Anti-CD137 construct, multispecific antibody and its use

Abstract

The present invention provides anti-CD137 constructs that bind to CD137, including multispecific anti-CD137 antibodies that have binding specificity for CD137 and one or more additional antigens, and methods of using the same. In some embodiments, the one or more additional antigens include epidermal growth factor receptor (EGFR).

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of International Patent Application No. PCT / CN2020 / 077148, filed on February 28, 2020, the entire content of which is incorporated herein by reference and for which priority is claimed.

[0002] This application relates to antibodies that bind to CD137 (including anti - CD137 monoclonal - specific antibodies and multispecific antibodies), methods of preparation, and their use, said use including the treatment of diseases or medical conditions.

Background Art

[0003] CD137 (also known as 4 - 1BB and TNFRSF9) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). Currently, from the understanding of CD137, its expression is usually activation - dependent and has been shown to be present in a wide subset of immune cells including activated NK and NKT cells, regulatory T cells, dendritic cells (DCs), stimulated mast cells, differentiating myeloid cells, monocytes, neutrophils, and eosinophils (Wang, 2009, Immunological Reviews 229:192 - 215). Furthermore, CD137 expression has been confirmed in tumor vasculature (Broll, 2001, Amer. J. Clin. Pathol. 115(4):543 - 549, Seaman, 2007, Cancer Cell 11:539 - 554) and endothelial cell sites of inflammation or atherosclerotic lesions (Drenkard, 2007 FASEB J. 21:456 - 463, Olofsson, 2008, Circulation 117:1292 - 1301). The ligand that stimulates CD137, namely CD137 ligand (4 - 1BBL), is expressed on activated antigen - presenting cells (APCs), myeloid progenitor cells, and hematopoietic stem cells.

[0004] Human CD137 is a 255 - amino - acid protein. The receptor is expressed on the cell surface in monomeric and dimeric forms and can trimerize with the CD137 ligand to activate downstream signals. 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). According to research, some CD137 agonist mAbs can exert antitumor effects in various models by increasing the expression of costimulatory molecules and significantly enhancing the cytolytic T lymphocyte response. CD137 agonist mAbs have been confirmed to be effective in preventive and therapeutic settings. Also, tumor models of CD137 monotherapy and combination therapy have established a persistent antitumor protective T - cell memory response (Lynch, 2008, Immunol Rev.22:277 - 286). In various autoimmune models generally recognized in this field (Vinay, 2006, J Mol Med 84:726 - 736), CD137 agonists have also been shown to inhibit autoimmune responses. Such dual activity of CD137 can provide potential antitumor activity and inhibit autoimmune side effects that may be associated with immunotherapies that disrupt immune tolerance.

[0005] All publications, patents, patent applications, and the entire content of the disclosed patent applications incorporated herein by reference.

Summary of the Invention

[0006] The present disclosure provides anti - CD137 constructs (e.g., anti - CD137 monoclonal antibodies and anti - CD137 multispecific antibodies), polynucleotides encoding the anti - CD137 constructs, kits, methods for modulating cell compositions, and methods for treating an individual using the anti - CD137 constructs. The present invention is based in part on the discovery of anti - CD137 monospecific and multispecific antibodies that exhibit improved safety and enhanced antitumor effects compared to conventional anti - CD137 antibodies in clinical trials.

[0007] The present disclosure provides a multispecific antibody that binds to CD137 and EGFR. In some embodiments, the multispecific antibody disclosed herein comprises a first antibody portion that binds to CD137 and a second antibody portion that binds to EGFR. In some embodiments, the first antibody portion comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein a) the V H comprises i) an HC-CDR1 comprising the amino acid sequence of GFX1X2X3DTYIX4 (SEQ ID NO: 177), wherein X1 = N or C, X2 = I, P, L or M, X3 = K, N, R, C or Q, and X4 = H or Q; ii) an HC-CDR2 comprising the amino acid sequence of X1IDPANGX2X3X4 (SEQ ID NO: 178), wherein X1 = K or R, X2 = N, G, F, Y, A, D, L, M or Q, X3 = S or T, and X4 = E or M; and iii) an HC-CDR3 comprising the amino acid sequence of GNLHYX1LMD (SEQ ID NO: 179), wherein X1 = Y, A or G, and b) the V Lis an LC-CDR1 comprising the amino acid sequence of KASQX1X2X3TYX4S (SEQ ID NO: 180), where X1 = A, P or T, X2 = I, T or P, X3 = N or A, and X4 = L, G or H; an LC-CDR2 comprising the amino acid sequence of RX1NRX2X3X4 (SEQ ID NO: 181), where X1 = A, Y, V or D, X2 = M, K, V or A, X3 = V, P, Y or G, and X4 = D or G; and an LC-CDR3 comprising the amino acid sequence of LQX1X2DFPYX3 (SEQ ID NO: 182), where X1 = Y, S or F, X2 = D, V, L, R, E or Q, and X3 = T or K.In some embodiments, HC-CDR1 comprises any one of the amino acid sequences of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 261, and 271, or a variant thereof comprising up to about 3 amino acid substitutions; HC-CDR2 comprises any one of the amino acid sequences of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 262, and 272, or a variant thereof comprising up to about 3 amino acid substitutions; HC-CDR3 comprises any one of the amino acid sequences of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 263, and 273, or a variant thereof comprising up to about 3 amino acid substitutions; LC-CDR1 comprises any one of the amino acid sequences of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 264, and 274, or a variant thereof comprising up to about 3 amino acid substitutions; LC-CDR2 comprises any one of the amino acid sequences of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 265, and 275, or a variant thereof comprising up to about 3 amino acid substitutions; and LC-CDR3 comprises any one of the amino acid sequences of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 266, and 276, or a variant thereof comprising up to about 3 amino acid substitutions.

[0008] In some embodiments, the first antibody portion cross-competes with a reference anti-CD137 construct for binding to CD137, and the reference anti-CD137 construct comprises a heavy chain variable region (V H )(the heavy chain variable region comprises HC-CDR1, HC-CDR2, and HC-CDR3 domains) and a light chain variable region (V L )(the light chain variable region comprises LC-CDR1, LC-CDR2, and LC-CDR3 domains), and it a) The V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:1, HC-CDR2 containing the amino acid sequence of SEQ ID NO:2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:3, and the V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:4, LC-CDR2 containing the amino acid sequence of SEQ ID NO:5, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:6, b) The V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:11, HC-CDR2 containing the amino acid sequence of SEQ ID NO:12, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:13, and the V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:14, LC-CDR2 containing the amino acid sequence of SEQ ID NO:15, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:16, c) The V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:21, HC-CDR2 containing the amino acid sequence of SEQ ID NO:22, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:23, and the V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:24, LC-CDR2 containing the amino acid sequence of SEQ ID NO:25, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:26, d) The V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:31, HC-CDR2 containing the amino acid sequence of SEQ ID NO:32, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:33, and the V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:34, LC-CDR2 containing the amino acid sequence of SEQ ID NO:35, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:36, e) The V Hcomprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:41, HC-CDR2 containing the amino acid sequence of SEQ ID NO:42, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:43, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:44, LC-CDR2 containing the amino acid sequence of SEQ ID NO:45, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:46, f) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:51, HC-CDR2 containing the amino acid sequence of SEQ ID NO:52, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:53, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:54, LC-CDR2 containing the amino acid sequence of SEQ ID NO:55, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:56, g) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:61, HC-CDR2 containing the amino acid sequence of SEQ ID NO:62, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:63, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:64, LC-CDR2 containing the amino acid sequence of SEQ ID NO:65, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:66, h) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:71, HC-CDR2 containing the amino acid sequence of SEQ ID NO:72, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:73, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:74, LC-CDR2 containing the amino acid sequence of SEQ ID NO:75, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:76, i) said V Hcomprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:81, HC-CDR2 containing the amino acid sequence of SEQ ID NO:82, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:83, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:84, LC-CDR2 containing the amino acid sequence of SEQ ID NO:85, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:86, j) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:91, HC-CDR2 containing the amino acid sequence of SEQ ID NO:92, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:93, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:94, LC-CDR2 containing the amino acid sequence of SEQ ID NO:95, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:96, k) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:101, HC-CDR2 containing the amino acid sequence of SEQ ID NO:102, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:103, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:104, LC-CDR2 containing the amino acid sequence of SEQ ID NO:105, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:106, l) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:111, HC-CDR2 containing the amino acid sequence of SEQ ID NO:112, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:113, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:114, LC-CDR2 containing the amino acid sequence of SEQ ID NO:115, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:116, m) said V Hcomprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:121, HC-CDR2 containing the amino acid sequence of SEQ ID NO:122, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:123, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:124, LC-CDR2 containing the amino acid sequence of SEQ ID NO:125, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:126, n) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:131, HC-CDR2 containing the amino acid sequence of SEQ ID NO:132, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:133, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:134, LC-CDR2 containing the amino acid sequence of SEQ ID NO:135, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:136, o) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:261, HC-CDR2 containing the amino acid sequence of SEQ ID NO:262, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:263, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:264, LC-CDR2 containing the amino acid sequence of SEQ ID NO:265, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:266, p) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:271, HC-CDR2 containing the amino acid sequence of SEQ ID NO:272, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:273, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:274, LC-CDR2 containing the amino acid sequence of SEQ ID NO:275, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:276, and is selected from the group consisting of

[0009] In some embodiments, the first antibody portion comprises a heavy chain variable region (V H )(the heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 domains) and a light chain variable region (V L )(the light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3 domains), where the V H and the V L are a) the V H comprises HC-CDR1 having the amino acid sequence of SEQ ID NO:1, HC-CDR2 having the amino acid sequence of SEQ ID NO:2, and HC-CDR3 having the amino acid sequence of SEQ ID NO:3, and the V L comprises LC-CDR1 having the amino acid sequence of SEQ ID NO:4, LC-CDR2 having the amino acid sequence of SEQ ID NO:5, and LC-CDR3 having the amino acid sequence of SEQ ID NO:6, b) the V H comprises HC-CDR1 having the amino acid sequence of SEQ ID NO:11, HC-CDR2 having the amino acid sequence of SEQ ID NO:12, and HC-CDR3 having the amino acid sequence of SEQ ID NO:13, and the V L comprises LC-CDR1 having the amino acid sequence of SEQ ID NO:14, LC-CDR2 having the amino acid sequence of SEQ ID NO:15, and LC-CDR3 having the amino acid sequence of SEQ ID NO:16, c) the V H comprises HC-CDR1 having the amino acid sequence of SEQ ID NO:21, HC-CDR2 having the amino acid sequence of SEQ ID NO:22, and HC-CDR3 having the amino acid sequence of SEQ ID NO:23, and the V L comprises LC-CDR1 having the amino acid sequence of SEQ ID NO:24, LC-CDR2 having the amino acid sequence of SEQ ID NO:25, and LC-CDR3 having the amino acid sequence of SEQ ID NO:26, d) the V Hcomprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:31, HC-CDR2 containing the amino acid sequence of SEQ ID NO:32, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:33, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:34, LC-CDR2 containing the amino acid sequence of SEQ ID NO:35, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:36, e) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:41, HC-CDR2 containing the amino acid sequence of SEQ ID NO:42, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:43, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:44, LC-CDR2 containing the amino acid sequence of SEQ ID NO:45, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:46, f) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:51, HC-CDR2 containing the amino acid sequence of SEQ ID NO:52, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:53, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:54, LC-CDR2 containing the amino acid sequence of SEQ ID NO:55, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:56, g) said V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:61, HC-CDR2 containing the amino acid sequence of SEQ ID NO:62, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:63, and said V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:64, LC-CDR2 containing the amino acid sequence of SEQ ID NO:65, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:66, h) said V Hcomprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:71, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:72, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:73, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:74, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:75, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:76, i) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:81, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:82, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:83, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:84, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:85, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:86, j) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:91, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:92, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:93, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:94, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:95, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:96, k) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:101, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:102, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:103, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:104, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:105, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:106, l) said V Hcomprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:111, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:112, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:113, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:114, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:115, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:116, m) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:121, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:122, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:123, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:124, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:125, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:126, n) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:131, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:132, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:133, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:134, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:135, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:136, o) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:261, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:262, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:263, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:264, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:265, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:266, p) said VH comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:271, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:272, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:273, and said V L is selected from comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:274, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:275, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:276.

[0010] In some embodiments, said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:11, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:13, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:15, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:22, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:23, and said V Lincludes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:25, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:31, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:32, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:33, and the V L includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:34, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:41, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:42, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:43, and the V L includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:44, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:45, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:51, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:52, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:53, and the V L includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:56. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:61, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:62, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:63, and the V LIt includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:64, LC-CDR2 containing the amino acid sequence of SEQ ID NO:65, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:66. In some embodiments, the V H It includes HC-CDR1 containing the amino acid sequence of SEQ ID NO:71, HC-CDR2 containing the amino acid sequence of SEQ ID NO:72, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:73, and the V L It includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:74, LC-CDR2 containing the amino acid sequence of SEQ ID NO:75, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:76. In some embodiments, the V H It includes HC-CDR1 containing the amino acid sequence of SEQ ID NO:81, HC-CDR2 containing the amino acid sequence of SEQ ID NO:82, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:83, and the V L It includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:84, LC-CDR2 containing the amino acid sequence of SEQ ID NO:85, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:86. In some embodiments, the V H It includes HC-CDR1 containing the amino acid sequence of SEQ ID NO:91, HC-CDR2 containing the amino acid sequence of SEQ ID NO:92, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:93, and the V L It includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:94, LC-CDR2 containing the amino acid sequence of SEQ ID NO:95, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:96. In some embodiments, the V H It includes HC-CDR1 containing the amino acid sequence of SEQ ID NO:101, HC-CDR2 containing the amino acid sequence of SEQ ID NO:102, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:103, and the V Lincludes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:104, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:105, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:106. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:111, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:112, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:113, and the V L includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:114, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:115, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:116. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:121, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:122, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:123, and the V L includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:124, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:125, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:126. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:131, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:132, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:133, and the V L includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:134, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:135, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:136. In some embodiments, the V H includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:141, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:142, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:143, and the V LIt includes an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:144, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:145, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:146. In some embodiments, the said V H includes an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:261, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:262, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:263, and the said V L includes an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:264, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:265, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:266. In some embodiments, the said V H includes an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:271, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:272, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:273, and the said V L includes an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:274, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:275, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:276.

[0011] In some examples, the said first antibody portion a) includes HC-CDR1, HC-CDR2, and HC-CDR3 which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the V H chain region having the sequence shown in SEQ ID NO:7, and LC-CDR1, LC-CDR2, and LC-CDR3 which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the V L chain region having the sequence shown in SEQ ID NO:8, and b) V HHC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 18 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and c) V having the sequences shown in SEQ ID NO: 27 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 28 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and d) V having the sequences shown in SEQ ID NO: 37 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 38 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and e) V having the sequences shown in SEQ ID NO: 47 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 48 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and f) V having the sequences shown in SEQ ID NO: 57 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 58 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and g) V having the sequences shown in SEQ ID NO: 67 respectively H HC - CDR1, HC - CDR2 and HC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 68 respectively L LC - CDR1, LC - CDR2 and LC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain region, and h) V having the sequences shown in SEQ ID NO: 77 respectively H HC - CDR1, HC - CDR2 and HC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 78 respectively L LC - CDR1, LC - CDR2 and LC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain region, and i) V having the sequences shown in SEQ ID NO: 87 respectively H HC - CDR1, HC - CDR2 and HC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 88 respectively L LC - CDR1, LC - CDR2 and LC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain region, and j) V having the sequences shown in SEQ ID NO: 97 respectively H HC - CDR1, HC - CDR2 and HC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 98 respectively L LC - CDR1, LC - CDR2 and LC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain region, and k) V having the sequences shown in SEQ ID NO: 107 respectively H HC - CDR1, HC - CDR2 and HC - CDR3 containing the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 108 respectively LLC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and l) V having the sequences shown in SEQ ID NO: 117 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 118 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and m) V having the sequences shown in SEQ ID NO: 127 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 128 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, or n) V having the sequences shown in SEQ ID NO: 137 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 138 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, and o) V having the sequences shown in SEQ ID NO: 267 respectively H HC-CDR1, HC-CDR2, and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain region, and V having the sequences shown in SEQ ID NO: 268 respectively L LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain region, or p) V having the sequences shown in SEQ ID NO: 277 respectively HHC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain region, and V which has the sequence shown in SEQ ID NO: 278 respectively L It includes LC-CDR1, LC-CDR2 and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain region.

[0012] In some embodiments, the first antibody portion is (a) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 7, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 8; (b) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 17, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 18; (c) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 27, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 28; (d) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 37, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 38; (e) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 47, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 48; (f) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 57, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 58; (g) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 67, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 68; (h) a heavy chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 77, and a light chain variable region containing an amino acid having the sequence shown in SEQ ID NO: 78; (i) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:87, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:88, (j) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:97, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:98, (k) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:107, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:108, (l) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:117, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:118, (m) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:127, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:128, (n) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:137, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:138, (o) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:267, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:268, or (p) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:277, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:278.

[0013] In some embodiments, the first antibody portion comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), where a) the V Hcomprises: i) an HC-CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 151-153, or a variant thereof comprising up to about 3 amino acid substitutions; ii) an HC-CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 154-156, or a variant thereof comprising up to about 3 amino acid substitutions; and iii) an HC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 157-159, or a variant thereof comprising up to about 3 amino acid substitutions; b) said V L comprises: i) an LC-CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 160-163, or a variant thereof comprising up to about 3 amino acid substitutions; ii) an HC-CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 164-166, or a variant thereof comprising up to about 3 amino acid substitutions; and iii) an HC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 167-169, or a variant thereof comprising up to about 3 amino acid substitutions.

[0014] In some embodiments, said first antibody portion comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein a) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 151, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 154, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 157, and said V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 160, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 164, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 167, b) said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 151, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 154, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 157, and said V Lcomprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:162, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:166, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:169, c) said V H comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:152, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:155, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:158, and said V L comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:163, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:166, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:169, d) said V H comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:153, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:156, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:159, and said V L comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:160, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:164, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:167, or e) said V H comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:153, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:156, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:159, and said V L comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:161, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:165, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:168.

[0015] In some embodiments, the first antibody moiety comprises an antibody or an antigen-binding fragment thereof selected from the group consisting of a full-length antibody, a multispecific antibody (e.g., a bispecific antibody), a single-chain Fv (scFv), a Fab fragment, a Fab' fragment, an F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a VH, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, and a tetrabody. In some embodiments, the first antibody moiety comprises a humanized anti-CD137 full-length antibody. In some embodiments, the first antibody moiety comprises a humanized anti-CD137 single-chain Fv fragment (scFv). In some embodiments, the first antibody moiety is a CD137 agonist antibody. H In some embodiments, the first antibody moiety comprises an anti-CD137 antibody moiety comprising an Fc region of a human immunoglobulin. In some embodiments, the Fc region is selected from the group consisting of the Fc regions of IgG, IgA, IgD, IgE, and IgM. In some embodiments, the Fc region is selected from the group consisting of the Fc regions of IgG1, IgG2, IgG3, and IgG4.

[0016] In some embodiments, the first antibody moiety binds to human CD137 and cynomolgus CD137. In some embodiments, the first antibody moiety does not bind to mouse CD137.

[0017] In some embodiments, the first antibody moiety comprises a single-chain Fv anti-CD137 fragment comprising a heavy-chain variable region (VH) and a light-chain variable region (VL), and the second antibody moiety comprises a full-length antibody that binds to EGFR and comprises two antibody heavy chains and two antibody light chains, wherein each heavy chain comprises a second heavy-chain variable region (VH), and each light chain comprises a second light-chain variable region (VL).

[0018] In some embodiments, the first antibody moiety H comprises a single-chain Fv anti-CD137 fragment comprising a heavy-chain variable region (VH) and a light-chain variable region (VL), and the second antibody moiety comprises a full-length antibody that binds to EGFR and comprises two antibody heavy chains and two antibody light chains, wherein each heavy chain comprises a second heavy-chain variable region (VH), and each light chain comprises a second light-chain variable region (VL). L ), and the second antibody moiety comprises a full-length antibody that binds to EGFR and comprises two antibody heavy chains and two antibody light chains, wherein each heavy chain comprises a second heavy-chain variable region (V H-2 ), and each light chain comprises a second light-chain variable region (V L-2) and wherein the anti-CD137 single-chain Fv fragment fuses with at least one of the heavy chain or the light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the C-terminus of each heavy chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the N-terminus of each heavy chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the C-terminus of each light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the N-terminus of each light chain of the full-length antibody.

[0019] In some embodiments, the V of the anti-CD137 single-chain Fv fragment H and V L fuse via a first peptide linker. In some embodiments, the first peptide linker contains about 4 to about 15 amino acids. In some embodiments, the first peptide linker contains a linker containing any one of the sequences of SEQ ID NOs: 232-260. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the full-length antibody via a second peptide linker. In some embodiments, the second peptide linker contains about 4 to about 15 amino acids. In some embodiments, the second peptide linker contains a linker containing any one of the sequences of SEQ ID NOs: 232-260.

[0020] In some embodiments, the second antibody moiety comprises an Fc region selected from an Fc region from IgG, IgA, IgD, IgE, IgM and hybrids of any combination thereof, and in some embodiments, the Fc region comprises a human Fc region. In some embodiments, the Fc region is selected from an Fc region from IgG1, IgG2, IgG3, IgG4 and hybrids of any combination thereof. In some embodiments, the Fc region comprises an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises an L234A mutation and an L235A mutation. In some embodiments, the Fc region comprises an IgG4 Fc region. In some embodiments, the IgG4 Fc region comprises an F234A mutation and an L235A mutation. In some embodiments, the IgG4 Fc region comprises an S228P mutation.

[0021] In some embodiments, the EGFR is human EGFR.

[0022] In some embodiments, the second antibody moiety comprises a full-length antibody that binds to EGFR and competes with an antibody or antibody fragment comprising a second heavy chain variable region (V H-3 ) and a second light chain variable region (V L-3 ) for the binding epitope of EGFR, wherein a) the V H-3 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:190, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:191, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:192, and b) the V L-3 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:193, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:194, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:195, b) the V H-3comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:198, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:199, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:200, and b) said V L-3 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:201, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:202, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:203, c) said V H-3 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:206, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:207, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:208, and b) said V L-3 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:209, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:210, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:211, d) said V H-3 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:214, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:215, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:216, and b) said V L-3 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:217, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:218, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:219, or e) said V H-3 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:222, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:223, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:224, and b) said V L-3 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:225, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:226, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:227.

[0023] In some embodiments, the second antibody portion binds to EGFR and comprises a full-length antibody comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), wherein a) the V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:190, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:191, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:192, and b) the V L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:193, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:194, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:195, b) the V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:198, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:199, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:200, and b) the V L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:201, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:202, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:203, c) the V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:206, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:207, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:208, and b) the V L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:209, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:210, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:211, d) the V H-2comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:214, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:215, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:216, and b) said V L-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:217, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:218, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:219, or e) said V H-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:222, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:223, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:224, and b) said V L-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:225, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:226, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:227.

[0024] In some embodiments, said V H-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:190, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:191, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:192, and said V L-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:193, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:194, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:195. In some embodiments, said V H-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:198, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:199, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:200, and said V L-2includes an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:201, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:202, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:203. In some embodiments, said V H-2 includes an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:206, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:207, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:208, and said V L-2 includes an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:209, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:210, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:211. In some embodiments, said V H-2 includes an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:214, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:215, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:216, and said V L-2 includes an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:217, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:218, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:219. In some embodiments, said V H-2 includes an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:222, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:223, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:224, and said V L-2 includes an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:225, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:226, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:227.

[0025] In some embodiments, a) said V H-2 includes an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:196, and / or said V L-2comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:197, and b) said V H-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:204, and / or said V L-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:205, c) said V H-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:212, and / or said V L-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:213, d) said V H-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:220, and / or said V L-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:221, e) said V H-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:228, and / or said V L-2 comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:229. In some embodiments, a) said V H-2 comprises the amino acid sequence of SEQ ID NO:196, said V L-2 comprises the amino acid sequence of SEQ ID NO:197, b) said V H-2 comprises the amino acid sequence of SEQ ID NO:204, said V L-2 comprises the amino acid sequence of SEQ ID NO:205, c) said V H-2 comprises the amino acid sequence of SEQ ID NO:212, said V L-2 comprises the amino acid sequence of SEQ ID NO:213, d) said V H-2 comprises the amino acid sequence of SEQ ID NO:220, said V L-2 comprises the amino acid sequence of SEQ ID NO:221, or e) said V H-2 comprises the amino acid sequence of SEQ ID NO:228, said V L-2It contains the amino acid sequence of SEQ ID NO: 229.

[0026] In some embodiments, the multispecific antibody is a) the first antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 121, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 122, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 123, and the V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 126; and b) the second antibody moiety comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), wherein the V H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 208, and the V L-2 comprises a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 210, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 211.

[0027] In some embodiments, the multispecific antibody is a) the first antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the V H comprises an HC-CDRl comprising the amino acid sequence of SEQ ID NO: 261, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 262, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 263, and the V LThe first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:264, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:265, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:266, and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) of the second antibody portion, wherein the V H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO:206, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO:207, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO:208, and the V L-2 comprises a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:209, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:210, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:211 of the second antibody portion.

[0028] In some embodiments, the multispecific antibody is a) the first antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:271, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:272, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:273, and the V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:274, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:275, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:276 of the first antibody portion, and b) a second antibody portion comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), wherein the V H-2comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO:206, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO:207, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO:208, said V L-2 comprises the second antibody moiety comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:209, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:210, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:211.

[0029] In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises an amino acid sequence having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 183, 185, 188, 189, 230, 231, 259, 260, 281 or 282. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises any one of the amino acid sequences of SEQ ID NO: 183, 185, 188, 189, 230, 231, 259, 260, 281 or 282. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 185. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 189. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 231.In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 259. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 260. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 281. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 282.

[0030] In some embodiments, the full-length anti-EGFR antibody included comprises a light chain comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 184, 186, or 187. In some embodiments, the full-length anti-EGFR antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 184. In some embodiments, the full-length anti-EGFR antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 186. In some embodiments, the full-length anti-EGFR antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 187.

[0031] The present disclosure further provides an immunoconjugate comprising any of the multispecific antibodies disclosed herein linked to a therapeutic agent or a marker. In some examples, the marker is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0032] The present disclosure further provides a pharmaceutical composition comprising any of the multispecific antibodies or any immunoconjugate disclosed herein and a pharmaceutically acceptable carrier agent.

[0033] The present disclosure further provides an isolated nucleic acid encoding any of the multispecific antibodies disclosed herein, a vector comprising any of the isolated nucleic acids disclosed herein, and an isolated host cell comprising any of the isolated nucleic acids or any of the vectors disclosed herein.

[0034] The present disclosure further provides a method for producing a multispecific antibody. In some embodiments, the method comprises: a) culturing any of the host cells disclosed herein under conditions effective to express the multispecific antibody; and b) obtaining the expressed multispecific antibody from the host cells.

[0035] The present disclosure further provides a method for treating or preventing a disease in an individual. In some embodiments, the method comprises administering to the individual an effective amount of any of the multispecific antibodies, any of the immunoconjugates, or any of the pharmaceutical compositions disclosed herein. In some embodiments, the disease is cancer or a tumor. In some embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, lung cancer, mesothelioma, endometrial cancer, cervical cancer, esophageal cancer, bladder cancer, salivary gland cancer, testicular cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, thymic cancer, adrenal cancer, head and neck cancer, brain cancer, thyroid cancer, sarcoma, myeloma, and leukemia. In some embodiments, the cancer or tumor is EGFR positive. In some embodiments, the cancer is lung cancer, colorectal cancer, or head and neck cancer.

[0036] In some embodiments, the multispecific antibody, immunoconjugate, or pharmaceutical composition is administered parenterally to the individual. In some embodiments, the multispecific antibody, immunoconjugate, or pharmaceutical composition is administered intravenously to the individual. In some embodiments, the subject is human.

[0037] The present disclosure provides any multispecific antibody disclosed herein for use as a drug. The present disclosure further provides any multispecific antibody disclosed herein for treating cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, lung cancer, mesothelioma, endometrial cancer, cervical cancer, esophageal cancer, bladder cancer, salivary gland cancer, testicular cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, thymic cancer, adrenal cancer, head and neck cancer, brain cancer, thyroid cancer, sarcoma, myeloma, and leukemia.

[0038] The present disclosure further provides a kit comprising any multispecific antibody, any immunoconjugate, any pharmaceutical composition, any nucleic acid, any vector, or any host cell disclosed herein. In some embodiments, the kit further comprises a manual for treating and / or preventing cancer or tumor.

Brief Description of the Drawings

[0039]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 4A

Figure 4B

Figure 5A

Figure 5B

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 8

Figure 9A

Figure 9B

Figure 10A

Figure 10B

Figure 11

Figure 12

Figure 13A

Figure 13B

Figure 14A

Figure 14B

Mode for Carrying Out the Invention

[0040] This application provides novel anti-CD137 constructs that specifically bind to CD137 (e.g., anti-CD137 scFvs, monoclonal antibodies, and multispecific antibodies that bind to tumor-associated antigens (TAAs)), methods for preparing the anti-CD137 constructs, and methods for using the constructs (e.g., methods for treating diseases or medical conditions, methods for modulating immune responses, or methods for modulating cell compositions). The present invention is based in part on the discovery of anti-CD137 monospecific and multispecific antibodies that exhibit improved safety and enhanced antitumor effects compared to conventional anti-CD137 antibodies in clinical trials.

[0041] I. Definitions The term "antibody" is used in its broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. The term "antibody moiety" refers to a full-length antibody or an antigen-binding fragment thereof.

[0042] Full-length antibodies contain two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as "V H " and "V L ", respectively. The variable regions in the two chains generally contain three highly variable loops, which are called complementarity-determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the convention of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997, Chothia 1985, Chothia 1987, Chothia 1989, Kabat 1987, Kabat 1991). The three CDRs of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit multiple effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0043] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, which includes, for example, bispecific antibodies, Fab, Fab’, F(ab’)2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized bispecific antibodies (ds bispecific antibodies), single-chain Fv (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed of antibody moieties containing one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not contain the complete antibody structure. The antigen-binding fragment can bind to an antigen to which the parental antibody or parental antibody fragment (e.g., parental scFv) binds. In some embodiments, the antigen-binding fragment may include one or more CDRs from a particular human antibody, and the CDRs are grafted into framework regions from one or more different human antibodies.

[0044] "Fv" is the smallest antibody fragment that contains the complete antigen recognition site and the antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain that are tightly non-covalently bound. From the folding of these two domains, six hypervariable loops (three loops from each of the heavy and light chains) are released, and these hypervariable loops provide the amino acid residues for antigen binding and confer the binding specificity of the antibody to the antigen. However, usually performed with lower affinity than the complete binding site, even a single variable domain (or half of the Fv containing only the three CDRs specific for the antigen) has the ability to recognize and bind the antigen.

[0045] "Single-chain Fv" (abbreviated as "sFv" or "scFv" as well) is an antibody fragment that contains V H and V L antibody domains linked in a single polypeptide chain. In some embodiments, the scFv polypeptide contains V H and V LFurther comprising a polypeptide linker between domains, which polypeptide linker enables the scFv to form the desired structure for antigen binding. For an overview of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994).

[0046] As used herein, the term "CDR" or "complementary determining region" is intended to mean the discontinuous antigen-binding sites found within the variable regions of the heavy and light chain polypeptides. These specific regions are described in Kabat et al., J. Biol. Chem., 252:6609-6616 (1977), Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008), Lefranc M.P. et al., Dev. Comp. Immunol., 27:55-77 (2003), Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where overlapping or subsets of amino acid residues are defined when making comparisons. However, the CDRs that refer to an antibody or a transplanted antibody or a variant thereof, when applying any one of the definitions, are intended to be within the scope of the terms defined and used herein. The amino acid residues covering the CDRs defined in each of the above references are placed in Table 1 below and used for comparison. CDR prediction algorithms and interfaces are known in the art and include, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008), Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The contents of the references cited in this section are hereby incorporated by reference in their entirety into this specification, used in this specification, and may be included in one or more claims of this specification.

[0047] [Table 1]

[0048] The expressions "for example, variable domain residue numbering in Kabat" or "for example, amino acid position numbering in Kabat" and variants thereof refer to the numbering system used for the heavy chain variable domain or the light chain variable domain of the antibody assembler of Kabat et al. above. Using this numbering system, the actual linear amino acid sequence may contain fewer or different amino acids corresponding to deletions or insertions in the FR or hypervariable regions (HVRs) of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (based on residue 52a of Kabat) and insertion residues after heavy chain FR residue 82 (e.g., based on residues 82a, 82b, and 82c of Kabat, etc.). By comparing the antibody sequence with the homologous region of the "standard" Kabat numbering sequence, the Kabat numbering of the residues of a given antibody can be determined.

[0049] Unless otherwise specifically described herein, the amino acid residues covering the CDRs of a full-length antibody (e.g., the anti-CD137 antibody disclosed herein) are defined based on the Kabat nomenclature of Kabat et al., and the residue numbering in immunoglobulin heavy chains, for example, in the Fc region, is the numbering of the EU index described by Kabat et al. and is defined based on the Kabat nomenclature for residues other than those covering the CDRs of any consensus sequence, where the modifications are based on experimental conditions. The "EU index described by Kabat" is the residue numbering of a human IgG1 EU antibody.

[0050] "Framework" or "FR" residues are those variable domain residues other than the CDR residues defined herein.

[0051] A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that includes minimal sequences derived from the non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody), where residues from the recipient hypervariable regions (HVRs) are replaced with residues from the hypervariable regions (donor antibody) of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) having the desired antigen specificity, affinity, and capacity. In some specific examples, framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. Also, a humanized antibody may include residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody includes substantially all of at least one and usually two variable domains, where all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin and all or substantially all of the FRs are of the human immunoglobulin sequence. A humanized antibody further optionally includes at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, (1986), Riechmann et al., Nature, 332:323-329 (1988), and Presta, Curr.Op.Struct.Biol. 2:593-596 (1992).

[0052] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or is prepared by any technique for the preparation of human antibodies disclosed herein. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies may be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Methods described by Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991), which are also applicable to the preparation of monoclonal antibodies, may be used. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared, for example, by administering an antigen to a transgenic animal (e.g., immunized xenomice) whose endogenous locus has been inactivated but which has been modified to produce such antibodies in response to antigen challenge (e.g., XENOMOUSE TM See U.S. Patent Nos. 6,075,181 and 6,150,584 related to the technology). Human antibodies produced by human B cell hybridoma technology are further described, for example, in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006).

[0053] The "percent amino acid sequence identity (%)" or "homology" related to the polypeptide and antibody sequence identified by this specification is defined as the percentage of amino acid residues in the candidate sequence that are the same as the amino acid residues in the polypeptide being compared, after alignment of the sequences (taking into account any conservative substitutions as part of sequence identity). For the purpose of determining the percent amino acid sequence identity, comparison can be achieved by various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. One of ordinary skill in the art can include any algorithm that achieves maximum comparison within the full length range of the sequences being compared and can determine appropriate parameters for use in the measurement comparison. However, for the purposes of this specification, the sequence comparison computer program MUSCLE is used to produce the value of percent amino acid sequence identity (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004, Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0054] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in one of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, when each of the positions in two DNA molecules is occupied by adenine, the molecules are homologous at this position. The percent homology between two sequences is a function obtained by dividing the number of matching or homologous positions shared by the two sequences by the number of positions being compared and multiplying by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Usually, the comparison is made when the two sequences are aligned to give maximum homology.

[0055] The term "constant domain" refers to a part of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another part of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain is the C H 1, C H 2 and C H 3 domains (collectively referred to as C H ), and the CHL (or C L ) domain of the light chain.

[0056] The "light chain" of an antibody (immunoglobulin) from any mammalian species can be designated as one of two clearly different types according to the amino acid sequence of its constant domain, called kappa ("κ") and lambda ("λ"), respectively.

[0057] The "CH1 domain" (also called "C1" of the "H1" domain) is usually from about amino acid 118 to about amino acid 215 (EU numbering system).

[0058] The "hinge region" is usually defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol., 22:161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the S-S bond between the heavy chains in the same positions.

[0059] The "CH2 domain" of the human IgG Fc region (also called the "C2" domain) is usually from about amino acid 231 to about amino acid 340. The CH2 domain is unique because it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete native IgG molecule. Presumably, the carbohydrate can provide an alternative to domain-domain pairing and contribute to the stabilization of the CH2 domain. Burton, Molec Immunol., 22:161-206 (1985).

[0060] The "CH3 domain" (also called the "C2" domain) includes the residue region near the C-terminus of the CH2 domain in the Fc region (i.e., from approximately amino acid residue 341 to the C-terminus of the antibody sequence (typically at amino acid residues 446 or 447 of IgG)).

[0061] As used herein, the "Fc region" or "fragment crystallizable region" is for defining the C-terminal region of an immunoglobulin heavy chain and includes the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, typically, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl-terminal end. For example, the C-terminal lysine (based on residue 447 in the EU numbering system) of the Fc region can be removed by, for example, during the production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of a complete antibody may include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which the K447 residue has not been removed, and a population of antibodies having a mixture of antibodies with or without the K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0062] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are native human FcRs. In addition, preferred FcRs are FcRs that bind to IgG antibodies (γ receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants or splice forms of these variants. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and the main difference is in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" as used herein covers other FcRs and includes FcRs that may be identified in the future.

[0063] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions have competitive binding to an antigen, they can bind to the same epitope within the antigen.

[0064] As used herein, when an equimolar concentration of a first antibody or fragment thereof is present, the first antibody or fragment thereof "competes" with the second antibody or fragment thereof for binding to the target antigen when the first antibody or fragment thereof inhibits the target antigen binding of the second antibody or fragment thereof by at least about 50% (e.g., any one of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa. In PCT Publication No. WO 03 / 48731, a high-throughput method for "sorting" these antibodies based on cross-competition of antibodies is described.

[0065] As used herein, the terms "specifically binds", "specifically recognizes" and "is specific for" refer to an interaction that is measurable and reproducible, e.g., the binding of a target to an antibody or antibody portion, which determines the presence of the target when a population of heteromolecules (including biomolecules) is present. For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to said target, and its affinity, binding, avidity and / or duration is longer than its binding to other targets. In some embodiments, the degree of binding to a target not related to the antibody is, for example, about 10% less than the degree of binding of the antibody to the target as measured by, e.g., radioimmunoassay (RIA). In some embodiments, the dissociation constant (K D ) of an antibody that specifically binds to a target is ≦ 10 -5 M, ≦ 10 -6 M, ≦ 10 -7 M, ≦ 10 -8 M, ≦ 10 -9 M, ≦ 10 -10 M, ≦ 10 -11 M, or ≦ 10 -12It is M. In some embodiments, the antibody specifically binds to an epitope on a protein conserved from different species of proteins. In some embodiments, specific binding may include, but is not limited to, exclusive binding. The binding specificity of the antibody or antigen-binding domain may be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -assay peptide scanning.

[0066] An "isolated" antibody (or construct) is an antibody that has been identified, isolated and / or recovered from the components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide does not associate with all other components in its production environment.

[0067] An "isolated" nucleic acid molecule encoding a construct, antibody or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminating nucleic acid molecule usually associated therewith in its production environment. Preferably, the isolated nucleic acid does not associate with all components related to the production environment. The form of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is different from the naturally occurring form or background. Thus, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that are naturally present in cells. The isolated nucleic acid contains this nucleic acid molecule that is usually contained in a cell containing the nucleic acid molecule, but this nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0068] The term "control sequence" refers to a desired DNA sequence for expressing a coding sequence operably linked thereto in a particular host organism. For example, control sequences applicable to prokaryotes include a promoter, an optional operator sequence and a ribosome binding site. It is known that eukaryotic cells utilize a promoter, a polyadenylation signal and an enhancer.

[0069] A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, when the DNA of a presequence or a secretory leader sequence is expressed as a preprotein involved in the secretion of a polypeptide, the DNA of this presequence or secretory leader sequence is operably linked to the DNA of this polypeptide. When a promoter or enhancer affects the transcription of a coding sequence, this promoter or enhancer is operably linked to this sequence. Or, when a ribosome binding site is positioned to facilitate translation, this ribosome binding site is operably linked to the coding sequence. Usually, "operably linked" means that the linked DNA sequences are contiguous and, in the case of a secretory leader, are contiguous and within the reading frame. However, an enhancer does not necessarily have to be contiguous. Ligation can be achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used based on conventional practice.

[0070] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. This term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the host cell genome into which they have been introduced. Some vectors can direct the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0071] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing foreign nucleic acid into a host cell. "Transfected" or "transformed" or "transduced" cells are cells transfected, transformed or transduced using foreign nucleic acid. This cell includes the primary target cell and its progeny.

[0072] "Host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which foreign nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may differ from that of the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the original transformed cell are included herein.

[0073] As used herein, "treatment" (or "treating") is a method for obtaining a beneficial or desired result, including clinical results. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviating one or more symptoms caused by a disease, reducing the degree of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the rate of progression of the disease, improving the disease state, providing (partial or complete) alleviation of the disease, reducing the dosage of one or more other medications required for the treatment of the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or extending the survival period. "Treatment" further encompasses reducing the pathological consequences (e.g., tumor volume) of cancer. The methods of this application contemplate any one or more of these aspects of treatment.

[0074] In the context of cancer, the term "treatment" includes any one or all of inhibition of cancer cell growth, inhibition of cancer cell replication, reduction of the overall tumor burden, and improvement of one or more symptoms associated with the disease.

[0075] The term "inhibition" (or "inhibit") refers to a decrease or cessation of any phenotypic feature, or a decrease or cessation in the incidence, degree or likelihood of this feature. Compared to a reference, "reduction" or "inhibition" refers to decreasing, lowering or preventing activity, function and / or amount. In some embodiments, "reduction" or "inhibition" refers to the ability to cause a general reduction of 20% or more. In another embodiment, "reduction" or "inhibition" refers to the ability to cause a general reduction of 50% or more. In yet another embodiment, "reduction" or "inhibition" refers to the ability to cause a general reduction of 75%, 85%, 90%, 95% or more.

[0076] As used herein, "reference" refers to any sample, standard or level used for comparison. A reference can be obtained from healthy and / or non-affected samples. In some embodiments, the reference is obtained from an untreated sample. In some embodiments, the reference is obtained from an unaffected or untreated sample of an individual. In some specific examples, the reference is obtained from one or more healthy individuals other than the individual or patient.

[0077] As used herein, "delaying the progression of a disease" refers to delaying, suppressing, decelerating, stabilizing, inhibiting and / or slowing down the progression of a disease (e.g., cancer). This delay may have different durations depending on the history of the disease and / or the individual being treated. Since the individual is not affected by the disease, a sufficient or significant delay may, in practice, include prevention. For example, it may be possible to delay advanced cancer (e.g., the progression of metastases).

[0078] As used herein, "prevention" includes providing prevention against the onset or recurrence of a disease in an individual who is at risk of developing the disease but has not been diagnosed as having the disease.

[0079] As used herein, to "inhibit" a function or activity means to reduce the function or activity compared to the same conditions other than the conditions or parameters of interest, or alternatively compared to other conditions. For example, an antibody that inhibits tumor growth reduced the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0080] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal, including but not limited to humans, cows, horses, cats, dogs, rodents or primates. In some embodiments, the individual is a human.

[0081] An "effective amount" of a drug refers to the amount that effectively achieves the required therapeutic or prophylactic effect within the required dosage and period. The specific dosage may vary depending on the particular drug selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the time of administration, the imaging target tissue and one or more of the physical delivery systems having it.

[0082] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist of the present application may vary depending on factors such as, for example, the disease state, age, sex and body weight of the individual and the ability of this substance / molecule, agonist or antagonist to elicit the desired response in the individual. The therapeutically effective amount is further an amount in which any of the toxic or adverse effects of this substance / molecule, agonist or antagonist are offset by the therapeutically beneficial effect. The therapeutically effective amount can be delivered in one or multiple administrations.

[0083] A "prophylactically effective amount" refers to the effective amount at the dosage and period required to achieve the desired prophylactic result. Typically, although not necessarily, the prophylactic dosage is used in a subject before or at the early stage of a disease, and such a prophylactically effective amount is smaller than the therapeutically effective amount.

[0084] The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to a preparation in a form that allows the biological activity of one or more active ingredients to be effective and that does not contain other ingredients having unacceptable toxicity to the individual to whom the preparation is administered. Such a preparation may be sterile.

[0085] "Pharmaceutically acceptable carrier agent" refers to a conventional non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier agent in the art used with a therapeutic agent, and the two together constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier agent is non-toxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier agent is suitable for the formulation employed.

[0086] A "sterile" preparation is sterile or substantially free of living microorganisms and their spores.

[0087] "Combined" administration with one or more other therapeutic agents includes simultaneous (parallel) and sequential administration or sequential administration in any order.

[0088] The term "parallel" is used herein to refer to administering two or more therapeutic agents, where at least a portion of the administrations overlap in time or the administration of one therapeutic agent is within a short time period relative to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered at any time interval not exceeding about 60 minutes (e.g., not exceeding any one of about 30, 15, 10, 5, or 1 minute).

[0089] As used herein, the term "sequential" is used to refer to the administration of two or more therapeutic agents, where, after the administration of one or more of the reagents has been discontinued, one or more other agents are subsequently administered. For example, the administration of two or more therapeutic agents is administered at time intervals greater than about 15 minutes (e.g., any one of about 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks or 1 month or longer).

[0090] As used herein, "administered in combination with" means administering a different therapy other than one therapy. Similarly, "administered in combination with" means administering a different therapy before, during, or after administering one therapy to an individual.

[0091] The term "package insert" generally refers to a manual included in the commercial packaging of a therapeutic product, and these manuals contain information regarding indications, methods of use, dosages, administration, combination therapies, contraindications and / or warnings for using such therapeutic products.

[0092] A "manufactured article" is any product (e.g., a package or container) or kit that contains at least one reagent, where the reagent is, for example, a drug for treating a disease or disorder (e.g., cancer) or a probe for specifically detecting a biomarker described herein. In some embodiments, the product or kit is distributed, circulated, or sold as a unit for performing the methods described herein.

[0093] It should be understood that the embodiments of the applications described herein include "consisting of" and "consisting essentially of".

[0094] As used herein, a reference to a "about" value or parameter includes (and describes) a variation to the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0095] As used herein, reference to a "not" value or parameter typically means and describes a "different" value or parameter. For example, a method that is not for treating cancer of type X means that the method is used for treating a cancer different from type X.

[0096] As used herein, the term "about X - Y" has the same meaning as "from about X to about Y".

[0097] As used in this specification and the appended claims, the singular forms "a", "or", and "the" include plural referents unless the context clearly dictates otherwise.

[0098] II.CD137(4-1BB) CD137 (4-1BB) is a member of the tumor necrosis factor receptor (TNF-R) gene family, which includes proteins involved in the regulation of cell proliferation, differentiation, and programmed cell death. CD137 is a 30 kDa type I membrane glycoprotein and is expressed as a 55 kDa homodimer. The receptor was first described in mice (B. Kwon et al., P.N.A.S. USA, 86:1963-7 (1989)) and later identified in humans (M. Alderson et al., Eur. J. Immunol., 24:2219-27 (1994), Z. Zhou et al., Immunol. Lett., 45:67 (1995)) (see also published PCT applications WO95 / 07984 and WO96 / 29348, and U.S. Patent No. 6,569,997, which are incorporated herein by reference (see SEQ ID NO:2)). The human and mouse forms of CD137 are 60% identical at the amino acid level. The presence of conserved sequences in the cytoplasmic domain and five other regions of the molecule indicates that these residues may be important for the function of the CD137 molecule (Z. Zhou et al., Immunol. Lett., 45:67 (1995)). CD137 is expressed mainly on lymphoid cells (e.g., activated T cells, activated natural killer (NK) cells, NKT cells, CD4+CD25+ regulatory T cells), and has also been shown to be expressed on activated thymocytes and intraepithelial lymphocytes. Additionally, CD137 has been shown to be expressed on cells of myeloid origin such as dendritic cells, monocytes, neutrophils, and eosinophils. Although the expression of CD137 is mainly restricted to immune / inflammatory cells, expression of CD137 on endothelial cells associated with small amounts of tissue at inflammatory sites and tumors has been described in the literature.

[0099] The functional activity of CD137 in T cells has been well characterized. It has already been demonstrated that, in the presence of suboptimal doses of anti-CD3, signaling through CD137 can induce T cell proliferation and cytokine synthesis (mainly IFN-γ) and inhibit activated cell death. These effects have been observed in both mouse and human T cells (W. Shuford et al., J. Exp. Med., 186(1):47-55 (1997), D. Vinay et al., Semin. Immunol, 10(6):481-9 (1998), D. Laderach et al., Int. Immunol., 14(10):1155-67 (2002)). In both humans and mice, co-stimulation enhances effector function by increasing both antigen specificity and the number of effector CD8+ T cells, for example, IFN-γ production and cytotoxicity. In the absence of anti-CD3 signaling, stimulation by CD137 does not alter T cell function, indicating that CD137 is a co-stimulatory molecule.

[0100] The use of an agonist anti-mouse CD137 monoclonal antibody in mice and in vivo efficacy studies suggested the role of CD137-targeted therapy in cancer treatment. In the paper by Melero et al., the agonist anti-mouse CD137 antibody resulted in cures in P815 mastocytoma tumors and in the low-immunogenic tumor model Agl04 (I. Melero et al., Nat. Med., 3(6):682-5 (1997)). Since selective in vivo depletion of each subset may lead to a decrease or complete loss of the anti-tumor effect, both CD4+ and CD8+ T cells and NK cells are required for the anti-tumor effect. It has also been demonstrated that minimal induction of the immune response is required for anti-CD137 therapy to be effective. Several researchers have demonstrated the feasibility of such an approach in cancer treatment using anti-CD137 antibodies (J. Kim et al., Cancer Res., 61(5):2031-7 (2001), O. Martinet et al., Gene Ther., 9(12):786-92 (2002), R. Miller et al., J. Immunol, 169(4):1792-800 (2002), R. Wilcox et al., Cancer Res., 62(15):4413-8 (2002)).

[0101] In addition to its role in immune development against cancer, experimental data further supports the use of CD137 agonist antibodies in the treatment of autoimmune and viral diseases (B. Kwon et al., Exp. Mol. Med., 35(1):8 - 16(2003), H. Salih et al., J. Immunol, 167(7):4059 - 66(2001), E. Kwon et al., P.N.A.S. USA, 96:15074 - 79(1999), J. Foell et al., N.Y. Acad. Sci., 987:230 - 5(2003), Y. Sun et al., Nat. Med., 8(12):1405 - 13(2002) S.K. Seo et al., Nat. Med.10;1099 - 94(2004)).

[0102] III. Anti - CD137 Construct In one aspect, the present invention provides a novel CD137 - specific construct (e.g., an isolated anti - CD137 construct) comprising an antibody portion that specifically binds to CD137. The specificity of the anti - CD137 construct originates from the anti - CD137 antibody portion that specifically binds to CD137 and is, for example, a full - length antibody or an antigen - binding fragment thereof. In some embodiments, the portion (e.g., the antibody portion) that is referred to as specifically binding to CD137 binds specifically to CD137 with an affinity that is at least about 10 - fold (e.g., at least about 10, 10 2 、10 3 、10 4 、10 5 、10 6 or 10 7 times any one of them) greater than its binding affinity for non - targets. In some embodiments, the non - target is an antigen other than CD137. The binding affinity can be determined by methods known in the art, such as ELISA, fluorescence - activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). K dcan be determined by methods known in the art, which may utilize, for example, surface plasmon resonance (SPR) assays on a Biacore instrument or the binding equilibrium exclusion method (KinExA) on a Sapidyne instrument.

[0103] Desired anti-CD137 constructs include, but are not limited to, anti-CD137 scFv, fusion proteins comprising an anti-CD137 antibody portion and a half-life extension domain (e.g., an Fc region, a domain that binds albumin), anti-CD137 monoclonal antibodies, multispecific anti-CD137 molecules (e.g., bispecific antibodies). The exemplary anti-CD137 constructs above do not mutually expand and are further discussed in each of the following sections.

[0104] In some embodiments, an anti-CD137 construct (e.g., anti-CD137 scFv) comprising an anti-CD137 antibody portion that specifically recognizes CD137 (e.g., human CD137) is provided, wherein the anti-CD137 antibody portion may be any one of the anti-CD137 antibody portions described herein.

[0105] In some embodiments, an anti-CD137 construct (e.g., anti-CD137 scFv) comprising an anti-CD137 antibody portion that binds to CD137 is provided, wherein the anti-CD137 antibody portion comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), and wherein a) the V His an HC-CDR1 comprising the amino acid sequence of GFX1X2X3DTYIX4 (SEQ ID NO: 177), where X1 = N or C, X2 = I, P, L or M, X3 = K, N, R, C or Q, X4 = H or Q; an HC-CDR2 comprising the amino acid sequence X1IDPANGX2X3X4 (SEQ ID NO: 178), where X1 = K or R, X2 = N, G, F, Y, A, D, L, M or Q, X3 = S or T, X4 = E or M; and an HC-CDR3 comprising the amino acid sequence GNLHYX1LMD (SEQ ID NO: 179), where X1 = Y, A or G, and b) said V L is an LC-CDR1 comprising the amino acid sequence of KASQX1X2X3TYX4S (SEQ ID NO: 180), where X1 = A, P or T, X2 = I, T or P, X3 = N or A, X4 = L, G or H; an LC-CDR2 comprising the amino acid sequence RX1NRX2X3X4 (SEQ ID NO: 181), where X1 = A, Y, V or D, X2 = M, K, V or A, X3 = V, P, Y or G, X4 = D or G; and an LC-CDR3 comprising the amino acid sequence LQX1X2DFPYX3 (SEQ ID NO: 182), where X1 = Y, S or F, X2 = D, V, L, R, E or Q, X3 = T or K.

[0106] In some embodiments, the anti-CD137 antibody portion that binds to CD137 comprises: a) an HC-CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 261, and 271, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions; b) an HC-CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 262, and 272, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions; c) an HC-CDR3 comprising any one of the amino acid sequences of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 263, and 273, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions; d) an LC-CDR1 comprising any one of the amino acid sequences of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 264, and 274, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions; e) an LC-CDR2 comprising any one of the amino acid sequences of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 265, and 275, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions; and f) an LC-CDR3 comprising any one of the amino acid sequences of SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 266, and 276, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 of the present application.

[0107] In some embodiments, an anti-CD137 construct comprising an anti-CD137 antibody portion is provided, wherein the anti-CD137 antibody portion cross-competes with a reference anti-CD137 construct for binding to CD137, and the anti-CD137 antibody portion comprises a heavy chain variable region (V H )(the heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 domains) and a light chain variable region (V L )(the light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3 domains), which is selected from the group consisting of: a) V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an HC-CDR2 having the amino acid sequence of SEQ ID NO:2, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an HC-CDR3 having the amino acid sequence of SEQ ID NO:3, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:4, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an LC-CDR2 having the amino acid sequence of SEQ ID NO:5, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an LC-CDR3 having the amino acid sequence of SEQ ID NO:6, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions; b) V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:11, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an HC-CDR2 having the amino acid sequence of SEQ ID NO:12, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an HC-CDR3 having the amino acid sequence of SEQ ID NO:13, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V Lis LC-CDR1 comprising the amino acid sequence of SEQ ID NO:14, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:15, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and c) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:23, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V L is LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and d) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:31, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:32, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:33, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V Lis LC-CDR1 comprising the amino acid sequence of SEQ ID NO:34, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:35, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:36, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and e) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:41, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:42, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:43, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and moreover V L is LC-CDR1 comprising the amino acid sequence of SEQ ID NO:44, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:45, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and f) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:51, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:52, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:53, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and moreover V Lcomprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:56, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and g) V H comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:61, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:62, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:63, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and moreover V L comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:64, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:65, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:66, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and h) V H comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:71, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:72, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:73, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and moreover V Lis LC-CDR1 comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:75, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:76, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and i) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:81, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:82, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:83, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V L is LC-CDR1 comprising the amino acid sequence of SEQ ID NO:84, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:85, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:86, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and j) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:91, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:92, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:93, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:94, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:95, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:96, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and k) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:101, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:102, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:103, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:104, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:105, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:106, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and l) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:111, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:112, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:113, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V Lis LC-CDR1 comprising the amino acid sequence of SEQ ID NO:114, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:115, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:116, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and m) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:121, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:122, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:123, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V L is LC-CDR1 comprising the amino acid sequence of SEQ ID NO:124, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:125, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:126, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and n) V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:131, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:132, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:133, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and V Lis LC-CDR1 comprising the amino acid sequence of SEQ ID NO:134, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR2 comprising the amino acid sequence of SEQ ID NO:135, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:136, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and o)V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:261, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR2 comprising the amino acid sequence of SEQ ID NO:262, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:263, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and and V L is LC-CDR1 comprising the amino acid sequence of SEQ ID NO:264, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR2 comprising the amino acid sequence of SEQ ID NO:265, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:266, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and p)V H is HC-CDR1 comprising the amino acid sequence of SEQ ID NO:271, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR2 comprising the amino acid sequence of SEQ ID NO:272, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:273, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:274, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:275, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:276, or a variant thereof comprising up to about 3 (e.g., 3, 2, or 1) amino acid substitutions.

[0108] In some embodiments, provided is an anti-CD137 construct (e.g., an anti-CD137 scFv) comprising an anti-CD137 antibody portion that binds to CD137, wherein the anti-CD137 antibody portion comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), and wherein a) the V H comprises i) an HC-CDR1 comprising the amino acid sequence of DTYIH or GFNIQDT, ii) an HC-CDR2 comprising the amino acid sequence of DPANGN, and iii) an HC-CDR3 comprising the amino acid sequence of GNLHYALMD, and b) the V L comprises i) an LC-CDR1 comprising the amino acid sequence of NTYLS, ii) an LC-CDR2 comprising the amino acid sequence of RVNRKV, and iii) an LC-CDR3 comprising the amino acid sequence of LQYLDFPY.

[0109] In some embodiments, provided is an anti-CD137 construct (e.g., an anti-CD137 scFv) comprising an anti-CD137 antibody portion that binds to CD137, wherein the anti-CD137 antibody portion comprises a) HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in the V H chain region having the sequences shown in SEQ ID No:7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 267, or 277, and b) V LIt includes LC-CDR1, LC-CDR2 and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain region.

[0110] In some embodiments, an anti-CD137 construct (e.g., anti-CD137 scFv) containing an anti-CD137 antibody portion that binds to CD137 is provided, and the anti-CD137 antibody portion is a) a V having the sequence shown in SEQ ID No: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 267 or 277 H chain region or a variant thereof, or a variant having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 267 or 277, and b) a V having the sequence shown in SEQ ID No: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 268 or 278 L chain region or a variant thereof, or a variant having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID O: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 268 or 278. In some embodiments, the V H chain region and the V L chain region are linked via a linker (e.g., a peptide linker).

[0111] In some embodiments, an anti-CD137 construct (e.g., anti-CD137 scFv) containing an anti-CD137 antibody portion that binds to CD137 is provided, and the anti-CD137 antibody portion is a V having the sequence shown in SEQ ID No: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 267 or 277 Hregion, and V having the sequences shown in SEQ ID No: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 268 or 278 L region.

[0112] In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises: (a) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:7 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:8; (b) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:17 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:18; (c) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:27 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:28; (d) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:37 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:38; (e) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:47 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:48; (f) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:57 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:58; (g) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:67 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:68; (h) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:77 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:78; (i) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:87 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:88; (j) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:97 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:98; (k) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:107 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:108; (l) a heavy chain variable region comprising amino acids having the sequence shown in SEQ ID NO:117 and a light chain variable region comprising amino acids having the sequence shown in SEQ ID NO:118; (m) SEQ IDA heavy chain variable region comprising an amino acid having the sequence shown in NO:127, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:128; (n) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:137, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:138; (o) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:267, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:268, or (p) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:277, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO:278.

[0113] In some embodiments, an anti-CD137 construct (e.g., anti-CD137 scFv) comprising an anti-CD137 antibody portion that binds to CD137 is provided, and the anti-CD137 antibody portion comprises a heavy chain (HC) having the sequence shown in SEQ ID No: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 269 or 279, and a light chain (LC) having the sequence shown in SEQ ID No: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 270 or 280.

[0114] In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:9, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:10. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:19, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:20. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:29, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:30. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:39, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:40. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:49, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:50. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:59, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:60. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:69, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:70.In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:79, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:80. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:89, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:90. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:99, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:100. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:109, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:110. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:119, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:120. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:129, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:130. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO:139, and a light chain comprising amino acids having the sequence shown in SEQ ID NO:140.In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO: 149 and a light chain comprising amino acids having the sequence shown in SEQ ID NO: 150. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO: 269 and a light chain comprising amino acids having the sequence shown in SEQ ID NO: 270. In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises a heavy chain comprising amino acids having the sequence shown in SEQ ID NO: 279 and a light chain comprising amino acids having the sequence shown in SEQ ID NO: 280.

[0115] In another aspect of the present application, there is provided an isolated anti-CD137 construct comprising an antibody portion that binds to CD137, said antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein a) said V H comprises i) an HC-CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 151-153, or a variant thereof comprising up to about 3 amino acid substitutions, ii) an HC-CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 154-156, or a variant thereof comprising up to about 3 amino acid substitutions, and iii) an HC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 157-159, or a variant thereof comprising up to about 3 amino acid substitutions, and b) said V Lcomprises: i) LC-CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 160-163, or a variant thereof comprising up to about 3 amino acid substitutions; ii) HC-CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 164-166, or a variant thereof comprising up to about 3 amino acid substitutions; and iii) HC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 167-169, or a variant thereof comprising up to about 3 amino acid substitutions.

[0116] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein said HC-CDR1 comprises the amino acid sequence SEQ ID NO: 151, said HC-CDR2 comprises the amino acid sequence SEQ ID NO: 154, and said HC-CDR3 comprises the amino acid sequence SEQ ID NO: 157, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein said LC-CDR1 comprises the amino acid sequence SEQ ID NO: 160, said LC-CDR2 comprises the amino acid sequence SEQ ID NO: 164, and said LC-CDR3 comprises the amino acid sequence SEQ ID NO: 167.

[0117] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein said HC-CDR1 comprises the amino acid sequence SEQ ID NO: 151, said HC-CDR2 comprises the amino acid sequence SEQ ID NO: 154, and said HC-CDR3 comprises the amino acid sequence SEQ ID NO: 157, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein said LC-CDR1 comprises the amino acid sequence SEQ ID NO: 162, said LC-CDR2 comprises the amino acid sequence SEQ ID NO: 166, and said LC-CDR3 comprises the amino acid sequence SEQ ID NO: 169.

[0118] In some embodiments, V Hcomprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein said HC-CDR1 comprises the amino acid sequence SEQ ID NO:152, said HC-CDR2 comprises the amino acid sequence SEQ ID NO:155, and said HC-CDR3 comprises the amino acid sequence SEQ ID NO:158, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein said LC-CDR1 comprises the amino acid sequence SEQ ID NO:163, said LC-CDR2 comprises the amino acid sequence SEQ ID NO:166, and said LC-CDR3 comprises the amino acid sequence SEQ ID NO:169.

[0119] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein said HC-CDR1 comprises the amino acid sequence SEQ ID NO:153, said HC-CDR2 comprises the amino acid sequence SEQ ID NO:156, and said HC-CDR3 comprises the amino acid sequence SEQ ID NO:159, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein said LC-CDR1 comprises the amino acid sequence SEQ ID NO:160, said LC-CDR2 comprises the amino acid sequence SEQ ID NO:164, and said LC-CDR3 comprises the amino acid sequence SEQ ID NO:167.

[0120] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein said HC-CDR1 comprises the amino acid sequence SEQ ID NO:153, said HC-CDR2 comprises the amino acid sequence SEQ ID NO:156, and said HC-CDR3 comprises the amino acid sequence SEQ ID NO:159, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein said LC-CDR1 comprises the amino acid sequence SEQ ID NO:161, said LC-CDR2 comprises the amino acid sequence SEQ ID NO:165, and said LC-CDR3 comprises the amino acid sequence SEQ ID NO:168.

[0121] In some embodiments based on any one of the anti-CD137 constructs described herein, the anti-CD137 antibody portion comprises: (a) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:170, and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:173; (b) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:170, and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:176; (c) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:171, and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:173; (d) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:171, and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:174; or (e) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:172, and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:175.

[0122] In some embodiments, the anti-CD137 construct may be a full length antibody, a bispecific antibody, a single chain Fv (scFv), a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VFv fragment ... H In some embodiments, the antibody or antigen-binding fragment thereof comprises or is selected from the group consisting of an antibody or antigen-binding fragment thereof selected from the group consisting of an Fc-Fv fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, and a tetrabody. In some embodiments, the antibody or antigen-binding fragment thereof is chimeric, human, partially humanized, fully humanized, or semi-synthetic. In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin isotype selected from the group consisting of IgG, IgM, IgA, IgD, and IgE. In some embodiments, the antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG1, IgG2, IgG3, or IgG4.

[0123] In some embodiments, the construct comprises a humanized anti-CD137 full-length antibody.

[0124] In some embodiments, the construct comprises a humanized anti-CD137 single-chain Fv fragment.

[0125] In some embodiments, the construct binds to human CD137. In some embodiments, the construct binds to mammalian CD137 (e.g., monkey CD137). In some embodiments, the construct binds to either human CD137 or monkey CD137. In some embodiments, the construct does not bind to mouse CD137.

[0126] As described above, the anti-CD137 construct disclosed herein comprises an anti-CD137 antibody portion that binds to CD137. In some embodiments, the anti-CD137 antibody portion binds to human and monkey CD137.

[0127] In some embodiments, the anti-CD137 antibody portion included in the anti-CD137 construct disclosed herein is a CD137 agonist, wherein the binding of the antibody portion to CD137 can enhance the immune signaling pathway mediated by CD137. In some embodiments, the binding of the antibody portion to CD137 cannot enhance the immune signaling pathway mediated by CD137 in the absence of cross-linking and / or clustering of the antibody portion / CD137 complex. In some embodiments, the binding of the antibody portion to CD137 cannot activate immune cells, such as T cells and / or NK cells. In some embodiments, the binding of the antibody portion to CD137 cannot activate immune cells, such as T cells and / or NK cells, in the absence of cross-linking and / or clustering of the antibody portion / CD137 complex. In some embodiments, the cross-linking and / or clustering of the antibody portion / CD137 complex may be mediated by a second portion of the anti-CD137 construct disclosed herein. In some embodiments, the cross-linking and / or clustering of the antibody portion / CD137 complex may be mediated by the binding of an Fc receptor to the Fc region of the anti-CD137 construct. In some embodiments, the cross-linking and / or clustering of the antibody portion / CD137 complex may be mediated by the binding of a second antibody portion of the anti-CD137 construct to a tumor-associated antigen (TAA).

[0128] In some embodiments, the antibody portion comprises an Fc region selected from the group consisting of Fc regions from IgG, IgA, IgD, IgE, IgM and any combination thereof and heteroconjugates. In some embodiments, the Fc region is derived from human IgG. In some embodiments, the Fc region comprises the Fc region of human IgG1, IgG2, IgG3, IgG4 or a combination or hybrid IgG. In some embodiments, the Fc region is the IgG1 Fc region. In some embodiments, the Fc region comprises the CH2 and CH3 domains of IgG1. In some embodiments, the Fc region is the IgG2 Fc region. In some embodiments, the Fc region comprises the CH2 and CH3 domains of IgG2. In some embodiments, the Fc region is the IgG4 Fc region. In some embodiments, the Fc region comprises the CH2 and CH3 domains of IgG4. Since the effector activity of IgG4 Fc is known to be lower than that of IgG1 or IgG2 Fc, it may be ideal for some uses. In some embodiments, the Fc region is derived from mouse immunoglobulin.

[0129] In some embodiments, the antibody portion comprises an Fc region. In some embodiments, the antibody portion is a scFv that fuses with the Fc region. In some embodiments, the antibody portion comprises a scFv that fuses with the Fc region via a peptide linker. In some embodiments, the Fc region is the human IgG1 Fc region. In some embodiments, the Fc region comprises one or more mutations to increase clearance or decrease the half-life.

[0130] In some embodiments, the Fc region comprises the hinge region (from Cys226), the immunoglobulin IgG heavy chain constant region including the IgG CH2 domain and the CH3 domain. As used herein, the term "hinge region" or "hinge sequence" refers to the amino acid sequence located between the linker and the CH2 domain. In some embodiments, the fusion protein comprises an Fc region that includes a hinge region. In some embodiments, the Fc region of the fusion protein starts from the hinge region and extends to the C-terminus of the IgG heavy chain. In some embodiments, the fusion protein comprises an Fc region that does not include a hinge region.

[0131] In some embodiments, the IgG CH2 domain starts from Ala231. In some embodiments, the CH3 domain starts from Gly341. It can be understood that the C-terminal Lys residue of human IgG may optionally be absent. Further to be understood is that conservative amino acid substitutions in the Fc region are considered to be within the scope of the present invention if they do not affect the required structure and / or stability of the Fc.

[0132] In some embodiments, each strand of the Fc region is fused to the same antibody moiety. In some embodiments, scFv-Fc comprises two identical scFvs as described herein, each fused to one strand of the Fc region. In some embodiments, scFv-Fc is a homodimer.

[0133] In some embodiments, the scFv-Fc comprises two different scFvs, each fused to a single chain of the Fc region. In some embodiments, the scFv-Fc is a heterodimer. Heterodimerization of the different polypeptides in the scFv-Fc can be facilitated by methods known in the art, which includes, but is not limited to, heterodimerization by the knob-in-hole technology. The structure and method of assembly of the knob-in-hole technology can be found, for example, in US5,821,333, US7,642,228, US2011 / 0287009 and PCT / US2012 / 059810, the entire content of which is incorporated herein by reference. This technique involves introducing a "knob" (or protrusion) by substituting a small amino acid residue with a large amino acid residue in the CH3 domain of one Fc, and introducing a "hole" (or cavity) by substituting one or more large amino acid residues with relatively small amino acid residues in the CH3 domain of another Fc. In some embodiments, one chain of the Fc region in the fusion protein contains a knob and the second chain of the Fc region contains a hole.

[0134] Preferred residues for forming the knob are typically naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferably, they are tryptophan and tyrosine. In one embodiment, the original residue for forming the knob has a small side chain volume, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. Exemplary amino acid substitutions in the CH3 domain for forming the knob include, but are not limited to, T366W, T366Y or F405W substitutions.

[0135] Preferred residues for forming the hole are usually naturally occurring amino acid residues, and preferably are selected from alanine (A), serine (S), threonine (T), and valine (V). In one embodiment, the original residue for forming the hole has a large side chain volume, for example, tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain for producing the hole include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions. In some embodiments, the knob includes a T366W substitution, and the hole includes a T366S / L368A / Y407V substitution. It should be understood that other modifications to the known Fc region in the art that contribute to heterodimerization are also contemplated and included in the present application.

[0136] Also contemplated are other scFv-Fc variants (including variants of the isolated anti-CD137 scFv-Fc, for example, full-length anti-CD137 antibody variants) that include any one of the variants described herein (e.g., Fc variants, effector function variants, glycosylation variants, cysteine engineering variants) or combinations thereof.

[0137] a) Antibody affinity The binding specificity of the antibody portion may be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM - assays and peptide scanning.

[0138] In some embodiments, the K D of the binding between the antibody portion and CD137 is about 10 -7 M to about 10 -12 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11M, about 10 -11 M to about 10 -12 M, about 10 -7 M to about 10 -12 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -7 M to about 10 -11 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M or about 10 -7 M to about 10 -9 M. In some embodiments, the K of the binding between the antibody portion and CD137 D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M is stronger than any one of them. In some embodiments, CD137 is human CD137.

[0139] In some embodiments, the K of the binding between the antibody portion and CD137 on is about 10 3 M -1 s -1 to about 10 8 M -1 s -1 about 10 3 M -1 s -1 to about 10 4 M -1 s -1 about 10 4 M -1 s -1 to about 10 5 M -1 s -1 about 10 5 M -1 s -1~about 10 6 M -1 s -1 、about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 or about 10 7 M -1 s -1 ~about 10 8 M -1 s -1 is. In some embodiments, the K of the binding between the antibody portion and CD137 on is about 10 3 M -1 s -1 ~about 10 5 M -1 s -1 、about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 、about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 、about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 、about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 or about 10 5 M -1 s -1 ~about 10 8 M -1 s -1 is. In some embodiments, the K of the binding between the antibody portion and CD137 on is about 10 3 M -1 s -1 、10 4 M -1 s -1 、10 5 M -1 s -1, 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 does not exceed any one of them. In some embodiments, CD137 is human CD137.

[0140] In some embodiments, the K of the binding between the antibody portion and CD137 off is about 1 s -1 ~ about 10 -6 s -1 , about 1 s -1 ~ about 10 -2 s -1 , about 10 -2 s -1 ~ about 10 -3 s -1 , about 10 -3 s -1 ~ about 10 -4 s -1 , about 10 -4 s -1 ~ about 10 -5 s -1 , about 10 -5 s -1 ~ about 10 -6 s -1 , about 1 s -1 ~ about 10 -5 s -1 , about 10 -2 s -1 ~ about 10 -6 s -1 , about 10 -3 s -1 ~ about 10 -6 s -1 , about 10 -4 s -1 ~ about 10 -6 s -1 , about 10 -2 s -1 ~ about 10 -5 s -1 or about 10 -3 s -1 ~ about 10 -5 s -1It is. In some embodiments, the K of the binding between the antibody moiety and CD137 off is about 1 s -1 10 -2 s -1 10 -3 s -1 10 -4 s -1 10 -5 s -1 or 10 -6 s -1 and is at least one of them. In some embodiments, CD137 is human CD137.

[0141] In some embodiments, the binding affinity of the anti-CD137 antibody moiety or anti-CD137 construct is higher (e.g., has a smaller Kd value) than that of a conventional anti-CD137 antibody (e.g., an anti-human CD137 antibody, e.g., BMS-663513 (urelumab) or PF-05082566 (utomilumab)).

[0142] b) Chimeric antibody or humanized antibody In some embodiments, the antibody moiety is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region originating from a mouse) and a human constant region. In some embodiments, the chimeric antibody is a "class-switch" antibody whose class or subclass has been modified from that of the parental antibody. A chimeric antibody includes its antigen-binding fragment.

[0143] In some embodiments, the chimeric antibody is a humanized antibody, which typically involves humanizing a non-human antibody to reduce its immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody includes one or more variable domains, where the HVRs, such as CDRs (or portions thereof), are derived from the non-human antibody and the FRs (or portions thereof) are of human antibody origin. The humanized antibody may optionally further include at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by the corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0144] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989), U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (where SDR (α-CDR) grafting was described), Padlan, Mol. Immunol. 28:489-498 (1991) (where "resurfacing" was described), Dall’Acqua et al., Methods 36:43-60 (2005) (where "FR shuffling" was described), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (where the "directed selection" method of FR shuffling was described).

[0145] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected by the "optimal fitting" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subclasses of light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and Presta et al., J. Immunol. 151:2623 (1993)), mature (somatic mutated) human framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0146] c) Human antibody In some embodiments, the antibody portion is a human antibody (referred to as a human domain antibody or human DAb). Human antibodies may be produced using different techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or DAbs) are known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0147] Human antibodies (e.g., human DAbs) can be prepared by administering an immunogen to a transgenic animal modified to produce a fully human antibody or a full antibody having a human variable region in response to an antigen attack. Such animals usually contain all or part of the human immunoglobulin locus, which replaces their endogenous immunoglobulin locus, or exists extrachromosomally, or is randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. See Lonberg, Nat. Biotech. 23:1117-1125 (2005) for an overview of methods for obtaining human antibodies from transgenic animals. Further, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE (商標) technology, U.S. Pat. No. 5,770,429, which describes the HuMab (登録商標) technology, U.S. Pat. No. 7,041,870, which describes the K-M MOUSE (登録商標) technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes the VelociMouse (登録商標) technology. The human variable regions from full antibodies produced by such animals may be further modified (e.g., by binding to different human constant regions).

[0148] Human antibodies (e.g., human DAbs) may be prepared by hybridoma-based methods. Human myelomas and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies (see, e.g., Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)) have been described. Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) further describe humans produced by human B cell hybridoma technology. Another method includes those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0149] Human antibodies (e.g., human DAbs) may be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences may then be joined to the required human constant domains. Descriptions of techniques for selecting human antibodies from antibody libraries are as follows.

[0150] d) antibodies derived from libraries In a combinatorial library, antibody moieties can be isolated by screening for antibodies having a desired activity or activities. For example, in the art, multiple methods for screening such libraries to produce phage display libraries and obtain antibodies having desired binding characteristics are known. Such methods are outlined, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (edited by O’Brien et al., Human Press, Totowa, NJ, 2001), and are further described in the literature of McCafferty et al., Nature 348:552-554, Clackson et al., Nature 352:624-628 (1991), Marks et al., J. Mol. Biol. 222:581-597 (1992), Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, editor, Human Press, Totowa, NJ, 2003), Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004), Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing single domain antibody libraries are described, see, for example, U.S. Patent No. 7,371,849.

[0151] In some phage display methods, by polymerase chain reaction (PCR), V H and V LClone each gene library, perform random recombination in the phage library, and screen for antigen-binding phages according to the description in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages usually display antibody fragments as scFv fragments or Fab fragments. Libraries from immunogens can provide high-affinity antibodies against the immunogen without constructing hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), natural libraries (e.g., obtained from humans) can be cloned without any immunization required to provide a single antibody source against a wide range of non-self and self antigens. Finally, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), unrearranged V gene fragments from cells can be cloned, encoding highly variable CDR3 regions using PCR primers containing random sequences, and a natural library can be synthesized by completing the rearrangement in vitro. Patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373 and U.S. Patent Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0152] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0153] e) Substitutions, insertions, deletions, and mutations In some embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. In Table 2, under the heading "Exemplary Substitutions", more substantial changes are provided and are further described below with reference to amino acid side-chain classes. Amino acid substitutions can be introduced into the target antibody and the products screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0154] [Table 2]

[0155] Amino acids can be grouped according to common side-chain characteristics: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0156] Non-conservative substitutions require the exchange of one member in one of these classes for another class.

[0157] One type of substitution variant relates to the substitution of one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Typically, the resulting variant is selected for further study and modified (e.g., improved) with respect to the parental antibody in terms of several biological properties (e.g., increased affinity, decreased immunogenicity) and / or substantially retains several biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which are generated using affinity maturation techniques (e.g., those described herein), such as phage display, which are easy to generate. Briefly, one or more HVR (or CDR) residues are mutated and the variant antibody is displayed on a phage and screened for a particular biological activity (e.g., binding affinity).

[0158] In the HVR (or CDR), modifications (e.g., substitutions) can be made, for example, to improve antibody affinity. Such modifications are made in HVR (or CDR) "hot spots" (i.e., residues encoded by codons that mutate frequently during the somatic maturation process) (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in the SDR (a-CDR), and the resulting variant V H or V LThe binding affinity can be tested. Affinity maturation by construction and re-selection from a secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O’Brien, Human Press, Totowa, NJ, (2001)). In some examples of affinity maturation, diversity is introduced into the variable genes selected for maturation by any one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is produced. Then, the library is screened to identify any antibody variants having the desired affinity. Another method of introducing diversity relates to the HVR orientation method, where some HVR (or CDR) residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify HVR (or CDR) residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are always targeted.

[0159] In some examples, substitutions, insertions or deletions may occur within one or more HVRs (or CDRs), provided that such modifications do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions as described herein) that do not substantially reduce binding affinity may be made in the HVR (or CDR). Such modifications may be at HVR “hot spots” or outside the CDR. The variant V provided above H In some examples of the VH sequences, each HVR (or CDR) is either unmodified or contains one, two or no more than three amino acid substitutions.

[0160] As described in Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying residues or regions of an antibody capable of performing targeted mutagenesis is called "alanine scanning mutagenesis." In such a method, target residues or groups of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified, and substitutions are made using neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. By introducing another substitution at the position of the amino acid, the functional sensitivity to the initial substitution can be demonstrated. Alternatively or additionally, the crystal structure of the antigen-antibody complex is for identifying the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or excluded as substitution candidates. The variants may be screened to determine whether they contain the desired attributes.

[0161] Insertions of amino acid sequences include fusions at the amino and / or carboxyl group termini within the range of polypeptides having lengths from one residue to one hundred or more residues, and insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion mutants of the antibody molecule include fusions of the N-terminal or C-terminal of the antibody with an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0162] f) Glycosylation variants In some embodiments, the antibody portion is modified to increase or decrease the degree of glycosylation of the construct. Addition or deletion of glycosylation sites to the antibody can be readily achieved by modifying the amino acid sequence to produce or remove one or more glycosylation sites.

[0163] When the antibody portion contains an Fc region (e.g., scFv-Fc), the carbohydrates linked thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually linked by N-linkage to Asn297 in the C H 2-domain. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide. In some embodiments, modifications can be made to the oligosaccharides in the antibody portion to produce antibody variants having some improved properties.

[0164] In some embodiments, the antibody moiety has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the total of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn297 measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 refers to the asparagine residue at approximately position 297 in the Fc region (EU number of the Fc region residue), however, due to minor sequence variations in the antibody, Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent No. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing afucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent No. US2003 / 0157108A1, Presta, L., and WO2004 / 056312A1, Adams et al., particularly Example 11), and knockout cell lines, for example, the α-1,6-fucosyltransferase gene FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107).

[0165] In some embodiments, the antibody portion has an oligosaccharide that is bisected, for example, where the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.) and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue linked to the Fc region in the oligosaccharide are further provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0166] g) Fc region variants In some embodiments, Fc region variants can be produced by introducing one or more amino acid modifications into the Fc region (e.g., scFv-Fc) of an antibody moiety. The Fc region variant may comprise one human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region), which sequence comprises amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0167] In some embodiments, the Fc region has some (but not all) effector functions, such that this fragment is a desirable candidate for applications where the half-life of the antibody moiety in vivo is important, but some effector functions (e.g., complement and ADCC) are not required or are detrimental. By performing in vitro and / or in vivo cytotoxicity assays, a decrease / depletion of CDC and / or ADCC activity can be confirmed. For example, by performing an Fc receptor (FcR) binding assay, it can be ensured that the antibody does not have FcγR binding ability (and thus may lack ADCC activity), but can retain FcRn binding ability. Primary cell NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). U.S. Patent No. 5,500,362 describes non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest (e.g., Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA, 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat′l Acad. Sci. USA, 82:1499-1502 (1985), see also 5,821,337 (Bruggemann, M. et al., J. Exp. Med., 166:1351-1361 (1987)). Alternatively, non-radioactive assays (e.g., ACTI for flow cytometry (商標)Non-radioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, California, and CytoTox 96 (登録商標) A non-radioactive cytotoxicity assay (see Promega, Madison, Wisconsin) may be used. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, in vivo, for example, in an animal model such as that described in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998), the ADCC activity of the target molecule may be evaluated. By performing a C1q binding assay, it can be confirmed that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, the binding of C1q and C3c to ELISA in WO2006 / 029879 and WO2005 / 100402. A CDC assay may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), Cragg, M.S. et al., Blood 101:1045-1052 (2003), and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may be performed by methods known in the art (see, for example, Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0168] Antibodies having reduced effector function include antibodies having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having two or more substitutions of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant in which residues 265 and 297 are substituted by alanine (U.S. Patent No. 7,332,581).

[0169] In this specification, several antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591 - 6604 (2001).)

[0170] In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the IgG1 Fc region contains the L234A mutation and / or the L235A mutation. In some embodiments, the Fc region is an IgG2 or IgG4 Fc region. In some embodiments, the Fc region is an IgG4 Fc region containing the S228P, F234A, and / or L235A mutations.

[0171] In some embodiments, the antibody portion contains an Fc region having one or more amino acid substitutions, and these substitutions (e.g., substitutions at positions 298, 333, and / or 334 (EU numbers of residues) within the Fc region) improve ADCC.

[0172] In some embodiments, modifications within the Fc region cause a modification (i.e., improvement or reduction) of C1q binding and / or complement - dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al., J. Immunol., 164:4178 - 4184 (2000).

[0173] In some embodiments, the antibody portion (e.g., scFv-Fc) variant comprises a variant Fc region, the variant Fc region comprising one or more amino acid substitutions that modify the half-life and / or the binding to the neonatal Fc receptor (FcRn). Antibodies having an extended half-life and improved binding to the neonatal Fc receptor (FcRn) are responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and are, for example, as described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more amino acid substitutions, where these substitutions modify the binding of the Fc region to FcRn. Such Fc variants include those variants having substitutions (e.g., substitution of Fc region residue 434) at one or more Fc region residues (U.S. Patent No. 7,371,826).

[0174] Further, see Duncan and Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351 regarding other examples of Fc region variants.

[0175] h) Cysteine engineered antibody variants In some embodiments, it may be necessary to produce a cysteine engineered antibody portion, e.g., a “thioMAb,” where one or more residues of the antibody are replaced by cysteine residues. In specific embodiments, the substituted residues are present at accessible sites of the antibody. By substituting those residues with cysteine residues, reactive thiol groups are located at accessible sites of the antibody and may be used to couple the antibody with other moieties, e.g., a drug moiety or a linker-drug moiety, to produce an immunoconjugate, as further described herein. In some embodiments, one or more of the residues of A118 (EU number) of the heavy chain and S400 (EU number) of the heavy chain Fc region may be replaced by cysteine residues. The cysteine engineered antibody portion may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0176] i) Antibody derivative In some embodiments, the antibody portions described herein may be further modified to include other non-proteinaceous portions known in the art and readily available. Suitable portions for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / prolylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may have advantages in preparation for stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers linked to the antibody may vary, and if more than one type of polymer is linked, they may be the same or different molecules. Generally, the number and / or type of polymer used for derivatization may be determined based on the following considerations, which include, but are not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used for determined diagnostic conditions, etc.

[0177] In some embodiments, the antibody portion may be further modified to include one or more bioactive proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biologically active" or "having biological activity" refers to a biological activity demonstrated in vivo to perform a specific function. For example, it may mean binding to a specific biomolecule (e.g., protein, DNA, etc.) and promoting or inhibiting the activity of such a biomolecule. In some embodiments, the bioactive protein or fragment thereof is a protein or polypeptide administered to a patient as an active pharmaceutical substance, a protein and polypeptide for the purpose of preventing, treating, and diagnosing a disease or disorder (e.g., an enzyme used in a diagnostic test or in vitro assay), and a protein and polypeptide administered to a patient to prevent a disease (e.g., a vaccine).

[0178] j) anti-CD137 scFv The anti-CD137 construct in some embodiments is a scFv (hereinafter referred to as "anti-CD137 scFv") that includes an anti-CD137 antibody portion described herein. The anti-CD137-scFv may include any one of the anti-CD137 antibody portions (see the "anti-CD137 antibody portion" section) described herein. In some embodiments, the anti-CD137 scFv has the V L (CD137)-L-V H (CD137) arrangement (from N-terminus to C-terminus). In some embodiments, the anti-CD137 scFv has the V H (CD137)-L-V L (CD137) arrangement (from N-terminus to C-terminus), where L is a linker (e.g., a peptide linker).

[0179] In some embodiments, the anti-CD137 scFv is chimeric, human, partially humanized, fully humanized, or semi-synthetic.

[0180] In some embodiments, the anti-CD137 VL and anti-CD137 VH in the scFv are linked via a linker (e.g., a peptide linker). In some embodiments, the linker comprises from about 4 to about 15 amino acids. In some embodiments, the linker is a GS linker. In some embodiments, the linker comprises any one of the sequences of SEQ ID No: 232-260.

[0181] k) Anti-CD137 fusion protein In some embodiments, the anti-CD137 construct comprises an anti-CD137 antibody portion and a half-life extending portion. In some embodiments, the half-life extending portion is an Fc region. In some embodiments, the half-life extending portion is an albumin binding portion (e.g., an albumin binding antibody portion).

[0182] In some embodiments, the half-life extending portion is an Fc region (e.g., any one of the Fc regions described herein or a variant thereof). The terms "Fc region", "Fc domain" or "Fc" refer to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain, including at least a portion of the constant region. The terms include native Fc regions and variant Fc regions. In some embodiments, the Fc region of a human IgG heavy chain extends from Cys226 to the carboxyl group terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present if it does not affect the structure or stability of the Fc region. Unless otherwise specified herein, amino acid residue numbering in IgG or the Fc region is based on the EU numbering system for antibodies (also referred to as the EU index), for example, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991.

[0183] In some embodiments, the Fc region is selected from the group consisting of the Fc regions of IgG, IgA, IgD, IgE, IgM, and any combination and heteroconjugate thereof. In some embodiments, the Fc region is selected from the group consisting of the Fc regions of IgG1, IgG2, IgG3, IgG4, and any combination and heteroconjugate thereof.

[0184] In some embodiments, compared to the corresponding wild-type Fc region, the Fc region has reduced effector function (e.g., by assaying the antibody-dependent cell-mediated cytotoxicity (ADCC) level, the effector function is reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95%).

[0185] In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc region is an IgG2 or IgG4 Fc region. In some embodiments, the Fc region is an IgG4 Fc region comprising an S228P, F234A and / or L235A mutation.

[0186] In some embodiments, the anti-CD137 antibody portion and the half-life extending portion are linked via a linker (e.g., any one of the linkers described in the "Linker" section).

[0187] In some embodiments, the anti-CD137 fusion protein further comprises a second antibody. In some embodiments, the second antibody binds to a tumor antigen (e.g., any one of the tumor antigens described herein).

[0188] l) Multispecific antibody This specification further provides a multispecific antibody (e.g., a bispecific antibody) that binds to both CD137 and a second antigen. In some embodiments, the second antigen is also CD137, but includes an epitope different from the anti-CD137 antibody portion described herein. In some embodiments, the second antigen is not CD137. In some embodiments, the second antigen is a tumor-associated antigen.

[0189] In some embodiments, the multispecific antibody comprises: a) a first antibody portion comprising any anti-CD137 construct described herein, and b) a second antibody portion that binds to a second antigen that is not CD137. In some embodiments, the second antigen includes a tumor-associated antigen.

[0190] In some embodiments, the first antibody portion comprises a single-chain Fv fragment that binds to CD137. In some embodiments, the second antibody portion comprises a full-length antibody that binds to a tumor-associated antigen and includes two antibody heavy chains and two antibody light chains, wherein each of the heavy chains comprises a second heavy-chain variable region (V H-2 ), and each of the light chains comprises a second light-chain variable region (V L-2 ), and wherein the anti-CD137 single-chain Fv fragment fuses with at least one of the heavy or light chains of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the C-terminus of the light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the N-terminus of the light chain of the full-length antibody.

[0191] In some embodiments, the multispecific antibody comprises: a) a first antibody portion comprising a single-chain Fv fragment that binds to CD137 and includes a first heavy-chain variable region (V H-1 ) and a first light-chain variable region (V L-1 ), and b) a second antibody portion that binds to a second antigen (e.g., a tumor-associated antigen) and includes a full-length antibody that includes two antibody heavy chains and two antibody light chains, wherein each of the heavy chains comprises a second heavy-chain variable region (V H-2 ), and each of the light chains comprises a second light-chain variable region (V L-2and a second antibody moiety comprising H-1 and V L-1 are fused to the full-length antibody via a first linker (e.g., a first peptide linker). In some embodiments, the first linker comprises about 1 to about 30 (e.g., about 4 to 20, about 3 to 20, about 6 to 18, or about 10 to 15) amino acids. In some embodiments, the first linker is a GS linker. In some embodiments, the linker has an amino acid sequence of any one of SEQ ID NOs: 232-260. In some embodiments, the full-length antibody comprises an Fc region selected from an Fc region from IgG, IgA, IgD, IgE, IgM, and any combination and hybrid thereof. In some embodiments, the Fc region is selected from an Fc region from IgG1, IgG2, IgG3, IgG4, and any combination and hybrid thereof. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG2 Fc region. In some embodiments, the IgG4 Fc region comprises an F234A mutation and an L235A mutation. In some embodiments, the IgG4 Fc region further comprises an S228P mutation.

[0192] In some embodiments, the multispecific antibody comprises: a) a first antibody portion comprising a full-length antibody comprising any of the anti-CD137 constructs described herein; and b) a second antibody portion comprising a single-chain Fv fragment that recognizes a second antigen (e.g., a tumor-associated antigen). In some embodiments, the single-chain Fv fragment is fused to one or two heavy chains of the anti-CD137 full-length antibody. In some embodiments, the single-chain Fv fragment is fused to the C-terminus of one or two heavy chains of the anti-CD137 full-length antibody. In some embodiments, the single-chain Fv fragment is fused to one or two N-termini of the heavy chain of the anti-CD137 full-length antibody. In some embodiments, the single-chain Fv fragment is fused to one or two light chains of the anti-CD137 full-length antibody. In some embodiments, the single-chain Fv fragment is fused to the C-terminus of one or two light chains of the anti-CD137 full-length antibody. In some embodiments, the single-chain Fv fragment is fused to the N-terminus of one or two anti-CD137 light chains of the full-length antibody.

[0193] As described herein, "tumor-associated antigen" refers to any antigen that is, for example, expressed at a level significantly higher (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% higher) in tumor cells (e.g., cancer cells) than in non-tumor cells (e.g., non-cancer cells).

[0194] Exemplary tumor-associated antigens recognizable by the second antibody portion described herein include, but are not limited to, alpha-fetoprotein (AFP), CA15-3, CA27-29, CA19-9, CA-125, calretinin, carcinoembryonic antigen, CD34, CD99, CD117, chromogranin, cytokeratin, myogenin, epithelial membrane protein (EMA), factor VIII, CD31 FL1, glial fibrillary acidic protein (GFAP), cystic disease fluid protein (GCDFP-15), HMB-45, human chorionic gonadotropin (hCG), inhibin, keratin, CD45, lymphocyte marker, MART-1 (Melan-A), Myo Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, S100 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, and vimentin.

[0195] In some embodiments, the tumor-associated antigen is selected from the group consisting of HER-2, EGFR, PD-L1, c-Met, B cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinases erb-B2, 3, 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), type 1 tyrosine kinase transmembrane receptor (ROR1), and any combination thereof. In some embodiments, the tumor-associated antigen is HER-2, EGFR, B7H3, c-Met, or PD-L1. In some embodiments, the tumor-associated antigen is selected from the group consisting of HER-2, EGFR, B7H3, c-Met, or PD-L1.

[0196] m) anti-CD137 x EGFR multispecific antibody In some embodiments, the multispecific antibodies disclosed herein comprise: a) a first antibody portion that is a single-chain Fv fragment that constitutes any of the anti-CD137 constructs disclosed herein; and b) a second antibody portion that binds to EGFR and comprises a full-length antibody comprising two antibody heavy chains and two antibody light chains, wherein each of the heavy chains comprises a second heavy-chain variable region (V H-2 ), and each of the light chains comprises a second light-chain variable region (V L-2 ), and wherein the anti-CD137 single-chain Fv fragment comprises a second antibody portion that fuses with the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the C-terminus of the heavy chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the N-terminus of the heavy chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the C-terminus of the light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the N-terminus of the light chain of the full-length antibody. In some embodiments, the EGFR is human EGFR. In some embodiments, the second antibody portion may be any full-length antibody disclosed in PCT / US2015 / 033402 (disclosed as International Publication WO2015184403, the content of which is incorporated herein by reference in its entirety). In some embodiments, the full-length antibody or anti-EGFR antibody portion that binds to EGFR competes with an antibody or antibody fragment comprising a third heavy-chain variable region (V H-3 ) and a third light-chain variable region (V L-3 ) for the binding epitope of EGFR. In some embodiments, the V H-3 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:190, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:191, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:192, and the V L-3includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:193, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:194, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:195. In some embodiments, the V H-3 includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:198, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:199, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:200, and the V L-3 includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:201, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:202, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:203. In some embodiments, the V H-3 includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:206, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:207, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:208, and the V L-3 includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:209, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:210, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:211. In some embodiments, the V H-3 includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:214, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:215, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:216, and the V L-3 includes LC-CDR1 comprising the amino acid sequence of SEQ ID NO:217, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:218, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:219. In some embodiments, the V H-3 includes HC-CDR1 comprising the amino acid sequence of SEQ ID NO:222, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:223, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:224, and the V L-3It includes an LC-CDR1 containing the amino acid sequence of SEQ ID NO:225, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:226, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:227.

[0197] In some embodiments, the said V H-2 includes an HC-CDR1 containing the amino acid sequence of SEQ ID NO:190, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:191, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:192, and the said V L-2 includes an LC-CDR1 containing the amino acid sequence of SEQ ID NO:193, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:194, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:195. In some embodiments, the said V H-2 includes an HC-CDR1 containing the amino acid sequence of SEQ ID NO:198, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:199, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:200, and the said V L-2 includes an LC-CDR1 containing the amino acid sequence of SEQ ID NO:201, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:202, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:203. In some embodiments, the said V H-2 includes an HC-CDR1 containing the amino acid sequence of SEQ ID NO:206, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:207, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:208, and the said V L-2 includes an LC-CDR1 containing the amino acid sequence of SEQ ID NO:209, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:210, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:211. In some embodiments, the said V H-2comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:214, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:215, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:216, and said V L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:217, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:218, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:219. In some embodiments, said V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:222, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:223, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:224, and said V L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:225, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:226, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:227.

[0198] In some embodiments, said V H-2 comprises the amino acid sequence of SEQ ID NO:196, 204, 212, 220 or 228 or a variant thereof having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO:196, 204, 212, 220 or 228, and / or said V L-2 comprises the amino acid sequence of SEQ ID NO:197, 205, 213, 221 or 229 or a variant thereof having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO:197, 205, 213, 221 or 229.

[0199] In some embodiments, said multispecific antibody is a) the first antibody moiety comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), said VH comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:121, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:122, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:123, wherein said V L is the first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:124, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:125, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:126, and b) a second antibody portion comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), wherein said V H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO:206, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO:207, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO:208, and said V L-2 is the second antibody portion comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:209, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:210, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:211.

[0200] In some embodiments, the multispecific antibody is a) the first antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:261, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:262, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:263, and said V L is the first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:264, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:265, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:266, and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2said second antibody portion comprising, and said V H-2 comprises a second HC-CDR1 having the amino acid sequence of SEQ ID NO:206, a second HC-CDR2 having the amino acid sequence of SEQ ID NO:207, and a second HC-CDR3 having the amino acid sequence of SEQ ID NO:208, and said V L-2 comprises a second LC-CDR1 having the amino acid sequence of SEQ ID NO:209, a second LC-CDR2 having the amino acid sequence of SEQ ID NO:210, and a second LC-CDR3 having the amino acid sequence of SEQ ID NO:211, and said second antibody portion.

[0201] In some embodiments, said multispecific antibody comprises: a) said first antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), said V H comprising an HC-CDRl having the amino acid sequence of SEQ ID NO:271, an HC-CDR2 having the amino acid sequence of SEQ ID NO:272, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:273, and said V L comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:274, an LC-CDR2 having the amino acid sequence of SEQ ID NO:275, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:276, and said first antibody portion; and b) said second antibody portion comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), said V H-2 comprising a second HC-CDR1 having the amino acid sequence of SEQ ID NO:206, a second HC-CDR2 having the amino acid sequence of SEQ ID NO:207, and a second HC-CDR3 having the amino acid sequence of SEQ ID NO:208, and said V L-2 comprising a second LC-CDR1 having the amino acid sequence of SEQ ID NO:209, a second LC-CDR2 having the amino acid sequence of SEQ ID NO:210, and a second LC-CDR3 having the amino acid sequence of SEQ ID NO:211, and said second antibody portion.

[0202] In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of the anti-EGFR full-length antibody, wherein the heavy chain of the anti-EGFR full-length antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of any one of SEQ ID NO: 183, 185, 188, 189, 230, 231, 259, 260, 281 or 282, or a variant of the amino acid sequence having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NO: 183, 185, 188, 189, 230, 231, 259, 260, 281 or 282. In some embodiments, the full-length antibody comprised in the second antibody moiety comprises a light chain, and the light chain comprises the amino acid sequence of any one of SEQ ID NO: 184, 186 or 187, or a variant of the amino acid sequence having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NO: 184, 186 or 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 183 and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 185 and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 188 and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 189 and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 184.In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 189, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 186. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 230, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 231, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 259, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 260, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 281, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 282, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187.

[0203] In some embodiments, a multispecific antibody is provided, which is a) an anti-CD137 single-chain Fv fragment that binds to CD137 as disclosed herein, wherein the single-chain Fv fragment comprises a first heavy-chain variable region (V H-1 ) and a first light-chain variable region (V L-1 ), wherein the VH-1 comprises: i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO:141, ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO:142, and iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO:143, wherein said V L is an anti-CD137 single-chain Fv fragment comprising: i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO:144, ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO:145, and iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO:146, and b) a full-length antibody that binds to EGFR and comprises two antibody heavy chains and two antibody light chains, wherein the full-length antibody that binds to EGFR comprises a second heavy-chain variable region (V H-2 ) and a second light-chain variable region (V L-2 ). In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the C-terminus of the heavy chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the N-terminus of the heavy chain of the full-length antibody.

[0204] In some embodiments, said V H-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:190, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:191, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:192, and said V L-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:193, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:194, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:195. In some embodiments, said V H-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:198, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:199, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:200, and said V L-2It includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:201, LC-CDR2 containing the amino acid sequence of SEQ ID NO:202, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:203. In some embodiments, the V H-2 includes HC-CDR1 containing the amino acid sequence of SEQ ID NO:206, HC-CDR2 containing the amino acid sequence of SEQ ID NO:207, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:208, and the V L-2 includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:209, LC-CDR2 containing the amino acid sequence of SEQ ID NO:210, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:211. In some embodiments, the V H-2 includes HC-CDR1 containing the amino acid sequence of SEQ ID NO:214, HC-CDR2 containing the amino acid sequence of SEQ ID NO:215, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:216, and the V L-2 includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:217, LC-CDR2 containing the amino acid sequence of SEQ ID NO:218, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:219. In some embodiments, the V H-2 includes HC-CDR1 containing the amino acid sequence of SEQ ID NO:222, HC-CDR2 containing the amino acid sequence of SEQ ID NO:223, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:224, and the V L-2 includes LC-CDR1 containing the amino acid sequence of SEQ ID NO:225, LC-CDR2 containing the amino acid sequence of SEQ ID NO:226, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:227.

[0205] In some embodiments, the V H-2comprises the amino acid sequence of SEQ ID NO: 196, 204, 212, 220 or 228, or a variant thereof having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 196, 204, 212, 220 or 228, and / or said V L-2 comprises the amino acid sequence of SEQ ID NO: 197, 205, 213, 221 or 229, or a variant thereof having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 197, 205, 213, 221 or 229.

[0206] In some embodiments, said V H-1 comprises the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 267 or 277, or a variant thereof having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 267 or 277, and / or said V L-1 comprises the amino acid sequence of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 268 or 278, or a variant thereof having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 268 or 278.

[0207] In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a full-length anti-EGFR antibody, wherein the heavy chain of the full-length anti-EGFR antibody fused to the anti-CD137 single-chain Fv fragment comprises an amino acid sequence of any one of SEQ ID NO: 183, 185, 188, 189, 230, 231, 259, 260, 281, or 282, or a variant of an amino acid sequence having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence of any one of SEQ ID NO: 183, 185, 188, 189, 230, 231, 259, 260, 281, or 282. In some embodiments, the full-length antibody comprised in the second antibody portion comprises a light chain, and the light chain comprises an amino acid sequence of any one of SEQ ID NO: 184, 186, or 187, or a variant of an amino acid sequence having at least about 80% (e.g., any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence of any one of SEQ ID NO: 184, 186, or 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 183, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 185, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 188, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 189, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 184.In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 189, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 186. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 230, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 231, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 259, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 260, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 281, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the multispecific antibody comprises an anti-EGFR heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence shown in SEQ ID NO: 282, and an anti-EGFR light chain comprising the amino acid sequence shown in SEQ ID NO: 187.

[0208] In some examples, the V of the anti-CD137 single-chain Fv fragment H-1 and V L-1It fuses with the full-length antibody via a first linker (e.g., a first peptide linker). In some embodiments, the first linker comprises about 1 to about 30 (e.g., about 4 to 20, about 3 to 20, about 6 to 18, or about 10 to 15) amino acids. In some embodiments, the first linker is a GS linker. In some embodiments, the linker has an amino acid sequence of any one of SEQ ID NOs: 232 - 260. In some embodiments, the anti-CD137 single-chain Fv fragment fuses with the full-length antibody via a second linker (e.g., a second peptide linker). In some embodiments, the second peptide linker comprises about 1 to about 30 (e.g., about 4 to 20, about 3 to 20, about 6 to 18, or about 10 to 15) amino acids. In some embodiments, the second peptide linker is a linker comprising any one of the sequences of SEQ ID NOs: 232 - 258.

[0209] In some embodiments, the full-length antibody has an Fc region selected from the Fc regions of IgG, IgA, IgD, IgE, IgM and hybrids of any combination thereof. In some embodiments, the Fc region is selected from the Fc regions of IgG1, IgG2, IgG3, IgG4 and hybrids of any combination thereof. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG2 Fc region. In some embodiments, the IgG4 Fc region comprises S228P, F234A and L235A mutations.

[0210] o) Multispecific antibody properties In some embodiments, the multispecific antibodies described herein have improved clinical properties relative to reference multispecific antibodies that bind CD137 and a second antigen (e.g., HER2, EGFR, PD-L1). In some embodiments, the multispecific antibody exhibits improved ADCC activity (e.g., ADCC-dependent cytotoxicity is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70% higher) compared to the ADCC activity of the reference multispecific antibody. In some embodiments, the multispecific antibody exhibits a greater antitumor effect (e.g., reducing tumor burden or improving survival rate by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70% or more) compared to the ADCC activity of the reference multispecific antibody. In some embodiments, the multispecific antibody exhibits less toxicity compared to the toxicity of the reference multispecific antibody.

[0211] In some embodiments, the multispecific antibody binds to both CD137 and a tumor-associated antigen, and the ADCC activity of tumor cells that are positive for or express high levels of the indicated tumor-associated antigen is improved (e.g., at least about 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 10-fold, 15-fold, 20-fold higher) compared to the ADCC activity of the reference multispecific antibody. In some embodiments, the EC50 for the cell-specific multispecific antibody does not exceed about 50%, 40%, 30%, 20%, 10% of the reference multispecific antibody.

[0212] In some embodiments, the multispecific antibody does not induce significant cytokine (e.g., IL-2, IFN-γ, TNF-α) release in individuals without cancer or tumors. In some embodiments, the multispecific antibody does not induce significant cytokine (e.g., IL-2, IFN-γ, TNF-α) release in non-cancerous tissues in individuals with cancer or tumors. In some embodiments, significant cytokine release is at least about 70%, 60%, 50%, 40%, 30% or 20% of the level of cytokine release induced by a reference multispecific antibody that binds to CD137 and the same second antigen. In some embodiments, the multispecific antibody induces less cytokine (e.g., IL-2, IFN-γ, TNF-α) release (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% less cytokine release) in individuals without cancer or tumors. In some embodiments, the multispecific antibody induces less cytokine (e.g., IL-2, IFN-γ, TNF-α) release (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% less cytokine release) in non-cancerous tissues in individuals with cancer or tumors.

[0213] n) tandem scFv The multispecific anti-CD137 molecule in some embodiments is a tandem scFv (also referred to herein as a "tandem scFv multispecific anti-CD137 antibody"), which comprises a first scFv and a second scFv, wherein the first scFv comprises an anti-CD137 antibody moiety (referred to herein as an "anti-CD137 scFv") that specifically recognizes CD137, and the second scFv specifically recognizes a second antigen. In some embodiments, the tandem scFv multispecific anti-CD137 antibody further comprises at least one (e.g., at least about 2, 3, 4, 5 or any one of more thereof) additional scFv.

[0214] In some embodiments, provided is a tandem scFv multispecific (e.g., bispecific) anti-CD137 antibody, which comprises a) a first scFv that specifically recognizes CD137, and b) a second scFv that specifically recognizes a second antigen (e.g., a tumor-associated antigen), wherein the tandem scFv multispecific anti-CD137 antibody is a tandem di-scFv or a tandem tri-scFv. In some embodiments, the tandem scFv multispecific anti-CD137 antibody is a tandem di-scFv. In some embodiments, the tandem scFv multispecific anti-CD137 antibody is a bispecific T cell engager. In some embodiments, the second scFv binds to a different CD137 epitope. In some embodiments, the second scFv specifically recognizes a second antigen that is not CD137. In some embodiments, the second scFv specifically recognizes a second antigen, e.g., a tumor-associated antigen. In some embodiments, the first anti-CD137 scFv is chimeric, human, partially humanized, fully humanized or semi-synthetic. In some embodiments, the second scFv is chimeric, human, partially humanized, fully humanized or semi-synthetic. In some embodiments, both the first and the second scFv are chimeric, human, partially humanized, fully humanized or semi-synthetic. In some embodiments, the tandem scFv multispecific anti-CD137 antibody further comprises at least one (e.g., any one of at least about 2, 3, 4, 5 or more) additional scFv. In some embodiments, the first anti-CD137 scFv and the second scFv are linked via a linker (e.g., a peptide linker). In some embodiments, the linker is (GGGGS) ncomprising the amino acid sequence, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, the linker comprises the amino acid sequence of TSGGGGS. In some embodiments, the first anti-CD137 scFv is linked to the N-terminus of the second scFv. In some embodiments, the first anti-CD137 scFv is linked to the C-terminus of the second scFv. In some embodiments, the tandem scFv multispecific (e.g., bispecific) anti-CD137 antibody further comprises a tag (e.g., a peptide tag for purification purposes). In some embodiments, the tag is linked to the N-terminus of the tandem scFv multispecific (e.g., bispecific) anti-CD137 antibody. In some embodiments, the tag is linked to the C-terminus of the tandem scFv multispecific (e.g., bispecific) anti-CD137 antibody. In some embodiments, the tag comprises the amino acid sequence of HHHHHH.

[0215] In some embodiments, the tandem scFv multispecific anti-CD137 antibody is a tandem di-scFv (referred to herein as a "tandem di-scFv bispecific anti-CD137 antibody") comprising two scFvs. The tandem di-scFv bispecific anti-CD137 antibody can be assembled in any arrangement, e.g., the V H and V L having, where X is a second antigen bound by the second scFv, and L1, L2, and L3 are optional linkers (e.g., peptide linkers). See the "Linker" section for all suitable linkers. In some embodiments, the linker (L1, L2, or L3) comprises the amino acid sequence of SRGGGGSGGGGSGGGGSLEMA. In some embodiments, the linker (L1, L2, or L3) is the (GGGGS) n sequence or (GGGGS) ncomprising an array, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, the linker (L1, L2 or L3) comprises the amino acid sequence of TSGGGGS. In some embodiments, the linker (L1, L2 or L3) comprises the amino acid sequence of GEGTSTGSGGSGGSGGAD.

[0216] V L (CD137)-L1-V H (CD137)-L2-V L (X)-L3-V H (X), V L (CD137)-L1-V H (CD137)-L2-V H (X)-L3-V L (X), V H (CD137)-L1-V L (CD137)-L2-V L (X)-L3-V H (X), V H (CD137)-L1-V L (CD137)-L2-V H (X)-L3-V L (X), V L (X)-L1-V H (X)-L2-V L (CD137)-L3-V H (CD137), V L (X)-L1-V H (X)-L2-V H (CD137)-L3-V L (CD137), V H (X)-L1-V L (X)-L2-V L (CD137)-L3-V H (CD137), V H (X)-L1-V L (X)-L2-V H (CD137)-L3-V L (CD137), V L (CD137)-L1-V H (X)-L2-V L (X)-L3-V H (CD137), V L (CD137)-L1-V L (X)-L2-V H (X)-L3-V H (CD137), V H (CD137)-L1-V H (X)-L2-V L (X)-L3-V L (CD137), V H (CD137)-L1-V L (X)-L2-V H (X)-L3-V L (CD137), V L (X)-L1-V H (CD137)-L2-V L (CD137)-L3-V H (X), V L (X)-L1-V L (CD137)-L2-V H (CD137)-L3-V H (X), V H (X)-L1-V H (CD137)-L2-V L (CD137)-L3-V L (X), or V H (X)-L1-V L (CD137)-L2-V H (CD137)-L3-V L (X).

[0217] o) Linker In some embodiments, the anti-CD137 constructs described herein include one or more linkers between two moieties (e.g., the anti-CD137 antibody moiety and the half-life extension moiety in the bispecific antibodies described herein, the anti-CD137 scFv and the full-length antibody). The length, flexibility and / or other properties of the one or more linkers used in the bispecific antibodies may have some effect on the properties, which include, but are not limited to, the affinity, specificity or binding to one or more specific antigens or epitopes. For example, a longer linker may be selected to ensure that two adjacent domains do not spatially interfere with each other. In some embodiments, the linker (e.g., a peptide linker) includes flexible residues (e.g., glycine and serine), whereby the adjacent domains can move freely relative to each other. For example, a glycine-serine dimer may be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0218] Other linker considerations include effects on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more stable or less stable and programmed degradation), rigidity, flexibility, effect on immunogenicity, modulation of antibody binding, ability to incorporate into micelles or liposomes, etc.

[0219] The coupling of the two parts can be accomplished by any of the following chemical reactions, which bind two molecules to a bispecific antibody as long as both of these two components retain their respective activities, for example, binding to CD137 and a second antigen respectively. This linkage may involve many chemical mechanisms, such as covalent bonding, affinity binding, insertion, coordination bonding and complexation. In some embodiments, the bond is a covalent bond. The covalent bond may be achieved by direct condensation of conventional side chains or incorporation of an external cross-linking molecule. In this case, many divalent or polyvalent linkers may be used to couple protein molecules. For example, representative coupling agents may include organic compounds, such as thioesters, carbodiimides, succinimidyl, diisocyanates, glutaraldehyde, diazophenyl and hexamethylenediamine. This list is not intended to be exhaustive of the various coupling agents known in the art and is merely an example of more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984), Jansen et al., Immunological Reviews 62:185-216 (1982), and Vitetta et al., Science 238:1098 (1987)).

[0220] The linkers applicable in the present application are those described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimidyl)). In some embodiments, the non-peptide linkers used herein are (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamide]hexanoate ester (Pierce Chemical Company, catalog number #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)propionamide]hexanoate ester (Pierce Chemical Company, catalog number #2165-G), (v) sulfo-NHS (N-hydroxysulfo-succinimidyl, Pierce Chemical Company, catalog number #24510) conjugated to EDC.

[0221] The linkers described herein can result in bispecific antibodies with different physicochemical properties because they contain components with different attributes. For example, the sulfo-NHS ester of alkyl carboxylate is more stable than that of aromatic carboxylate. The solubility of the linker containing NHS ester is lower than that of sulfo-NHS ester. Also, the linker SMPT contains a sterically hindered disulfide bond and can form an antibody fusion protein with enhanced stability. This is because disulfide bonds are usually less stable than other bonds, and when the disulfide bond is cleaved in vitro, there are fewer available antibody fusion proteins. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. When used in combination with sulfo-NHS, carbodiimide coupling (e.g., EDC) forms an ester that is more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0222] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of an antibody having only a heavy chain may be used as a linker. See, for example, WO1996 / 34103.

[0223] The peptide linker may have any suitable length. In some embodiments, the length of the peptide linker is at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker does not exceed about any one of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. In some embodiments, the length of the peptide linker is any one of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0224] The basic technical feature of such a peptide linker is that the peptide linker does not contain any polymerization activity. The features of the peptide linker (which include the lack of secondary structure promoting effect) are known in the art and are described, for example, in Dall’Acqua et al. (Biochem. (1998) 37, 9266 - 9273), Cheadle et al. (Mol Immunol (1992) 29, 21 - 30) and Raag and Whitlow (FASEB (1995) 9(1), 73 - 80). For the “peptide linker”, particularly preferably, the amino acid is Gly. Also, a peptide linker that does not promote any secondary structure is preferred. The linkage of domains to each other may be provided, for example, by genetic engineering. Methods for preparing fused and functionally linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are well known in the art (for example, WO99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience], New York 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0225] The peptide linker may be a stable linker, which is not cleaved by proteases, particularly matrix metalloproteases (MMP).

[0226] The linker may be a flexible linker. Exemplary flexible linkers are glycine polymers (G) n , glycine - serine polymers (for example, (GS) n , (GSGGS) n , (GGGGS) n and (GGGS) nincluding, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Since glycine and glycine-serine polymers are relatively unstructured, they can function as neutral tethers between components. Glycine clearly penetrates much more phi-psi space even relative to alanine and is much less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). The design of antibody fusion proteins may include a linker that is flexible overall or in part, whereby the linker may include a flexible linker portion and one or more portions that impart a less flexible structure to provide the desired antibody fusion protein structure, as will be recognized by those skilled in the art.

[0227] p) Immunoconjugate Also provided herein are immunoconjugates, said immunoconjugates comprising any of the anti-CD137 constructs (e.g., bispecific antibodies) described herein conjugated to a therapeutic agent or a marker. In some embodiments, said marker is selected from the group consisting of radioisotopes, fluorescent dyes and enzymes.

[0228] q) Nucleic acid Nucleic acid molecules encoding the anti-CD137 constructs or anti-CD137 antibody portions described herein were also considered. In some embodiments, nucleic acids (or a group of nucleic acids) encoding full-length anti-CD137 antibodies were provided. In some embodiments, nucleic acids (or a group of nucleic acids) encoding anti-CD137 scFv were provided. In some embodiments, nucleic acids (or a group of nucleic acids) encoding anti-CD137 Fc fusion proteins were provided. In some embodiments, nucleic acids (or a group of nucleic acids) encoding multispecific anti-CD137 molecules (e.g., multispecific anti-CD137 antibodies or bispecific anti-CD137 antibodies) or polypeptide portions thereof were provided. In some embodiments, the nucleic acids (or a group of nucleic acids) encoding the anti-CD137 constructs described herein may further comprise a nucleic acid sequence encoding a peptide tag (e.g., a protein purification tag, e.g., a His tag, an HA tag).

[0229] This specification further contemplates isolated host cells comprising anti-CD137 constructs, isolated nucleic acids encoding anti-CD137 construct polypeptide components, or vectors comprising nucleic acids encoding anti-CD137 construct polypeptide components described herein.

[0230] This application further includes variants of these nucleic acid sequences. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the anti-CD137 constructs or anti-CD137 antibody portions of this application under at least moderately stringent hybridization conditions.

[0231] The present invention further provides vectors inserted with the nucleic acids of the present invention.

[0232] Using the standard gene delivery protocol, the nucleic acids of the invention can also be used in nucleic acid immunization and gene therapy. Methods used for gene delivery are known in the art. See, for example, U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In some embodiments, the invention provides gene therapy vectors.

[0233] The nucleic acid can be cloned into multiple types of vectors. For example, the nucleic acid can be cloned into a vector, which includes, but is not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.

[0234] Alternatively, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Viruses that can function as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, U.S. Pat. No. 6,326,193).

[0235] III. Preparation Methods In some embodiments, provided are methods for preparing an anti-CD137 construct or antibody portion that binds to CD137, and compositions produced in the process of preparing the anti-CD137 construct or antibody portion, such as polynucleotides, nucleic acid constructs, vectors, host cells or media. The anti-CD137 constructs or antibody portions or compositions described herein can generally be prepared by the following plurality of methods described and are specifically described by specific examples.

[0236] Antibody Expression and Production The antibodies described herein (including anti-CD137 monoclonal antibodies, anti-CD137 bispecific antibodies and anti-CD137 antibody portions) can be prepared by any method known in the art, including the methods described below and in the specific examples.

[0237] Monoclonal Antibodies Monoclonal antibodies are obtained from a substantially homogeneous group of antibodies, i.e., each antibody constituting the group is identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates the characteristic of the antibody that it is not a mixture of individual antibodies. For example, the monoclonal antibody may be prepared by the hybridoma method first described by Kohler et al. (Nature, 256:495 (1975)), or by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal, e.g., a hamster or llama, is immunized as described above to induce lymphocytes that produce or can produce antibodies that specifically bind to the protein for immunization. Alternatively, lymphocytes may be immunized in vitro. Then, the lymphocytes are fused with myeloma cells using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 (Academic Press, 1986)). See also the camel immunization in Specific Example 1.

[0238] The immunizing agent typically includes an antigen protein or a fusion mutant thereof. Usually, when human-derived cells are desired, peripheral blood lymphocytes ("PBL") are used, or when non-human mammalian sources are desired, spleen cells or lymph node cells are used. Then, to form hybridoma cells, the lymphocytes are fused with an immortalized cell line using a suitable fusing agent (e.g., polyethylene glycol). Goding, Monoclonal Antibodies: Principles and Practice, (Academic Press, 1986), pages 59-103).

[0239] The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Usually, a rat or mouse myeloma cell line is used. The hybridoma cells thus prepared are seeded and grown in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, when the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the medium for hybridomas typically contains hypoxanthine, methotrexate, and thymidine (HAT medium), which are substances that inhibit the growth of HGPRT-deficient cells.

[0240] Preferred immortalized myeloma cell lines are those that fuse efficiently, are selected, support stable high-level production of antibodies by antibody-producing cells, and are sensitive to media (e.g., HAT medium). Here, preferably, they are murine myeloma cell lines, such as MOPC-21 and MPC-11 mouse tumors obtained from the Salk Institute Cell Distribution Center, San Diego, California, USA, and cell lines originating from SP-2 cells (and its derivatives, e.g., X63-Ag8-653) obtained from the American Type Culture Collection, Manassas, Virginia, USA. Human myelomas and mouse-human heteromyeloma cell lines are also described as being used for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0241] Assay for the production of monoclonal antibodies against an antigen in the medium in which hybridoma cells grow. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays, e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0242] It may be assayed whether a monoclonal antibody against a desired antigen is present in the medium for culturing hybridoma cells. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, the binding affinity can be determined by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0243] After identifying hybridoma cells capable of producing antibodies having the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Also, hybridoma cells can be grown in vivo as tumors in mammals.

[0244] The monoclonal antibody secreted by the subclone is appropriately isolated from the medium, ascites or serum by conventional immunoglobulin purification methods (e.g., protein A-agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography).

[0245] Alternatively, monoclonal antibodies may be prepared by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567 and as described herein. Using conventional methods (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of a murine antibody), DNA encoding a monoclonal antibody can be readily isolated and sequenced. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and these vectors can be transfected into host cells that do not additionally produce immunoglobulins (e.g., E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells) to synthesize the monoclonal antibody in such recombinant host cells. Commentaries on the recombinant expression of DNA encoding antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

[0246] In another embodiment, antibodies can be isolated from an antibody phage library produced using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies using phage libraries, respectively. Subsequent publications have described chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)) and the production of high affinity (nM range) human antibodies by combinatorial infection and in vivo recombination. Thus, these techniques are viable alternatives to the traditional monoclonal antibody hybridoma technology for isolating monoclonal antibodies.

[0247] Furthermore, for example, a mouse sequence homologous to the coding sequences of the human heavy and light chain constant domains can be replaced (U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or the DNA may be modified, such as by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Usually, such a non-immunoglobulin polypeptide replaces the constant domain of an antibody or replaces the variable domain of one antigen-binding site of an antibody to produce a chimeric bivalent antibody containing one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0248] The monoclonal antibodies described herein may be monovalent, and their preparation is well known in the art. For example, one method relates to the recombinant expression of an immunoglobulin light chain and a modified heavy chain. Usually, to prevent cross-linking of the heavy chains, the heavy chains are cleaved at any point in the Fc region. Alternatively, the relevant cysteine residues may be replaced with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also suitable for the preparation of monovalent antibodies. Digestion of antibodies to produce antibody fragments (especially Fab fragments) may be accomplished using conventional techniques known in the art.

[0249] Also, chimeric or hybrid antibodies can be prepared in vitro using methods known in synthetic protein chemistry (including those methods related to cross-linking agents). For example, immunotoxins can be constructed by disulfide bond exchange reactions or by forming thioether bonds. Specific examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0250] Also, for the production of monoclonal antibodies, see specific examples.

[0251] Multispecific antibody This specification further provides a method for preparing the multispecific antibodies (e.g., bispecific antibodies) described herein. The multispecific antibodies may be prepared using any method known in the art or described herein (e.g., Specific Examples 1 and 3).

[0252] The methods for preparing the multispecific antibodies of the present application include those described in WO2008119353 (Genmab), WO2011131746 (Genmab) and the report by van der Neut-Kolfschoten et al. (Science. September 14, 2007; 317(5844):1554-7). Examples of other platforms for preparing bispecific antibodies include, but are not limited to, BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc engineering IgGl (Xencor) or DuoBody.

[0253] Traditional methods, such as the hybridizing hybridoma and chemical conjugate methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) may also be used. Co-expression of two components in a host cell (e.g., the heavy chain of a full-length antibody against a tumor-associated antigen fused with an anti-CD137 scFv and the light chain of the antibody against the tumor-associated antigen) may result in the presence of a mixture of antibody products in addition to the desired bispecific antibody in vitro, and the multispecific antibody may be isolated, for example, by affinity chromatography or similar methods.

[0254] Nucleic acid molecules encoding antibody portions In some embodiments, provided is a polynucleotide encoding any one of the anti-CD137 constructs or antibody portions described herein. In some embodiments, provided is a polynucleotide prepared using any of the methods described herein. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding the heavy or light chain of an antibody portion (e.g., an anti-CD137 antibody portion). In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding the heavy and light chains of an antibody portion (e.g., an anti-CD137 antibody portion). In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain, and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain. In some embodiments, provided is a nucleic acid molecule encoding a scFv (e.g., an anti-CD137 scFv).

[0255] In some such embodiments, the heavy and light chains are expressed as two isolated polypeptides from one nucleic acid molecule or two isolated nucleic acid molecules. In some embodiments, for example, where the antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising the linked heavy and light chains.

[0256] In some embodiments, the polynucleotide encoding the heavy or light chain of an antibody portion (e.g., an anti-CD137 antibody portion) comprises a nucleotide sequence encoding a leader sequence that, upon translation, is located at the N-terminus of the heavy or light chain. As described above, the leader sequence may be a native heavy or light chain leader sequence or another heterologous leader sequence.

[0257] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA.

[0258] Nucleic acid molecules can be constructed using conventional recombinant DNA techniques in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0259] Nucleic acid construct In some embodiments, a nucleic acid construct comprising any polynucleotide described herein is provided. In some embodiments, a nucleic acid construct prepared using any method described herein is provided.

[0260] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the polynucleotide. In some embodiments, the polynucleotide corresponds to a gene, where the promoter is the wild-type promoter of the gene.

[0261] Vector In some embodiments, a vector comprising any polynucleotide encoding the heavy chain and / or light chain of any antibody moiety (e.g., anti-CD137 antibody moiety) described herein or a nucleic acid construct described herein is provided. In some embodiments, a vector prepared using any method described herein is provided. A vector comprising a polynucleotide encoding any anti-CD137 construct (e.g., antibody, scFv, fusion protein) or other form of construct (e.g., anti-CD137 scFv) described herein is further provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector comprises a first polynucleotide sequence encoding the heavy chain and a second polynucleotide sequence encoding the light chain. In some embodiments, the heavy chain and the light chain are expressed as two separate polypeptides from the vector. In some embodiments, the heavy chain and the light chain are expressed as part of a single polypeptide, e.g., when the antibody is an scFv.

[0262] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain, and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into a host cell in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, the first vector and the second vector are transfected into a host cell at a molar ratio or mass ratio of 5:1 to 1:5. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0263] In some embodiments, a vector optimized for polypeptide expression in CHO or cells of CHO origin or NSO cells is selected. Exemplarily, such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880 - 889 (2004).

[0264] host cell In some embodiments, host cells are provided that contain any of the polypeptides, nucleic acid constructs, and / or vectors described herein. In some embodiments, host cells are provided that are prepared using any of the methods described herein. In some embodiments, the host cells can produce any of the antibody moieties described herein under fermentation conditions.

[0265] In some embodiments, the antibody portions described herein (e.g., anti-CD137 antibody portions) can be expressed in prokaryotic cells (e.g., bacterial cells), or eukaryotic cells (e.g., fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells). Such expression may be performed, for example, according to methods known in the art. Exemplary eukaryotic cells for expressing polypeptides include, but are not limited to, COS cells (including COS 7 cells), 293 cells (including 293-6E cells), CHO cells (including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells), PER.C6 (登録商標) cells (Crucell), and NSO cells. In some embodiments, the antibody portions described herein (e.g., anti-CD137 antibody portions) can be expressed in yeast. See, for example, U.S. Patent No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications on the heavy and / or light chains of the antibody portion. For example, in some embodiments, CHO cells produce polypeptides with a higher degree of sialylation than the same polypeptides produced in 293 cells.

[0266] Introduction of one or more nucleic acids into the desired host cell may be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting and exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transfected transiently or stably within the desired host cell based on any suitable method.

[0267] The present invention further provides a host cell comprising any polynucleotide or vector described herein. In some embodiments, the present invention provides a host cell comprising an anti-CD137 antibody. Any host cell capable of overexpressing heterologous DNA may be used for the purpose of isolating a gene encoding a desired antibody, polypeptide, or protein. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or Bacillus subtilis) and yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Kluyveromyces lactis).

[0268] In some embodiments, the antibody moiety is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009), Spirin, Trends Biotechnol. 22:538-45 (2004), Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0269] Medium In some embodiments, a medium comprising any antibody moiety, polynucleotide, nucleic acid construct, vector, and / or host cell described herein is provided. In some embodiments, a medium prepared using any method described herein is provided.

[0270] In some embodiments, the medium comprises hypoxanthine, aminopterin, and / or thymidine (e.g., HAT medium). In some embodiments, the medium is serum-free. In some embodiments, the medium contains serum. In some embodiments, the medium is D-MEM or RPMI-1640 medium.

[0271] Purification of the antibody moiety An anti-CD137 construct (e.g., an anti-CD137 monoclonal antibody or bispecific antibody) may be purified by any suitable method. Such methods include, but are not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the ROR1 ECD and the constant region of the antibody. For example, Protein A, Protein G, Protein A / G, or an antibody affinity column may bind to the constant region and may be used to purify an anti-CD137 construct that includes an Fc region. Hydrophobic interaction chromatography, e.g., a butyl column or a phenyl column, may also be applied to the purification of some polypeptides, e.g., antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be applied to the purification of some polypeptides, e.g., antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophobic interaction / anion exchange, hydrophobic interaction / cation exchange, etc.) may also be applied to the purification of some polypeptides, e.g., antibodies. A number of methods for purifying polypeptides are known in the art.

[0272] IV. Methods of Modulating Cell Compositions Any anti-CD137 construct described herein (including any multispecific antibody) may be used in a method of modulating a cell composition (e.g., a T cell composition). The method includes contacting the cell composition with the anti-CD137 construct. In some embodiments, the contacting or at least a portion of the contacting is performed ex vivo. In some embodiments, the contacting or at least a portion of the contacting is performed in vivo.

[0273] Contact In some embodiments, the contact is carried out in the presence of an agent. In some embodiments, the agent binds to CD3 (e.g., anti-CD3 antibody). In some embodiments, the agent binds to CD28 (e.g., anti-CD28 antibody). In some embodiments, the agent includes an agent that binds to CD3 and an agent that binds to CD28. In some embodiments, the agent is a cytokine (e.g., IL-2, IFNγ). In some embodiments, the agent is one or more (e.g., one, two, three, four, or five agents) selected from the group consisting of an agent that binds to CD3, CD28, an agent that binds to IL-2, TNF-α, and an agent that binds to IFNγ. In some embodiments, the agent includes an agent that binds to CD3 (e.g., anti-CD3 antibody), IL-2, and IFNγ.

[0274] In some embodiments, the concentration of the agent (e.g., anti-CD3 antibody) is at least about 0.01 μg / ml, 0.02 μg / ml, 0.03 μg / ml, 0.05 μg / ml, 0.075 μg / ml, 0.1 μg / ml, 0.125 μg / ml, 0.25 μg / ml, 0.5 μg / ml, or 1 μg / ml.

[0275] In some embodiments, the contact is carried out for at least about 1 hour, 2 hours, 4 hours, 8 hours, or overnight. In some embodiments, the contact is carried out for at least about 1 day, 2 days, or 3 days. In some embodiments, the contact is carried out for less than about 24 hours, 12 hours, or 8 hours. In some embodiments, the contact is carried out for less than about 14 days, 10 days, 7 days, 5 days, or 3 days. In some embodiments, the contact is carried out for about 0 - 48 hours, 1 - 24 hours, 2 - 20 hours, 4 - 16 hours, or 8 - 12 hours.

[0276] In some embodiments, the contact is carried out at a temperature of about 0 - 20°C. In some embodiments, the contact is carried out at a temperature of about 2°C - 8°C.

[0277] Cell composition In some embodiments, the cell composition comprises immune cells (e.g., human immune cells). In some embodiments, the immune cells comprise T cells (e.g., enriched T cells, e.g., the cells in the composition have at least 50%, 60%, 70%, 80%, 90% or 95% T cells). In some embodiments, the T cells are enriched CD4+ T cells (e.g., the T cells in the composition have at least 50%, 60%, 70%, 80%, 90% or 95% CD4+ T cells). In some embodiments, the T cells are enriched CD8+ T cells (e.g., the T cells in the composition have at least 50%, 60%, 70%, 80%, 90% or 95% CD8+ T cells). In some embodiments, the T cells comprise regulatory T cells (Treg cells). For example, the T cells comprise at least 2.5%, 5%, 7.5%, 10%, 15% or 20% regulatory T cells. In some embodiments, the T cells are cysteine engineering T cells, which comprise a recombinant receptor (e.g., a chimeric antigen receptor). In some embodiments, the immune cells comprise NK cells (e.g., enriched NK cells, e.g., the cells in the composition have at least 50%, 60%, 70%, 80%, 90% or 95% NK cells). In some embodiments, the cells in the composition comprise cytokine-induced killer (CIK) cells. In some embodiments, the cells comprise any one or more types of immune cells, such as B cells, dendritic cells or macrophages.

[0278] In some embodiments, the cells are pre-treated or treated simultaneously with an agent. In some embodiments, the agent binds to CD3 (e.g., anti-CD3 antibody). In some embodiments, the agent binds to CD28 (e.g., anti-CD28 antibody). In some embodiments, the agent comprises an agent that binds to CD3 and an agent that binds to CD28. In some embodiments, the agent is a cytokine (e.g., IL-2, IFNγ). In some embodiments, the agent comprises one or more (e.g., one, two, three, four, or five agents) agents selected from the group consisting of an agent that binds to CD3, CD28, an agent that binds to IL-2, TNF-α, and IFNγ.

[0279] In some embodiments, the concentration of the agent (e.g., anti-CD3 antibody) is at least about 0.01 μg / ml, 0.02 μg / ml, 0.03 μg / ml, 0.05 μg / ml, 0.075 μg / ml, 0.1 μg / ml, 0.125 μg / ml, 0.25 μg / ml, 0.5 μg / ml, or 1 μg / ml.

[0280] In some embodiments, after the contact, the cells in the composition are administered to a subject.

[0281] V. Methods for Treating or Modulating an Individual's Immune Response This specification further provides a method for treating an individual disease or disorder or modulating an individual's immune response. The method comprises administering to a subject (e.g., a mammal, e.g., a human) any anti-CD137 construct described herein.

[0282] In some embodiments, further provided is a method for treating an individual disease or disorder or modulating an individual's immune response, comprising administering to the subject an effective amount of an anti-CD137 construct disclosed herein. In some embodiments, the construct is any multispecific antibody described herein.

[0283] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, glioblastoma, ovarian cancer, lung cancer (e.g., NSCLC), oropharyngeal cancer, colorectal cancer, breast cancer, head and neck cancer, or leukemia (e.g., AML). In some embodiments, the individual is human. In some embodiments, the antibody portion is chimeric or humanized. In some embodiments, the antibody portion is a full-length antibody. In some embodiments, the antibody portion has an isotype selected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, IgA, IgD, and IgE. In some embodiments, the effective amount of the anti-CD137 construct is from about 0.005 μg / kg to about 5 μg / kg of the total body weight of the individual. In some embodiments, the antibody agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.

[0284] In some embodiments, the individual is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In some embodiments, the individual is less than about 60 years old (e.g., including any one of less than about 50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, the individual age is greater than about 60 years old (including, for example, any one of greater than 70, 80, 90, or 100 years old). In some embodiments, the individual is diagnosed as having one or more of the diseases or disorders described herein (e.g., cancer, autoimmune disease, or transplantation), or having a genetic predisposition thereto. In some embodiments, the individual has one or more risk factors associated with one or more of the diseases or disorders described herein.

[0285] Regulation of immune response In some embodiments, modulation of the immune response comprises modulating a cell population in an individual. In some embodiments, the cell population is a T cell population. In some embodiments, the cell population is dendritic cells. In some embodiments, the cell population is macrophages. In some embodiments, the cell population is B cells. In some embodiments, the cell population is NK cells. In some groups, the cell population is effector T cells and / or memory T cells. In some embodiments, the cell population is effector / memory T cells defined by a phenotype with high CD44 and low CD62L.

[0286] In some embodiments, the modulation comprises promoting the proliferation of the cell population. In some embodiments, the proliferation of the cell population is increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after administration of the anti-CD137 construct as compared to the proliferation of reference cells after administration of a control construct (e.g., an antibody that does not bind to CD137). In some embodiments, the proliferation of the cell population is increased by at least about 1-fold, 1.2-fold, 1.5-fold, 1.7-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, or 6-fold after administration of the anti-CD137 construct as compared to the proliferation of reference cells after administration of a control construct (e.g., an antibody that does not bind to CD137).

[0287] Use of anti-CD137 multispecific antibodies In some embodiments, provided is a method of treating a disease or disorder (e.g., cancer) in an individual, comprising administering to the individual an effective amount of the multispecific antibody disclosed herein.

[0288] In some embodiments, provided is a method of treating a tumor or cancer, comprising administering to an individual an effective amount of the multispecific antibody disclosed herein.

[0289] Use of CD137 x EGFR multispecific cells In some embodiments, provided is a method of treating EGFR+ cancer in an individual, comprising administering to the individual a CD137 x EGFR bispecific antibody (e.g., any CD137 x EGFR bispecific antibody described herein).

[0290] In some embodiments, the cancer is EGFR positive. In some embodiments, the cancer is EGFR 高 positive. In some embodiments, the EGFR positive cancer or EGFR 高 positive cancer is selected from breast cancer, lung cancer, gastric cancer, ovarian cancer, melanoma, head and neck cancer, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, and cervical cancer.

[0291] In some embodiments, the EGFR positive cancer or EGFR 高 positive cancer is breast cancer or gastric cancer. In some embodiments, the EGFR 高 positive cancer has an average EGFR expression equal to or greater than that of SK-Hep1 cells.

[0292] In some embodiments, the EGFR 高 positive cancer has an average EGFR expression that is at least 50%, 75%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 20-fold, 25-fold, or 30-fold higher than the average EGFR expression of SKBR3 cells. In some embodiments, the cancer has an average EGFR expression (MFI) of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500. In some embodiments, the EGFR 高 positive cells have an EGFR expression level equivalent to that of Sk-Hep1 cells (e.g., within about 50% to about 200%, about 67% to about 150%, about 75% to about 125% of its expression level).

[0293] In some embodiments, the CD137 x EGFR bispecific antibody is administered to an individual (e.g., a human) at a dosage of about 0.001 mg / kg to about 10 mg / kg (e.g., about 0.001 mg / kg to about 0.01 mg / kg, e.g., about 0.01 mg / kg to about 0.1 mg / kg, e.g., about 0.1 mg / kg to about 1 mg / kg, e.g., about 1 mg / kg to about 10 mg / kg). In some embodiments, the CD137 x EGFR bispecific antibody is administered to an individual (e.g., a human) at a dosage of about 0.01 mg / kg to about 0.2 mg / kg. In some embodiments, the CD137 x EGFR bispecific antibody is administered to an individual at a frequency of about once to three times a week (e.g., about once, twice, or three times a week). In some embodiments, the CD137 x EGFR bispecific antibody is administered over at least one, two, or three weeks.

[0294] Disease or disorder The anti-CD137 construct described herein may be used for the treatment of any disease or disorder. In some examples, the disease or disorder is an infection (e.g., a bacterial or viral infection). In some examples, the disease or disorder is an autoimmune disorder. In some examples, the disease or disorder is cancer. In some examples, the disease or disorder is transplantation.

[0295] In some examples, the anti-CD137 construct is used in a method for treating cancer. Cancers treatable using any of the methods described herein include tumors that are not vascularized or not sufficiently vascularized and vascularized tumors. As described in the present application, the types of cancers treatable with the anti-CD137 construct include, but are not limited to, epithelial cancers, blastomas, sarcomas, benign and malignant tumors, and malignancies (e.g., sarcomas, epithelial cancers, and melanomas). Adult and pediatric tumors / cancers are also included. In some examples, the cancer is a solid tumor.

[0296] In each embodiment, the cancer is early cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, remission cancer, recurrent cancer, cancer in adjuvant therapy, cancer in neoadjuvant therapy, or cancer that is substantially difficult to treat.

[0297] Specific examples of cancers that can be treated by the method of the present application include, but are not limited to, anal cancer, astrocytoma (e.g., cerebellum and brain), basal cell carcinoma, bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brainstem glioma, cerebellar or brain astrocytoma (e.g., astrocytoma, malignant glioma, medulloblastoma, and glioblastoma)), breast cancer, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), esophageal cancer, eye cancer (e.g., intraocular melanoma and retinoblastoma), gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hepatocellular carcinoma (liver) cancer (e.g., hepatic epithelial cancer and hepatocellular tumor), leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoid tumor (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, myelodysplastic syndrome, nasopharyngeal cancer, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, peritoneal cancer, pituitary tumor, lymphoma, rectal cancer, renal cancer, renal pelvic cancer and ureteral cancer (transitional cell carcinoma), rhabdomyosarcoma, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, tuberous sclerosis, and post-transplant lymphoproliferative disorder (PTLD).

[0298] In some embodiments, the cancer is selected from the group consisting of melanoma, glioblastoma, ovarian cancer, lung cancer (e.g., NSCLC), oropharyngeal cancer, colorectal cancer, breast cancer, head and neck cancer, or leukemia (e.g., AML).

[0299] The administration method of the anti-CD137 construct The dosage of an anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) administered to an individual to treat a disease or disorder described herein can vary depending on the particular anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody), the mode of administration, and the type of disease or disorder being treated. In some embodiments, the type of the disease or condition is cancer. In some embodiments, the effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount effective to produce an objective response (e.g., a partial response or a complete response). In some embodiments, the effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount sufficient to produce a complete response in an individual. In some embodiments, the effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount sufficient to produce a partial response in an individual. In some embodiments, the effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount sufficient to produce a total response rate greater than any one of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% in a population of individuals being treated with the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody). For example, the response of an individual to treatment by the methods described herein can be determined based on the RECIST level.

[0300] In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount sufficient to extend the progression-free survival of an individual. In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount sufficient to extend the overall survival of an individual. In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount sufficient to generate a clinical benefit in any one greater than about 50%, 60%, 70%, or 77% of a population of individuals treated with the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody).

[0301] In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody), alone or in combination with a second, third, and / or fourth agent, is an amount sufficient to reduce tumor size, reduce the number of cancer cells, or reduce the tumor growth rate by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to the corresponding tumor size, number of cancer cells, or tumor growth rate of the same subject prior to treatment, or the corresponding activity of other subjects not receiving treatment (e.g., receiving placebo treatment). The magnitude of the effector can be measured using standard methods such as, for example, in vitro assays using purified enzymes, cell-based assays, animal models, or human trials.

[0302] In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount lower than the level that induces a toxicological effector (i.e., an effector higher than a clinically acceptable toxicity level), or is an amount at a level where potential side effects can be controlled or tolerated when the composition is administered to an individual.

[0303] In some embodiments, the effective amount of the anti-CD137 construct (e.g., anti-CD137 monoclonal or bispecific antibody) is an amount close to the maximum tolerated dose (MTD) of the composition based on the same dosing regimen. In some embodiments, the effective amount of the anti-CD137 construct (e.g., anti-CD137 monoclonal or bispecific antibody) is greater than any one of about 80%, 90%, 95% or 98% of the MTD.

[0304] In some embodiments, the effective amount of the anti-CD137 construct (e.g., anti-CD137 monoclonal or bispecific antibody) is an amount that decelerates or inhibits the progression of the disease or disorder (e.g., decelerates or inhibits by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%) compared to an untreated individual. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is an infection.

[0305] In some embodiments, the effective amount of the anti-CD137 construct (e.g., anti-CD137 monoclonal or bispecific antibody) is an amount that reduces the side effects (autoimmune response) of the condition (e.g., transplantation) (e.g., reduces by at least about 5%, 10%, 15%, 20%, 30%, 40% or 50%) compared to an untreated individual.

[0306] In some embodiments of any of the above aspects, the effective amount of the anti-CD137 construct (e.g., anti-CD137 monoclonal or bispecific antibody) is in the range of about 0.001 μg / kg to about 100 mg / kg of total weight, for example, about 0.005 μg / kg to about 50 mg / kg, about 0.01 μg / kg to about 10 mg / kg or about 0.01 μg / kg to about 1 mg / kg.

[0307] In some embodiments, the treatment comprises administering the anti-CD137 construct one or more times (e.g., administering the anti-CD137 construct about two, three, four, five, six, seven, eight, nine, or ten times). In some embodiments, it is administered twice within about one week. In some embodiments, the second administration is performed at least about 1, 2, 3, 4, 5, 6, or 7 days after the completion of the first administration. In some embodiments, the second administration is performed about 1 - 14 days, 1 - 10 days, 1 - 7 days, 2 - 6 days, or 3 - 5 days after the completion of the first administration. In some embodiments, the anti-CD137 construct is administered about 1 - 3 times per week (e.g., about once per week, about twice per week, or about three times per week).

[0308] The anti-CD137 construct can be administered to an individual (e.g., a human) by various routes, including intravenous, intraarterial, intraperitoneal, intralung, oral, inhalation, intracapsular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, the anti-CD137 construct is included in a pharmaceutical composition when administered to an individual. In some embodiments, a sustained / continuous release formulation of the composition may be used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered orally.

[0309] Combination therapy The present application further provides a method of treating a disease or disorder (e.g., cancer) by administering an anti-CD137 construct to an individual, wherein the method further comprises administering a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy for treating the disease or disorder. In some embodiments, the second agent or therapy comprises a chemotherapeutic agent. In some embodiments, the second agent or therapy comprises surgery. In some embodiments, the second agent or therapy comprises radiation therapy. In some embodiments, the second agent or therapy comprises immunotherapy. In some embodiments, the second agent or therapy comprises hormone therapy. In some embodiments, the second agent or therapy comprises an angiogenesis inhibitor. In some embodiments, the second agent or therapy comprises a tyrosine kinase inhibitor. In some embodiments, the second agent or therapy comprises an infectious agent.

[0310] In some embodiments, the anti-CD137 construct is administered simultaneously with the second agent or therapy. In some embodiments, the anti-CD137 construct is administered concurrently with the second agent or therapy. In some embodiments, the anti-CD137 construct is administered sequentially with the second agent or therapy. In some embodiments, the anti-CD137 construct is administered in the same unit dosage form as the second agent or therapy. In some embodiments, the anti-CD137 construct is administered in a different unit dosage form than the second agent or therapy.

[0311] In some embodiments, the second agent or therapy is an agent that binds to EGFR (e.g., trastuzumab or trastuzumab emtansine). In some embodiments, the second agent or therapy is an agent that binds to EGFR. In some embodiments, the second agent or therapy targets PD-L1 or PD-1 (e.g., an anti-PD-1 antibody). In some embodiments, the second agent is an agent that targets CTLA-4 (e.g., an anti-CTLA-4 antibody). In some embodiments, the second agent includes T cells (e.g., CAR T cells). In some embodiments, the second agent includes cytokines. In some embodiments, the second agent or therapy includes carboplatin, paclitaxel, and / or radiation therapy. In some embodiments, the second agent or therapy includes a vaccine, e.g., an HPV vaccine. In some embodiments, the second agent or therapy includes an EGFR inhibitor (e.g., cetuximab). In some embodiments, the second agent or therapy includes an anti-tumor enzyme inhibitor (e.g., irinotecan). See the exemplary combination therapies in Table 3 below.

[0312]

Table 3

[0313] VI. Compositions, Kits, and Products This specification further provides a composition (e.g., a formulation) comprising any one of the anti-CD137 constructs or anti-CD137 antibody portions described herein, a nucleic acid encoding the antibody portion, a vector comprising the nucleic acid encoding the antibody portion, or a host cell comprising the nucleic acid or vector.

[0314] Suitable anti-CD137 construct formulations described herein can be obtained in the form of lyophilized formulations or aqueous solutions by mixing the anti-CD137 construct or anti-CD137 antibody portion having the desired purity with an optional pharmaceutically acceptable carrier agent, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th Edition Osol, A. Ed. (1980)). Acceptable carrier agents, excipients, or stabilizers are non-toxic to the receptor at the dosages and concentrations employed, and include buffers (e.g., phosphates, citrates and other organic acids, antioxidants including ascorbic acid and methionine), preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens such as methylparaben or propylparaben, catechin, resorcinol, cyclohexanol, 3-pentanol, and metacresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., zinc protein complexes), and / or TWEEN (商標) , PLURONICS (商標) or nonionic surfactants such as polyethylene glycol (PEG). Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted to high protein concentrations with a suitable diluent, and the reconstituted formulation can be administered subcutaneously to an individual to be imaged, diagnosed or treated herein.

[0315] Formulations for in vivo administration need to be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0316] A kit comprising any one of the anti-CD137 constructs or anti-CD137 antibody portions described herein is further provided. The kit may be used in any method for modulating or treating the cell compositions described herein.

[0317] In some embodiments, kits comprising anti-CD137 constructs that bind to CD137 are provided.

[0318] In some embodiments, the kit further comprises a device capable of delivering the anti-CD137 construct to an individual. For example, one type of device for parenteral delivery use is a syringe for injecting the composition in vivo into a subject. Aspiration devices may also be used for some applications.

[0319] In some embodiments, the kit further comprises a therapeutic agent for treating a disease or disorder (e.g., cancer, infectious disease, autoimmune disease or transplantation).

[0320] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit can optionally provide other components such as buffer solutions and explanatory information.

[0321] Accordingly, the present application further provides a product. The product may include a container and a marker or a packaging insert that is in or related to the container. Suitable containers include vials (e.g., sealed vials), bottles, cans, flexible packages, and the like. Generally, the container may contain the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The marker or packaging insert indicates that the composition is used for imaging, diagnosing, or treating a specific disease condition in an individual. The marker or packaging insert further includes instructions for administering the composition to an individual and imaging the individual. The marker can indicate recombinant and / or usage instructions. The container containing the composition may be a multi-use vial that allows repeated administration (e.g., 2 - 6 administrations) of the reconstituted formulation. A packaging insert generally refers to the manual included in the commercial packaging of a diagnostic product, and these manuals contain information regarding the indications, usage methods, dosages, administrations, contraindications, and / or warnings for using such diagnostic products. Further, the product may further include a second container, and the container contains a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0322] The kit or product may include a plurality of unit doses of the composition and a usage manual, packaged in an amount sufficient for storage and use in pharmacies (e.g., hospital pharmacies and dispensing pharmacies).

[0323] Those skilled in the art will recognize that multiple embodiments are possible within the scope and spirit of the present invention. The present inv...

Claims

1. A bispecific antibody that binds to CD137 and EGFR, comprising a first antibody portion that binds to CD137 and a second antibody portion that binds to EGFR, The first antibody portion includes a heavy chain variable region (V Ｈ ) containing HC-CDR1, HC-CDR2, and HC-CDR3 domains, and a light chain variable region (V Ｌ ) containing LC-CDR1, LC-CDR2, and LC-CDR3 domains, and the heavy chain variable region and the light chain variable region consist of the group consisting of the following i) said V Ｈ comprises an HC - CDR1 containing the amino acid sequence of SEQ ID NO: 121, an HC - CDR2 containing the amino acid sequence of SEQ ID NO: 122, and an HC - CDR3 containing the amino acid sequence of SEQ ID NO: 123, and said V Ｌ comprises an LC - CDR1 containing the amino acid sequence of SEQ ID NO: 124, an LC - CDR2 containing the amino acid sequence of SEQ ID NO: 125, and an LC - CDR3 containing the amino acid sequence of SEQ ID NO: 126, ii) said V Ｈ comprises an HC - CDR1 containing the amino acid sequence of SEQ ID NO: 261, an HC - CDR2 containing the amino acid sequence of SEQ ID NO: 262, and an HC - CDR3 containing the amino acid sequence of SEQ ID NO: 263, and said V Ｌ comprises an LC - CDR1 containing the amino acid sequence of SEQ ID NO: 264, an LC - CDR2 containing the amino acid sequence of SEQ ID NO: 265, and an LC - CDR3 containing the amino acid sequence of SEQ ID NO: 266, selected from the second antibody portion comprises a second heavy chain variable region (VH-2) and a second light chain variable region (VL-2), and the VH-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 208, and the VL-2 comprises a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 210, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 211, a bispecific antibody.

2. The first antibody portion is (i) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 127, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 128, (ii) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 267, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 268, or (iii) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 277, and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 278, the bispecific antibody according to claim 1.

3. The first antibody portion includes an antibody or an antigen-binding fragment thereof selected from the group consisting of a full-length antibody, a multispecific antibody, a single-chain Fv (scFv), a Fab fragment, a Fab' fragment, an F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv) ２ , V Ｈ H, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, and a tetrabody, the multispecific antibody according to claim 1 or 2.

4. The bispecific antibody according to claim 3, wherein the bispecific antibody is a bispecific antibody.

5. The first antibody portion is a CD137 agonist antibody, the bispecific antibody according to any one of claims 1 to 4.

6. The second antibody portion comprises an Fc region selected from the group consisting of an Fc region from IgG1, IgG2, IgG3, IgG4 and any combination thereof and hybrids, the bispecific antibody according to any one of claims 1 to 5.

7. The Fc region comprises an IgG1 Fc region, the bispecific antibody according to claim 6.

8. The IgG1 Fc region comprises an L234A mutation and an L235A mutation, the bispecific antibody according to claim 7.

9. The multispecific antibody according to claim 6, wherein the Fc region comprises an IgG4 Fc region.

10. The multispecific antibody according to claim 9, wherein the IgG4 Fc region comprises an F234A mutation and an L235A mutation.

11. The multispecific antibody according to claim 9 or 10, wherein the IgG4 Fc region further comprises an S228P mutation.

12. The V Ｈ－２ comprises the amino acid sequence of SEQ ID NO: 212, and the V Ｌ－２ comprises the amino acid sequence of SEQ ID NO: 213, The multispecific antibody according to claim 1.

13. a) said first antibody portion comprising a heavy chain variable region (V Ｈ ) and a light chain variable region (V Ｌ ), wherein said V Ｈ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 121, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 122, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 123, and said V Ｌ comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 126; and said first antibody portion b) the second heavy chain variable region (V Ｈ－２ ) and the second light chain variable region (V Ｌ－２ ) of the second antibody portion, wherein the V Ｈ－２ comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 208, and the V Ｌ－２ comprises a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 210, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 211, and the multispecific antibody according to any one of claims 1 to 12, comprising the second antibody portion.

14. a) the first antibody portion comprising a heavy chain variable region (V Ｈ ) and a light chain variable region (V Ｌ ), wherein said V Ｈ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 261, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 262, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 263, and wherein said V Ｌ comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 264, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 265, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 266, the first antibody portion; b) said second antibody moiety comprising a second heavy chain variable region (V Ｈ－２ ), and a second light chain variable region (V Ｌ－２ ), wherein said V Ｈ－２ comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 208, ＼ and said V Ｌ－２ comprises a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 210, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 211; a multispecific antibody according to any one of claims 1 to 13.

15. i) A heavy chain of an anti-EGFR full-length antibody fused with a single-chain Fv fragment against CD137 comprising an amino acid sequence having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 183, 185, 188, 189, 230, 231, 259, 260, 281 or 282; and ii) A light chain of an anti-EGFR full-length antibody comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 187, the multispecific antibody according to any one of claims 1 to 14.

16. i) A heavy chain of an anti-EGFR full-length antibody fused with a single-chain Fv fragment against CD137 comprising any one of the amino acid sequences of SEQ ID NOs: 183, 185, 188, 189, 230, 231, 259, 260, 281 or 282; and ii) A light chain of an anti-EGFR full-length antibody comprising the amino acid sequence of SEQ ID NO: 187, the multispecific antibody according to any one of claims 1 to 15.

17. An immunoconjugate comprising the multispecific antibody according to any one of claims 1 to 16 linked to a therapeutic agent or a marker.

18. The immunoconjugate according to claim 17, wherein the marker is selected from the group consisting of a radioisotope, a fluorescent dye and an enzyme.

19. A pharmaceutical composition comprising the multispecific antibody according to any one of claims 1 to 16 or the immunoconjugate according to claim 17 or 18 and a pharmaceutically acceptable carrier.

20. An isolated nucleic acid encoding the multispecific antibody according to any one of claims 1 to 16.

21. A vector comprising the isolated nucleic acid according to claim 20.

22. An isolated host cell comprising the isolated nucleic acid according to claim 20 or the vector according to claim 21.

23. A method for producing a multispecific antibody, comprising: a) culturing the isolated host cell according to claim 22 under conditions effective for expressing the multispecific antibody; and b) obtaining the expressed multispecific antibody from the host cell. **Claim 24** A cancer therapeutic agent comprising the multispecific antibody according to any one of claims 1 to 16. **Claim 25** The cancer is selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, lung cancer, mesothelioma, endometrial cancer, cervical cancer, esophageal cancer, bladder cancer, salivary gland cancer, testicular cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, thymic cancer, adrenal cancer, head and neck cancer, brain cancer, thyroid cancer, sarcoma, myeloma and leukemia. The cancer therapeutic agent according to claim 24. **Claim 26** A kit comprising the multispecific antibody according to any one of claims 1 to 16, the immunoconjugate according to claim 17 or 18, the pharmaceutical composition according to claim 19, the nucleic acid according to claim 20, the vector according to claim 21 or the host cell according to claim 22. **Claim 27** The kit according to claim 26, further comprising a manual for treating and / or preventing cancer or tumor.

Images