respectively) demonstrated potent antitumor activity in the CT26 tumor model, with 5 / 8 mice treated with AG10131 (1 mg / kg group) and 6 / 8 mice treated with AG10131 (5 mg / kg group) showing complete responses over 60 days before rechallenge with CT26 tumor cells. Furthermore, these mice remained tumor-free after rechallenge with the same tumor cells, indicating that specific antitumor memory developed in these mice.

[0269] To test this hypothesis, splenocytes were collected from these tumor-rejecting re-challenged mice and control mice and co-cultured with mitomycin C-arrested CT26 tumor cells in vitro for 7 days to expand tumor-specific memory T cells. These splenocytes were then recovered and mixed with fluorescently labeled live CT26 tumor cells at different E / T ratios for 4 h, 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 splenocytes from tumor-rejecting re-challenged mice previously treated with AG10058 and AG10131.

[0270] Example 11

[0271] AG10131-IgG4 does not induce ADCC effect

[0272] Using EasySep human CD8 + Human CD8 T cell enrichment kit (StemCell Technologies) was used to isolate human CD8 T cells from peripheral blood of healthy donors. + T cells were then stimulated with PMA (50 ng / ml) + ionomycin (1 uM) for 18 hours in vitro. These activated CD8 + T cells were used as target cells. NK cells from different healthy donors were isolated using a human NK isolation kit (StemCell Technologies) and used as effector cells. For antibody-dependent cellular cytotoxicity (ADCC) assays, effector cells (NK) and target cells (activated CD8 +T cells) were mixed in a 96-well plate at a ratio of 5:1 and cultured for 4 hours. The supernatant from each well was then collected and the fluorescence signal was detected by a plate reader SpectraMax i3x (Ex 488nm, Em 520nm). Isotype hIgG4 mAb was used as a negative control, while humanized OKT3 (anti-CD3 hIgG1 from Novoprotein) was used as a positive control. The % lysis was then calculated using the following formula: % lysis = [(experimental release) - Ave (target + NK)] / [Ave (target Max) - Ave (target only)] × 100% ( Figure 15 ).

[0273] Example 12

[0274] Developmental characteristics of antibodies

[0275] For developmental evaluation, purified AG10058, AG10059, AG10131, and AC1097 were exchanged into PB buffer (20 mM PB, 150 mM NaCl, pH 7.0). All experiments, including filtration, centrifugation, and accelerated stress testing, were performed in PB buffer. For all SEC-HPLC analyses, a TSKgel column (Tosoh Bioscience G3000SWxl) was used.

[0276] 12a. Solubility

[0277] 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. Samples (10 μg each) were then assayed by SEC-HPLC to detect high molecular weight (HMW) aggregates. As shown in the chromatogram ( Figure 16 ), no increase in HMW aggregates was observed at high concentration (20 mg / ml) for all antibodies tested.

[0278] Table 6: Antibody solubility

[0279]

[0280] 12b. Antibody Solubility under Accelerated Stress Conditions

[0281] Antibody solubility was also examined under accelerated stress conditions and the results are summarized in Table 7. All antibodies remained stable after six cycles of freezing (-80°C) and thawing (room temperature) ( Figure 17 After seven days at 50°C, there was little change in HMW aggregates or LMW fragments ( Figure 17In longer-term time-course experiments (40°C for up to 28 days), all antibodies remained stable with no significant increase in HMW aggregates or LMW fragments ( Figure 17 ).

[0282] Table 7: Changes in HMW under accelerated conditions

[0283]

[0284] Furthermore, thermal stability measurements by differential scanning calorimetry (DSC) showed that AG10131 and AG10058 were stable up to at least about 59°C. The midpoint of the transition, Tm (the characteristic temperature at which the unfolding transition occurs for almost all protein domains), was approximately Figure 18 and shown in Table 8 below.

[0285] Table 8: Thermal stability measured by DSC

[0286] Tm starting point (℃) Tm1(℃) Tm2(℃) AG10058 61.5 67.3 76.9 AG10131 59.3 67.6 81.5

[0287] Furthermore, the highest achievable concentrations after centrifugation exceeded 180 mg / mL and 220 mg / mL for AG10131 and AG10058, respectively.

[0288] Example 13

[0289] Safety profile in relevant species: mice and cynomolgus monkeys

[0290] 13a. Repeated-dose toxicity study of AG10131 in normal C57BL / 6 mice.

[0291] Repeated dose toxicity of AG10131 is carried out in normal C57BL / 6 mice. Vehicle, AG10131 (100 mg / kg) were administered intraperitoneally (ip) (10 mL / kg) on ​​the 1st, 4th, 8th and 11th days. Five female mice (7-8 weeks old) were included in each group. The abnormal behavior and symptoms of mice were monitored every day, and food intake and body weight were measured every day. On the 14th day, the animals were euthanized for autopsy and other analysis. Blood was collected from each animal, and 2 blood samples per group were used for hematological analysis (RBC, platelets, WBC, WBC classification) and 3 other blood samples in the group were 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 stored in FFPE: heart, lung, thymus, liver, spleen and kidney. FFPE blocks of liver tissue were prepared, sectioned, and stained with H&E for histopathological analysis.

[0292] No abnormal behavior or unplanned animal deaths were observed throughout the in-life period of the study. AG10131 did not affect food intake and body weight compared to vehicle treatment. Necropsy also did not reveal any obvious lesions in the mice treated with AG10131. Hematological analysis did not reveal any significant changes in the 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.

[0293] 13b. Repeated-dose study of AG10131 in cynomolgus monkeys

[0294] Repeated dosing studies of AG10131 were performed in normal cynomolgus monkeys. Human IgG4 isotype control (10 mg / kg), AG10131 (0.5 and 10 mg / kg) were administered intravenously (iv) (1 mL / kg) on ​​days 0, 7, 14, and 22. One male and one female cynomolgus monkey (3 to 5 years old) were included in each group. The animals were monitored daily for abnormal behavior and clinical signs, and food intake was measured daily. Body weight was measured on days (-15), (-5) before dosing, and on days 6, 13, 18, and 26 after dosing. Hematology and blood chemistry parameters were measured on days (-12) and (-5) before dosing and on days 7, 14, 19, and 27 after dosing (10 mg / kg group only), and urinalysis was performed on days (-12) before dosing, (-5) and on days 6, 13, and 18 after dosing. Animals in the 10 mg / kg group were euthanized on day 27 for autopsy and other analyses. Major organs were removed and weighed. FFPE liver tissue blocks were prepared, sectioned, and stained with H&E for histopathological analysis.

[0295] No abnormal behavior or unplanned animal deaths were observed in any group throughout the in-life period of the study. Treatment with 10 mg / kg of AG10131 did not affect food intake and body weight compared to vehicle treatment. No clinical signs, including injection site reactions, were noted. Autopsies did not show any obvious lesions or weight abnormalities in all organs examined in cynomolgus monkeys treated with 10 mg / kg of AG10131. Hematology, blood chemistry, and urine parameters were also within normal ranges in all treatment groups ( Figure 21 After repeated administration of AG10131 at 10 mg / kg, histopathological analysis of the liver did not show any obvious abnormalities, including lymphocytic infiltration ( Figure 22Overall, AG10131 was well tolerated in cynomolgus monkeys at doses up to 10 mg / kg per week and no overt toxicity was detected.

[0296] Example 14

[0297] Pharmacokinetics of AG10131 in cynomolgus monkeys

[0298] 14a. Pharmacokinetics of AG10131 in cynomolgus monkeys

[0299] The pharmacokinetic study of AG10131 was conducted in naive cynomolgus monkeys. Three dose levels of AG10131 (10 mg / kg, 30 mg / kg, and 100 mg / kg) were administered intravenously to three groups of monkeys. Each group consisted of three males and three females. Serum samples were collected before 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.

[0300] AG10131 was rapidly cleared on day 14 (336 hr) in 12 of the 16 animals, 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, 2 more animals from the high dose group showed rapid clearance. Serum concentrations in these 14 animals were low or below the limit of quantification. This is consistent with the observation that antidrug antibodies were produced 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 ranges from 7.3 to 8.8 days.

[0301] 14b. Pharmacokinetics of AG10131 in rats

[0302] The pharmacokinetic study of AG10131 was conducted in naive SD rats. Three dose levels of AG10131 (10 mg / kg, 30 mg / kg, and 100 mg / kg) were administered intravenously to three groups of animals. Each group consisted of 15 males and 15 females. Serum samples were collected from three animals at each time point: pre-dose, 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 post-dose. The serum concentration of AG10131 was determined by ELISA and the data were analyzed by Phoenix Professional V6.3.

[0303] 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.

[0304] 14c. Pharmacokinetics of AG10131 in mice

[0305] The pharmacokinetic study of AG10131 was conducted in BALB / c mice of approximately 8 weeks of age. Three female BALB / c mice in each dosing group were injected intravenously with test antibodies including AG10131 at 1 mg / kg via the tail vein. Blood samples (~100 u1 per sample) were collected at 1 h, 8, 48, 168 and 336 hours after administration. Blank control blood was collected from three naive female mice that were not administered the antibody. The serum concentration of each test antibody, including AG10131, was determined by ELISA, where anti-human IgG (Fc specific) antibodies were used for capture and HRP-labeled anti-human IgG (Fab specific) antibodies were used for detection.

[0306] All tested antibodies, including the isotype control (AG10154), two benchmark antibodies (AC1020 and AC1021), and three Adagene antibodies (AG10131, AG10058, and AG10059), exhibited comparable pharmacokinetics in mice ( Figure 25 ). Sequence Listing <110> Adagene Inc. <120> Anti-CD137 molecules and their uses <130> 695402000340 <140> Not yet allocated <141> at the same time <160> 865 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (1)..(1) <223> X1: F or Y <220> <221> X2 <222> (4)..(4) <223> X2: S or T <220> <221> X3 <222> (5)..(5) <223> X3: G or N or S <220> <221> X4 <222> (7)..(7) <223> X4: A or 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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (4)..(4) <223> X1: S or T <220> <221> X2 <222> (7)..(7) <223> X2: H or Y <220> <221> X3 <222> (8) <223> X3: H or Y <220> <221> X4 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (6) <223> X1: G or S <220> <221> X2 <222> (9)..(9) <223> X2: A or G <220> <221> X3 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (7)..(7) <223> X1: A or D or Y <220> <221> X2 <222> (8) <223> X2: D or G <220> <221> X3 <222> (11)..(11) <223> X3: R or S or Y <220> <221> X4 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (3) <223> X1: D or E <220> <221> X2 <222> (9)..(9) <223> X2: N or S <220> <221> X3 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (3) <223> X1: A or G or S or V or Y <220> <221> X2 <222> (7)..(7) <223> X2: A or D or S or Y <220> <221> X3 <222> (9)..(9) <223> X3: D or G or S <220> <221> X4 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (3) <223> X1: E or G <220> <221> X2 <222> (5)..(5) <223> X2: E or S <220> <221> X3 <222> (6) <223> X3: D or T <220> <221> X4 <222> (7)..(7) <223> X4: A or T or V <220> <221> X5 <222> (9)..(9) <223> X5: A or I or L or T or V <220> <221> X6 <222> (14)..(14) <223> X6: A or 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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (1)..(1) <223> X1: Q or R <220> <221> X2 <222> (5)..(5) <223> X2: D or G or S <220> <221> X3 <222> (6) <223> X3: I or V <220> <221> X4 <222> (7)..(7) <223> X4: G or R or S or T <220> <221> X5 <222> (8) <223> X5: P or R or S or T <220> <221> X6 <222> (9)..(9) <223> X6: A or D or F or S or V or Y <220> <221> X7 <222> (10)..(10) <223> X7: L or V <220> <221> X8 <222> (11)..(11) <223> X8: A or 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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (1)..(1) <223> X1: A or D <220> <221> X2 <222> (4)..(4) <223> X2: N or S or T <220> <221> X3 <222> (5)..(5) <223> X3: L or R <220> <221> X4 <222> (6) <223> X4: A or E or Q <220> <221> X5 <222> (7)..(7) <223> X5: S or T <220> <221> X6 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (5)..(5) <223> X1: A or G or S or Y <220> <221> X2 <222> (7)..(7) <223> X2: Q or S or Y <220> <221> X3 <222> (8) <223> X3: I or L or T or Y <220> <221> X4 <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 organism <220> <223> synthetic sequence <220> <221> X1 <222> (3) <223> X1: E or Q <220> <221> X2 <222> (5)..(5) <223> X2: P or S or Y <220> <221> X3 <222> (6) <223> X3: D or L or S or T or Y <220> <221> X4 <222> (7)..(7) <223> X4: D or E or H or S or T <220> <221> X5 <222> (8) <223> X5: D or L or T or W <220> <221> X6 <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 organism <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 organism <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 organism <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 organism <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 <212> PRT <213> Artificial organism <220> <223> synthetic sequence <400> 17 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 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 107 <212> PRT <213> Artificial organism <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 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 <210> 19 <211> 120 <212> PRT <213> Artificial organism <220> <223> synthetic sequence <400> 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 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 organism <220> <223> Synthetic sequence <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 organism <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 organism <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 organism <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<0​​​​​​​​​ 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 organism <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 organism <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 organism <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 organism <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 organism <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 organism <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 organism <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 organism <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 organism <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 organism <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[[ID=ll]] <212> PRT <213> Artificial organism <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 organism <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 organism <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 100 105 <210> 37 <211> 124 <212> PRT <213> Artificial organism <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 organism <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 organism <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 organism <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 organism <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​​​​​​​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 organism <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 organism <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 organism <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 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 45 <211> 123 <212> PRT <213> Artificial organism <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<00019s40><210> 46 <211> 107 <212> PRT <213> Artificial organism <220> <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 100 105 <210> 47 <211> 123 <212> PRT <213> Artificial organism <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 organism <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 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 organism <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 �5 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[[ID=

[23] ] <210> 50 <211> 107 <212> PRT <213> Artificial organism <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 organism <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 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 organism <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 organism <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[[ID=⑨]] <211> 107 <212> PRT <213> Artificial organism <220> <223> Synthetic sequence <400> 54[[ID=②1]] 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 Note: In the translation, for the tags like etc., they are preserved as they are because they seem to be some kind of specific identifiers in the patent text format and might have specific meanings within that context. Also, for the Chinese characters in the amino acid sequences, they are translated according to the standard amino acid names in English. For the <xxx> tags with text that seems to be some kind of sequence or structure identifiers, they are kept unchanged as they might be crucial for the patent's specific content understanding and indexing. ⑨ and ②1 are just the numbers in the original text that are likely part of a sequence or identifier system and are maintained as is. If there is a specific rule or convention for these numbers in the original language's patent system, it would be important to follow that more precisely in a real - world patent translation scenario. Here, since no further information is given, they are just left as they are to match the original text's structure. <210> 55 <211> 124 <212> PRT <213> Artificial organism <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 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 Ala Gly Asp Trp Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 107 <212> PRT <213> Artificial organism <220> <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 organism <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 organism <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 organism <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 organism <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 organism <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 [[ID=S38]] 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 organism <220> <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 organism <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 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 64 <211> 110 <212> PRT <213> Artificial organism <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 40 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 organism <220> <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 organism <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 organism <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 [[ID=​​​​​​​​​​​​ 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 organism <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[[ID=2^3]] <210> 70 <211> 107 <212> PRT <213> Artificial organism <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 2^0 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 organism <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 organism <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 organism <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<00,02542>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 organism <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 organism <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<x 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 organism <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 organism <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 organism <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 organism <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 organism <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 <213> Artificial organism <220> <223> Synthetic sequence <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Ala 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 organism <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 organism <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 organism <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 organism <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 organism <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 organism <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 organism <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 e100 105 <210> 89 <211> 124 <212> PRT <213> Artificial organism <220> <223> Synthetic sequence <400> 89 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

注:这里的“e100”可能有误,推测是“100”,按照100翻译

Claims

1. An isolated antibody that binds to the extracellular domain of human CD137, or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising 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: 712, HVR-H2 consisting of the amino acid sequence of SEQ ID NO: 736, and HVR-H3 consisting of the amino acid sequence of SEQ ID NO: 760; and (ii) the light chain variable region comprises HVR-L1 consisting of the amino acid sequence of SEQ ID NO:784, HVR-L2 consisting of the amino acid sequence of SEQ ID NO:808, and HVR-L3 consisting of the amino acid sequence of SEQ ID NO:

832.

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: 61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

62.

3. The antibody or antigen-binding fragment of claim 2, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 619, and the light chain comprises the amino acid sequence of SEQ ID NO:

620.

4. The antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antigen-binding fragment has a K of 100 nM or less as measured by surface plasmon resonance. D Binds human CD137.

5. The antibody or antigen-binding fragment of claim 4, wherein the antibody or antigen-binding fragment has a K of 50 nM or less as measured by surface plasmon resonance. D Binds human CD137.

6. The antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey CD137.

7. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the activity of human CD137 expressed on human cells is reduced when contacted with the antibody or antigen-binding fragment.

8. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment has a half-maximal inhibitory concentration (IC) of 100 nM or less for blocking the in vitro binding of human CD137 to human CD137L. 50 ).

9. The antibody or antigen-binding fragment of any one of claims 1-3, wherein when the antibody or antigen-binding fragment is provided at a concentration of 1 μM or greater, the antibody or antigen-binding fragment completely blocks the in vitro binding of human CD137 to human CD137L.

10. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the activity of human CD137 expressed on human cells is increased when contacted with the antibody or antigen-binding fragment.

11. The antibody or antigen-binding fragment of claim 10, wherein contacting human cells expressing CD137 with the antibody or antigen-binding fragment results in increased NF-κB-dependent transcription.

12. The antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody comprises a human IgG2 Fc region.

13. The antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody comprises a human IgG4 Fc region.

14. The antibody or antigen-binding fragment of claim 13, wherein the human IgG4 Fc region comprises an S241P mutation, wherein numbering is according to Kabat.

15. A polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1-14. A vector comprising the polynucleotide according to claim 15 .

17. The vector of claim 16, wherein the vector is an expression vector.

18. A host cell comprising the polynucleotide of claim 15 or the vector of claim 16 or 17.

19. A method for preparing an antibody or an antigen-binding fragment, comprising culturing the host cell of claim 18 under conditions suitable for producing the antibody or the antigen-binding fragment.

20. The method of claim 19, further comprising recovering the antibody or antigen-binding fragment produced by the cell.

21. An isolated antibody or antigen-binding fragment thereof that binds to the extracellular domain of human CD137, which is prepared by the method of claim 19 or 20.

22. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-14 and 21, and a pharmaceutically acceptable carrier.

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