Anti-CD137 constructs, multispecific antibodies and uses thereof

Abstract

The present invention relates to 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 them. In some embodiments, the one or more additional antigens include human epidermal growth factor receptor 2 (HER2).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2020 / 077146, filed February 28, 2020, the entire contents of which are incorporated herein by reference and claim priority thereto.

[0002] This application relates to antibodies that bind to CD137 (including anti-CD137 monospecific and multispecific antibodies), methods of preparation and uses thereof, including the treatment of diseases or conditions. [Background technology]

[0003] CD137 (also known as 4-1BB and TNFRSF9) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). Current understanding of CD137 indicates that its expression is generally activation-dependent and is present on a broad subpopulation of immune cells, including activated NK and NKT cells, regulatory T cells, dendritic cells (DCs), stimulated mast cells, differentiating bone marrow cells, monocytes, neutrophils, and eosinophils (Wang, 2009, Immunological Reviews 229:192-215). 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 in endothelial cells at sites of inflammation or atherosclerosis (Drenkard, 2007 FASEB J. 21:456-463; Olofsson, 2008, Circulation 117:1292-1301). The stimulatory ligand for CD137, i.e., 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 both monomeric and dimeric forms and may trimerize with CD137 ligands to activate downstream signals. Studies on mouse and human T cells have demonstrated that CD137 promotes cell proliferation, survival, and enhanced cytokine production (Croft, 2009, Nat Rev Immunol 9:271-285). Studies have shown that several CD137 agonist mAbs can exert antitumor effects in various models by increasing the expression of costimulatory molecules and significantly enhancing cytolytic T lymphocyte responses. CD137 agonist mAbs have been shown to be effective in both preventive and therapeutic settings. Furthermore, tumor models using CD137 monotherapy and combination therapy have established durable antitumor protective T cell memory responses (Lynch, 2008, Immunol Rev. 22:277-286). CD137 agonists have also been shown to inhibit autoimmune responses in a variety of autoimmune models commonly accepted in the art (Vinay, 2006, J Mol Med 84:726-736). This dual activity of CD137 can provide potential antitumor activity while also inhibiting autoimmune side effects that may be associated with immunotherapies that break immune tolerance.

[0005] All publications, patents, patent applications, and the entire contents of the disclosed patent applications cited in this application are hereby incorporated 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 individuals with the anti-CD137 constructs. The present invention is based, in part, on the discovery of anti-CD137 monospecific and multispecific antibodies that have shown improved safety and enhanced anti-tumor efficacy compared to conventional anti-CD137 antibodies in clinical trials.

[0007] The present disclosure provides multispecific antibodies that bind to CD137 and HER2. In some examples, the multispecific antibodies disclosed herein comprise a first antibody portion that binds to CD137 and a second antibody portion that binds to HER2. In some examples, the first antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), wherein a) said 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 Lcomprises: i) an LC-CDR1 comprising the amino acid sequence of KASQX1X2X3TYX4S (SEQ ID NO: 180), wherein X1 = A, P, or T, X2 = I, T, or P, X3 = N, A, and X4 = L, G, or H; ii) an LC-CDR2 comprising the amino acid sequence of RX1NRX2X3X4 (SEQ ID NO: 181), wherein X1 = A, Y, V, or D, X2 = M, K, V, or A, X3 = V, P, Y, or G, and X4 = D or G; and iii) an LC-CDR3 comprising the amino acid sequence of LQX1X2DFPYX3 (SEQ ID NO: 182), wherein X1 = Y, S, or F, X2 = D, V, L, R, E, or Q, and X3 = T, or K.In some embodiments, HC-CDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 231, and 241, or a variant thereof comprising up to about three amino acid substitutions; HC-CDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 232, and 242, or a variant thereof comprising up to about three amino acid substitutions; and HC-CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 231, and 241, or a variant thereof comprising up to about three amino acid substitutions. LC-CDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 233 and 243, or a variant thereof comprising up to about three amino acid substitutions; LC-CDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 234 and 244, or a variant thereof comprising up to about three amino acid substitutions; LC-CDR2 comprises the amino acid sequence of any one of SEQ ID NOs: and LC-CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 235 and 245, or a variant thereof containing up to about three amino acid substitutions, and LC-CDR4 comprises the amino acid sequence of any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 236 and 246, or a variant thereof containing up to about three 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), which is selected from: a) The above 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 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; b) The above 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 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; c) 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 L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26; d) The above V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36; e) The above V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; f) Said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56; g) the V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 63, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 66; h) the V H comprises 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 Lcomprises 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) The above 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 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) The above 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 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) the 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 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 H comprises 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 Lcomprises 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 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) the 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 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) The above V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; p) the V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 241, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 242, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 243, andL comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:244, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:245, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:246.

[0009] In some embodiments, the first antibody portion 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 ) wherein the light chain variable region comprises an LC-CDR1, an LC-CDR2, and an LC-CDR3 domain, H and the aforementioned V L is selected from the following: a) The above 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 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; b) The above 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 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; c) 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 Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26; d) The above V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36; e) The above V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; f) Said V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56; g) the V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 63, and Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 66; h) the V H comprises 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 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) The above 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 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) The above 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 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) the 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 Lcomprises 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 H comprises 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 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 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) the 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 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) The above V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233, andL comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; p) the V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 241, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 242, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 243, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:244, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:245, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:246.

[0010] In some embodiments, the first antibody portion 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 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 L In some embodiments, the first antibody portion 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, the first antibody portion 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), Hcomprises 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 L In some embodiments, the first antibody portion 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, the first antibody portion 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), 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 L In some embodiments, the first antibody portion comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:25, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the first antibody portion 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), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and LIn some embodiments, the first antibody portion comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:34, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the first antibody portion 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), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, and L In some embodiments, the first antibody portion comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:44, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:45, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46. In some embodiments, the first antibody portion 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), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53, and L In some embodiments, the first antibody portion comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:56. In some embodiments, the first antibody portion 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), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 63, and L In some embodiments, the first antibody portion comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:64, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:65, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:66. In some embodiments, the first antibody portion 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), H comprises 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 L In some embodiments, the first antibody portion 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), 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 LIn some embodiments, the first antibody portion 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. In some embodiments, the first antibody portion 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), 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 L In some embodiments, the first antibody portion 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. In some embodiments, the first antibody portion 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), 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 L In some embodiments, the first antibody portion 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), H comprises 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 L In some embodiments, the first antibody portion 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. In some embodiments, the first antibody portion 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), 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 L In some embodiments, the first antibody portion 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. In some embodiments, the first antibody portion 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), 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 Lcomprises 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. In some embodiments, the first antibody portion 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), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 141, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 142, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 143, and L In some embodiments, the first antibody portion comprises 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 first antibody portion 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), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233, and L In some embodiments, the first antibody portion comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:234, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:235, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:236. In some embodiments, the first antibody portion 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), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 241, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 242, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 243, and L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:244, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:245, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:246.

[0011] In some embodiments, the first antibody portion comprises: a) V, each having the sequence shown in SEQ ID NO:7 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 8. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; b) V, each having the sequence shown in SEQ ID NO: 17 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 18. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; c) V, each having the sequence shown in SEQ ID NO: 27 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 28. LLC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; d) V, each having the sequence shown in SEQ ID NO: 37 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 38. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; e) V, each having the sequence shown in SEQ ID NO: 47 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 48. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; f) V, each having the sequence shown in SEQ ID NO: 57 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, which have the sequences shown in SEQ ID NO: 58, respectively. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; g) V, each having the sequence shown in SEQ ID NO: 67 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, which have the sequences shown in SEQ ID NO: 68, respectively. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; h) V, each having the sequence shown in SEQ ID NO: 77 HHC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, which have the sequences shown in SEQ ID NO: 78, respectively. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; i) V, each having the sequence shown in SEQ ID NO: 87 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 88. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; j) V, each having the sequence shown in SEQ ID NO: 97 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 98. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; k) V, each having the sequence shown in SEQ ID NO: 107 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, which have the sequences shown in SEQ ID NO: 108, respectively. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; l) V, each having the sequence shown in SEQ ID NO: 117 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, which have the sequences shown in SEQ ID NO: 118, respectively. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; m) V, each having the sequence shown in SEQ ID NO: 127 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, which have the sequences shown in SEQ ID NO: 128, respectively. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; n) V, each having the sequence shown in SEQ ID NO: 137 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, which have the sequences shown in SEQ ID NO: 138, respectively. L LC-CDR1, LC-CDR2 and LC-CDR3 containing amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; o) V, each having the sequence shown in SEQ ID NO: 237 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 238. L LC-CDR1, LC-CDR2 and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; or p) V, each having the sequence shown in SEQ ID NO: 247 H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region, and V, each having the sequence shown in SEQ ID NO: 248. L The chain region includes LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3.

[0012] In some embodiments, the first antibody portion comprises: (a) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:8; (b) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:17, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:18; (c) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:27, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:28; (d) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:37, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:38; (e) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:47, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:48; (f) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:57, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:58; (g) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:67, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:68; (h) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:77, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:78; (i) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:87, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:88; (j) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:97, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:98; (k) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 107, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 108; (l) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 117, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 118; (m) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 127, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 128; (n) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 137, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 138; (o) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:237, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:238; or (p) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:247, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:248.

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

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

[0015] In some embodiments, the first antibody portion is a full-length antibody, a multispecific antibody (e.g., 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 V H In some embodiments, the first antibody portion comprises an antibody or antigen-binding fragment thereof selected from the group consisting of a CD137 antibody fragment, 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 portion comprises a humanized anti-CD137 full-length antibody. In some embodiments, the first antibody portion comprises a humanized anti-CD137 single-chain Fv fragment (scFv). In some embodiments, the first antibody portion is a CD137 agonist antibody.

[0016] In some embodiments, the first antibody portion comprises an anti-CD137 antibody portion comprising a human immunoglobulin Fc region. In some embodiments, the Fc region is selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In some embodiments, the Fc region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions.

[0017] In some embodiments, the first antibody portion binds to human CD137 and monkey CD137, hi some embodiments, the first antibody portion does not bind to mouse CD137.

[0018] In some embodiments, the first antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L and a second antibody portion comprising an anti-CD137 single-chain Fv fragment comprising a second heavy chain variable region (V), and the second antibody portion comprises a full-length antibody that binds to HER2 and comprises two antibody heavy chains and two antibody light chains, wherein the heavy chains each comprise a second heavy chain variable region (V H-2 ), and the light chains each comprise a second light chain variable region (V L-2 ), wherein the anti-CD137 single-chain Fv fragment is fused to at least one of the heavy chain or light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the C-terminus of each heavy chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the N-terminus of each heavy chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the C-terminus of each light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to 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 is fused to the full-length antibody via a first peptide linker. In some embodiments, the first peptide linker comprises about 4 to about 15 amino acids. In some embodiments, the first peptide linker comprises a linker comprising the sequence of any one of SEQ ID NOs: 206-230. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the full-length antibody via a second peptide linker. In some embodiments, the second peptide linker comprises about 4 to about 15 amino acids. In some embodiments, the second peptide linker comprises a linker comprising the sequence of any one of SEQ ID NOs: 206-230.

[0020] In some embodiments, the second antibody portion comprises an Fc region selected from the Fc region of IgG, IgA, IgD, IgE, IgM, and any combination and hybrid thereof. In some embodiments, the Fc region comprises a human Fc region. In some embodiments, the Fc region is selected from the Fc region of IgG1, IgG2, IgG3, IgG4, and any combination and hybrid 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 HER2 is human HER2.

[0022] In some embodiments, the second antibody portion binds to HER2 and comprises a third heavy chain variable region (V H-3 ) and the third light chain variable region (V L-3 ) a full-length antibody that competes with an antibody or antibody fragment comprising: a) said V H-3 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194; and b) said V L-3 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0023] In some embodiments, the second antibody portion comprises a full-length antibody that binds to HER2 and comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), wherein a) said V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194; and b) said V L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0024] In some embodiments, the V H-2 comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:202, and / or L-2 comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:203. H-2 comprises the amino acid sequence of SEQ ID NO: 202, L-2 comprises the amino acid sequence of SEQ ID NO:203.

[0025] In some embodiments, the multispecific antibody comprises: a) the first antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), 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, L a) a 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 heavy chain variable region (V H-2) and a second light chain variable region (V L-2 ) and H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, L-2 comprises a second antibody portion comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0026] In some embodiments, the multispecific antibody comprises: a) the first antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233, L a) a first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:234, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:235, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:236; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) and H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, L-2 comprises a second antibody portion comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0027] In some embodiments, the multispecific antibody comprises: a) the first antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 241, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 242, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 243, L a) a first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:244, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:245, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:246; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) and H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, L-2 comprises a second antibody portion comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0028] In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 183, 184, 204, 205, 251, 252, 253, or 254. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 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, 184, 204, 205, 251, 252, 253, or 254.

[0029] In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length 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 the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 184. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 251. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 252. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 253. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the heavy chain of the anti-HER2 full length antibody, wherein the heavy chain of the anti-HER2 full length antibody fused to the anti-CD137 single chain Fv fragment comprises the amino acid sequence of SEQ ID NO: 254. In some embodiments, the included anti-HER2 full length antibody comprises a light chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 185.In some embodiments, the anti-HER2 full-length antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:185.

[0030] The present disclosure further provides an immunoconjugate comprising any of the multispecific antibodies disclosed herein linked to a therapeutic agent or marker, in some embodiments, the marker is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0031] The present disclosure further provides a pharmaceutical composition comprising any multispecific antibody or any immunoconjugate disclosed herein and a pharmaceutically acceptable carrier agent.

[0032] The present disclosure further provides isolated nucleic acids encoding any of the multispecific antibodies disclosed herein, vectors comprising any of the isolated nucleic acids disclosed herein, and isolated host cells comprising any of the isolated nucleic acids or any of the vectors disclosed herein.

[0033] The present disclosure further provides methods for producing a multispecific antibody, in some embodiments, the methods comprising: 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.

[0034] 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 multispecific antibody, any immunoconjugate, or any pharmaceutical composition disclosed herein. In some embodiments, the disease is cancer or 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, thymus cancer, adrenal cancer, head and neck cancer, brain cancer, thyroid cancer, sarcoma, myeloma, and leukemia. In some embodiments, the cancer or tumor is HER2-positive. In some embodiments, HER2 expression in the cancer or tumor is higher than the HER2 expression level in SK-Hep1. In some embodiments, the cancer is breast cancer or gastric cancer.

[0035] In some embodiments, the multispecific antibody, immunoconjugate, or pharmaceutical composition is administered to the individual parenterally. In some embodiments, the multispecific antibody, immunoconjugate, or pharmaceutical composition is administered to the individual intravenously. In some embodiments, the subject is a human.

[0036] The present disclosure provides any of the multispecific antibodies disclosed herein for use as a drug. The present disclosure further provides any of the multispecific antibodies 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, thymus cancer, adrenal cancer, head and neck cancer, brain cancer, thyroid cancer, sarcoma, myeloma, and leukemia.

[0037] 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 comprising instructions for treating and / or preventing cancer or tumors. [Brief explanation of the drawings]

[0038] [Figure 1A] Figure 1 shows the binding affinity of exemplary anti-CD137 antibody clone 2-9 variants to human CD137 and cynomolgus monkey CD137. Figure 1A shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1B shows the binding of clone 2-9 IgG2wt to cynomolgus monkey CD137. Figure 1C shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1D shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1E shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1F shows the binding of clone 2-9-2 IgG4wt to human CD137. [Figure 1B] Figure 1 shows the binding affinity of exemplary anti-CD137 antibody clone 2-9 variants to human CD137 and cynomolgus monkey CD137. Figure 1A shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1B shows the binding of clone 2-9 IgG2wt to cynomolgus monkey CD137. Figure 1C shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1D shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1E shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1F shows the binding of clone 2-9-2 IgG4wt to human CD137. [Figure 1C]Figure 1 shows the binding affinity of exemplary anti-CD137 antibody clone 2-9 variants to human CD137 and cynomolgus monkey CD137. Figure 1A shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1B shows the binding of clone 2-9 IgG2wt to cynomolgus monkey CD137. Figure 1C shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1D shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1E shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1F shows the binding of clone 2-9-2 IgG4wt to human CD137. [Figure 1D] Figure 1 shows the binding affinity of exemplary anti-CD137 antibody clone 2-9 variants to human CD137 and cynomolgus monkey CD137. Figure 1A shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1B shows the binding of clone 2-9 IgG2wt to cynomolgus monkey CD137. Figure 1C shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1D shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1E shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1F shows the binding of clone 2-9-2 IgG4wt to human CD137. [Figure 1E] Figure 1 shows the binding affinity of exemplary anti-CD137 antibody clone 2-9 variants to human CD137 and cynomolgus monkey CD137. Figure 1A shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1B shows the binding of clone 2-9 IgG2wt to cynomolgus monkey CD137. Figure 1C shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1D shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1E shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1F shows the binding of clone 2-9-2 IgG4wt to human CD137. [Figure 1F]Figure 1 shows the binding affinity of exemplary anti-CD137 antibody clone 2-9 variants to human CD137 and cynomolgus monkey CD137. Figure 1A shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1B shows the binding of clone 2-9 IgG2wt to cynomolgus monkey CD137. Figure 1C shows the binding of clone 2-9 IgG2wt to human CD137. Figure 1D shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1E shows the binding of clone 2-9-1 IgG1 SELF to human CD137. Figure 1F shows the binding of clone 2-9-2 IgG4wt to human CD137. [Figure 2A] Luciferase activity in an NF-κB reporter gene assay is shown in the absence (2A) or presence (2B) of FcγRIIB-expressing 293F cells. Reference Ab 1 and reference Ab 2 are reference anti-CD137 antibodies described in Example 2. αCD137 Ab clones represent different anti-CD137 monoclonal antibodies. [Figure 2B] Luciferase activity in an NF-κB reporter gene assay is shown in the absence (2A) or presence (2B) of FcγRIIB-expressing 293F cells. Reference Ab 1 and reference Ab 2 are reference anti-CD137 antibodies described in Example 2. αCD137 Ab clones represent different anti-CD137 monoclonal antibodies. [Figure 3A] Luciferase activity in an NF-κB reporter gene assay is shown in the absence (3A) or presence (3B and 3C) of 293F cells expressing FcγRIIB. Reference Ab 1 and Reference Ab 2 are reference anti-CD137 antibodies described in Example 2. 2-9-1 IgG1 SELF contains an IgG1 Fc with mutations S267E / L328F. 2-9-1 IgG2 SELF contains an IgG2 Fc with mutations S267E / L328F. 2-9 IgG2wt contains wild-type human IgG2 Fc. 2-9-2 IgG4 contains wild-type human IgG4 Fc. [Figure 3B] Luciferase activity in an NF-κB reporter gene assay is shown in the absence (3A) or presence (3B and 3C) of 293F cells expressing FcγRIIB. Reference Ab 1 and Reference Ab 2 are reference anti-CD137 antibodies described in Example 2. 2-9-1 IgG1 SELF contains an IgG1 Fc with mutations S267E / L328F. 2-9-1 IgG2 SELF contains an IgG2 Fc with mutations S267E / L328F. 2-9 IgG2wt contains wild-type human IgG2 Fc. 2-9-2 IgG4 contains wild-type human IgG4 Fc. [Figure 3C] Luciferase activity in an NF-κB reporter gene assay is shown in the absence (3A) or presence (3B and 3C) of 293F cells expressing FcγRIIB. Reference Ab 1 and Reference Ab 2 are reference anti-CD137 antibodies described in Example 2. 2-9-1 IgG1 SELF contains an IgG1 Fc with mutations S267E / L328F. 2-9-1 IgG2 SELF contains an IgG2 Fc with mutations S267E / L328F. 2-9 IgG2wt contains wild-type human IgG2 Fc. 2-9-2 IgG4 contains wild-type human IgG4 Fc. [Figure 4A] Figure 4 shows the production of IFN-γ (4A) and IL2 (4B) by PBMCs obtained from donors in the presence of different concentrations of different anti-CD137 antibodies. Reference Ab 1 and Reference Ab 2 are the reference anti-CD137 antibodies described in Example 2. 2-9-1 IgG1 SELF contains an IgG1 Fc and mutations S267E / L328F. 2-9-1 IgG2 SELF contains an IgG2 Fc and mutations S267E / L328F. 2-9 IgG2 wt contains wild-type human IgG2 Fc. 2-9-2 IgG4 contains wild-type human IgG4 Fc. [Figure 4B]Figure 4 shows the production of IFN-γ (4A) and IL2 (4B) by PBMCs obtained from donors in the presence of different concentrations of different anti-CD137 antibodies. Reference Ab 1 and Reference Ab 2 are the reference anti-CD137 antibodies described in Example 2. 2-9-1 IgG1 SELF contains an IgG1 Fc and mutations S267E / L328F. 2-9-1 IgG2 SELF contains an IgG2 Fc and mutations S267E / L328F. 2-9 IgG2 wt contains wild-type human IgG2 Fc. 2-9-2 IgG4 contains wild-type human IgG4 Fc. [Figure 5A] Figure 5 shows the results of an in vivo study of the 2-9 mutant in an MC38 mouse colon cancer model. Figure 5A shows tumor growth curves for the vector control group and the treatment group. Figure 5B shows the weight changes of mice during treatment. [Figure 5B] Figure 5 shows the results of an in vivo study of the 2-9 mutant in an MC38 mouse colon cancer model. Figure 5A shows tumor growth curves for the vector control group and the treatment group. Figure 5B shows the weight changes of mice during treatment. [Figure 6A] Figure 6 shows the results of an in vivo study of utomilumab analogues in an MC38 mouse colon cancer model. Figure 6A shows the tumor growth curves for the IgG control group and the treatment group. Figure 6B shows the weight changes of mice during treatment. [Figure 6B] Figure 6 shows the results of an in vivo study of utomilumab analogues in an MC38 mouse colon cancer model. Figure 6A shows the tumor growth curves for the IgG control group and the treatment group. Figure 6B shows the weight changes of mice during treatment. [Figure 7A] An exemplary bispecific antibody design is shown, in which an anti-CD137 scFv is fused to a full-length antibody that binds generally to a tumor-associated antigen (7A) and specifically to HER2 (7B). αTAA represents the antibody against the tumor-associated antigen. αCD137 represents the anti-CD137 scFv. [Figure 7B]An exemplary bispecific antibody design is shown, in which an anti-CD137 scFv is fused to a full-length antibody that binds generally to a tumor-associated antigen (7A) and specifically to HER2 (7B). αTAA represents the antibody against the tumor-associated antigen. αCD137 represents the anti-CD137 scFv. [Figure 8A] Figure 8 shows the binding affinity of exemplary anti-CD137 x HER2 bispecific antibodies to CD137 (8A) and HER2 (8B), as measured, for example, by an Octet bispecific binding assay. αHER2 represents a full-length anti-HER2 monoclonal antibody derived from the amino acid sequence of trastuzumab. 2-9scFv_αHER2-HC-C represents a bispecific antibody in which an anti-CD137scFv derived from clone 2-9 is fused to the C-terminus of the heavy chain of the full-length anti-HER2 antibody. [Figure 8B] Figure 8 shows the binding affinity of exemplary anti-CD137 x HER2 bispecific antibodies to CD137 (8A) and HER2 (8B), as measured, for example, by an Octet bispecific binding assay. αHER2 represents a full-length anti-HER2 monoclonal antibody derived from the amino acid sequence of trastuzumab. 2-9scFv_αHER2-HC-C represents a bispecific antibody in which an anti-CD137scFv derived from clone 2-9 is fused to the C-terminus of the heavy chain of the full-length anti-HER2 antibody. [Figure 9A] Figure 9 shows the binding affinity of exemplary anti-CD137 x HER2 bispecific antibodies and anti-CD137 monoclonal antibodies to CD137 (9A) and HER2 (9B), measured, for example, by whole cell binding assays. αHER2 represents an anti-HER2 monoclonal antibody derived from the amino acid sequence of trastuzumab. αEGFR represents an anti-EGFR monoclonal antibody. "αCD137scFv-αHER2-IgG1" and "αCD137scFv-αHER2-IgG4-FALA" represent bispecific antibodies in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody with an IgG1 or IgG4 isotype (with the Fc FALA mutation), respectively. [Figure 9B]Figure 9 shows the binding affinity of exemplary anti-CD137 x HER2 bispecific antibodies and anti-CD137 monoclonal antibodies to CD137 (9A) and HER2 (9B), measured, for example, by whole cell binding assays. αHER2 represents an anti-HER2 monoclonal antibody derived from the amino acid sequence of trastuzumab. αEGFR represents an anti-EGFR monoclonal antibody. "αCD137scFv-αHER2-IgG1" and "αCD137scFv-αHER2-IgG4-FALA" represent bispecific antibodies in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody with an IgG1 or IgG4 isotype (with the Fc FALA mutation), respectively. [Figure 10A] Figure 10A shows the cross-linking effect of an exemplary bispecific antibody that binds both CD137 and HER2. Figure 10A illustrates the experimental steps, showing that the cross-linking effect is triggered in the presence of an anti-CD137 x HER2 bispecific antibody (bottom panel, left panel) but not in the presence of an anti-CD137 monoclonal antibody (top panel, left panel). Figures 10B-10C show the CD137 activation levels in 293T cells after contact with HER2-high NCI-N87 cells or HER2-low SK-Hep1 cells in the presence of anti-CD137 x HER2 bispecific antibodies with various formats (HC-C, HC-N, LC-C, LC-N). αCD137-αHER2-HC-C represents a bispecific antibody in which an anti-CD137 scFv derived from clone 2-9 is fused to the C-terminus of the heavy chain of a full-length anti-HER2 antibody. αCD137-αHER2-HC-N represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the N-terminus of the heavy chain of a full-length anti-HER2 antibody. αCD137-αHER2-LC-C represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the C-terminus of the light chain of a full-length anti-HER2 antibody. αCD137-αHER2-LC-N represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the N-terminus of the light chain of a full-length anti-HER2 antibody. [Figure 10B]Figure 10A shows the cross-linking effect of an exemplary bispecific antibody that binds both CD137 and HER2. Figure 10A illustrates the experimental steps, showing that the cross-linking effect is triggered in the presence of an anti-CD137 x HER2 bispecific antibody (bottom panel, left panel) but not in the presence of an anti-CD137 monoclonal antibody (top panel, left panel). Figures 10B-10C show the CD137 activation levels in 293T cells after contact with HER2-high NCI-N87 cells or HER2-low SK-Hep1 cells in the presence of anti-CD137 x HER2 bispecific antibodies with various formats (HC-C, HC-N, LC-C, LC-N). αCD137-αHER2-HC-C represents a bispecific antibody in which an anti-CD137 scFv derived from clone 2-9 is fused to the C-terminus of the heavy chain of a full-length anti-HER2 antibody. αCD137-αHER2-HC-N represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the N-terminus of the heavy chain of a full-length anti-HER2 antibody. αCD137-αHER2-LC-C represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the C-terminus of the light chain of a full-length anti-HER2 antibody. αCD137-αHER2-LC-N represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the N-terminus of the light chain of a full-length anti-HER2 antibody. [Figure 10C]Figure 10A shows the cross-linking effect of an exemplary bispecific antibody that binds both CD137 and HER2. Figure 10A illustrates the experimental steps, showing that the cross-linking effect is triggered in the presence of an anti-CD137 x HER2 bispecific antibody (bottom panel, left panel) but not in the presence of an anti-CD137 monoclonal antibody (top panel, left panel). Figures 10B-10C show the CD137 activation levels in 293T cells after contact with HER2-high NCI-N87 cells or HER2-low SK-Hep1 cells in the presence of anti-CD137 x HER2 bispecific antibodies with various formats (HC-C, HC-N, LC-C, LC-N). αCD137-αHER2-HC-C represents a bispecific antibody in which an anti-CD137 scFv derived from clone 2-9 is fused to the C-terminus of the heavy chain of a full-length anti-HER2 antibody. αCD137-αHER2-HC-N represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the N-terminus of the heavy chain of a full-length anti-HER2 antibody. αCD137-αHER2-LC-C represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the C-terminus of the light chain of a full-length anti-HER2 antibody. αCD137-αHER2-LC-N represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to the N-terminus of the light chain of a full-length anti-HER2 antibody. [Figure 11A]Figure 11 shows the cross-linking effect of an exemplary anti-CD137 x HER2 bispecific antibody with or without an Fc mutation. Figure 11A shows the CD137 activation level in 293T cells after contact with HER2-high SKBR3 cells. Figure 11B shows the CD137 activation level in 293T cells after contact with HER2-low SK-Hep1 cells. The anti-CD137 x HER2 bispecific antibody used in this example has an HC-C format. "αCD137scFv-αHER2-IgG1" and "αCD137scFv-αHER2-IgG4-FALA" represent bispecific antibodies in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody (with an Fc FALA mutation) of the IgG1 or IgG4 isotype derived from trastuzumab. "αCD137 mAb-IgG2" represents a monoclonal anti-CD137 antibody derived from clone 2-9, which has an IgG2 isotype. [Figure 11B] Figure 11 shows the cross-linking effect of an exemplary anti-CD137 x HER2 bispecific antibody with or without an Fc mutation. Figure 11A shows the CD137 activation level in 293T cells after contact with HER2-high SKBR3 cells. Figure 11B shows the CD137 activation level in 293T cells after contact with HER2-low SK-Hep1 cells. The anti-CD137 x HER2 bispecific antibody used in this example has an HC-C format. "αCD137scFv-αHER2-IgG1" and "αCD137scFv-αHER2-IgG4-FALA" represent bispecific antibodies in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody (with an Fc FALA mutation) of the IgG1 or IgG4 isotype derived from trastuzumab. "αCD137 mAb-IgG2" represents a monoclonal anti-CD137 antibody derived from clone 2-9, which has an IgG2 isotype. [Figure 12]Figure 1 shows the IFNγ and IL-2 production of effector cells after contact with HER2-high SKBR3 cells or HER2-low MDA-MB-231 cells in the presence of various concentrations of anti-CD137 x HER2 bispecific antibody or CD137 monoclonal antibody. The anti-CD137 x HER2 bispecific antibody used in this example has an HC-C format. "αCD137scFv-αHER2-IgG1" and "αCD137scFv-αHER2-IgG4-FALA" represent bispecific antibodies in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody of the IgG1 or IgG4 isotype (with the Fc FALA mutation). [Figure 13] Figure 1 shows tumor volume changes in LoVo / hPBMC xenografted NOD-SCID mice after administration of a) an anti-CD137 x HER2 bispecific antibody (αCD137scFv-αHER2-IgG4-FALA HCC-L7, 10 mg / kg) and b) a combination of an anti-CD137 monoclonal antibody (2-9 mAb, 7.5 mg / kg) and an anti-HER2 monoclonal antibody (αHER2, 7.5 mg / kg), compared to vehicle control. "αCD137scFv-αHER2-IgG4-FALA" represents a bispecific antibody in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody with an IgG4 isotype (containing the Fc FALA mutation). [Figure 14A]Figure 14 shows the tumor volume change in OE19 / hPBMC xenografted NOD-SCID mice after administration of a) anti-CD137 x HER2 bispecific antibody (αCD137scFv-αHER2-IgG4-FALA HCC-L7, 0.4 mg / kg), b) a combination of anti-CD137 monoclonal antibody (2-9 mAb, 0.3 mg / kg) and anti-HER2 monoclonal antibody (αHER2, 0.3 mg / kg), and c) anti-HER2 monoclonal antibody (αHER2, 0.3 mg / kg) compared to vehicle control. "αCD137scFv-αHER2-IgG4-FALA" represents the bispecific antibody, in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody with an IgG4 isotype (containing the Fc FALA mutation). Figure 14A shows the tumor growth curves for each group. FIG. 14B shows the individual tumor volumes for each treatment group on day 21. [Figure 14B] Figure 14 shows the tumor volume change in OE19 / hPBMC xenografted NOD-SCID mice after administration of a) anti-CD137 x HER2 bispecific antibody (αCD137scFv-αHER2-IgG4-FALA HCC-L7, 0.4 mg / kg), b) a combination of anti-CD137 monoclonal antibody (2-9 mAb, 0.3 mg / kg) and anti-HER2 monoclonal antibody (αHER2, 0.3 mg / kg), and c) anti-HER2 monoclonal antibody (αHER2, 0.3 mg / kg) compared to vehicle control. "αCD137scFv-αHER2-IgG4-FALA" represents the bispecific antibody, in which the anti-CD137 scFv derived from clone 2-9 is fused to a full-length anti-HER2 antibody with an IgG4 isotype (containing the Fc FALA mutation). Figure 14A shows the tumor growth curves for each group. FIG. 14B shows the individual tumor volumes for each treatment group on day 21. DETAILED DESCRIPTION OF THE INVENTION

[0039] 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 a disease or condition, modulating an immune response, or modulating a cellular composition). The present invention is based, in part, on the discovery of anti-CD137 monospecific and multispecific antibodies that have shown improved safety and enhanced anti-tumor efficacy compared to conventional anti-CD137 antibodies in clinical trials.

[0040] I. Definition The term "antibody" is used in its broadest sense and covers a variety of 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 portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0041] A full-length antibody contains 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 are called "V" and "V" respectively. H " and "V LThe variable regions in the two chains generally contain three highly variable loops, referred to as complementarity-determining regions (CDRs) (light chain (LC) CDRs comprising LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs comprising 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 conventions 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 interspersed with flanking segments called framework regions (FRs), which are even more conserved than the CDRs and form a scaffold supporting 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 antibody classes or isotypes are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains. Some major antibody classes are divided into subclasses, e.g., IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgG1 (α1 heavy chain), or IgG2 (α2 heavy chain).

[0042] The term "antigen-binding fragment" as used herein refers to an antibody fragment, including, for example, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain Fvs (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from antibody portions comprising one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the entire antibody structure. An antigen-binding fragment can bind to the same antigen as the parent antibody or parent antibody fragment (e.g., the parent scFv). In some examples, an antigen-binding fragment can comprise one or more CDRs from a particular human antibody, wherein the CDRs are grafted onto framework regions from one or more different human antibodies.

[0043] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. Folding of the two domains releases six hypervariable loops (three loops from each heavy and light chain), which provide amino acid residues for antigen binding and confer binding specificity to the antibody for the antigen. However, even a single variable domain (or half an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to antigen, although usually with lower affinity than the complete binding site.

[0044] "Single-chain Fv" (also abbreviated as "sFv" or "scFv") refers to VFvs linked to a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment comprising a V H and V LThe scFv further comprises a polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0045] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the discontinuous antigen-binding sites found within the variable regions of 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 MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where overlapping or subsets containing amino acid residues are defined when compared with each other. However, it is intended that the application of any one definition to refer to an antibody, grafted antibody, or variant thereof, falls within the scope of the term as defined and used herein. The amino acid residues covering the CDRs defined in each of the above references are listed in Table 1 below 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 incorporated by reference in their entirety, used herein, and may be included in one or more claims herein.

[0046] [Table 1]

[0047] The phrases "variable domain residue numbering, e.g., according to Kabat" or "amino acid position numbering, e.g., according to Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the antibody assembler of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or hypervariable region (HVR) of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (based on Kabat residue 52a) and an inserted residue after heavy chain FR residue 82 (e.g., based on Kabat residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by comparing the antibody sequence to the "standard" Kabat numbered sequence at the regions of homology.

[0048] Unless otherwise specified herein, the amino acid residues covering the CDRs of a full-length antibody (e.g., an anti-CD137 antibody disclosed herein) are defined according to the Kabat nomenclature of Kabat et al., supra, and residue numbering in an immunoglobulin heavy chain, e.g., an Fc region, is that of the EU index as described in Kabat et al., supra, except for the amino acid residues covering the CDRs of any shared sequence, which are defined according to the Kabat nomenclature, with modifications based on experimental conditions. The "EU index as described in Kabat" is the residue numbering of the human IgG1 EU antibody.

[0049] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0050] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies, which contain minimal sequence derived from the non-human antibody. Most often, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a recipient hypervariable region (HVR) are replaced by residues from a hypervariable region (donor antibody) from a non-human species (e.g., mouse, rat, rabbit, or non-human primate) having the desired antigen specificity, affinity, and capacity. In some embodiments, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Humanized antibodies usually comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that 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).

[0051] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been prepared by any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies may be generated using various techniques known in the art, including phage display libraries. See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also usable for preparing monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals (e.g., immunized xenomice) that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (e.g., XENOMOUSE). (商標) (See U.S. Patent Nos. 6,075,181 and 6,150,584 for related techniques.) For human antibodies produced by human B cell hybridoma technology, see further, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006).

[0052] "Percent (%) amino acid sequence identity" or "homology" between a polypeptide and an antibody sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to 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 purposes of determining percent amino acid sequence identity, comparisons can be performed using a variety of methods within the skill of the art, including publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring comparisons, including any algorithm that maximizes comparison over the entire length of the sequences being compared. However, for purposes of this specification, the sequence comparison computer program MUSCLE is used to generate percent amino acid sequence identity values ​​(Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0053] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., when a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, multiplied 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. Comparisons are usually performed when the two sequences are aligned to maximize homology.

[0054] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin, the variable domain, and that contains the antigen-binding site. The constant domain is H 1. C H 2 and C H 3 Domains (C H ) and light chain CHL (or C L ) domain.

[0055] The "light chains" of any antibody (immunoglobulin) of any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), depending on the amino acid sequence of their constant domains.

[0056] The "CH1 domain" (also called "C1" for "H1" domain) is typically from about amino acid 118 to about amino acid 215 (EU numbering system).

[0057] The "hinge region" is usually defined as the region of IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol., 22:161-206 (1985)). 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 bonds between the heavy chains in the same positions.

[0058] The "CH2 domain" of the human IgG Fc region (also called the "C2" domain) typically spans from about amino acid 231 to about amino acid 340. The CH2 domain is unique because it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact, native IgG molecules. Speculation suggests that the carbohydrates may provide an alternative for domain-domain pairing and contribute to the stabilization of the CH2 domain. Burton, Molec Immunol., 22:161-206 (1985).

[0059] The "CH3 domain" (also called the "C2" domain) comprises the region of residues in the Fc region that are closest to the C-terminus of the CH2 domain (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residue 446 or 447 for IgG).

[0060] As used herein, the terms "Fc region" or "fragment crystallizable region" are used to define the C-terminal region of an immunoglobulin heavy chain, and include native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is typically defined as stretching from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus. For example, the C-terminal lysine (residue 447 in the EU numbering system) of the Fc region can be removed during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a complete antibody composition can include antibodies with all K447 residues removed, antibodies with K447 residues intact, and mixtures 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.

[0061] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR. A preferred FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants or splice forms of these variants. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences and are primarily distinguished by their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting 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" herein encompasses other FcRs, including those identified in the future.

[0062] As used herein, the term "epitope" refers to the specific atom or amino acid group on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions can bind to the same epitope in an antigen if they have competitive binding to the antigen.

[0063] As used herein, a first antibody or fragment thereof "competes" with a second antibody or fragment thereof for binding to a target antigen when, in the presence of equimolar concentrations of the first antibody or fragment thereof, the first antibody or fragment thereof inhibits target antigen binding of the second antibody or fragment thereof by at least about 50% (e.g., at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), or vice versa. PCT Publication No. WO 03 / 48731 describes a high-throughput method for "boxing" antibodies based on their cross-competition.

[0064] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as the binding of a target to an antibody or antibody portion, which determines the presence of the target in the presence of heteromolecules (including biomolecules). 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 the target with a longer affinity, avidity, readiness, and / or duration than binding to other targets. In some examples, the extent of binding of an antibody to an unrelated target is about 10% less than the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some examples, the dissociation constant (K) of an antibody that specifically binds to a target is about 10% less than the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). D )≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In some embodiments, specific binding may include, but is not limited to, exclusive binding. The binding specificity of an antibody or antigen-binding domain may be determined experimentally by methods known in the art. Such methods include Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE. TM - Including, but not limited to, assays and peptide scanning.

[0065] An "isolated" antibody (or construct) is an antibody that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free of association with all other components of its production environment.

[0066] 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 contaminant nucleic acid molecule normally associated with it in its production environment. Preferably, an isolated nucleic acid is free from association with all components of the production environment. The form of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is different from its naturally occurring form or background. Thus, an isolated nucleic acid molecule is different from a nucleic acid encoding the polypeptides and antibodies described herein that is naturally present in a cell. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0067] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences applicable to prokaryotes include promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0068] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to DNA for a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned favorably for translation. Typically, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0069] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0070] 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. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with foreign nucleic acid. This cell includes the primary target cell and its progeny.

[0071] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of transfers. The nucleic acid content of the progeny may differ from that of the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0072] As used herein, "treatment" or "treating" refers to a method for obtaining beneficial or desired results (including clinical results). For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the rate of disease progression, ameliorating the disease state, providing partial or complete relief from the disease, reducing the dosage of one or more other drugs required to treat the disease, slowing the progression of the disease, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" further covers reducing the pathological consequences of cancer (e.g., tumor volume). The methods of this application contemplate any one or more of these aspects of treatment.

[0073] In the context of cancer, the term "treatment" includes any one or all of inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0074] The term "inhibition" or "inhibit" refers to the reduction or cessation of any phenotypic characteristic, or to the reduction or cessation of the incidence, degree, or likelihood of this characteristic. Compared to a reference, "reduction" or "inhibition" refers to a decrease, decrease, or prevention of an activity, function, and / or amount. In some embodiments, "reduction" or "inhibition" refers to the ability to cause a general decrease of 20% or more. In other embodiments, "reduction" or "inhibition" refers to the ability to cause a general decrease of 50% or more. In yet other embodiments, "reduction" or "inhibition" refers to the ability to cause a general decrease of 75%, 85%, 90%, 95%, or more.

[0075] As used herein, "reference" refers to any sample, standard, or level used for comparison. A reference may be obtained from a healthy and / or undiseased sample. In some embodiments, a reference may be obtained from an untreated sample. In some embodiments, a reference may be obtained from a undiseased or untreated sample from an individual. In some embodiments, a reference may be obtained from one or more healthy individuals who are not the individual or patient.

[0076] As used herein, "delaying disease progression" refers to delaying, inhibiting, slowing, stabilizing, inhibiting, and / or slowing the progression of a disease (e.g., cancer). The delay may have different lengths of time depending on the history of the disease and / or the individual being treated. A sufficient or significant delay may, in effect, include prevention, since the individual is free from the disease. For example, advanced cancer (e.g., the progression of metastases) may be delayed.

[0077] As used herein, "prevention" includes providing protection against the onset or recurrence of a disease in individuals who may be susceptible to the disease but who have not been diagnosed with the disease.

[0078] As used herein, to "inhibit" a function or activity is to decrease the function or activity relative to the same conditions other than the condition or parameter of interest, or alternatively, relative to another condition. For example, an antibody that inhibits tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0079] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0080] An "effective amount" of a drug refers to an amount that effectively achieves a desired therapeutic or prophylactic effect at a required dosage and for a required period of time. The specific dosage may vary depending on one or more of the particular drug selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the duration of administration, the tissue to be imaged, and the physical delivery system therewith.

[0081] 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 weight of the individual and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the substance / molecule, agonist, or antagonist are counteracted by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations.

[0082] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, such a prophylactically effective amount will be less than the therapeutically effective amount.

[0083] The terms "drug formulation" and "drug composition" refer to a formulation in a form that allows the biological activity of one or more active ingredients to be effective and that does not contain other ingredients that are unacceptably toxic to the individual to whom the formulation is administered. Such formulations may be sterile.

[0084] "Pharmaceutically acceptable carrier agent" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier agent conventional in the art used with a therapeutic agent, which together constitute a "pharmaceutical composition" for administration to an individual. Pharmaceutically acceptable carrier agents are non-toxic to recipients at the dosages and concentrations employed and are compatible with the other ingredients of the formulation. Pharmaceutically acceptable carrier agents are suitable for the formulation employed.

[0085] A "sterile" formulation is aseptic or substantially free of living microorganisms and their spores.

[0086] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.

[0087] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time or the administration of one therapeutic agent is within a short window of the administration of another therapeutic agent. For example, the two or more therapeutic agents are administered any time apart not exceeding about 60 minutes (e.g., not exceeding any one of about 30, 15, 10, 5, or 1 minutes).

[0088] The term "sequential" is used herein to refer to the administration of two or more therapeutic agents, where administration of one or more agents is discontinued followed by administration of one or more other agents. For example, the administration of the two or more therapeutic agents is separated by a time interval of greater than about 15 minutes (e.g., about 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).

[0089] As used herein, "in combination with" refers to the administration of one therapy other than another. Similarly, "in combination with" refers to the administration of one therapy before, during, or after administering another therapy to an individual.

[0090] The term "packaging insert" refers to a manual typically included in the commercial packaging of a therapeutic product, which manual contains information regarding the indications for using such therapeutic product, methods of use, dosage, administration, concomitant therapy, contraindications and / or warnings.

[0091] An "article of manufacture" is any product (e.g., a package or container) or kit that includes at least one reagent, such as 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, sold, or sold as a unit for performing a method described herein.

[0092] It is to be understood that the examples of the application described herein include "consisting of" and "consisting essentially of."

[0093] As used herein, references to "about" a value or parameter include (and describe) variations on that value or parameter itself. For example, a statement about "about X" includes a statement of "X."

[0094] As used herein, reference to a value or parameter that is "not" generally means and describes a "different" value or parameter. For example, a method is not for treating cancer type X means that the method is used to treat a cancer different from type X.

[0095] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0096] 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.

[0097] II.CD137(4-1BB) CD137 (4-1BB) is a member of the tumor necrosis 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 expressed as a 55 kDa homodimer. The receptor was first described in mice (B. Kwon et al., PNASUSA, 86:1963-7 (1989)) and subsequently 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 WO 95 / 07984 and WO 96 / 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. Conserved sequences appear in the cytoplasmic domain and five other regions of the molecule, indicating that these residues may be important for the function of the CD137 molecule (Z. Zhou et al., Immunol. Lett., 45:67 (1995)). CD137 is primarily expressed on lymphoid cells (e.g., activated T cells, activated natural killer (NK) cells, NKT cells, and CD4+CD25+ regulatory T cells), and has also been shown to be expressed on activated thymocytes and intraepithelial lymphocytes. CD137 has also been shown to be expressed on cells of myeloid origin, such as dendritic cells, monocytes, neutrophils, and eosinophils. Although CD137 expression is primarily restricted to immune / inflammatory cells, reports have described its expression on endothelial cells associated with minor tissues at sites of inflammation and in tumors.

[0098] The functional activities of CD137 on T cells have been well characterized. Previously, it was demonstrated that suboptimal doses of anti-CD3 can signal through CD137 to induce T cell proliferation and cytokine synthesis (primarily 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, costimulation enhances effector functions, such as IFN-γ production and cytotoxicity, by increasing the number of antigen-specific and effector CD8+ T cells. In the absence of anti-CD3 signaling, stimulation with CD137 does not alter T cell function, indicating that CD137 is a costimulatory molecule.

[0099] In vivo efficacy studies using agonistic anti-mouse CD137 monoclonal antibodies in mice have suggested the potential for CD137-targeted therapy in cancer treatment. In a paper by Melero et al., agonistic anti-mouse CD137 antibodies induced cures in P815 mastocytoma tumors and in the low-immunogenic tumor model Ag104 (I. Melero et al., Nat. Med., 3(6):682-5 (1997)). Antitumor activity requires both CD4+ and CD8+ T cells and NK cells, as selective in vivo depletion of each subpopulation can result in reduced or complete loss of antitumor activity. It has also been demonstrated that induction of a minimal immune response is necessary for anti-CD137 therapy to be effective. Several researchers have demonstrated the feasibility of such methods 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)).

[0100] In addition to their role in the development of immunity against cancer, experimental data also support 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., PNASUSA, 96:15074-79 (1999); J. Foell et al., NY 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)).

[0101] III. Anti-CD137 Constructs In one embodiment, the present invention provides novel CD137-specific constructs (e.g., isolated anti-CD137 constructs) comprising an antibody portion that specifically binds to CD137. The specificity of the anti-CD137 constructs derives from the anti-CD137 antibody portion, e.g., a full-length antibody or an antigen-binding fragment thereof, that specifically binds to CD137. In some embodiments, the referenced portion (e.g., antibody portion) that specifically binds to CD137 is characterized in that the portion has a binding affinity that is at least about 10-fold (e.g., at least about 10, 10) greater than the binding affinity for a non-target. 2 , 10 3 , 10 4 , 10 5 , 10 6 or 10 7 The term "specifically binds to CD137" refers to binding with an affinity of at least 100% (including any one of 100% and 100% of the total binding affinity). In some embodiments, the non-target is an antigen other than CD137. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). dcan be determined by methods known in the art, such as, for example, using a surface plasmon resonance (SPR) assay on a Biacore instrument or using kinetic exclusion assay (KinExA) on a Sapidyne instrument.

[0102] Desirable anti-CD137 constructs include, but are not limited to, anti-CD137 scFvs, fusion proteins comprising an anti-CD137 antibody portion and a half-life-prolonging domain (e.g., an Fc region, a domain that binds albumin), anti-CD137 monoclonal antibodies, and multispecific anti-CD137 molecules (e.g., bispecific antibodies). The above exemplary anti-CD137 constructs are not mutually exclusive and are further discussed in each section below.

[0103] In some embodiments, an anti-CD137 construct (e.g., an anti-CD137 scFv) is provided that comprises an anti-CD137 antibody portion that specifically recognizes CD137 (e.g., human CD137), wherein the anti-CD137 antibody portion may be any one of the anti-CD137 antibody portions described herein.

[0104] In some embodiments, an anti-CD137 construct (e.g., an anti-CD137 scFv) is provided that includes an anti-CD137 antibody portion that binds to CD137, the anti-CD137 antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), wherein a) said V Hcomprises: 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) said V L comprises: i) an LC-CDR1 comprising the amino acid sequence of KASQX1X2X3TYX4S (SEQ ID NO: 180), wherein X1 = A, P, or T, X2 = I, T, or P, X3 = N, A, and X4 = L, G, or H; ii) an LC-CDR2 comprising the amino acid sequence of RX1NRX2X3X4 (SEQ ID NO: 181), wherein X1 = A, Y, V, or D, X2 = M, K, V, or A, X3 = V, P, Y, or G, and X4 = D or G; and iii) an LC-CDR3 comprising the amino acid sequence of LQX1X2DFPYX3 (SEQ ID NO: 182), wherein X1 = Y, S, or F, X2 = D, V, L, R, E, or Q, and X3 = T, or K.

[0105] In some embodiments, the anti-CD137 antibody portion that binds to CD137 comprises: a) an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 231, and 241, or a variant thereof comprising up to about three (e.g., three, two, or one) amino acid substitutions; b) an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 232, and 242, or a variant thereof comprising up to about three (e.g., three, two, or one) amino acid substitutions; and c) an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 232, and 242, or a variant thereof comprising up to about three (e.g., three, two, or one) amino acid substitutions. d) an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 233, and 243, or a variant thereof containing up to about three (e.g., three, two, or one) amino acid substitutions; d) an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 234, and 244, or a variant thereof containing up to about three (e.g., three, two, or one) amino acid substitutions; and e) an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 234, and 244, or a variant thereof containing up to about three (e.g., three, two, or one) amino acid substitutions. and f) an LC-CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 235, and 245, or a variant thereof comprising up to about three (e.g., three, two, or one) amino acid substitutions; and f) an LC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 236, and 246, or a variant thereof comprising up to about three (e.g., three, two, or one) 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.

[0106] In some embodiments, an anti-CD137 construct is provided that includes an anti-CD137 antibody portion, 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 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), which is selected from the group consisting of: a) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about three (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:4, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and c) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:23, or a variant thereof comprising up to about three (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: 24, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; d) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and e) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about three (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: 44, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 46, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and f) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and g) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 63, or a variant thereof comprising up to about three (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: 64, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 65, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 66, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and h) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 81, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 82, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 83, or a variant thereof comprising up to about three (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: 84, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 86, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and j) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:91, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:92, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:93, or a variant thereof comprising up to about three (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 three (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 three (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 three (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 three (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 three (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 three (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 three (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 three (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 three (e.g., 3, 2, or 1) amino acid substitutions; H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:111, or a variant thereof comprising up to about three (e.g., three, two, or one) amino acid substitutions. and an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, or a variant thereof comprising up to about three (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 three (e.g., 3, 2, or 1) amino acid substitutions; and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 115, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 116, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 121, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 122, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 123, or a variant thereof comprising up to about three (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: 124, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 126, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 131, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 132, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 133, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and V LLC-CDR1 comprising the amino acid sequence of SEQ ID NO: 134, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 135, or a variant thereof comprising up to about three (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 three (e.g., 3, 2, or 1) amino acid substitutions; o) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233, or a variant thereof comprising up to about three (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: 234, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 236, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 241, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 242, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 243, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and V Lcomprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:244, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:245, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:246, or a variant thereof comprising up to about three (e.g., 3, 2, or 1) amino acid substitutions.

[0107] In some embodiments, an anti-CD137 construct (e.g., an anti-CD137 scFv) is provided that includes an anti-CD137 antibody portion that binds to CD137, the anti-CD137 antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), wherein a) said 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.

[0108] In some embodiments, an anti-CD137 construct (e.g., an anti-CD137 scFv) is provided that includes an anti-CD137 antibody portion that binds to CD137, the anti-CD137 antibody portion comprising: a) a VF having the sequence set forth in SEQ ID No: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 237, or 247, respectively; H a) HC-CDR1, HC-CDR2 and HC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 in the chain region; and b) V having the sequences shown in SEQ ID Nos: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 238 or 248, respectively. LThe chain region includes LC-CDR1, LC-CDR2, and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2, and CDR3.

[0109] In some embodiments, an anti-CD137 construct (e.g., an anti-CD137 scFv) is provided that includes an anti-CD137 antibody portion that binds to CD137, the anti-CD137 antibody portion comprising: a) a VF having a sequence set forth in SEQ ID No: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 237, or 247; H a) a V strand region or variant thereof, or variant having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 237, or 247; and b) a V strand region or variant thereof, or variant having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 238, or 248. L In some embodiments, the V strand region or variant thereof, or variant having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOs: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 238, or 248. H Chain region and V L The chain regions are joined via linkers (eg, peptide linkers).

[0110] In some embodiments, an anti-CD137 construct (e.g., an anti-CD137 scFv) is provided that includes an anti-CD137 antibody portion that binds to CD137, wherein the anti-CD137 antibody portion has a V sequence set forth in SEQ ID No: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 237, or 247. Hregion, and V having the sequence shown in SEQ ID No: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 238 or 248 L Includes the area.

[0111] 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:7 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:8; (b) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:17 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:18; (c) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:27 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:28; (d) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:37 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:38; (e) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:47 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:48; and (f) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:57 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:57. (g) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:67 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:68; (h) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:77 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:78; (i) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:87 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:88; (j) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:97 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:98; (k) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:107 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:108; (l) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:117 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:118. (m) a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:118;(n) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 137 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 138; (o) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 237 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 238; or (p) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 247 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 248.

[0112] In some embodiments, an anti-CD137 construct (e.g., an anti-CD137 scFv) is provided that comprises an anti-CD137 antibody portion that binds to CD137, wherein the anti-CD137 antibody portion comprises a heavy chain (HC) having the sequence set forth in SEQ ID No: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 239, or 249, and a light chain (LC) having the sequence set forth in SEQ ID No: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 240, or 250.

[0113] 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 set forth in SEQ ID NO:9 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:19 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:29 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:39 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:49 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:59 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:69 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:79 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:89 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:99 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO: 109 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO: 119 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO: 129 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO:139 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO: 149 and a light chain comprising amino acids having the sequence set forth 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 set forth in SEQ ID NO: 239 and a light chain comprising amino acids having the sequence set forth in SEQ ID NO: 240. 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 set forth in SEQ ID NO: 249 and a light chain comprising amino acids having the sequence set forth in SEQ ID NO: 250.

[0114] 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 the light chain variable region (V L ), wherein a) said V H comprises i) an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 151-153, or a variant thereof comprising up to about three amino acid substitutions; ii) an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 154-156, or a variant thereof comprising up to about three amino acid substitutions; and iii) an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 157-159, or a variant thereof comprising up to about three amino acid substitutions; and b) said V Lcomprises i) an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 160-163 or a variant thereof comprising up to about three amino acid substitutions; ii) an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 164-166 or a variant thereof comprising up to about three amino acid substitutions; and iii) an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 167-169 or a variant thereof comprising up to about three amino acid substitutions.

[0115] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1 comprises the amino acid sequence SEQ ID NO: 151, the HC-CDR2 comprises the amino acid sequence SEQ ID NO: 154, and the HC-CDR3 comprises the amino acid sequence SEQ ID NO: 157; and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1 comprises the amino acid sequence SEQ ID NO: 160, the LC-CDR2 comprises the amino acid sequence SEQ ID NO: 164, and the LC-CDR3 comprises the amino acid sequence SEQ ID NO: 167.

[0116] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1 comprises the amino acid sequence SEQ ID NO: 151, the HC-CDR2 comprises the amino acid sequence SEQ ID NO: 154, and the HC-CDR3 comprises the amino acid sequence SEQ ID NO: 157; and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1 comprises the amino acid sequence SEQ ID NO: 162, the LC-CDR2 comprises the amino acid sequence SEQ ID NO: 166, and the LC-CDR3 comprises the amino acid sequence SEQ ID NO: 169.

[0117] In some embodiments, V Hcomprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1 comprises the amino acid sequence SEQ ID NO: 152, the HC-CDR2 comprises the amino acid sequence SEQ ID NO: 155, and the HC-CDR3 comprises the amino acid sequence SEQ ID NO: 158, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1 comprises the amino acid sequence SEQ ID NO: 163, the LC-CDR2 comprises the amino acid sequence SEQ ID NO: 166, and the LC-CDR3 comprises the amino acid sequence SEQ ID NO: 169.

[0118] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1 comprises the amino acid sequence SEQ ID NO: 153, the HC-CDR2 comprises the amino acid sequence SEQ ID NO: 156, and the HC-CDR3 comprises the amino acid sequence SEQ ID NO: 159, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1 comprises the amino acid sequence SEQ ID NO: 160, the LC-CDR2 comprises the amino acid sequence SEQ ID NO: 164, and the LC-CDR3 comprises the amino acid sequence SEQ ID NO: 167.

[0119] In some embodiments, V H comprises HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1 comprises the amino acid sequence SEQ ID NO: 153, the HC-CDR2 comprises the amino acid sequence SEQ ID NO: 156, and the HC-CDR3 comprises the amino acid sequence SEQ ID NO: 159, and V L comprises LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1 comprises the amino acid sequence SEQ ID NO: 161, the LC-CDR2 comprises the amino acid sequence SEQ ID NO: 165, and the LC-CDR3 comprises the amino acid sequence SEQ ID NO: 168.

[0120] 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.

[0121] 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 V H The antibody or antigen-binding fragment thereof may comprise or consist of an antibody or antigen-binding fragment thereof selected from the group consisting of H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, and 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.

[0122] In some examples, the construct comprises a humanized anti-CD137 full-length antibody.

[0123] In some examples, the construct comprises a humanized anti-CD137 single chain Fv fragment.

[0124] 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 both human CD137 and monkey CD137. In some embodiments, the construct does not bind to mouse CD137.

[0125] As described above, the anti-CD137 constructs disclosed herein comprise an anti-CD137 antibody portion that binds to CD 137. In some embodiments, the anti-CD137 antibody portion binds to human and monkey CD137.

[0126] In some embodiments, the anti-CD137 antibody portion included in the anti-CD137 constructs disclosed herein is a CD137 agonist, wherein binding of the antibody portion to CD137 can enhance a CD137-mediated immune signaling pathway. In some embodiments, binding of the antibody portion to CD137 cannot enhance a CD137-mediated immune signaling pathway in the absence of cross-linking and / or clustering of the antibody portion / CD137 complex. In some embodiments, binding of the antibody portion to CD137 cannot activate immune cells, e.g., T cells and / or NK cells, in the absence of cross-linking and / or clustering of the antibody portion / CD137 complex. In some embodiments, cross-linking and / or clustering of the antibody portion / CD137 complex may be mediated by a second portion of the anti-CD137 constructs disclosed herein. In some embodiments, cross-linking and / or clustering of the antibody moiety / CD137 complex may be mediated by binding of an Fc receptor to the Fc region of the anti-CD137 construct, hi some embodiments, cross-linking and / or clustering of the antibody moiety / CD137 complex may be mediated by binding of a second antibody moiety of the anti-CD137 construct to a tumor-associated antigen (TAA).

[0127] 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 and heteroconjugate thereof. 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 an IgG1 Fc region. In some embodiments, the Fc region comprises the CH2 and CH3 domains of IgG1. In some embodiments, the Fc region is an IgG2 Fc region. In some embodiments, the Fc region comprises the CH2 and CH3 domains of IgG2. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region comprises the CH2 and CH3 domains of IgG4. IgG4 Fc is known to have lower effector activity than IgG1 or IgG2 Fc, and may therefore be ideal for some uses. In some embodiments, the Fc region is derived from a mouse immunoglobulin.

[0128] In some embodiments, the antibody portion comprises an Fc region. In some embodiments, the antibody portion is an scFv fused to an Fc region. In some embodiments, the antibody portion comprises an scFv fused to an Fc region via a peptide linker. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region comprises one or more mutations to increase clearance or decrease half-life.

[0129] In some embodiments, the Fc region comprises an immunoglobulin IgG heavy chain constant region, including the hinge region (from Cys226), 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 the hinge region. In some embodiments, the Fc region of the fusion protein begins at 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 the hinge region.

[0130] In some embodiments, the IgG CH2 domain starts at Ala231. In some embodiments, the CH3 domain starts at Gly341. It is understood that the C-terminal Lys residue of human IgG may optionally be absent. It should further be understood that conservative amino acid substitutions in the Fc region are considered within the scope of the present invention if they do not affect the necessary structure and / or stability of the Fc.

[0131] In some embodiments, each chain of the Fc region is fused to the same antibody moiety. In some embodiments, an scFv-Fc comprises two identical scFvs described herein, each fused to one chain of the Fc region. In some embodiments, an scFv-Fc is a homodimer.

[0132] In some embodiments, an scFv-Fc comprises two different scFvs, each fused to one chain of an Fc region. In some embodiments, the scFv-Fc is a heterodimer. Heterodimerization of different polypeptides in an scFv-Fc can be promoted by methods known in the art, including, but not limited to, heterodimerization using the knobs-in-holes technique. The structure and assembly methods for the knobs-in-holes technique can be found, for example, in U.S. Pat. No. 5,821,333, U.S. Pat. No. 7,642,228, U.S. Pat. No. 2011 / 0287009, and PCT Publication No. PCT / US2012 / 059810, the entire contents of which are incorporated herein by reference. This technique involves the steps of introducing a "knob" (or protrusion) in the CH3 domain of one Fc by replacing a small amino acid residue with a larger one, and introducing a "hole" (or cavity) in the CH3 domain of another Fc by replacing one or more large amino acid residues with a smaller one. In some embodiments, one chain of the Fc domain in the fusion protein comprises a knob and the second chain of the Fc domain comprises a hole.

[0133] 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 preferred are tryptophan and tyrosine. In one example, the original residues for forming the knob have small side chain volumes, such as 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.

[0134] Preferred residues for forming holes are typically naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T), and valine (V). In one example, the original residue for forming a hole has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain to generate holes include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions. In some examples, the knob comprises a T366W substitution and the hole comprises a T366S / L368A / Y407V substitution. It should be understood that other modifications to the Fc region known in the art that contribute to heterodimerization are also contemplated and included in the present application.

[0135] Other scFv-Fc variants (including variants of isolated anti-CD137 scFv-Fc, e.g., full-length anti-CD137 antibody variants) that comprise any one of the variants described herein (e.g., Fc variants, effector function variants, glycosylation variants, cysteine ​​engineered variants) or combinations thereof are also contemplated.

[0136] a) Antibody affinity The binding specificity of an antibody moiety may be determined experimentally by methods known in the art, such as Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE. TM - Including, but not limited to, assays and peptide scanning.

[0137] In some embodiments, the K of binding between the antibody moiety and CD137 D is about 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -7 M~about 10 -10 M, about 10 -8 M~about 10 -10 M or about 10 -7 M~about 10 -9 In some embodiments, the K of binding between the antibody moiety and CD137 is M. D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M. In some embodiments, the CD137 is human CD137.

[0138] In some embodiments, the K of binding between the antibody moiety and CD137 on is about 10 3 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 3 M -1 s -1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~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 In some embodiments, the K of binding between the antibody moiety and CD137 is 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 In some embodiments, the K of binding between the antibody moiety and CD137 is 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 In some embodiments, the CD137 is human CD137.

[0139] In some embodiments, the K of binding between the antibody moiety and CD137 off is approximately 1 s -1 ~about 10 -6 s -1 , about 1s -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 1s -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 -1In some embodiments, the K of binding between the antibody moiety and CD137 is off is approximately 1 s -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 -6 s -1 In some embodiments, the CD137 is human CD137.

[0140] In some examples, the binding affinity of the anti-CD137 antibody portion 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)).

[0141] b) Chimeric or humanized antibodies In some embodiments, the antibody portion 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 of murine origin) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0142] In some embodiments, a chimeric antibody is a humanized antibody, typically a non-human antibody that has been humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody, and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody may optionally further comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody derived from the HVR residues), e.g., to restore or improve antibody specificity or affinity.

[0143] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are also 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. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (where SDR (a-CDR) grafting was described), Padlan, Mol. Immunol. 28:489-498 (1991) (where "surface remodification" 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 "guided selection" methods of FR shuffling were described).

[0144] Human framework regions that can be used for humanization include framework regions selected by the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)), framework regions derived from shared sequences of human antibodies of a particular subclass of light or heavy chain variable region (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol. 151:2623 (1993))), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and See Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0145] c) Human antibodies In some embodiments, the antibody moiety is a human antibody (also called 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, US 20090307787 A1, U.S. Patent No. 8,754,287, US 20150289489 A1, US 20100122358 A1, and WO 2004049794.

[0146] Human antibodies (e.g., human DAbs) can be prepared by administering an immunogen to transgenic animals that have been modified to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Further, see, e.g., XENOMOUSE. TM US Patent Nos. 6,075,181 and 6,150,584, describing the technology, HuMab (登録商標) U.S. Patent No. 5,770,429, KM MOUSE, describes the technology (登録商標) US Patent No. 7,041,870 describing the technology, and VelociMouse (登録商標) See US 2007 / 0061900, which describes the technology. The human variable regions from whole antibodies produced by such animals may be further modified (e.g., by attaching to different human constant regions).

[0147] Human antibodies (e.g., human DAbs) may be prepared using hybridoma-based methods. Human myeloma and murine human heteromyeloma cell lines for producing human monoclonal antibodies have been described (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)). Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) further describe human antibodies produced by human B cell hybridoma technology. Other methods include, for example, those described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes 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).

[0148] Human antibodies (e.g., human DAbs) may be produced by isolating Fv clone variable domain sequences selected from a human phage display library. These variable domain sequences may then be combined with the necessary human constant domains. A description of techniques for selecting human antibodies from antibody libraries follows.

[0149] d) Library-derived antibodies Antibody moieties can be isolated by screening combinatorial libraries for antibodies with a desired activity or activities. For example, several methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and in 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, ed., 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 have been described, see, e.g., U.S. Pat. No. 7,371,849.

[0150] In some phage display methods, V H and V LGene libraries can be cloned individually, randomly recombined into phage libraries, and antigen-binding phages can be screened as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages usually display antibody fragments as scFv or Fab fragments. Libraries from immune sources can provide high-affinity antibodies to immunogens without constructing hybridomas. Alternatively, natural libraries (e.g., obtained from humans) can be cloned without the need for any immunization, providing a single source of antibodies against a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J., 12:725-734 (1993). Finally, natural libraries can be synthesized by cloning unrearranged V gene fragments from cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and completing the reassortment in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373 and U.S. Patent Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0151] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0152] e) Substitutions, insertions, deletions and mutations In some examples, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 2 and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into a target antibody and the products screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0153] [Table 2]

[0154] Amino acids may be grouped according to general side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) alkaline: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0155] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0156] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). The resulting variants are typically selected for further study and modify (e.g., improve) some biological property relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or substantially retain some biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which is easy to generate, for example, using phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR (or CDR) residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0157] Modifications (e.g., substitutions) can be made in HVRs (or CDRs) to, for example, improve antibody affinity. Such modifications can be made in HVR (or CDR) "hot spots" (i.e., residues encoded by codons frequently mutated during the somatic maturation process) (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in SDRs (a-CDRs), resulting in mutant V H or V LThe binding affinity of the antibody can be tested. Affinity maturation by construction and reselection from a secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien, ed., Human Press, Totowa, NJ, (2001)). In some embodiments 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), producing a secondary library. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves the HVR-directed method, in which several 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 frequently targeted.

[0158] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs (or CDRs), so long as such modifications do not substantially reduce the antibody's ability to bind antigen. For example, conservative modifications (e.g., conservative substitutions according to the present disclosure) may be made in an HVR (or CDR) that do not substantially reduce binding affinity. Such modifications may be outside of an HVR "hotspot" or CDR. The variants V provided above may be H In some embodiments of the H sequence, each HVR (or CDR) is unaltered or contains no more than one, two, or three amino acid substitutions.

[0159] As described in Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying antibody residues or regions amenable to targeted mutagenesis is called "alanine scanning mutagenesis." In such methods, a target residue or group of residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) is identified, and substitution with neutral or negatively charged amino acids (e.g., alanine or polyalanine) is performed to determine whether the antibody-antigen interaction is affected. Further substitutions at the amino acid position can be introduced to demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as substitution candidates. Mutants can be screened to determine whether they contain desired attributes.

[0160] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0161] 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 an antibody can be readily accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0162] When the antibody moiety includes an Fc region (e.g., scFv-Fc), the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, high-contact angle oligosaccharides, which are typically N-linked to the Fc region C. H The oligosaccharide is linked to Asn297 of the IgG2 domain. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose attached to the GlcNAc in the "stem" of the IgG2 domain. In some embodiments, modifications can be made to the oligosaccharide in the antibody moiety to produce antibody variants with improved properties.

[0163] In some embodiments, the antibody portion comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies 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 sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue at about position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variation in antibodies, Asn297 may also 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, e.g., US Patent Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.)). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 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 defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent No. US 2003 / 0157108 A1; Presta, L., and WO 2004 / 056312 A1; Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0164] In some embodiments, the antibody portion has a bisected oligosaccharide, for example, where a high-contact angle 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 WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Further provided are antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. Such antibody variants may have improved ADCC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0165] g) Fc region mutants In some examples, Fc region variants can be produced by introducing one or more amino acid modifications into the Fc region of an antibody portion (e.g., scFv-Fc). The Fc region variants may comprise a single human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region), which sequence comprises amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0166] In some embodiments, the Fc region possesses some (but not all) effector functions, making the fragment a desirable candidate for applications in which in vivo half-life of the antibody portion is important, but some effector functions (e.g., complement and ADCC) are unnecessary or deleterious. Reduced / depleted CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody does not bind FcγR (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. 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). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, 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), U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med., 166:1351-1361 (1987)). Alternatively, non-radioactive assays (e.g., ACTI for flow cytometry) can be used. (商標)Non-radioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, CA, and CytoTox 96) (登録商標) Non-radioactive cytotoxicity assays (see Promega, Madison, Wisconsin) may also be used. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the target molecule may be assessed in vivo, e.g., in an animal model such as that described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0167] Antibodies with reduced effector function (U.S. Pat. No. 6,737,056) include antibodies with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329. Such Fc mutants include Fc mutants with substitutions at two or more 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 with alanine (U.S. Pat. No. 7,332,581).

[0168] Described herein are several antibody variants with improved or decreased binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). 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.

[0169] In some embodiments, the antibody portion comprises an Fc region with one or more amino acid substitutions, wherein these substitutions (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) within the Fc region) improve ADCC.

[0170] In some embodiments, modifications occur in the Fc region that result in modified (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 164:4178-4184 (2000).

[0171] In some embodiments, the antibody portion (e.g., scFv-Fc) variant comprises a variant Fc region, which comprises one or more amino acid substitutions that alter half-life and / or binding to the neonatal Fc receptor (FcRn). Antibodies with 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)), e.g., as described in US 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more amino acid substitutions, where these substitutions alter binding of the Fc region to FcRn. Such Fc variants include those variants with substitutions at one or more Fc region residues (eg, substitution of Fc region residue 434) (US Pat. No. 7,371,826).

[0172] See also Duncan and Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0173] h) Cysteine ​​Engineered Antibody Variants In some examples, this may involve the production of cysteine-engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of the antibody are replaced with cysteine ​​residues. In specific examples, the substituted residues are located at accessible sites of the antibody. By replacing these residues with cysteine ​​residues, reactive thiol groups are located at accessible sites of the antibody, which may be used to couple the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. In some examples, any one or more residues of A118 (EU numbering) of the heavy chain and S400 (EU numbering) of the heavy chain Fc region may be replaced with cysteine ​​residues. Cysteine-engineered antibody moieties may be produced, for example, as described in U.S. Pat. No. 7,521,541.

[0174] i) Antibody derivative In some embodiments, the antibody moieties described herein may be further modified to include other non-proteinaceous moieties known in the art and readily available. Moieties suitable 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 / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) with dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in formulations due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one type of polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used for a determined diagnostic condition, etc.

[0175] In some embodiments, the antibody portion may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biologically active" or "having biological activity" refers to a biological activity exhibited in vivo to perform a specific function. For example, it may mean binding to a specific biological molecule (e.g., protein, DNA, etc.) and promoting or inhibiting the activity of such a biological molecule. In some embodiments, biologically active proteins or fragments thereof include proteins or polypeptides administered to a patient as active drug substances, proteins and polypeptides for purposes of prevention or treatment and diagnosis of disease or conditions (e.g., enzymes used in diagnostic tests or in vitro assays), and proteins and polypeptides administered to a patient to prevent disease (e.g., vaccines).

[0176] j) Anti-CD137 scFv In some embodiments, the anti-CD137 construct is an scFv (hereinafter referred to as "anti-CD137 scFv") comprising an anti-CD137 antibody moiety described herein. The anti-CD137-scFv may comprise any one of the anti-CD137 antibody moieties (see "anti-CD137 antibody moiety" section) described herein. In some embodiments, the anti-CD137 scFv comprises a V L (CD137)-LV H (CD137). In some embodiments, the anti-CD137 scFv has the following configuration (N-terminus to C-terminus): V H (CD137)-LV L (CD137) (from N-terminus to C-terminus), and L is a linker (eg, a peptide linker).

[0177] In some embodiments, the anti-CD137 scFv is chimeric, human, partially humanized, fully humanized, or semi-synthetic.

[0178] In some embodiments, the anti-CD137 V scFv Land anti-CD137 V H are linked via a linker (e.g., a peptide linker). In some embodiments, the linker comprises about 4 to about 15 amino acids. In some embodiments, the linker is a GS linker. In some embodiments, the linker comprises the sequence of any one of SEQ ID Nos: 206-230.

[0179] k) anti-CD137 fusion protein In some embodiments, the anti-CD137 construct comprises an anti-CD137 antibody portion and a half-life extending moiety. In some embodiments, the half-life extending moiety is an Fc region. In some embodiments, the half-life extending moiety is an albumin-binding moiety (e.g., an albumin-binding antibody portion).

[0180] In some embodiments, the half-life extending moiety 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 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 an IgG or Fc region is based on the EU numbering system for antibodies (also referred to as the EU index), as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0181] In some embodiments, the Fc region is selected from the group consisting of an IgG, IgA, IgD, IgE, IgM Fc region, and any combination and heteroconjugate thereof, hi some embodiments, the Fc region is selected from the group consisting of an IgG1, IgG2, IgG3, IgG4 Fc region, and any combination and heteroconjugate thereof.

[0182] In some embodiments, the Fc region has reduced effector function (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% reduced effector function as assayed for antibody-dependent cell cytotoxicity (ADCC) levels) compared to a corresponding wild-type Fc region.

[0183] 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.

[0184] In some embodiments, the anti-CD137 antibody moiety and the half-life extending moiety are linked via a linker (eg, any one of the linkers described in the "linker" section).

[0185] In some embodiments, the anti-CD137 fusion protein further comprises a second antibody, which in some embodiments binds to a tumor antigen (e.g., any one of the tumor antigens described herein).

[0186] l) Multispecific antibodies Further provided herein are multispecific antibodies (e.g., bispecific antibodies) that bind to both CD137 and a second antigen. In some embodiments, the second antigen is also CD137, but comprises a different epitope than the anti-CD137 antibody moiety described herein. In some embodiments, the second antigen is not CD137. In some embodiments, the second antigen is a tumor-associated antigen.

[0187] In some embodiments, the multispecific antibody comprises a) a first antibody portion comprising any of the anti-CD137 constructs described herein, and b) a second antibody portion that binds to a non-CD137 second antigen, which in some embodiments comprises a tumor-associated antigen.

[0188] 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 comprises two antibody heavy chains and two antibody light chains, wherein the heavy chains each comprise a second heavy chain variable region (V H-2 ), and the light chains each comprise a second light chain variable region (V L-2 ), wherein the anti-CD137 single-chain Fv fragment is fused to at least one of the heavy chain or light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the C-terminus of the light chain of the full-length antibody. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the N-terminus of the light chain of the full-length antibody.

[0189] In some embodiments, the multispecific antibody comprises: a) a first heavy chain variable region (V) that binds to CD137; H-1 ) and the first light chain variable region (V L-1 and b) a second antibody portion comprising a full-length antibody that binds to a second antigen (e.g., a tumor-associated antigen) and that comprises two antibody heavy chains and two antibody light chains, wherein the heavy chains each comprise a second heavy chain variable region (V H-2 ), and the light chains each comprise a second light chain variable region (V L-2and a second antibody portion comprising: a first antibody moiety comprising a C-terminus of one or two heavy chains of a full-length antibody; a second antibody moiety comprising a C-terminus of one or two heavy chains of a full-length antibody; a second antibody moiety comprising a C-terminus of one or two light ... H-1 and V L-1 is 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 amino acids (e.g., about 4 to 20, about 3 to 20, about 6 to 18, or about 10 to 15). In some embodiments, the first linker is a GS linker. In some embodiments, the linker has the amino acid sequence of any one of SEQ ID NOs: 206-230. In some embodiments, the full-length antibody comprises an Fc region selected from the Fc regions of IgG, IgA, IgD, IgE, IgM, and any combination and hybrid thereof. In some embodiments, the Fc region is selected from the Fc regions of 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.

[0190] 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 recognizing 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 full-length anti-CD137 antibody. In some embodiments, the single-chain Fv fragment is fused to the C-terminus of one or two heavy chains of the full-length anti-CD137 antibody. In some embodiments, the single-chain Fv fragment is fused to the N-terminus of one or two heavy chains of the full-length anti-CD137 antibody. In some embodiments, the single-chain Fv fragment is fused to one or two light chains of the full-length anti-CD137 antibody. In some embodiments, the single-chain Fv fragment is fused to the C-terminus of one or two light chains of the full-length anti-CD137 antibody. In some embodiments, the single chain Fv fragment is fused to the N-terminus of one or two anti-CD137 light chains of a full-length antibody.

[0191] As described herein, a "tumor-associated antigen" refers to, for example, any antigen whose expression in tumor cells (e.g., cancer cells) is significantly higher (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% higher) than that in non-tumor cells (e.g., non-cancer cells).

[0192] Exemplary tumor-associated antigens recognizable by the second antibody moieties described herein include 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), Myoglobin (MYC), and IL-1. These include, but are not limited to, Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, S100 proteins, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, and vimentin.

[0193] In some embodiments, the tumor-associated antigen is 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 kinase 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), inclusion protein 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), tyrosine kinase transmembrane receptor type 1 (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.

[0194] m) Anti-CD137 x HER2 multispecific antibody In some examples, the multispecific antibodies disclosed herein comprise: a) a first antibody portion that is a single-chain Fv fragment comprising any of the anti-CD137 constructs disclosed herein; and b) a second antibody portion that binds to HER2 and comprises a full-length antibody comprising two antibody heavy chains and two antibody light chains, each of the heavy chains comprising a second heavy chain variable region (V H-2 ), and the light chains each comprise a second light chain variable region (V L-2 ), wherein the anti-CD137 single chain Fv fragment comprises a second antibody moiety fused to a full-length antibody. 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. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the C-terminus of the light chain of the full-length antibody. In some embodiments, the anti-CD137 single chain Fv fragment is fused to the N-terminus of the light chain of the full-length antibody. In some embodiments, the HER2 is human HER2. In some embodiments, the full-length antibody or anti-HER2 antibody moiety that binds to HER2 comprises a third heavy chain variable region (V) for a binding epitope of HER2. H-3 ) and the third light chain variable region (V L-3 ) wherein a) said V H-3 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194; and b) said V L-3 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 196, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 198, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 200. H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, L-2comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0195] In some embodiments, the multispecific antibody comprises: a) a heavy chain variable region (V H ) and the light chain variable region (V L ) and 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, L a) a 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 heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) and H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, L-2 comprises a second antibody portion comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0196] In some embodiments, the multispecific antibody comprises: a) a heavy chain variable region (V H ) and the light chain variable region (V L ) and Hcomprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233, L a) a first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:234, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:235, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:236; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) and H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, L-2 comprises a second antibody portion comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0197] In some embodiments, the multispecific antibody comprises: a) a heavy chain variable region (V H ) and the light chain variable region (V L ) and H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 241, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 242, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 243, L a) a first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:244, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:245, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:246; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2) and H-2 comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, L-2 comprises a second antibody portion comprising a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200.

[0198] In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO:202 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:202, and / or L-2 comprises the amino acid sequence of SEQ ID NO:203 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:203. H-2 comprises the amino acid sequence of SEQ ID NO: 202, L-3 comprises the amino acid sequence of SEQ ID NO:203.

[0199] In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of the anti-HER2 full-length antibody, wherein the heavy chain of the anti-HER2 full-length antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of any one of SEQ ID NOs: 183, 184, 204, 205, 251, 252, 253, or 254, or a variant of an amino acid sequence having at least about 80% sequence identity (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) to the amino acid sequence of any one of SEQ ID NOs: 183, 184, 204, 205, 251, 252, 253, or 254. In some embodiments, the full-length antibody comprised in the second antibody portion comprises a light chain, and the light chain comprises the amino acid sequence of SEQ ID NO:185 or a variant of the amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:185.

[0200] In some embodiments, a multispecific antibody is provided, comprising: 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 the first light chain variable region (V L-1 ), wherein said V H-1 comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 141, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 142, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 143, Li) an anti-CD137 single-chain Fv fragment comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 144, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 145, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 146; and b) a full-length antibody that binds to HER2 and comprises two antibody heavy chains and two antibody light chains, wherein the anti-CD137 single-chain Fv fragment is fused to the heavy chain of the full-length antibody, and the full-length antibody that binds to HER2 comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) and a full-length antibody comprising: a) said V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194; and b) said V L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:196, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:198, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:200. 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.

[0201] In some embodiments, the 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, 237, or 247, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 237, or 247, and / or L-1includes the amino acid sequence of SEQ ID NO:8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 238, or 248, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 238, or 248.

[0202] In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO:202 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:202, and / or L-2 includes the amino acid sequence of SEQ ID NO:203 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:203.

[0203] In some embodiments, the V H-3 comprises the amino acid sequence of SEQ ID NO:202 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:202, and / or L-3 includes the amino acid sequence of SEQ ID NO:203 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:203.

[0204] In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of the anti-HER2 full-length antibody, wherein the heavy chain of the anti-HER2 full-length antibody fused to the anti-CD137 single-chain Fv fragment comprises the amino acid sequence of any one of SEQ ID NOs: 183, 184, 204, 205, 251, 252, 253, and 254, or a variant of an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 183, 184, 204, 205, 251, 252, 253, and 254. In some embodiments, the full-length antibody comprised in the second antibody portion comprises a light chain, and the light chain comprises the amino acid sequence of SEQ ID NO:185 or a variant of the amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:185.

[0205] In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:183, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185. In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:184, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185. In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:204, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185. In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:205, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185. In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:251, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185. In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:252, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185. In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:253, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185. In some embodiments, the multispecific antibody comprises an anti-HER2 heavy chain fused to an anti-CD137 scFv comprising the amino acid sequence set forth in SEQ ID NO:254, and an anti-HER2 light chain comprising the amino acid sequence set forth in SEQ ID NO:185.

[0206] In some embodiments, the V of the anti-CD137 single chain Fv fragment H-1 and V L-1is fused to the heavy chain of a 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 amino acids (e.g., about 4 to 20, about 3 to 20, about 6 to 18, or about 10 to 15). In some embodiments, the first linker is a GS linker. In some embodiments, the linker has the amino acid sequence of any one of SEQ ID NOs: 260-230. In some embodiments, the anti-CD137 single-chain Fv fragment is fused to the heavy chain of a 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 amino acids (e.g., about 4 to 20, about 3 to 20, about 6 to 18, or about 10 to 15). In some embodiments, the second peptide linker is a linker comprising the sequence of any one of SEQ ID NOs: 206-230.

[0207] In some embodiments, the full-length antibody has an Fc region selected from the Fc regions of IgG, IgA, IgD, IgE, IgM, and any combination and hybrid thereof. In some embodiments, the Fc region is selected from the Fc regions of 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 S228P, F234A, and L235A mutations.

[0208] o) Multispecific antibody properties In some embodiments, the multispecific antibodies described herein have improved clinical properties relative to a reference multispecific antibody that binds CD137 and a second antigen (e.g., HER2, EGFR, PD-L1). In some embodiments, the multispecific antibodies exhibit 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 antibodies exhibit a greater anti-tumor effect (e.g., reducing tumor burden or improving survival 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 antibodies exhibit less toxicity compared to the toxicity of the reference multispecific antibody.

[0209] In some embodiments, the multispecific antibody binds to both CD137 and a tumor-associated antigen, and exhibits improved ADCC activity against tumor cells positive for or expressing high levels of the indicated tumor-associated antigen compared to the ADCC activity of a reference multispecific antibody (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 than TAA expression in non-tumor cells). In some embodiments, the EC50 for the cell-specific multispecific antibody is not more than about 50%, 40%, 30%, 20%, or 10% of that of the reference multispecific antibody.

[0210] 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 tissue in individuals with cancer or tumors. In some embodiments, significant cytokine release is a cytokine release level that is at least about 70%, 60%, 50%, 40%, 30%, or 20% of the level 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 tissue in individuals with cancer or tumors.

[0211] n) tandem scFv In some embodiments, the multispecific anti-CD137 molecule 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 portion (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 any one of 2, 3, 4, 5, or more) additional scFvs.

[0212] In some embodiments, a tandem scFv multispecific (e.g., bispecific) anti-CD137 antibody is provided, comprising: 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 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 non-CD137 second antigen. 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 second scFvs 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., at least about any one of 2, 3, 4, 5, or more) additional scFvs. 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) nwhere 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.

[0213] In some embodiments, the tandem scFv multispecific anti-CD137 antibody is a tandem bi-scFv comprising two scFvs (herein referred to as a "tandem bi-scFv bispecific anti-CD137 antibody"). A tandem bi-scFv bispecific anti-CD137 antibody can be comprised of V- and V-terminal fragments assembled in any configuration, for example, the configurations listed below (from N-terminus to C-terminus). H and V L where X is a second antigen bound by the second scFv, and L1, L2, and L3 are optional linkers (e.g., peptide linkers). For all suitable linkers, see the "Linker" section. In some embodiments, the linker (L1, L2, or L3) comprises the amino acid sequence SRGGGGSGGGGSGGGGSLEMA. In some embodiments, the linker (L1, L2, or L3) comprises the amino acid sequence (GGGGS) n Either a sequence or (GGGGS) nIn some embodiments, the linker (L1, L2, or L3) comprises the amino acid sequence TSGGGGS. In some embodiments, the linker (L1, L2, or L3) comprises the amino acid sequence GEGTSTGSGGSGGSGGAD.

[0214] 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), In L (CD137)-L1-V H (X)-L2-V L (X)-L3-V H (CD137) In L (CD137)-L1-V L (X)-L2-V H (X)-L3-V H (CD137) In H (CD137)-L1-V H (X)-L2-V L (X)-L3-V L (CD137) In H (CD137)-L1-V L (X)-L2-V H (X)-L3-V L (CD137) In L (X)-L1-V H (CD137)-L2-V L (CD137)-L3-V H (X)、 In L (X)-L1-V L (CD137)-L2-V H (CD137)-L3-V H (X)、 In H (X)-L1-V H (CD137)-L2-V L (CD137)-L3-V L (X)、or In H (X)-L1-V L (CD137)-L2-V H (CD137)-L3-V L (X)。

[0215] 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 half-life extending moiety in a bispecific antibody described herein, or the anti-CD137 scFv and full-length antibody). The length, flexibility, and / or other properties of the linker(s) used in the bispecific antibody can affect its properties, including, but not limited to, affinity, specificity, or binding to one or more particular 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) so that adjacent domains can move freely relative to each other. For example, a glycine-serine dyad 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.

[0216] Other linker considerations include 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 be incorporated into micelles or liposomes, etc.

[0217] Coupling of the two moieties can be accomplished by any chemical reaction that combines the two molecules into a bispecific antibody, e.g., binds CD137 and two antigens, respectively, as long as both components retain their respective activities. This linkage can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. In some embodiments, the bond is covalent. Covalent bonding can be achieved by traditional direct condensation of side chains or by incorporating an external crosslinking molecule. In this case, many bivalent or polyvalent linkers can be used to couple the protein molecules. For example, representative coupling agents can 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, but rather is exemplary of some of the 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)).

[0218] Linkers applicable in this application are 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 examples, non-peptide linkers used herein include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)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)propionamido]hexanoate (Pierce Chemical Co., catalog number #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chemical Co., catalog number #2165-G), (v) sulfo-NHS (N-hydroxysulfo-succinimidyl, Pierce Chemical Co., catalog number #24510) conjugated to EDC.

[0219] The linkers described herein contain components with different properties, potentially resulting in bispecific antibodies with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS esters are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, allowing the formation of antibody fusion proteins with enhanced stability. Disulfide bonds are generally more unstable than other bonds, and disulfide bonds are cleaved in vitro, resulting in less available antibody fusion protein. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. When used in combination with sulfo-NHS, carbodiimide coupling (e.g., EDC) forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0220] 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, WO 1996 / 34103.

[0221] 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 any one of about 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, and in some embodiments, the length of the peptide linker is any one of about 1 to about 10 amino acids, about 1 to about 20 amino acids, about 1 to about 30 amino acids, about 5 to about 15 amino acids, about 10 to about 25 amino acids, about 5 to about 30 amino acids, about 10 to about 30 amino acids, about 30 to about 50 amino acids, about 50 to about 100 amino acids, or about 1 to about 100 amino acids.

[0222] The essential technical feature of such a peptide linker is that it does not contain any polymerization activity. The characteristics of peptide linkers, including the lack of secondary structure-promoting activity, 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 "peptide linkers," the amino acid is particularly preferably Gly. Also preferred are peptide linkers that do not promote any secondary structure. The connection of domains to each other may be provided, for example, by genetic engineering. Methods for preparing fused, operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 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).

[0223] The peptide linker may be a stable linker, which is not cleaved by proteases, in particular matrix metalloproteases (MMPs).

[0224] The linker may be a flexible linker. An exemplary flexible linker is a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n , (GGGGS) n and (GGGS) n(where n is an integer of at least 1), including glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can function as neutral tethers between components. Glycine clearly penetrates 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)). Those skilled in the art will recognize that the design of antibody fusion proteins may include fully or partially flexible linkers, whereby the linker may include a flexible linker portion and one or more portions that confer less flexible structure to provide the desired antibody fusion protein structure.

[0225] p) Immunoconjugates Provided herein further include immunoconjugates, wherein the immunoconjugates comprise any of the anti-CD137 constructs (e.g., multispecific antibodies) described herein linked to a therapeutic agent or marker. In some embodiments, the marker is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0226] q) Nucleic acid Nucleic acid molecules encoding the anti-CD137 constructs or anti-CD137 antibody portions described herein are also contemplated. In some examples, a nucleic acid (or collection of nucleic acids) encoding a full-length anti-CD137 antibody is provided. In some examples, a nucleic acid (or collection of nucleic acids) encoding an anti-CD137 scFv is provided. In some examples, a nucleic acid (or collection of nucleic acids) encoding an anti-CD137 Fc fusion protein is provided. In some examples, a nucleic acid (or collection of nucleic acids) encoding a multispecific anti-CD137 molecule (e.g., a multispecific anti-CD137 antibody or a bispecific anti-CD137 antibody) or a polypeptide portion thereof is provided. In some examples, a nucleic acid (or collection of nucleic acids) encoding an anti-CD137 construct 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).

[0227] The present specification further contemplates isolated host cells comprising an anti-CD137 construct, an isolated nucleic acid encoding an anti-CD137 construct polypeptide component, or a vector comprising a nucleic acid encoding an anti-CD137 construct polypeptide component described herein.

[0228] The present application further includes variants of these nucleic acid sequences, for example, variants that include nucleotide sequences that hybridize to a nucleic acid sequence encoding an anti-CD137 construct or anti-CD137 antibody portion of the present application under at least moderately stringent hybridization conditions.

[0229] The present invention further provides a vector into which the nucleic acid of the present invention is inserted.

[0230] Using standard gene delivery methods, the nucleic acids of the present invention can also be used in nucleic acid immunization and gene therapy. Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties. In some embodiments, the present invention provides gene therapy vectors.

[0231] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0232] 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 chronic viruses. Suitable vectors typically contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584, WO 01 / 29058, U.S. Patent No. 6,326,193).

[0233] III. Preparation method In some examples, methods for preparing anti-CD137 constructs or antibody portions that bind to CD137 are provided, as well as compositions produced during the preparation of the anti-CD137 constructs or antibody portions, such as polynucleotides, nucleic acid constructs, vectors, host cells, or media. The anti-CD137 constructs, antibody portions, or compositions described herein can be prepared by several methods, which are generally described below and specifically described by specific examples.

[0234] 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 those described below and in the specific examples.

[0235] Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical except for possible, naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of individual antibodies. For example, the monoclonal antibodies may be prepared using the hybridoma method first described by Kohler et al. (Nature, 256:495 (1975)), or may be prepared by recombinant DNA methods (U.S. Pat. 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 elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing protein. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). See also camel immunization in Example 1.

[0236] The immunizing agent typically contains the antigen protein or a fusion variant thereof. Usually, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent (e.g., polyethylene glycol) to form a hybridoma cell. (Goding, Monoclonal Antibodies: Principles and Practice, (Academic Press, 1986), pp. 59-103).

[0237] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells thus prepared are seeded and grown in an appropriate medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells are deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma typically contains hypoxanthine, methotrexate, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells.

[0238] Preferred immortalized myeloma cell lines are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to the culture medium (e.g., HAT medium), preferably murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors obtained from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 cells (and their derivatives, e.g., X63-Ag8-653) obtained from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for use in producing 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)).

[0239] The culture medium in which the hybridoma cells are grown is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0240] The culture medium in which the hybridoma cells are cultured may be assayed for the presence of monoclonal antibodies against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody is determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0241] After hybridoma cells capable of producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0242] The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification techniques (e.g., protein A-agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0243] Monoclonal antibodies may also be prepared by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 and herein. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of binding specifically to the heavy and light chain genes encoding murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, and these vectors can be transfected into host cells that do not additionally produce immunoglobulins (e.g., E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells), and the monoclonal antibodies are synthesized in such recombinant host cells. Commentary articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Rev. 130:151-188 (1992).

[0244] In another example, antibodies can be isolated from antibody phage libraries generated 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, respectively, using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) and combinatorial infection and in vivo recombination as a strategy to construct very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). As such, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0245] Additionally, the DNA may be modified, for example, by substituting the coding sequences for the homologous murine sequences for the human heavy and light chain constant domains (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody or for the variable domains of one antigen-binding site of an antibody to produce a chimeric bivalent antibody comprising one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0246] The monoclonal antibodies described herein may be monovalent, and their preparation is well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. Typically, the heavy chain is truncated at a point in the Fc region to prevent cross-linking of the heavy chain. Alternatively, the relevant cysteine ​​residue may be substituted with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Antibody purification to produce antibody fragments (particularly Fab fragments) may be completed using routine techniques known in the art.

[0247] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed by disulfide bond exchange or thioether bond formation. Specific examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0248] Also see the specific examples for the production of monoclonal antibodies.

[0249] multispecific antibodies The present specification further provides methods for preparing the multispecific antibodies (e.g., bispecific antibodies) described herein. Multispecific antibodies may be prepared using any method known in the art or described herein (e.g., Examples 1 and 3).

[0250] Methods for preparing multispecific antibodies of the present application include those described in WO 2008119353 (Genmab), WO 2011131746 (Genmab) and the report by van der Neut-Kolfschoten et al. (Science. 2007 Sep. 14;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-engineered IgG1 (Xencor) or DuoBody.

[0251] Traditional methods, such as hybridization hybridoma and chemical conjugation 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., a heavy chain of an anti-tumor associated antigen full-length antibody fused to an anti-CD137 scFv and a light chain of an anti-tumor associated antigen antibody) may result in the presence of a mixture of antibody products other than the desired bispecific anti-CD137 antibody, which may then be isolated, for example, by affinity chromatography or similar methods.

[0252] Nucleic acid molecules encoding antibody portions In some embodiments, a polynucleotide encoding any one of the anti-CD137 constructs or antibody moieties described herein is provided. In some embodiments, a polynucleotide prepared using any of the methods described herein is provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody moiety (e.g., an anti-CD137 antibody moiety). In some embodiments, a nucleic acid molecule comprises polynucleotides encoding both the heavy chain and the light chain of an antibody moiety (e.g., an anti-CD137 antibody moiety). 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, a nucleic acid molecule encoding an scFv (e.g., an anti-CD137 scFv) is provided.

[0253] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides from a single nucleic acid molecule or from two separate nucleic acid molecules. In some embodiments, for example, when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising the linked heavy and light chains.

[0254] In some examples, a polynucleotide encoding a heavy or light chain of an antibody moiety (e.g., an anti-CD137 antibody moiety) comprises a nucleotide sequence encoding a leader sequence that is located at the N-terminus of the heavy or light chain upon translation. As described above, the leader sequence may be a native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0255] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA.

[0256] The nucleic acid molecule can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0257] Nucleic Acid Constructs In some examples, nucleic acid constructs are provided that include any of the polynucleotides described herein. In some examples, nucleic acid constructs are provided that are prepared using any of the methods described herein.

[0258] 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, wherein the promoter is the wild-type promoter of the gene.

[0259] vector In some embodiments, vectors are provided that include any polynucleotide encoding the heavy and / or light chain of any antibody portion described herein (e.g., an anti-CD137 antibody portion) or a nucleic acid construct described herein. In some embodiments, vectors prepared using any of the methods described herein are provided. Further provided are vectors that include a polynucleotide encoding any anti-CD137 construct (e.g., an antibody, scFv, fusion protein) or other form of construct described herein (e.g., an anti-CD137 scFv). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector includes a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, for example, when the antibody is an scFv.

[0260] In some embodiments, a first vector comprises a polynucleotide encoding a heavy chain, and a second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, the first vector and the second vector are transfected into host cells in a molar 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 to the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain to the vector encoding the light chain.

[0261] In some embodiments, a vector is selected that is optimized for polypeptide expression in CHO or cells of CHO origin or NSO cells. Illustratively, such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0262] host cell In some embodiments, a host cell is provided that comprises any of the polypeptides, nucleic acid constructs, and / or vectors described herein. In some embodiments, a host cell is provided that is prepared using any of the methods described herein. In some embodiments, the host cell is capable of producing any of the antibody portions described herein under fermentation conditions.

[0263] In some examples, an antibody portion (e.g., an anti-CD137 antibody portion) described herein 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 carried out, for example, according to methods known in the art. Exemplary eukaryotic cells for expressing polypeptides include 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 cells, and the like. (登録商標) Examples of suitable eukaryotic host cells include, but are not limited to, yeast cells (Crucell), and NSO cells. In some embodiments, an antibody portion described herein (e.g., an anti-CD137 antibody portion) can be expressed in yeast. See, e.g., U.S. Patent No. US 2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to 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.

[0264] Introduction of one or more nucleic acids into desired host cells 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 exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transfected instantaneously or stably into desired host cells according to any suitable method.

[0265] The present invention further provides host cells comprising any of the polynucleotides or vectors described herein. In some embodiments, the present invention provides host cells comprising an anti-CD137 antibody. Any host cell capable of overexpressing heterologous DNA may be used for the purpose of isolating genes encoding antibodies, polypeptides, or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., Escherichia coli or Bacillus subtilis) and yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Kluyveromyces lactis).

[0266] In some embodiments, the antibody portion 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), and Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0267] Culture medium In some examples, media are provided that include any of the antibody portions, polynucleotides, nucleic acid constructs, vectors, and / or host cells described herein. In some examples, media are provided that are prepared using any of the methods described herein.

[0268] In some embodiments, the medium contains hypoxanthine, aminopterin, and / or thymidine (e.g., HAT medium). In some embodiments, the medium does not contain serum. In some embodiments, the medium contains serum. In some embodiments, the medium is D-MEM or RPMI-1640 medium.

[0269] Purification of antibody moieties Anti-CD137 constructs (e.g., anti-CD137 monoclonal antibodies or bispecific antibodies) may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the ROR1 ECD and the constant region of antibodies. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns may be used to purify anti-CD137 constructs that bind to the constant region and include an Fc region. Hydrophobic interaction chromatography, such as a butyl column or a phenyl column, may also be applied to the purification of some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be applied to the purification of some polypeptides, such as 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, such as antibodies. Numerous methods for purifying polypeptides are known in the art.

[0270] IV. Methods of Modulating Cellular Composition Any of the anti-CD137 constructs described herein (including any multispecific antibodies) 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 an anti-CD137 construct. In some embodiments, the contacting, or at least a portion of the contacting, occurs ex vivo. In some embodiments, the contacting, or at least a portion of the contacting, occurs in vivo.

[0271] contact In some embodiments, the contacting occurs in the presence of an agent. In some embodiments, the agent binds to CD3 (e.g., an anti-CD3 antibody). In some embodiments, the agent binds to CD28 (e.g., an 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 includes one or more (e.g., one, two, three, four, or five) agents selected from the group consisting of an agent that binds to CD3, an agent that binds to CD28, IL-2, TNF-α, and IFNγ. In some embodiments, the agent includes an agent that binds to CD3 (e.g., an anti-CD3 antibody), IL-2, and IFNγ.

[0272] In some embodiments, the concentration of the agent (e.g., an 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.

[0273] In some embodiments, the contacting is for at least about 1 hour, 2 hours, 4 hours, 8 hours, or overnight. In some embodiments, the contacting is for at least about 1 day, 2 days, or 3 days. In some embodiments, the contacting is for less than about 24 hours, 12 hours, or 8 hours. In some embodiments, the contacting is for less than about 14 days, 10 days, 7 days, 5 days, or 3 days. In some embodiments, the contacting is for about 0-48 hours, 1-24 hours, 2-20 hours, 4-16 hours, or 8-12 hours.

[0274] In some embodiments, the contacting is carried out at a temperature of about 0-20° C. In some embodiments, the contacting is carried out at a temperature of about 2-8° C.

[0275] 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 are 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 are 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 are 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-engineered 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.

[0276] In some embodiments, the cells are pretreated or co-treated with an agent. In some embodiments, the agent binds to CD3 (e.g., an anti-CD3 antibody). In some embodiments, the agent binds to CD28 (e.g., an 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 includes 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, an agent that binds to CD28, IL-2, TNF-α, and IFNγ.

[0277] In some embodiments, the concentration of the agent (e.g., an 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.

[0278] In some embodiments, after said contacting, the cells in said composition are administered to the individual.

[0279] V. Methods of Treating or Modulating an Individual's Immune Response Further provided herein are methods of treating a disease or condition in an individual or modulating an immune response in an individual, the methods comprising administering to an individual (e.g., a mammal, e.g., a human) any of the anti-CD137 constructs described herein.

[0280] Further provided in some embodiments is a method of treating a disease or condition in an individual or modulating the immune response of an individual, comprising administering to said individual an effective amount of an anti-CD137 construct disclosed herein, wherein the construct is any of the multispecific antibodies described herein.

[0281] In some embodiments, the disease or condition 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 about 0.005 μg / kg to about 5 μg / kg of the individual's total body weight. In some embodiments, the antibody agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.

[0282] In some embodiments, the individual is a mammal (e.g., a human, a non-primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In some embodiments, the individual is under about 60 years of age (e.g., under about any one of 50, 40, 30, 25, 20, 15, or 10 years of age). In some embodiments, the individual is greater than about 60 years of age (inclusive of any one of 70, 80, 90, or 100 years of age). In some embodiments, the individual is diagnosed with or has a genetic predisposition to one or more diseases or disorders described herein (e.g., cancer, an autoimmune disease, or a transplant). In some embodiments, the individual has one or more risk factors associated with one or more diseases or disorders described herein.

[0283] Regulation of immune responses In some embodiments, modulating an immune response comprises modulating a population of cells in an individual. In some embodiments, the population of cells is a T cell population. In some embodiments, the population of cells is a dendritic cell. In some embodiments, the population of cells is a macrophage. In some embodiments, the population of cells is a B cell. In some embodiments, the population of cells is a NK cell. In some embodiments, the population of cells is an effector T cell and / or a memory T cell. In some embodiments, the population of cells is an effector / memory T cell phenotypically defined by high CD44 and low CD62L.

[0284] In some embodiments, the modulation comprises promoting proliferation of the cell population. In some embodiments, proliferation of the cell population after administration of the anti-CD137 construct is increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to proliferation of reference cells after administration of a control construct (e.g., an antibody that does not bind to anti-CD137). In some embodiments, proliferation of the cell population after administration of the anti-CD137 construct 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 compared to proliferation of reference cells after administration of a control construct (e.g., an antibody that does not bind to CD137).

[0285] Use of anti-CD137 polyspecific antibodies In some embodiments, provided are methods of treating a disease or condition (e.g., cancer) in an individual, comprising administering to said individual an effective amount of a multispecific antibody disclosed herein.

[0286] In some embodiments, methods are provided for treating tumors or cancer, comprising administering to an individual an effective amount of a multispecific antibody disclosed herein.

[0287] Use of CD137 x HER2 multispecific antibodies In some examples, methods are provided for treating HER2+ cancer in an individual, comprising administering to said individual a CD137 x HER2 multispecific antibody (e.g., any of the CD137 x HER2 bispecific antibodies described herein).

[0288] In some embodiments, the cancer is HER2 positive. 高 In some embodiments, the HER2-positive cancer or HER2 高 The cancer is selected from breast cancer, gastric cancer, ovarian cancer, melanoma, head and neck cancer, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma and cervical cancer.

[0289] In some embodiments, the HER2-positive cancer or HER2 高 The cancer is breast cancer or gastric cancer. 高 The cancers have average HER2 expression equal to or greater than that of NCI-N87 or SKBR3 cells.

[0290] In some embodiments, the HER2 高 The cancer has an average HER2 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 HER2 expression of SK-Hep1 cells or MDA-MB-231 cells. In some embodiments, the cancer has an average HER2 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 HER2 高 The cells have a HER2 expression level equivalent to that of NCI-N87 cells or SKBR3 cells (for example, within about 50% to about 200%, about 67% to about 150%, or about 75% to about 125% of that).

[0291] Disease or illness The anti-CD137 constructs described herein may be used to treat any disease or condition. In some embodiments, the disease or condition is an infection (e.g., a bacterial infection or a viral infection). In some embodiments, the disease or condition is an autoimmune disorder. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is transplantation.

[0292] In some embodiments, the anti-CD137 constructs are used in methods for treating cancer. Cancers that can be treated using any of the methods described herein include unvascularized or poorly vascularized tumors and vascularized tumors. As described herein, cancer types that can be treated with anti-CD137 constructs include, but are not limited to, epithelial carcinomas, blastomas, sarcomas, benign and malignant tumors, and malignant tumors (e.g., sarcomas, epithelial carcinomas, and melanomas). Adult tumors / cancers and pediatric tumors / cancers are also included. In some embodiments, the cancer is a solid tumor.

[0293] In each embodiment, the cancer is an early stage cancer, a non-metastatic cancer, a primary cancer, an advanced cancer, a locally advanced cancer, a metastatic cancer, a cancer in remission, a recurrent cancer, a cancer in adjuvant therapy, a cancer in neoadjuvant therapy, or a substantially incurable cancer.

[0294] Specific examples of cancers that can be treated by the methods of the present application include anal cancer, astrocytoma (e.g., cerebellum and brain), basal cell carcinoma, bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., astrocytoma, malignant glioma, meloblastoma, 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, and hepatocellular (liver) cancer (e.g., hepatocellular carcinoma and hepatocellular carcinoma). , leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoid tumors (e.g., lymphocele), medulloblastoma, melanoma, mesothelioma, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, peritoneal cancer, pituitary tumor, lymphoma, rectal cancer, kidney cancer, renal pelvis cancer and ureter 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).

[0295] 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).

[0296] Methods for administering the anti-CD137 constructs 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 disease or disorder is cancer. In some embodiments, an 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, an 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, an 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, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount sufficient to produce an overall response rate of 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 treated with the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody). For example, an individual's response to treatment with the methods described herein can be determined based on RECIST levels.

[0297] In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is sufficient to extend progression-free survival in an individual. In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is sufficient to extend overall survival in an individual. In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is sufficient to produce a clinical benefit in greater than about 50%, 60%, 70%, or 77% of an individual population treated with the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody).

[0298] 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 decrease tumor growth rate by at least about any one of 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 in the same subject before treatment, or compared to the corresponding activity in another subject not receiving treatment (e.g., receiving a placebo). 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 studies.

[0299] In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is below a level that induces toxic effects (i.e., effects that are greater than a clinically acceptable level of toxicity) or is at a level that allows potential side effects to be managed or tolerated when the composition is administered to an individual.

[0300] In some embodiments, the effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is close to the maximum tolerated dose (MTD) of the composition based on the same dosing regimen, and in some embodiments, the effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is greater than about 80%, 90%, 95%, or 98% of the MTD.

[0301] In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount that slows or inhibits progression of the disease or condition (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%) compared to an individual not receiving treatment. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an infection.

[0302] In some embodiments, an effective amount of the anti-CD137 construct (e.g., an anti-CD137 monoclonal or bispecific antibody) is an amount that reduces the side effects (e.g., autoimmune response) of a disease condition (e.g., transplant) (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50%) compared to an individual not receiving the treatment.

[0303] 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, e.g., 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.

[0304] In some embodiments, the treatment comprises one or more administrations of the anti-CD137 construct (e.g., about two, three, four, five, six, seven, eight, nine, or ten administrations of the anti-CD137 construct). In some embodiments, the two administrations are administered within about one week. In some embodiments, the second administration is administered at least about 1, 2, 3, 4, 5, 6, or 7 days after the first administration is completed. In some embodiments, the second administration is administered about 1-14 days, 1-10 days, 1-7 days, 2-6 days, or 3-5 days after the first administration is completed. 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).

[0305] The anti-CD137 construct can be administered to an individual (e.g., a human) by a variety of routes, including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesical, 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, sustained continuous release formulations 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.

[0306] Combination therapy The present application further provides methods of treating a disease or condition (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 condition. In some embodiments, the second agent or therapy comprises a chemotherapy 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.

[0307] In some embodiments, the anti-CD137 construct is administered simultaneously with a second agent or therapy. In some embodiments, the anti-CD137 construct is administered concurrently with a second agent or therapy. In some embodiments, the anti-CD137 construct is administered sequentially with a 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.

[0308] In some embodiments, the second agent or therapy is an agent that binds to HER2 (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 comprises a T cell (e.g., a CAR T cell). In some embodiments, the second agent comprises a cytokine. In some embodiments, the second agent or therapy comprises carboplatin, paclitaxel, and / or radiation therapy. In some embodiments, the second agent or therapy comprises a vaccine, e.g., an HPV vaccine. In some embodiments, the second agent or therapy comprises an EGFR inhibitor (e.g., cetuximab). In some embodiments, the second agent or therapy comprises an anti-tumor enzyme inhibitor (e.g., irinotecan). See exemplary combination therapies in Table 3 below.

[0309] [Table 3]

[0310] VI. Compositions, Kits and Articles of Manufacture The present specification further provides compositions (e.g., formulations) comprising any one of the anti-CD137 constructs or anti-CD137 antibody portions described herein, nucleic acids encoding the antibody portions, vectors comprising nucleic acids encoding the antibody portions, or host cells comprising the nucleic acids or vectors.

[0311] Suitable anti-CD137 construct formulations described herein can be obtained in the form of a lyophilized formulation or aqueous solution by mixing the anti-CD137 construct or anti-CD137 antibody portion having the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations used, and include buffers (e.g., phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine), preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parahydroxybenzoates such as methylparaben or propylparaben, catechin, resorcinol, cyclohexanol, 3-pentanol, and meta-cresol). low molecular weight (fewer 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 dextrins; 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 a non-ionic surfactant such as polyethylene glycol (PEG). Lyophilized formulations suitable for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the individual being imaged, diagnosed, or treated herein.

[0312] Formulations for in vivo administration must be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0313] Further provided are kits comprising any one of the anti-CD137 constructs or anti-CD137 antibody moieties described herein, which may be used in any of the methods for modulating or treating cell compositions described herein.

[0314] In some examples, kits are provided that contain anti-CD137 constructs that bind to CD137.

[0315] 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 into a subject in vivo. Aspiration devices may also be used in some uses.

[0316] In some embodiments, the kit further comprises a therapeutic agent for treating a disease or condition (eg, cancer, an infectious disease, an autoimmune disease, or a transplant).

[0317] The kits of the present application are in suitable packaging, including but not limited to vials, bottles, cans, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kits can optionally provide other components, such as buffers and instructional information.

[0318] Therefore, the present application further provides an article of manufacture. The article of manufacture may include a container and a marker or packaging insert on or associated with the container. Suitable containers include vials (e.g., sealed vials), bolts, cans, flexible packaging, etc. Typically, the container contains 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 injection needle). The marker or packaging insert indicates that the composition is used for imaging, diagnosing, or treating a particular 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 instructions for recombination and / or use. 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 package insert typically refers to a manual included in the commercial packaging of a diagnostic product, which contains information regarding the indications, methods of use, dosage, administration, contraindications, and / or warnings for using such a diagnostic product. The product may also further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0319] The kit or product may include multiple unit doses of the composition packaged in an amount sufficient for storage and use in a pharmacy (eg, a hospital pharmacy and a compounding pharmacy) and instructions for use.

[0320] Those skilled in the art will recognize that multiple embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail with reference to the following non-limiting examples. It should be understood that the following examples further illustrate the present invention but should not be construed as in any way limiting its scope. [Table 4-1] Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 [Table 4-19] [Table 4-20] [Table 4-21] [Example]

[0321] The following specific examples are merely examples of the present application and should not be construed as limiting the present application in any way. The following specific examples and detailed description are offered by way of illustration and not by way of limitation.

[0322] Example 1: Production of anti-CD137 antibodies A. Production of anti-CD137 antibodies from mouse hybridomas Fully human monoclonal antibodies against the human CD137 (4-1BB) receptor were produced in BALB / c mice. The extracellular domain of human CD137 in RIBI adjuvant (Ribi Immunochemical) was used to immunize BALB / c mice. Prior to fusion, the same amount of antigen was used to boost the mice intravenously (iv). Lymph nodes from immunized mice with sufficient huCD137 antibody titers were fused with mouse myeloma cells using standard methods.

[0323] Hybridoma screening. Binding to huCD137 was detected by ELISA. To identify hybridomas secreting anti-human CD137 antibodies, ELISA plates (Corning) were coated overnight at 4°C with 1 μg / ml human CD137-Fc fusion protein in PBS. The plates were then washed three times with PBS containing 0.5% Tween-20 (PBS-T) and blocked with 5% milk in PBS-T for 60 minutes at room temperature. Thirty microliters of supernatant diluted 1:3 in PBS was added to the plates and incubated at ambient temperature for 1-2 hours. The plates were then washed as described above, and antibody binding was detected with goat F(ab')2 anti-human IgG conjugated to horseradish peroxidase (HRP). The plates were developed with TMB and read at 450 nm. The amino acid sequences of the optimal anti-CD137 hybridoma antibodies (cloning numbers 29.39, 44.21, 3.10, 30.19, and 6.62) identified from the screening are shown in the sequence listing (SEQ ID NO: 151-176) and Table 4.

[0324] [Table 5]

[0325] B. Humanization of Anti-CD137 Antibodies A representative clone, 29.39, was selected for framework humanization. Briefly, the clone's sequence was subjected to Igblast to search a database of human germline genes. The desired germline sequence was selected, and mutations were performed on the framework sequence to change it from mouse to human. For the heavy chain, human germline IGHV1-46*01 was used, and the following mutations were made in the framework: E1Q, K3Q, M5V, E6Q, L11V, 20V, T23K, K38R, R40A, E42G, I48M, D60A, L61Q, N62K, A67V, I69M, A71R, S75T, N76S, A78V, L80M, Q81E, T83R, S111V, L112S, and E113S. For the light chain, human germline IGKV1-12*01 was used with the following framework mutations: V3Q, M11V, Y12S, L15V, E17D, F36Y, S43A, T46L, Q69T, Y71F, S72T, D79Q, Y80P, M83F, G84A, and I85T. The construct was cloned into an expression vector and antibody protein was produced in SS320 cells.

[0326] C. Affinity Maturation, Selection and Modification Affinity maturation was performed on the humanized 29.39 clones. Primers were designed to mutate single amino acids in each CDR region. Mutation libraries were prepared using assembly PCR and cloned into phagemid vectors. Library quality was assessed by DNA sequencing of transformed TG1 cells and clones. Phage production was performed using helper phage, and phage panning was performed using streptavidin-conjugated Dynabeads coated with biotinylated human CD137 ECD or cynomolgus monkey CD137 ECD. After three rounds of panning, the panning products were eluted for infection of SS320 cells, and colonies were picked and cultured in 2YT medium with IPTG. The binding affinity of the Fab in the supernatant was tested by ELISA assay. Positive clones against human and cynomolgus monkey CD137 were selected. The best 14 binders (clone numbers 3, 9, 23, 25, 33, 35, 5, 6, 17, 18, 19, 20, 2-9, and 2-11) and their CDRs (Kabat), VH, VL, heavy chain (HC), and light chain (LC) are shown in the sequence listing (SEQ ID NOS: 1-140). The shared sequences of these CDRs (slightly altered Kabat), VH, VL, heavy chain (HC), and light chain (LC) are shown in the sequence listing (SEQ ID NOS: 141-150). Subclones 2-9 containing different IgG subtypes were also synthesized. Their CDR regions (Kabat), VH, VL, heavy chain (HC), and light chain (LC) are shown in the sequence listing (SEQ ID NOS: 231-250).

[0327] Example 2: Characterization of exemplary anti-CD137 antibodies A. Affinity Assessment Biolayer interferometry was used to measure the binding kinetics of anti-CD137 antibodies, measuring their association and dissociation constants. Binding kinetics were measured at 30°C on a FortieBio Octet Red 96 and analyzed using FortieBio Data Analysis 9.0 software. An anti-human IgG Fc (AHC) sensor was used to capture anti-CD137 antibodies or control antibodies. During data processing, the kinetic buffer hole alone was set as a reference hole for background subtraction. For each antibody-antigen binding event, data were fitted using a 1:1 Langmuir model with global fitting for Rmax coupling for association and dissociation. Human CD137-his is a recombinant CD137 antigen (Sino Biological, Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 10041-H08H) with a polyhistidine tag fused to the C-terminus of the human CD137 antigen. Cynomolgus CD137-his is a recombinant CD137 antigen (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 90847-K02H-100) with a polyhistidine tag fused to the C-terminus of cynomolgus CD137 antigen.

[0328] As shown in Fig...

Claims

1. A multispecific antibody that binds to CD137 and HER2, comprising a first antibody portion that binds to CD137 and a second antibody portion that binds to HER2; The first antibody portion comprises a heavy chain variable region (V) comprising HC-CDR1, HC-CDR2 and HC-CDR3 domains. Ｈ ) and a light chain variable region (V) comprising LC-CDR1, LC-CDR2 and LC-CDR3 domains. Ｌ wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of: i) 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; 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; ii) the V Ｈ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233; Ｌ comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; is selected from The second antibody portion comprises a second heavy chain variable region (V Ｈ－２ ) and a second light chain variable region (V Ｌ－２ ) and V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194; Ｌ－２ A multispecific antibody comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 196, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 198, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

200.

2. The first antibody portion comprises: (i) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 127, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 128; or (ii) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 237, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 238; 2. The multispecific antibody of claim 1 , comprising:

3. The first antibody portion may be a full-length antibody, a multispecific antibody (e.g., 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 , V Ｈ 3. The multispecific antibody of claim 1 or 2, comprising an antibody or antigen-binding fragment thereof selected from the group consisting of H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, and tetrabody.

4. The multispecific antibody of any one of claims 1 to 3, wherein the first antibody portion is a CD137 agonist antibody.

5. The multispecific antibody of any one of claims 1 to 4, wherein the second antibody portion comprises an Fc region selected from the group consisting of Fc regions from IgG1, IgG2, IgG3, IgG4, and any combination and hybrid thereof.

6. The multispecific antibody of claim 5 , wherein the Fc region comprises an IgG1 Fc region.

7. The multispecific antibody of claim 6 , wherein the IgG1 Fc region comprises an L234A mutation and an L235A mutation.

8. The multispecific antibody of claim 5 , wherein the Fc region comprises an IgG4 Fc region.

9. The multispecific antibody of claim 8 , wherein the IgG4 Fc region comprises an F234A mutation and an L235A mutation.

10. The multispecific antibody of claim 8 or 9, wherein the IgG4 Fc region further comprises a S228P mutation.

11. The V Ｈ－２ comprises the amino acid sequence of SEQ ID NO: 202, Ｌ－２ The multispecific antibody according to any one of claims 1 to 10, comprising the amino acid sequence of SEQ ID NO:

203.

12. a) Heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) and 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, Ｌ a 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; b) a second heavy chain variable region (V Ｈ－２ ) and a second light chain variable region (V Ｌ－２ ) and Ｈ－２ comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, Ｌ－２ a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

200.

13. a) Heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) and Ｈ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 231, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 232, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 233; Ｌ a first antibody portion comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; b) a second heavy chain variable region (V Ｈ－２ ) and a second light chain variable region (V Ｌ－２ ) and Ｈ－２ comprises a second HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a second HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a second HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 194, Ｌ－２ a second LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 196, a second LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 198, and a second LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

200.

14. i) a heavy chain of an anti-HER2 full-length antibody fused to an anti-CD137 single-chain Fv fragment comprising any one of the amino acid sequences of SEQ ID NOs: 183, 184, 204, 205, 251, 252, or 254; ii) a light chain of an anti-HER2 full-length antibody comprising the amino acid sequence of SEQ ID NO:

185.

15. 15. An immunoconjugate comprising the multispecific antibody of any one of claims 1 to 14 linked to a therapeutic agent or marker.

16. The immunoconjugate of claim 15 , wherein the marker is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

17. 17. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 14 or an immunoconjugate according to claim 15 or 16 and a pharmaceutically acceptable carrier agent.

18. An isolated nucleic acid encoding a multispecific antibody according to any one of claims 1 to 14.

19. A vector comprising the isolated nucleic acid of claim 18.

20. 20. An isolated host cell comprising the isolated nucleic acid of claim 18 or the vector of claim 19.

21. 1. A method for producing a multispecific antibody, comprising: a) culturing the isolated host cell of claim 20 under conditions that effectively express the multispecific antibody; b) obtaining the expressed multispecific antibody from said host cell.

22. The multispecific antibody according to any one of claims 1 to 14, for use as a drug.

23. The multispecific antibody according to any one of claims 1 to 14, for use in the treatment of cancer.

24. 24. The multispecific antibody of claim 23, wherein 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, thymus cancer, adrenal cancer, head and neck cancer, brain cancer, thyroid cancer, sarcoma, myeloma and leukemia.

25. 21. A kit comprising a multispecific antibody according to any one of claims 1 to 14, an immunoconjugate according to claim 15 or 16, a pharmaceutical composition according to claim 17, a nucleic acid according to claim 18, a vector according to claim 19 or a host cell according to claim 20.

26. 26. The kit of claim 25, further comprising a manual for treating and / or preventing cancer or tumors.

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