Anti-CD137 antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human CD137, multispecific antibodies and antigen-binding fragments thereof that specifically bind to human GPC3 and CD137, pharmaceutical compositions comprising the antibodies, and uses of the antibodies, multispecific antibodies, or compositions for treating diseases such as cancer.

Description

[Technical Field]

[0001] Disclosed herein are antibodies that specifically bind to human CD137 (TNF receptor superfamily member 9 (TNFRSF9)), multispecific antibodies that bind to human CD137 or antigen-binding fragments thereof, compositions comprising the antibodies, and methods of use for the treatment of cancer. [Background technology]

[0002] Glypican-3 (GPC3) belongs to the heparan sulfate proteoglycan (HSPG) family and contains a 60-70 kD core protein that is attached to the surface of the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The carboxy terminus of GPC3 is modified with heparan sulfate side chains (Filmus J et al., J. Clin. Inv. 2001;108:497-501).

[0003] The specific expression of GPC3 in tumor cells has attracted widespread attention. GPC3 is expressed in hepatocellular carcinoma (HCC), the most common type of liver cancer. Notably, its expression is not detected in non-malignant tissues. Overexpression of GPC3 has also been reported in hepatoblastoma, lung squamous cell carcinoma (LSCC), and other cancers. Therefore, GPC3 is suitable as a tumor antigen for targeted therapy (Li N et al., Trends Cancer. 2018;4:741-54; Ho M, et al., Eur J Cancer. 2011;47:333-8; Moek et al., Am. J. Pathol. 2018;188(9):1973-1981).

[0004] CD137 (also known as TNFRSF9 / 41BB) is a costimulatory molecule belonging to the TNFRSF family. It was discovered by T cell factor screening in murine helper and cytotoxic cells stimulated with concanavalin A. It was identified in 1989 as an inducible gene expressed in antigen-primed but not resting T cells (Kwon et al., Proc. Natl. Acad. Sci. USA. 1989;86:1963-1967). Furthermore, it is known to be expressed on dendritic cells (DCs), natural killer cells (NK) (Vinay et al., Mol. Cancer Ther. 2012;11:1062-1070), activated CD4+ and CD8+ T lymphocytes, eosinophils, natural killer T cells (NKT), and mast cells (Kwon et al., 1989 supra; Vinay D., Int. J. Hematol. 2006;83:23-28). CD137 maintains and enhances immune effector functions by inducing Th1 cytokine production (Bartkowiak et al., Front Oncol. 2015;5:117; Shuford et al., J Exp Med. 1997;186:47-55). CD137 signaling leads to increased expression of pro-survival molecules through NF-κB pathway activation when CD137 binds to its only ligands (CD137L, 4-1BBL, or TNFSF9) (Wang et al., Immunol Rev. 2009;229:192-215).

[0005] The anti-CD137 antibodies urelumab (BMS-663513), which binds to CRD I of CD137, and utomilumab (PF-05082566), which binds to CRDs III and IV of CD137, show potential as cancer therapeutics due to their ability to activate cytotoxic T cells and increase interferon-gamma (IFN-γ) production. The mechanism underlying tumor regression by these antibodies is their enhancing effect on immune cell responses to cancer. Specifically, anti-CD137 antibodies stimulate and activate effector T lymphocytes (e.g., by stimulating CD8+ T lymphocytes to produce IFN-γ) and enhance the production of NKT and APCs (e.g., macrophages).

[0006] Urelumab demonstrated promising results in preclinical experiments and early clinical trials (Sznol et al., Clin. Oncol. 2008;26(Suppl. 15)). However, later studies demonstrated hepatotoxicity, resulting in a pause in development of this antibody until February 2012 (Segal et al., Clin. Cancer Res. 2017;23:1929-1936). The hepatotoxicity was primarily due to the S100A4 protein secreted by tumor and stromal cells, and dose-limiting studies of urelumab to 8 mg or 0.1 mg / kg per patient every 3 weeks revived interest in this antibody (Segal et al., Clin. Cancer Res. 2017;23:1929-1936).

[0007] In contrast to urelumab, utomilumab has shown a more favorable safety profile, with early studies showing no hepatotoxicity or other dose-limiting factors (Segal et al., J. Clin. Oncol. 2014;32(Suppl. 15)). Results reported from a phase I trial of utomilumab as monotherapy showed a favorable safety profile (Segal et al., Clin. Cancer Res. 2018;24:1816-1823). The differences between the two antibodies are speculated to be due to their different binding sites on the CD137 receptor.

[0008] There are no approved therapeutic antibodies against CD137, and there remains an unmet medical need for CD137-targeting therapies. Furthermore, anti-TAAxCD137 multispecific antibodies that recruit immune cells to tumor-associated antigen (TAA)-expressing cancers are useful for cancer treatment. Summary of the Invention

[0009] The present disclosure includes antibodies and antigen-binding fragments that specifically bind to human CD137. Furthermore, the CD137 VHH domain fragments disclosed herein can be used to construct multispecific antibodies with other modalities, such as TAAs, immune checkpoints, or immune stimulators. CD137 antibodies may be used alone or in combination with other modalities to treat or prevent cancer, autoimmune diseases, or infectious diseases.

[0010] The present disclosure is also directed to multispecific anti-GPC3xCD137 antibodies and antigen-binding fragments thereof.

[0011] The present disclosure encompasses the following embodiments.

[0012] Embodiment 1. An antibody or antigen-binding fragment thereof that specifically binds to human CD137, (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35); (2) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35); or (3) The antibody or antigen-binding fragment thereof specifically binds to a human CD137 dimer comprising or consisting of a first human CD137 monomer and a second human CD137 monomer, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope on the first human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67, and the antibody or antigen-binding fragment thereof specifically binds to an epitope on the second human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and / or the antibody or antigen-binding fragment thereof binds to a human CD137 dimer and promotes human CD137 clustering.

[0013] Embodiment 2. An antibody or antigen-binding fragment thereof that specifically binds to human CD137, (i) a heavy chain variable region (VH) comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, or (ii) The antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 10, and (c) HCDR3 of SEQ ID NO: 3.

[0014] Embodiment 3. An antibody or antigen-binding fragment thereof according to any one of Embodiments 1 and 2, (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or (v) the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 4.

[0015] Embodiment 4. The antibody or antigen-binding fragment thereof of Embodiment 3, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 4 are inserted, deleted, or substituted.

[0016] Embodiment 5. An antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, comprising: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15, or (v) the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO: 4.

[0017] Embodiment 6. The antibody or antigen-binding fragment thereof of any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0018] Embodiment 7. The antibody or antigen-binding fragment thereof of any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type.

[0019] Embodiment 8. The antibody or antigen-binding fragment thereof of any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0020] Embodiment 9. An antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof has reduced or no glycosylation or is hypofucosylated.

[0021] Embodiment 10. The antibody or antigen-binding fragment thereof of any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises an increase in bisecting GlcNac structures.

[0022] Embodiment 11. The antibody or antigen-binding fragment thereof of any one of the preceding embodiments, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 53.

[0023] Embodiment 12. The antibody or antigen-binding fragment thereof of any one of the preceding embodiments, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function and / or extended half-life, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO: 20.

[0024] Embodiment 13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0025] Embodiment 14. A method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 12, or the pharmaceutical composition of Embodiment 13.

[0026] Embodiment 15. The method of embodiment 14, wherein the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0027] Embodiment 16. The method of any one of Embodiments 14-15, wherein the antibody or antigen-binding fragment thereof is administered in combination with another therapeutic agent.

[0028] Embodiment 17. The method of embodiment 16, wherein the therapeutic agent is an anti-PD-1 antibody.

[0029] Embodiment 18. The method of embodiment 17, wherein the anti-PD1 antibody is tislelizumab.

[0030] Embodiment 19: At least a first antigen-binding domain that specifically binds to a human tumor-associated antigen (TAA), and A multispecific antibody or antigen-binding fragment thereof comprising at least a second antigen-binding domain that specifically binds to human CD137, wherein the second antigen-binding domain comprises: (1) an antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35); (2) an antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35); or (3) The multispecific antibody or antigen-binding fragment thereof, which is an antibody or antigen-binding fragment thereof that specifically binds to a human CD137 dimer comprising or consisting of a first human CD137 monomer and a second human CD137 monomer, and which specifically binds to an epitope on the first human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67, and specifically binds to an epitope on the second human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and / or binds to the human CD137 dimer and promotes human CD137 clustering.

[0031] Embodiment 20. A multispecific antibody or antigen-binding fragment thereof, comprising at least a first antigen-binding domain that specifically binds to a human tumor-associated antigen (TAA) and at least a second antigen-binding domain that specifically binds to human CD137, wherein the second antigen-binding domain is: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3; or (ii) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO: 3.

[0032] Embodiment 21. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 20, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 4.

[0033] Embodiment 22. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 21, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15, or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 4.

[0034] Embodiment 23. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 22, wherein the TAA is GPC3.

[0035] Embodiment 24. A multispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to human glypican 3 (GPC3) and a second antigen-binding domain that specifically binds to human CD137.

[0036] Embodiment 25. The multispecific antibody or antigen-binding fragment thereof of embodiment 24, wherein the second antigen-binding domain that specifically binds to human CD137 is: (1) an antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35); (2) an antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35); or (3) The multispecific antibody or antigen-binding fragment thereof, which is an antibody or antigen-binding fragment thereof that specifically binds to a human CD137 dimer comprising or consisting of a first human CD137 monomer and a second human CD137 monomer, and which specifically binds to an epitope on the first human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67, and specifically binds to an epitope on the second human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and / or binds to the human CD137 dimer and promotes human CD137 clustering.

[0037] Embodiment 26: The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 24 to 25, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3; or (ii) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO: 3.

[0038] Embodiment 27 The multispecific antibody or antigen-binding fragment thereof of Embodiment 26, wherein the second antigen-binding domain that specifically binds to human CD137 is: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 4.

[0039] Embodiment 28. The multispecific antibody or antigen-binding fragment thereof of Embodiment 27, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 4 are inserted, deleted, or substituted.

[0040] Embodiment 29. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 24 to 28, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 17; (ii) A heavy chain variable region (VH) containing SEQ ID NO: 11, (iii) A heavy chain variable region (VH) containing SEQ ID NO: 13, (iv) A heavy chain variable region (VH) containing SEQ ID NO: 15, or (v) A heavy chain variable region (VH) containing SEQ ID NO: 4, wherein the multispecific antibody or an antigen-binding fragment thereof comprises the same.

[0041] Embodiment 30. The multispecific antibody or an antigen-binding fragment thereof according to any one of Embodiments 19 to 29, wherein the first antigen-binding domain specifically binding to human GPC3 is (a) A heavy chain variable region (VH) containing HCDR1 of SEQ ID NO: 45, (b) HCDR2 of SEQ ID NO: 46, and (c) HCDR3 of SEQ ID NO: 47, and (d) A light chain variable region (VL) containing LCDR1 of SEQ ID NO: 48, (e) LCDR2 of SEQ ID NO: 49, and (f) LCDR3 of SEQ ID NO: 50, wherein the multispecific antibody or an antigen-binding fragment thereof comprises the same.

[0042] Embodiment 31. The multispecific antibody or an antigen-binding fragment thereof according to any one of Embodiments 19 to 30, wherein the first antigen-binding domain specifically binding to human GPC3 is A heavy chain variable region (VH) containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 41, and a light chain variable region (VL) containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 43, wherein the multispecific antibody or an antigen-binding fragment thereof comprises the same.

[0043] Embodiment 32. The multispecific antibody or an antigen-binding fragment thereof according to Embodiment 31, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NO: 41 or SEQ ID NO: are inserted, deleted or substituted, and the multispecific antibody or an antigen-binding fragment thereof comprises the same.

[0044] Embodiment 33. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 32, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 41 and a light chain variable region (VL) comprising SEQ ID NO: 43.

[0045] Embodiment 34: The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 33, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises: (a) a heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 45, (b) an HCDR2 of SEQ ID NO: 46, and (c) an HCDR3 of SEQ ID NO: 47; and (d) a light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 48, (e) an LCDR2 of SEQ ID NO: 49, and (f) an LCDR3 of SEQ ID NO: 50; The second antigen-binding domain that specifically binds to human CD137 is (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3; or (ii) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO: 3.

[0046] Embodiment 35: The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 34, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 41 and a light chain variable region (VL) comprising SEQ ID NO: 43; The second antigen-binding domain that specifically binds to human CD137 is (i) a heavy chain variable region (VH) comprising SEQ ID NO: 4; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15, or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 17.

[0047] Embodiment 36. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 35, wherein the multispecific antibody or antigen-binding fragment thereof is a monoclonal antibody, chimeric antibody, humanized antibody, engineered human antibody, single-chain antibody (scFv), Fab fragment, Fab' fragment, or F(ab')2 fragment.

[0048] Embodiment 37: The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 36, wherein the first antigen-binding domain that specifically binds to human GPC3 is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a single-domain antibody, a Fab fragment, a Fab' fragment, or a F(ab')2 fragment; The multispecific antibody or antigen-binding fragment thereof, wherein the second antigen-binding domain that specifically binds to human CD137 is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a single-domain antibody, a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0049] Embodiment 38. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 37, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody.

[0050] Embodiment 39. The multispecific antibody or antigen-binding fragment thereof of Embodiment 38, wherein the bispecific antibody is in a 2+2 format.

[0051] Embodiment 40. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 39, wherein the multispecific antibody or antigen-binding fragment thereof comprises a linker of SEQ ID NO: 60 to SEQ ID NO: 101.

[0052] Embodiment 41. The multispecific antibody or antigen-binding fragment thereof of embodiment 40, wherein the linker is SEQ ID NO: 62.

[0053] Embodiment 42. The multispecific antibody or antigen-binding fragment thereof of embodiment 40, wherein the linker is SEQ ID NO: 67.

[0054] Embodiment 43. A multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 42, comprising a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a kappa or lambda light chain constant region; The multispecific antibody or antigen-binding fragment thereof, wherein the heavy chain constant region comprises a CH1 and / or Fc domain.

[0055] Embodiment 44. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 43, wherein the multispecific antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0056] Embodiment 45. A multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 44, wherein the multispecific antibody or antigen-binding fragment thereof has reduced or no glycosylation or is hypofucosylated.

[0057] Embodiment 46. A multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 45, comprising an increase in bisecting GlcNac structures.

[0058] Embodiment 47. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 46, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO: 53.

[0059] Embodiment 48. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 47, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function and / or extended half-life, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO: 20.

[0060] Embodiment 49. The multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 48, comprising an Fc domain, wherein the Fc domain is IgG4 Fc.

[0061] Embodiment 50. A multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 49, a) the heavy chain variable region (VH) of the first antigen-binding domain that specifically binds to human GPC3, the CH1 domain, the Fc domain, and the heavy chain variable region (VH) of the second antigen-binding domain that specifically binds to human CD137 are arranged in an N-terminal to C-terminal direction in the first polypeptide; Optionally, the C-terminus of the Fc domain is linked to the N-terminus of the heavy chain variable region (VH) of the second antigen-binding domain via a linker; and b) the multispecific antibody or antigen-binding fragment thereof, wherein the light chain variable region (VL) and the first light chain constant region of the first antigen-binding domain that specifically binds to human GPC3 are arranged in an N-terminal to C-terminal direction within the second polypeptide.

[0062] Embodiment 51. A multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 50, (i) a first polypeptide of SEQ ID NO: 25 and a second polypeptide of SEQ ID NO: 23; (ii) a first polypeptide of SEQ ID NO: 21 and a second polypeptide of SEQ ID NO: 23; (iii) a first polypeptide of SEQ ID NO: 33 and a second polypeptide of SEQ ID NO: 23; (iv) a first polypeptide of SEQ ID NO: 27 and a second polypeptide of SEQ ID NO: 23; (v) a first polypeptide of SEQ ID NO: 29 and a second polypeptide of SEQ ID NO: 23, or (vi) the multispecific antibody or antigen-binding fragment thereof, comprising a first polypeptide of SEQ ID NO: 31 and a second polypeptide of SEQ ID NO: 23.

[0063] Embodiment 52. A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 19 to 51 and a pharmaceutically acceptable carrier.

[0064] Embodiment 53. A method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 19 to 51, or the pharmaceutical composition of Embodiment 52.

[0065] Embodiment 54 The method of embodiment 53, wherein the cancer is an advanced or metastatic solid tumor.

[0066] Embodiment 55. The method of any one of embodiments 53-54, wherein the cancer expresses GPC3.

[0067] Embodiment 56. The method of any one of embodiments 53 to 55, wherein the cancer is liver cancer, lung cancer, gastric cancer, germ cell tumor, thyroid cancer, pancreatic cancer, ovarian cancer, skin cancer, kidney cancer, esophageal cancer, atypical teratoid rhabdoid tumor of the brain, or undifferentiated synovial sarcoma.

[0068] Embodiment 57 The method of embodiment 56, wherein the liver cancer is hepatoblastoma or hepatocellular carcinoma (HCC).

[0069] Embodiment 58 The method of embodiment 56, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

[0070] Embodiment 59. The method of embodiment 58, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.

[0071] Embodiment 60 The method of embodiment 58, wherein the non-small cell lung cancer is GPC3+ squamous non-small cell lung cancer.

[0072] Embodiment 61 The method of embodiment 56, wherein the gastric cancer is alpha-fetoprotein+ (AFP+) gastric cancer.

[0073] Embodiment 62 The method of embodiment 56, wherein the kidney cancer is Wilms' tumor.

[0074] Embodiment 63 The method of embodiment 56, wherein the esophageal cancer is esophageal squamous cell carcinoma.

[0075] Embodiment 64 The method of embodiment 56, wherein the esophageal cancer is GPC3+ esophageal squamous cell carcinoma.

[0076] Embodiment 65 The method of embodiment 56, wherein the germ cell tumor is a yolk sac tumor or a non-dysgerminoma.

[0077] Embodiment 66. The method of any one of Embodiments 53 to 65, wherein the multispecific antibody or antigen-binding fragment thereof, or the pharmaceutical composition is administered in combination with another therapeutic agent.

[0078] Embodiment 67. The method of embodiment 66, wherein the therapeutic agent is an anti-PD1 or anti-PDL1 antibody.

[0079] Embodiment 68. The method of embodiment 67, wherein the anti-PD1 antibody is tislelizumab.

[0080] Embodiment 69. An isolated nucleic acid encoding the antibody, multispecific antibody, or antigen-binding fragment thereof of any one of Embodiments 1 to 12 and 19 to 51.

[0081] Embodiment 70. A vector comprising the nucleic acid of embodiment 69.

[0082] Embodiment 71. A host cell comprising the nucleic acid of embodiment 69 or the vector of embodiment 70.

[0083] Embodiment 72. A process for producing a multispecific antibody or antigen-binding fragment thereof, comprising culturing the host cell of embodiment 71 and recovering the antibody or antigen-binding fragment thereof from the culture.

[0084] In some embodiments, the present disclosure provides anti-CD137 antibodies or antigen-binding fragments thereof that exhibit specific binding and high affinity to human CD137 and cynomolgus monkey CD137, exhibit excellent overall biophysical properties, e.g., Tm or Tagg, exhibit excellent pharmacokinetics, and / or are capable of binding to CD137 dimers and promoting CD137 clustering.

[0085] In some embodiments, the present disclosure provides anti-CD137 antibodies or antigen-binding fragments thereof having at least one or more of the following characteristics: (1) It exhibits specific binding and high affinity to human CD137 and cynomolgus monkey CD137, (2) Excellent pharmacokinetics. (3) having good overall biophysical properties, e.g., Tm or Tag, and / or having good overall stability; (4) It is a humanized antibody with low immunogenicity risk in humans, and (5) It can bind to CD137 dimers and promote CD137 clustering.

[0086] In some embodiments, the present disclosure provides anti-CD137 antibodies or antigen-binding fragments thereof that are humanized antibodies that maintain specific binding and high affinity for human CD137 and cynomolgus monkey CD137, and exhibit excellent overall biophysical properties and / or stability, while maintaining a low risk of immunogenicity in humans. In some embodiments, the present disclosure provides anti-CD137 antibodies or antigen-binding fragments thereof that exhibit specific binding and high affinity for human CD137 and cynomolgus monkey CD137, and / or that can bind to CD137 dimers and promote CD137 clustering (e.g., via CDR residues).

[0087] In some embodiments, the present disclosure provides an anti-GPC3xCD137 multispecific antibody or antigen-binding fragment thereof having at least one or more of the following characteristics: (1) It exhibits specific binding and high affinity to human CD137 and cynomolgus monkey CD137, (2) It has specific binding and high affinity to human GPC3 and cynomolgus monkey GPC3, and exhibits high affinity for a wide range of GPC expression (low to high expression). (3) Induction of T cell activation, including cytokine release (e.g., IFN-γ or IL-2) and T cell killing activity, in a GPC3-dependent manner, and reduction of T cell activation or T cell killing activity in the absence of GPC3-expressing cells; (4) Induction of T cell activation and potent T cell killing activity against a wide range of GPC3 expressing cells (low, medium, and high expression). (5) effectively inhibiting tumor growth when administered alone; (7) induce synergistic effects (e.g., tumor growth inhibition and / or tumor-free rate) when administered with an anti-PD-1 antibody; (8) Excellent pharmacokinetics. (9) having good overall biophysical properties, e.g., Tm or Tag, and / or stability; (10) It can bind to CD137 dimers and promote CD137 clustering. [Brief explanation of the drawings]

[0088] [Figure 1] We demonstrate that BGA-9612 bound to huCD137 and partially competed with 20 μg / ml CD137L in comparison to urelumab (BMS-663513) by ELISA. [Figure 2] Figure 1 shows a comparison of FACS binding affinity between BGA-9612 and other humanized VHHs in human CD137-overexpressing HuT78 cells. [Figure 3] FIG. 1 is a schematic representation of the design of the tumor-targeting GPC3xCD137 multispecific antibody format. [Figure 4A] 1 shows a flow cytometric binding assay of BE-774 to CD137-overexpressing cells Hut78 / CD137, demonstrating the binding of BE-774 to native huCD137 expressed on the cell surface. [Figure 4B] 1 shows a flow cytometric binding assay of BE-774 to GPC3-expressing cells HepG2, demonstrating the binding of BE-774 to native huGPC3 expressed on the cell surface. [Figure 5]Figures A-B demonstrate BE-774- and BE-653-induced T cell activation when cocultured with GPC3-positive tumor cells, HepG2. Figure A is a schematic representation of CD137 (41BB) activation upon costimulation of huPBMCs with BE-774 or BE-653 and an OS8-expressing hepatocellular carcinoma (HCC) cell line. Figure B shows dose-dependent cytokine release induced by BE-774 and BE-653 in PBMCs cocultured with HepG2 cells but not with GPC3-negative cells. [Figure 6] Figures A-B demonstrate that BE-774 and BE-653 enhanced T cell killing activity against GPC3-positive tumor cells, HepG2. Figure A shows a schematic diagram of CD137 (4-1BB) activation by costimulating huPBMCs with BE-774 or BE-653 in combination with the EpCAM / CD3 bispecific T cell engager (BiTE), which provides the first signal for T cell activation. Figure B shows that BE-774 and BE-653 dose-dependently enhanced T cell killing activity against GPC3-expressing cells, but not against GPC3-negative cells. [Figure 7] PK profiles of BE-933 and BE-774 in cynomolgus monkeys. [Figure 8] PK profiles of BE-933 and BE-774 in the hFcRn mouse model. [Figure 9] Panels A and B show the binding of BE-915 to human CD137 overexpressed in Hut78 (FIG. 9B) and human GPC3 expressed in HepG2 (FIG. 9A). [Figure 10] 1 shows the binding specificity of BE-915 to CD137 and other TNFRSF members. [Figure 11] Figures 11A and 11B show that BE-915 cross-competes with CD137L for binding to human CD137. CD137L blocks the binding of BE-915 to CD137 expressed on HuT78 (Figure 11A). BE-915 blocks the binding of CD137L to CD137 expressed on HuT78 (Figure 11B). [Figure 12]Figures A-C show that BE-915 induces the release of IL-2 and IFN-γ from human PBMCs. Figure A shows a schematic diagram of CD137 activation by costimulation of huPBMCs with BE-915 and an OS8-expressing hepatocellular carcinoma (HCC) cell line. Figures B-C show that BE-915 induces dose-dependent cytokine release in PBMCs in a GPC3 expression-dependent manner. PBMCs from two donors were tested. [Figure 13] Figures A-C show that BE-915 induces T cell killing activity in human PBMCs. Figure A shows a schematic diagram of CD137 activation by costimulating huPBMCs with BE-915 in combination with the EpCAM / CD3 bispecific T cell engager (BiTE), which provides the first signal for T cell activation. Figures B-C show that BE-915 dose-dependently enhanced T cell killing activity against GPC3-expressing cells, but not against GPC3-negative cells. PBMCs from two donors were tested. [Figure 14] 1 shows the pharmacokinetic profile of BE-915 in cynomolgus monkeys after iv injection (5 mg / kg, N=2). [Figure 15] Shows the efficacy of BE-915 monotherapy in the MC38 / hGPC3 model in humanized CD137 knock-in mice. [Figure 16] Figure 1 shows the efficacy of the combination of BE-915 and anti-PD-1 antibody in the LL / 2 / hGPC3 model in humanized CD137 knock-in mice. [Figure 17] Figure 1 shows a schematic diagram showing the partial competitive binding of VHH(BGA-2524) to CD137L for CD137. The crystal structure of VHH(BGA-2524) / CD137 was superimposed with the CD137L / CD137 complex (PDB: 6MGP) via the CD137 CRD1 and CRD2 domains. CD137, CD137L, and VHH(BGA-2524) are colored black, white, and gray, respectively. [Figure 18]VHH(BGA-2524) binds to CD137 dimers. Crystal structure analysis shows that VHH(BGA-2524) has the ability to bind to CD137 dimers and promote CD137 clustering. Each monomer of the CD137 dimer is shown in white or gray, and VHH(BGA-2524) is shown in black schematic on the surface (left). The epitope of VHH(BGA-2524) is shown in black on the surface of the CD137 dimer (right, BGA-2524 has been removed). [Figure 19] Atomic interactions on the binding surface of the VHH(BGA-2524) / CD137 complex are shown. The binding interface between VHH(BGA-2524) and CD137 identifies certain key residues of VHH(BGA-2524) (paratope residues, amino acids underlined) and CD137 (epitope residues). Each monomer of the CD137 dimer is shown as a white or gray schematic diagram covered by a transparent surface, with the CRD1, CRD2, and CRD3 domains indicated by lines, respectively. Paratope residues are indicated by black lines, and amino acids are underlined (most of the framework regions have been removed). DETAILED DESCRIPTION OF THE INVENTION

[0089] The present disclosure provides anti-CD137 antibodies and antigen-binding fragments thereof, as well as multispecific antibodies or antigen-binding fragments thereof that recognize CD137 as one antigen and at least one tumor-associated antigen (TAA) as the other antigen. The present disclosure also provides anti-GPC3xCD137 multispecific antibodies and antigen-binding fragments thereof. Furthermore, the present disclosure provides antibodies that have desirable pharmacokinetic properties, desirable biophysical properties, and other desirable attributes, and thus can be used to reduce the likelihood of cancer or treat cancer. The present disclosure also provides pharmaceutical compositions comprising the antibodies, as well as methods for producing and using such pharmaceutical compositions, for the prevention and treatment of cancer and related disorders.

[0090] I. Anti-GPC3 antibody The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to GPC3. In one embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof is present in a concentration of 1×10 -6 M to 1 x 10 -10 Binding affinity (K D In another embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof specifically binds to human GPC3 at a concentration of about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M or approximately 1 x 10 -10 Binding affinity (K D ) binds to human GPC3.

[0091] In one embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising, according to Kabat numbering, (a) an HCDR1 of SEQ ID NO: 45, (b) an HCDR2 of SEQ ID NO: 46, (c) an HCDR3 of SEQ ID NO: 47, and (d) an LCDR1 of SEQ ID NO: 48, (e) an LCDR2 of SEQ ID NO: 49, and (f) an LCDR3 of SEQ ID NO: 50. A light chain variable region (VL) comprising:

[0092] In another embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 41, and LCDR1, LCDR2, and LCDR3 from the light chain variable region (VL) set forth in SEQ ID NO: 43.

[0093] In another embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises deletions, insertions, or substitutions of 1, 2, 3, 4, or 5 or fewer amino acids in the CDRs, preferably conservative amino acid substitutions, while maintaining binding specificity and affinity.

[0094] In another embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 41, and a light chain variable region (VL) comprising an amino acid sequence that is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 43. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids are inserted, deleted, or substituted (optionally with conservative amino acid substitutions) within SEQ ID NO: 41 or SEQ ID NO: 43. In another embodiment, such changes are within the framework regions of the variable region. In another embodiment, an anti-GPC3 antibody or antigen-binding fragment thereof having such changes maintains binding specificity and affinity.

[0095] In another embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising SEQ ID NO: 41 and a light chain variable region (VL) comprising SEQ ID NO: 43.

[0096] In another embodiment, the anti-human GPC3 antibody or antigen-binding fragment thereof exhibits cross-species binding activity to cynomolgus monkey GPC3.

[0097] II. Anti-CD137 antibody [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human CD137. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof generated as described below.

[0098] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67 of human CD137 (SEQ ID NO: 35), optionally wherein the epitope is determined by X-ray diffraction.

[0099] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35), optionally wherein the epitope is determined by X-ray diffraction.

[0100] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to a human CD137 dimer comprising, or consisting of, a first human CD137 monomer and a second human CD137 monomer, and the antibody or antigen-binding fragment thereof specifically binds to a human CD137 dimer comprising, consisting essentially of, or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67. The antibody or antigen-binding fragment thereof specifically binds to an epitope on a second human CD137 monomer (SEQ ID NO: 35), the epitope comprising, consisting essentially of, or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, optionally the epitope is determined by X-ray diffraction, and optionally the antibody or antigen-binding fragment thereof binds to a human CD137 dimer and promotes human CD137 clustering.

[0101] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising one or more amino acid residues selected from the group consisting of Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35).

[0102] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising one or more amino acid residues selected from the group consisting of Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35).

[0103] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to a human CD137 dimer comprising, or consisting of, a first human CD137 monomer and a second human CD137 monomer, and the antibody or antigen-binding fragment thereof comprises one or more amino acids selected from the group consisting of Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67. The antibody or antigen-binding fragment thereof specifically binds to an epitope on a first human CD137 monomer (SEQ ID NO: 35) comprising one or more amino acid residues selected from the group consisting of Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and optionally, the antibody or antigen-binding fragment thereof binds to a human CD137 dimer and promotes human CD137 clustering.

[0104] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising one or more amino acid residues selected from the group consisting of Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67, Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35).

[0105] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35).

[0106] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35).

[0107] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof specifically binds to a human CD137 dimer comprising or consisting of a first human CD137 monomer and a second human CD137 monomer, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope on the first human CD137 monomer (SEQ ID NO: 35) consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67, and the antibody or antigen-binding fragment thereof specifically binds to an epitope on the second human CD137 monomer (SEQ ID NO: 35) consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and optionally, the antibody or antigen-binding fragment thereof binds to the human CD137 dimer and promotes human CD137 clustering.

[0108] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, wherein the antibody or antigen-binding fragment thereof comprises a paratope comprising one or more amino acid residues selected from the group consisting of Asn31, Tyr32, Ala33, Trp52, Ser53, Tyr55, His57, Leu98, Lys99, Tyr100, Pro101, Thr104, Thr106, Tyr109 (natural sequence order), or Asn31, Tyr32, Ala33, Trp52, Ser54, Tyr56, His58, Leu96, Lys97, Tyr98, Pro99, Thr100B, Thr100D, Tyr102 (Kabat nomenclature).

[0109] In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to human CD137 and binds primarily to the flanks of the CRD2 domain of (e.g., human) CD137 via CDR residues (e.g., Asn31, Tyr32, Ala33, Trp52, Ser54, Tyr56, His58, Leu96, Lys97, Tyr98, Pro99, Thr100B, Thr100D, Tyr102 (Kabat nomenclature) of human CD137 VHH).

[0110] In some embodiments, the epitope of human CD137 bound by an antibody or antigen-binding fragment thereof that specifically binds to human CD137 of the present disclosure is determined by X-ray diffraction.

[0111] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to human CD137, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VH domain having the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17 (Table 1). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to human CD137, wherein the antibodies or antigen-binding fragments comprise an HCDR comprising the amino acid sequence of any one of the HCDRs listed in Table 1. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to human CD137, wherein the antibodies comprise one, two, three, or more HCDRs (or alternatively consist of one, two, three, or more HCDRs) comprising the amino acid sequence of any of the HCDRs listed in Table 1.

[0112] In one embodiment, the antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:10, or selected from SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, or selected from SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59.

[0113] In one embodiment, the anti-CD137 antibody or antigen-binding fragment thereof comprises: (i) HCDR1 (heavy chain complementarity determining region 1), HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 4; (ii) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 8; (iii) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 6; (iv) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 11; (v) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 13; (vi) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 15; or (vii) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 17.

[0114] In one embodiment, the anti-CD137 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, according to Kabat numbering; or (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO: 3.

[0115] Other antibodies or antigen-binding fragments thereof of the present disclosure include amino acid alterations in the CDR regions that have a percent identity of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the CDR regions disclosed in Table 1. In some embodiments, this includes amino acid alterations (insertions, deletions, or substitutions, optionally conservative amino acid substitutions) in which no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acids are altered in the CDR regions when compared to the CDR regions set forth in the sequences in Table 1, while maintaining binding specificity and affinity.

[0116] Other antibodies of the disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been altered but have percent identities to the sequences set forth in Table 1 of at least 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, optionally without altering the corresponding sequences of the CDRs. In some embodiments, it comprises alterations in amino acid sequence such that no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids are changed in the variable regions (e.g., framework regions of the variable regions) when compared to the variable regions set forth in the sequences in Table 1, while retaining therapeutic activity / binding specificity / affinity, and optionally, the corresponding sequences of the CDRs are unchanged. In some embodiments, it comprises an alteration in amino acid sequence, such that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids have been changed, e.g., inserted, deleted or substituted (optionally, conservative amino acid substitutions), in the variable region (e.g., framework region of the variable region) when compared to the variable region shown in the sequence set forth in Table 1, while retaining therapeutic activity / binding specificity / affinity, and optionally, the corresponding sequences of the CDRs are unchanged.

[0117] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, wherein amino acids F37, Y47, G49, and I94 (Kabat numbering) within the framework regions are retained.

[0118] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO: 3, wherein amino acids F37, Y47, G49, and I94 (Kabat numbering) within the framework regions are retained.

[0119] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, comprising a VH domain having an amino acid sequence set forth in Table 1, or a variant thereof, wherein HCDR1, HCDR2, and HCDR3 are unchanged, and amino acids F37, Y47, G49, and I94 (Kabat numbering) within the framework regions are retained.

[0120] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CD137, comprising a heavy chain variable region comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17, wherein HCDR1, HCDR2, and HCDR3 are unchanged, and amino acids F37, Y47, G49, and I94 (Kabat numbering) within the framework regions are retained.

[0121] In another embodiment, the present disclosure provides a method for producing a 1×10 -6 M to 1 x 10 -10 Binding affinity (K D In another embodiment, the anti-CD137 antibody or antigen-binding domain thereof binds to human CD137 at about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 m or approximately 1 x 10 -10 Binding affinity (K D ) binds to human CD137.

[0122] The present disclosure also provides nucleic acid sequences encoding the VH and full-length heavy chains of antibodies that specifically bind to human CD137. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0123] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CD137 antibodies listed in Table 1. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies in binding assays. The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to CD137 demonstrates that the test antibody can compete with that antibody or antigen-binding fragment thereof for binding to CD137. Without being bound by any one theory, such antibodies may bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on CD137 as the competing antibody or antigen-binding fragment thereof. In certain embodiments, an antibody that binds to the same epitope on CD137 as an antibody or antigen-binding fragment thereof of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0124] In some embodiments, the anti-CD137 antibody comprises at least one antigen-binding site, at least the variable region. In some embodiments, the anti-CD137 antibody comprises an antigen-binding fragment derived from a CD137 antibody described herein. In some embodiments, the anti-CD137 antibody is isolated or recombinant. In some embodiments, the anti-CD137 antibody also encompasses multispecific antibodies that target CD137 as at least one arm and other antigen(s) as another arm(s).

[0125] III. Anti-CD137 multispecific antibody In one embodiment, the anti-CD137 antibodies disclosed herein can be used to construct multispecific antibodies with other modalities such as human tumor-associated antigens (TAAs), immune checkpoints, or immune stimulators.

[0126] In one embodiment, the anti-CD137 antibodies disclosed herein can be incorporated into an anti-CD137×TAA multispecific antibody, where the TAA is an antibody or fragment thereof directed against any human tumor-associated antigen. The antibody molecule is a multispecific antibody molecule, e.g., comprising multiple antigen-binding domains, where at least one antigen-binding domain sequence specifically binds to a human TAA as a first antigen / epitope and a second antigen-binding domain sequence specifically binds to human CD137 as a second antigen / epitope. In one embodiment, the multispecific antibody comprises a third, fourth, or fifth antigen-binding domain. In one embodiment, the multispecific antibody is a bispecific, trispecific, or tetraspecific antibody. In each example, the multispecific antibody comprises at least one anti-TAA antigen-binding domain and at least one anti-CD137 antigen-binding domain.

[0127] In one embodiment, a multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds to only two antigens. A bispecific antibody comprises a first antigen-binding domain that specifically binds to a TAA and a second antigen-binding domain that specifically binds to human CD137. This includes bispecific antibodies comprising a heavy chain variable domain and a light chain variable domain that specifically bind to a TAA, and a heavy chain variable domain that specifically binds to human CD137. In some embodiments, a bispecific antibody comprises an antigen-binding fragment, which can be a Fab, F(ab')2, Fv, single-chain Fv (scFv), or single-domain antibody.

[0128] In some embodiments, the second antigen-binding domain that specifically binds human CD137 comprises an anti-CD137 antibody disclosed in Section II.

[0129] In one embodiment, the multispecific antibody of the present disclosure is administered in a concentration of 1 x 10 -6 M to 1 x 10 -10 Binding affinity (K D In another embodiment, the multispecific antibodies of the disclosure bind to human TAA and / or human CD137 at a concentration of about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 m or approximately 1 x 10 -10 Binding affinity (K D ) binds to human TAA and / or human CD137.

[0130] In one embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein a first antigen-binding domain specifically binds to a human TAA and a second antigen-binding domain that specifically binds to human CD137 comprises (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, or (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO: 3.

[0131] In another embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein a first antigen-binding domain specifically binds to a human TAA and a second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 15; or (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 4.

[0132] In another embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein a first antigen-binding domain specifically binds to a human TAA and a second antigen-binding domain that specifically binds to human CD137 comprises (i) a heavy chain variable region (VH) comprising SEQ ID NO: 4, (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11, (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13, (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15, or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 17.

[0133] In one embodiment, the TAA is human GPC3. First antigen-binding domains that specifically bind to human GPC3 include the anti-GPC3 antibodies disclosed in Section I.

[0134] The present disclosure provides multivalent antibodies (e.g., tetravalent antibodies) having at least two antigen-binding domains, which can be readily produced by recombinant expression of nucleic acids encoding the antibody polypeptide chains. The multivalent antibodies herein contain three to eight, preferably four, antigen-binding domains that specifically bind to at least two antigens.

[0135] IV. Anti-GPC3xCD137 multispecific antibody In one embodiment, the anti-GPC3 and anti-CD137 antibodies disclosed herein can be combined into an anti-GPC3xCD137 multispecific antibody. The antibody molecule is a multispecific antibody molecule, e.g., comprising multiple antigen-binding domains, where at least one antigen-binding domain sequence specifically binds to GPC3 as a first epitope / antigen and a second antigen-binding domain sequence specifically binds to CD137 as a second epitope / antigen. In one embodiment, the multispecific antibody comprises a third, fourth, or fifth antigen-binding domain. In one embodiment, the multispecific antibody is a bispecific antibody, trispecific antibody, or tetraspecific antibody. In each example, the multispecific antibody comprises at least one anti-GPC3 antigen-binding domain and at least one anti-CD137 antigen-binding domain.

[0136] In one embodiment, a multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds to only two antigens. A bispecific antibody comprises a first antigen-binding domain that specifically binds to human GPC3 and a second antigen-binding domain that specifically binds to CD137. This includes bispecific antibodies comprising a heavy chain variable domain and a light chain variable domain that specifically bind to human GPC3 as a first epitope / antigen and a heavy chain variable domain that specifically binds to human CD137 as a second epitope / antigen. In some embodiments, a bispecific antibody comprises an antigen-binding fragment, which may be a Fab, F(ab')2, Fv, single-chain Fv (scFv), or single-domain antibody.

[0137] The present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to human glypican 3 (GPC3) and a second antigen-binding domain that specifically binds to human CD137.

[0138] Examples of first antigen-binding domains that specifically bind to human glypican 3 (GPC3) include the anti-GPC3 antibodies described in Section I. Examples of second antigen-binding domains that specifically bind to human CD137 include the anti-CD137 antibodies disclosed in Section II.

[0139] In one embodiment, the multispecific antibody of the present disclosure is administered in a concentration of 1 x 10 -6 M to 1 x 10 -10 Binding affinity (K D In another embodiment, the multispecific antibodies of the present disclosure bind to human GPC3 and / or human CD137 at a concentration of about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 m or approximately 1 x 10 -10 Binding affinity (K D ) and binds to human GPC3 and / or human CD137.

[0140] In one embodiment, a multispecific antibody of the present disclosure binds specifically to human GPC3 and exhibits high affinity for both human GPC3 and monkey GPC3. In another embodiment, a multispecific antibody of the present disclosure binds specifically to human CD137. In another embodiment, a multispecific antibody of the present disclosure exhibits high affinity for both human CD137 and monkey CD137.

[0141] In one embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein a first antigen-binding domain that specifically binds to human GPC3 comprises a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 45, (b) an HCDR2 of SEQ ID NO: 46, and (c) an HCDR3 of SEQ ID NO: 47, according to Kabat numbering, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 48, (e) an LCDR2 of SEQ ID NO: 49, and (f) an LCDR3 of SEQ ID NO: 50; and a second antigen-binding domain that specifically binds to human CD137 comprises (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, or (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO: 3, according to Kabat numbering.

[0142] In another embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein a first antigen-binding domain that specifically binds to human GPC3 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 41 and a light chain variable region (VL) comprising SEQ ID NO: 43, and a second antigen-binding domain that specifically binds to human CD137 comprises (i) a heavy chain variable region (VH) comprising SEQ ID NO: 4, (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11, (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13, (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15, or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 17.

[0143] In another embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein the multispecific antibody or antigen-binding fragment is (i) BE-933 comprising a first polypeptide of SEQ ID NO:27 and a second polypeptide of SEQ ID NO:23; (ii) BE-774 comprising a first polypeptide of SEQ ID NO:25 and a second polypeptide of SEQ ID NO:23; (iii) BE-653 comprising a first polypeptide of SEQ ID NO:29 and a second polypeptide of SEQ ID NO:23; (iv) BE-915 comprising a first polypeptide of SEQ ID NO:21 and a second polypeptide of SEQ ID NO:23; (v) BE-647 comprising a first polypeptide of SEQ ID NO:31 and a second polypeptide of SEQ ID NO:23; or (vi) BE-621 comprising a first polypeptide of SEQ ID NO:33 and a second polypeptide of SEQ ID NO:23.

[0144] Other multispecific antibodies or antigen-binding fragments thereof of the present disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been altered, but have percent identities of at least 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% to the sequences described herein (e.g., the CDRs are not altered). In some embodiments, it comprises changes in amino acids such that no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids are changed in the variable regions (e.g., framework regions) when compared to the variable regions described herein, while retaining therapeutic activity / binding specificity / affinity.

[0145] V. Other Format and module ratio In one embodiment, the anti-CD137 antibodies disclosed herein can be used to construct multispecific antibodies with other modalities, such as TAA, immune checkpoint, or immune stimulator. To illustrate the format and ratio, a TAA (e.g., GPC3) is used as an example below. The description of GPC3 in the following embodiments can also be applied to other TAAs.

[0146] The multispecific antibodies of the present disclosure may be in different formats. In one embodiment, the multispecific antibodies of the present disclosure have the following formats: (1) Format A provides a symmetric IgG-like multispecific molecule with a Fab x VH configuration. An anti-huCD137 VH domain antibody is fused to the C-terminus of the Fc (CH3 domain) of an anti-GPC3 antibody, with a linker between them, as shown in Figure 3. (2) Format B also provides a symmetric IgG-like multispecific molecule with a Fab x VH configuration. An anti-huCD137 VH domain antibody is fused to the C-terminus of the light chain (CK) of an anti-GPC3 antibody, with a linker between them. (3) Format C provides a symmetric VH antibody-like multispecific molecule with a Fab x VH configuration. The Fab region of an anti-GPC3 antibody is fused to the N-terminus of the VH of an anti-huCD137 VH domain Ab, with a linker between them. (4) Format D also provides a symmetric IgG-like multispecific molecule with a Fab x VH configuration. The anti-huCD137 VH domain antibody was fused to the N-terminus of the heavy chain (VH) of the anti-GPC3 antibody using a linker in between. In one embodiment, the multispecific antibody is in Format A shown in Figure 3.

[0147] The multispecific antibodies of the present disclosure can be constructed with different module ratios, such as 1:1. In one embodiment, an inactive Fc can be used for the multispecific antibody, and Zymeworks' Azymetric™ platform can be utilized to assemble a Fab x VH configuration, in which ZW1 mutations (chain A: T350V / L351Y / F405A / Y407V, chain B: T350V / T366L / K392L / T394W) can be introduced into the CH3 domain of the heavy chain to enable efficient heterodimer formation (Von Kreudenstein et al., (2013) Mabs 5(5):646-54, incorporated by reference in its entirety). In one aspect, a specific ratio activates CD137 in a GPC3-dependent manner but does not activate CD137 in the absence of GPC3.

[0148] In one embodiment, the multispecific antibody or antigen-binding fragment thereof comprises: a) a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable region (such as a first heavy chain variable region), a CH1 domain, an Fc domain, and a second heavy chain variable region (such as a second heavy chain variable region), wherein the C-terminus of the Fc domain is optionally linked to the N-terminus of the second heavy chain variable region via a linker; and b) a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable region (such as a first light chain variable region) and a first light chain constant region, wherein the first heavy chain variable region and the first light chain variable region form a first antigen-binding domain that specifically binds to human GPC3, and the second heavy chain variable region forms a second antigen-binding domain that specifically binds to human CD137. In another embodiment, the multispecific antibody or antigen-binding fragment thereof comprises two of the first polypeptides and two of the second polypeptides.

[0149] Linker It is also understood that the domains and / or regions of the polypeptide chains of a multispecific antibody can be separated by linker regions of various lengths. In some embodiments, antigen-binding domains are separated from each other, from the CL, CH1, hinge, CH2, CH3, or the entire Fc region by linker regions. For example, VL1-CL-(linker)VH2-CH1. For example, such linker regions can comprise a random assortment of amino acids or a limited set of amino acids. Such linker regions can be flexible or rigid (see, e.g., US2009 / 0155275, incorporated by reference in its entirety).

[0150] Multispecific antibodies have been synthesized via dimerization devices such as leucine zippers (Kostelny et al., J. Immunol. 1992;148:1547-53, de Kruifetal J. Biol. Chem. 1996;271:7630-4) and Ig C / CH1 domains (Muller et al., FEBS Lett. 1998;422:259-64), in the form of bispecific antibodies (Holliger et al., Proc. Nat. Acad. Sci. USA. 1993;90:6444-8, Zhu et al., Bio / Technology (NY) 1996;14:192-6), Fab-scFv fusions (Schoonjans et al., J. Immunol. 2000;165:7050-7), and miniantibody formats (Packet et al., Biochemistry 1992;31:1579-84; Packet et al., Bio / Technology 1993;11:1271-7) by genetically fusing two single-chain Fv (scFv) or Fab fragments with or without a flexible linker (Mallender et al., J. Biol. Chem. 1994;269:199-206; Packet et al., Proc. Natl. Acad. Sci. USA 1995;92:7021-5; Zapata et al., Protein Eng. 1995;8:1057-62). Each reference cited in this paragraph is incorporated herein by reference in its entirety.

[0151] The multispecific antibodies disclosed herein comprise a linker region of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues between one or more of their antigen-binding domains, CL domains, CH1 domains, hinge region, CH2 domains, CH3 domains, or Fc region. In some embodiments, the amino acids glycine and serine comprise the linker region.In another embodiment, the linker is selected from the group consisting of GS (SEQ ID NO:97), GGS (SEQ ID NO:98), GSG (SEQ ID NO:99), SGG (SEQ ID NO:100), GGG (SEQ ID NO:101), GGGS (SEQ ID NO:60), SGGG (SEQ ID NO:61), GGGGS (SEQ ID NO:62), GGGGSGS (SEQ ID NO:63), GGGGSGS (SEQ ID NO:64), GGGGSGGS (SEQ ID NO:65), GGGGSGGGGS (SEQ ID NO:66), GGGSGGGGSGGGGS (SEQ ID NO:67), AK TTPKLEEGEFSEAR (SEQ ID NO: 68), AKTTPKLEEGEFSEARV (SEQ ID NO: 69), AKTTPKLGG (SEQ ID NO: 70), SAKTTPKLGG (SEQ ID NO: 71), AKTTPKLEEGEFSEARV (SEQ ID NO: 72), SAKTTP (SEQ ID NO: 73), SAKTTPKLGG (SEQ ID NO: 74), RADAAP (SEQ ID NO: 75), RADAAPTVS (SEQ ID NO: 76), RADAAAAAGGPGS (SEQ ID NO: 77), RADAAAA(G4S ) 4 (SEQ ID NO: 78), SAKTTP (SEQ ID NO: 79), SAKTTPKLGG (SEQ ID NO: 80), SAKTTPKLEEGEFSEARV (SEQ ID NO: 81), ADAAP (SEQ ID NO: 82), ADAAPTVSIFPP (SEQ ID NO: 83), TVAAP (SEQ ID NO: 84), TVAAPSVFIFPP (SEQ ID NO: 85), QPKAAP (SEQ ID NO: 86), QPKAAPSVTLFPP (SEQ ID NO: 87), AKTTPP (SEQ ID NO: 88), AKTTPPSVTPLAP (SEQ ID NO: No. 89), AKTTAP (SEQ ID NO: 90), AKTTAPSVYPLAP (SEQ ID NO: 91), ASTKGP (SEQ ID NO: 92), ASTKGPSVFPLAP (SEQ ID NO: 93), GENKVEYAPALMALS (SEQ ID NO: 94), GPAKELTPLKEAKVS (SEQ ID NO: 95), and GHEAAAVMQVQYPAS (SEQ ID NO: 96), or any combination thereof (see WO 2007 / 024715, incorporated by reference in its entirety).

[0152] Dimerization-specific amino acids In one embodiment, the multispecific antibody comprises at least one dimerization-specific amino acid change. The dimerization-specific amino acid change results in "knobs-into-holes" interactions, increasing the assembly of correct multivalent antibodies. The dimerization-specific amino acid can be in the CH1 domain or the CL domain, or a combination thereof. The dimerization-specific amino acid can be used to pair a CH1 domain with another CH1 domain (CH1-CH1) and a CL domain with another CL domain (CL-CL), and can be found in at least WO2014082179, WO2015181805 family, and WO2017059551, each of which is incorporated by reference in its entirety. The dimerization-specific amino acid can be in the Fc domain or combined with a dimerization-specific amino acid in the CH1 or CL domain. In one embodiment, the present disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair.

[0153] Fc region modification The Fc region, if present, may be a wild-type Fc region of the IgG1, IgG2, IgG3, or IgG4 subclass.

[0154] In one embodiment, the antibody, multispecific antibody or antigen-binding fragment thereof comprises an IgG1 or IgG4 Fc domain with reduced effector function. In another embodiment, the Fc domain comprises the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 19. In another embodiment, the IgG1 Fc comprises the mutations E233P, L234A, L235A, G236del, and P329A.

[0155] In one embodiment, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain with extended half-life. In another embodiment, the multispecific antibody or antigen-binding fragment thereof comprises an IgG1 Fc domain into which YTE mutations (M252Y / S254T / T256E, EU numbering as set forth in US7658921, incorporated by reference in its entirety) located in CH2 of the IgG Fc region have been introduced.

[0156] In one embodiment, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain with reduced effector function and extended half-life, hi another embodiment, the Fc domain comprises the amino acid sequence of SEQ ID NO: 20.

[0157] In another embodiment, the antibodies of the disclosure have potent Fc-mediated effector function, where the antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells expressing a TAA (e.g., GPC3).

[0158] In yet another embodiment, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be substituted with a different amino acid residue, resulting in an antibody with altered affinity for an effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al., each of which is incorporated by reference in its entirety.

[0159] In another embodiment, one or more amino acid residues can be substituted with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al., which is incorporated by reference in its entirety.

[0160] In yet another embodiment, one or more amino acid residues are altered to modify the antibody's ability to fix complement. This approach is described, for example, in publication WO 94 / 29351 by Bodmer et al., which is incorporated by reference in its entirety. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues include, but are not limited to, the heavy chain constant regions of the IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the kappa isotype, as described by Jefferis et al., MAbs. 2009;1:332-338, which is incorporated by reference in its entirety.

[0161] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcγ receptor. This approach is described, for example, in WO 00 / 42072 by Presta, which is incorporated by reference in its entirety. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 2001;276:6591-6604), which is incorporated by reference in its entirety.

[0162] In yet another embodiment, the glycosylation of the multispecific antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks or has reduced glycosylation). Altering the glycosylation can, for example, increase the affinity of the antibody for an "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such techniques are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al., each of which is incorporated by reference in its entirety.

[0163] Additionally or alternatively, antibodies can be generated with altered types of glycosylation (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures). Such altered glycosylation patterns have been demonstrated to enhance the ADCC ability of antibodies. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al., incorporated by reference in its entirety, describes a cell line in which the FUT8 gene, encoding fucosyltransferase, has been functionally disrupted such that antibodies expressed in such cell lines exhibit hypofucosylation. Publication WO 03 / 035835 by Presta, which is incorporated by reference in its entirety, describes a variant CHO cell line, Lecl3 cells, which has a reduced ability to attach fucose to Asn(297)-linked sugars, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., J. Biol. Chem. 2002;277:26733-26740, which is incorporated by reference in its entirety). WO 99 / 54342 by Umana et al., which is incorporated by reference in its entirety, describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 1999;17:176-180, which is incorporated by reference in its entirety).

[0164] In another aspect, when reduced ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, et al., MAbs. 2010;2:181-189, the entire contents of which are incorporated by reference). However, native IgG4 has been found to be less stable under stress conditions, such as in acidic buffers or at elevated temperatures (Angal, S. Mol Immunol. 1993;30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry,37:9266-9273; Aalberse et al., Immunol. 2002;105:9-19, the entire contents of which are incorporated by reference). Reduced ADCC can be achieved by operably linking an antibody to an IgG4 Fc that has been engineered with a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of antibodies as biologics, one of the more undesirable intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form half-antibodies, which generates bispecific antibodies in vivo via a process called "Fab arm exchange" (Van der Neut Kolfschoten M, et al., Science. 2007;317:1554-157, incorporated by reference in its entirety). Mutation of serine to proline at position 228 (EU numbering system) appeared to be inhibitory to IgG4 heavy chain separation (Angal, S. Mol Immunol. 1993;30:105-108, incorporated by reference in its entirety; Aalberse et al., Immunol. 2002;105:9-19, incorporated by reference in its entirety).Some amino acid residues in the hinge and gamma Fc region have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al., Proc. Natl. Acad. Sci. USA. 1991; 88: 9036-9040; Mukherjee, J. et al., FASEB J. 1995; 9: 115-119; Armour, KL et al., Eur J Immunol. 1999; 29: 2613-2624; Clynes, RA et al., 2000 Nature Medicine, 6: 443-446; Arnold JN, Annu Rev Immunol. 2007; 25: 21-50, each of which is incorporated by reference in its entirety). Furthermore, some IgG4 isoforms that occur rarely in the human population may also result in different physicochemical properties (Brusco, A. et al., Eur J Immunogenet. 1998;25:349-55; Aalberse et al., Immunol. 2002;105:9-19, each of which is incorporated by reference in its entirety). To generate multispecific antibodies with low ADCC and CDC but good stability, the hinge and Fc regions of human IgG4 can be modified to introduce a number of alterations. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88 of U.S. Patent No. 8,735,553 to Li et al., which is incorporated by reference in its entirety.

[0165] In another embodiment, an antibody of the disclosure comprises a human IgG4 Fc domain with an S228P and / or R409K substitution (according to the EU numbering system).

[0166] antibody production Antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including, but not limited to, recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as a mammalian host cell, a bacterial host cell, a yeast host cell, an insect host cell, etc.

[0167] The present disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding a heavy or light chain variable region or a segment comprising a complementarity determining region described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region or light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:42, or SEQ ID NO:44.

[0168] The polynucleotides of the present disclosure can encode the variable region sequences of anti-TAA (e.g., GPC3)xCD137 antibodies. They can also encode both the variable and constant regions of the antibodies. Some of the polynucleotide sequences encode polypeptides containing both the heavy and light chain variable regions of one of the exemplified anti-TAA (e.g., GPC3)xCD137 antibodies.

[0169] The present disclosure further provides polynucleotides encoding the anti-GPC3xCD137 antibodies described herein. In some aspects, the polynucleotide encoding the first or second polypeptide of the anti-GPC3xCD137 antibody has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 24. In some embodiments, the polynucleotides described herein can be codon-optimized for expression in host cells, for example, eukaryotic cells, more particularly mammalian cells (e.g., CHO cells).

[0170] The present disclosure also provides expression vectors and host cells for producing the antibodies herein, e.g., anti-CD137 antibodies, and anti-TAA (e.g., GPC3)xCD137 antibodies. The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under the control of an inducing condition. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of antibodies or antigen-binding fragments. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be increased by incorporating enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 1994;20:125, and Bittner et al., Meth. Enzymol. 1987;153:516, each of which is incorporated by reference in its entirety). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0171] Host cells for harboring and expressing antibody chains can be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors, typically containing expression control sequences compatible with the host cell (e.g., an origin of replication), can also be made in these prokaryotic hosts. Additionally, a variety of well-known promoters exist, such as any number of lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or promoter systems derived from phage lambda. Promoters typically control expression (optionally with operator sequences) and contain ribosome binding site sequences for initiating and completing transcription and translation. Other microbes, such as yeast, can also be used to express antibodies. Insect cells can also be used in conjunction with baculovirus vectors. In another embodiment, mammalian host cells are used to express and produce the antibodies of the present disclosure. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring exogenous expression vectors. These include any normal mortal, or normal or abnormal immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is reviewed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987, which is incorporated by reference in its entirety.Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, an enhancer (see, e.g., Queen et al., Immunol. Rev. 1986;89:49-68, incorporated by reference in its entirety), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or tunable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0172] Bispecific antibody production The current standard for engineered heterodimeric antibody Fc domains is the knobs-into-holes (KiH) design, which introduces mutations at the core CH3 domain interface. The resulting heterodimers have reduced CH3 melting temperatures (below 69°C). In contrast, the Zymeworks Azymetric™ platform (supra) heterodimeric Fc design has a thermal stability of 81.5°C, comparable to the wild-type CH3 domain.

[0173] Pharmaceutical Composition Also provided are compositions, such as pharmaceutical preparations, comprising an antibody or antigen-binding fragment thereof of the present invention, or a polynucleotide comprising a sequence encoding the antibody or antigen-binding fragment thereof. In certain embodiments, the composition comprises one or more antibodies or antigen-binding fragments thereof of the present invention, or one or more polynucleotides comprising a sequence encoding one or more antibodies or antigen-binding fragments thereof of the present invention. These compositions can further comprise a suitable carrier, e.g., a pharmaceutically acceptable excipient such as a buffer, which are known in the art.

[0174] The compositions disclosed herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Suitable forms depend on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of an injectable or infusible solution. One suitable mode of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0175] Methods of detection and diagnosis The antibodies or antigen-binding fragments of the present disclosure are useful in a variety of applications, including, but not limited to, methods for detecting CD137 or GPC3. In one embodiment, the antibodies or antigen-binding fragments are useful for detecting the presence of CD137 or GPC3 in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In other embodiments, such tissues include normal tissues and / or cancerous tissues that express CD137 or GPC3 at higher levels than other tissues.

[0176] In one aspect, the present disclosure provides a method for detecting the presence of CD137 or GPC3 in a biological sample. In a specific aspect, the method comprises contacting the biological sample with the antibody of the present disclosure under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, a urine, tissue, sputum, or blood sample.

[0177] Also included is a method for diagnosing disorders associated with GPC3 expression. In a specific embodiment, the method comprises contacting test cells with an anti-GPC3xCD137 antibody, determining (either quantitatively or qualitatively) the expression level of GPC3 expressed by the test cells by detecting binding of the anti-GPC3xCD137 antibody to the GPC3 polypeptide, and comparing the expression level by the test cells with the GPC3 expression level in control cells (e.g., normal cells or non-GPC3-expressing cells of the same tissue origin as the test cells), wherein a higher level of GPC3 expression in the test cells compared to the control cells indicates the presence of a disorder associated with GPC3 expression.

[0178] VI. Treatment method Anti-CD137 antibody The antibody or antigen-binding fragment of the present disclosure is useful in various applications, including but not limited to, methods for treating CD137-related disorders or diseases.In one embodiment, the CD137-related disorders or diseases is cancer.In the case of CD137xTAA multispecific antibodies, the cancer can be specific to TAA, and CD137 acts to recruit immune cells to TAA-expressing tumors.

[0179] In one aspect, the present disclosure provides a method for treating cancer. In certain aspects, the method comprises administering a therapeutically effective amount of an anti-CD137 antibody or antigen-binding fragment thereof, or a CD137-containing multispecific antibody, or a pharmaceutical composition thereof, to a patient in need thereof. In another aspect, the present disclosure provides an anti-CD137 antibody or antigen-binding fragment or multispecific antibody, or a pharmaceutical composition for use in treating cancer. In another aspect, the present disclosure provides the use of an anti-CD137 antibody or antigen-binding fragment, a multispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition in the manufacture of a medicament for treating cancer.

[0180] Cancers include, but are not limited to, gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0181] Anti-GPC3xCD137 multispecific antibody The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for treating GPC3-associated disorders or diseases. In one embodiment, the GPC3-associated disorder or disease is cancer.

[0182] In one aspect, the present disclosure provides a method for treating cancer. In a specific aspect, the method comprises administering a therapeutically effective amount of an anti-GPC3xCD137 antibody or antigen-binding fragment, or a pharmaceutical composition thereof, to a patient in need thereof. In another aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition for use in treating cancer. In another aspect, the present disclosure provides the use of a multispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition in the manufacture of a medicament for treating cancer.

[0183] In one embodiment, the cancer expresses GPC3. In one embodiment, the cancer is an advanced or metastatic solid tumor.

[0184] Cancers may include, but are not limited to, any one or more of liver cancer, lung cancer, gastric cancer, germ cell tumors, thyroid cancer, pancreatic cancer, ovarian cancer, skin cancer, kidney cancer (e.g., Wilms' tumor), esophageal cancer, atypical teratoid rhabdoid tumor of the brain, and undifferentiated synovial sarcoma. In some embodiments, the liver cancer is hepatoblastoma or hepatocellular carcinoma (HCC). In another embodiment, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). In another embodiment, the non-small cell lung cancer is squamous non-small cell lung cancer. In another embodiment, the non-small cell lung cancer is GPC3+ squamous non-small cell lung cancer. In another embodiment, the gastric cancer is alpha-fetoprotein positive (AFP+) gastric cancer. In another embodiment, the kidney cancer is Wilms' tumor. In another embodiment, the esophageal cancer is esophageal squamous cell carcinoma. In another embodiment, the esophageal cancer is GPC3+ esophageal squamous cell carcinoma. In another embodiment, the germ cell tumor is a yolk sac tumor or a non-dysgerminoma.

[0185] others The antibodies or antigen-binding fragments disclosed herein can be administered by any suitable means, for example, parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration if localized treatment is desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time periods, bolus administration, and pulse infusion.

[0186] The antibodies or antigen-binding fragments of the present disclosure can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical professionals. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will vary depending on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above.

[0187] For the prevention or treatment of disease, the appropriate dosage of an antibody or antigen-binding fragment of the disclosure will vary depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0188] VII. Combination Therapy In one embodiment, the anti-CD137 antibodies or anti-CD137-containing multispecific antibodies of the present disclosure, such as anti-CD137xTAA antibodies or anti-GPC3xCD137 antibodies, may be used in combination with other therapeutic agents.

[0189] Other therapeutic agents include, for example, other immune checkpoint antibodies. Such immune checkpoint antibodies may include anti-PD1 antibodies. Anti-PD1 antibodies may include, but are not limited to, tislelizumab, pembrolizumab, or nivolumab. Tislelizumab is disclosed in US Pat. No. 8,735,553. Pembrolizumab (formerly known as MK-3475), disclosed in US Pat. No. 8,354,509 and US Pat. No. 8,900,587, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of PD1 receptor ligands PD-L1 and PD-L2. Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in US Pat. No. 8,008,449 and WO 2006 / 121168.

[0190] Other immune checkpoint antibodies for combination with the anti-CD137 antibodies or anti-CD137-containing multispecific antibodies of the present disclosure may include anti-TIGIT antibodies, including, but not limited to, the anti-TIGIT antibodies disclosed in WO2019 / 129261.

[0191] In one embodiment, the present disclosure provides the combined use of an anti-CD137 antibody or anti-CD137-containing multispecific antibody of the present disclosure (e.g., an anti-CD137xTAA antibody or anti-GPC3xCD137 antibody) and an anti-PD-1 antibody (such as tislelizumab or other anti-PD-1 antibody described above) in the manufacture of a medicament for the treatment of cancer, such as the cancers described above. In another embodiment, the present disclosure provides a combination of an anti-CD137 antibody or anti-CD137-containing multispecific antibody of the present disclosure (e.g., an anti-CD137xTAA antibody or anti-GPC3xCD137 antibody) and an anti-PD-1 antibody (such as tislelizumab or other anti-PD-1 antibody described above) for use in the treatment of cancer, such as the cancers described above.

[0192] Combination therapy can refer to and include any one of the following: - simultaneous administration of such combination therapy to a patient in need of such treatment (where such components are formulated together in a single dosage form that releases said components to said patient substantially simultaneously); - substantially simultaneous administration of such combination to a patient in need of treatment (where such components are formulated separately from one another in separate dosage forms which are taken by the patient at substantially the same time, at the same time that the components are released to the patient at substantially the same time); - sequential administration of such combination therapy to a patient in need of treatment, where such components are formulated separately from one another in separate dosage forms which are taken sequentially by the patient with a substantial time interval between each administration, while the components are released to the patient at substantially different times; and - Sequential administration of such combination to a patient in need of treatment (where such components are formulated together into a single dosage form which releases such components in a controlled manner, whereby the components are released to the patient simultaneously and / or at different times, simultaneously, sequentially and / or overlappingly, and each part may be administered by either the same or different routes).

[0193] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art.

[0194] As used in this specification, including the appended claims, singular terms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0195] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0196] As used herein, the term "anti-cancer agent" refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0197] The terms "CD137" or "TNFRSF9," "ILA," "41BB," or "4-1BB" refer to costimulatory molecules belonging to the TNFRSF family. The nucleic acid sequence of human CD137 is set forth in SEQ ID NO: 36, based on GenBank sequence accession number NM_001561.4. The amino acid sequence of human CD137 is SEQ ID NO: 35.

[0198] The term "glypican 3" (GPC3) is also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, and SGBS1. The amino acid sequence of human GPC3 (SEQ ID NO: 51) can also be found in NCBI Reference Sequence: NP_004475.1. The nucleic acid sequence of human GPC3 is set forth in SEQ ID NO: 52.

[0199] As used herein, the terms "administration," "administering," "treating," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, or diagnostic agent or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell and contact of a reagent with a fluid when such fluid is in contact with the cell. The terms "administration" and "treatment" also refer to treatment of a cell with a reagent, diagnostic agent, binding compound, or with another cell, e.g., in vitro and ex vivo. The term "subject" as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., a rat, mouse, dog, cat, rabbit), and most preferably a human. In one aspect, treating any disease or disorder refers to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating a disease or disorder, either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. In yet another embodiment, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of a disease or disorder.

[0200] The term "subject" in the context of this disclosure is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disorder described herein).

[0201] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen at multiple sites through non-covalent forces. Generally, the more interactions, the stronger the affinity.

[0202] The term "antibody," as used herein, refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0203] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies, human engineered antibodies, single-chain antibodies (scFv), single-domain antibodies, Fab fragments, Fab' fragments, or F(ab')2 fragments. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Furthermore, antibodies include derivatives of antibodies, such as those linked directly or indirectly to another agent (e.g., another drug) or conjugated to another drug. The term "antibody" as used herein includes monospecific antibodies, bispecific antibodies, and multispecific antibodies.

[0204] The term "chimeric antibody" refers to a molecule composed of domains derived from different species, i.e., in which the variable domains of an antibody derived from one host species (e.g., mouse, rabbit, llama, etc.) are fused with the constant domains of an antibody derived from a different species (e.g., human).

[0205] In some embodiments, the anti-GPC3 antibody comprises at least one antigen-binding site, at least a variable region. In some embodiments, the anti-GPC3 antibody comprises an antigen-binding fragment derived from the GPC3 antibody described herein. In some embodiments, the anti-GPC3 antibody is isolated or recombinant.

[0206] In some embodiments, the anti-CD137 antibody comprises at least one antigen-binding site, at least the variable region. In some embodiments, the anti-CD137 antibody comprises an antigen-binding fragment derived from a CD137 antibody described herein. In some embodiments, the anti-CD137 antibody is isolated or recombinant.

[0207] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies with different amino acid sequences within the variable domains, particularly the complementarity-determining regions (CDRs), which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those of skill in the art. See, for example, Kohler et al., Nature. 1975; 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, 1993. The antibodies disclosed herein can be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, IgA, and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High-titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into pristine-primed mice, such as Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluid or from the culture supernatant using column chromatography methods well known to those skilled in the art.

[0208] Generally, the basic structural unit of an antibody comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Furthermore, human heavy chains are usually classified as α, δ, ε, γ, or μ, and antibody isotypes are defined as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and heavy chains also contain a "D" region of approximately 10 amino acids.

[0209] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0210] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called "complementarity-determining regions (CDRs)," which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res. 2001;29:205-206; Chothia and Lesk, J. Mol. Biol. 1987;196:901-917; Chothia et al., Nature. 1989;342:877-883; Chothia et al., J. Mol. Biol. 1992;227:799-817; Al-Lazikani et al., J. Mol. Biol. 1997;273:927-748; ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist. 1999;7,132-136; Lefranc, M.-P. et al., Dev. Comp. Immunol., 27,55-77(2003) (see "IMGT" numbering scheme)).Definitions of antigen-binding sites are also described in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996), and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989), Martin et al., Methods Enzymol., 203:121-153 (1991), and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.In IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0211] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. A hypervariable region comprises amino acid residues from the "CDRs" (e.g., LCDR1, LCDR2, and LCDR3 of the light-chain variable domain and HCDR1, HCDR2, and HCDR3 of the heavy-chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining antibody CDR regions by sequence); see also Chothia and Lesk, J. Mol. Biol. 1987;196:901-917 (defining antibody CDR regions by structure). The terms "framework" or "FR" residues refer to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0212] Unless otherwise indicated, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as single-chain Fvs (ScFvs), nanobodies formed from antibody fragments, and multispecific antibodies.

[0213] As used herein, an antibody "specifically binds" to a target protein means that the antibody exhibits preferential binding to that target relative to other proteins, although this specificity does not require absolute binding specificity. The terms "specifically bind" or "selectively bind" an antibody are used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding fragment, and refer to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biologics, such as a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice as much as background levels and does not specifically bind in significant amounts to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least 10 times as much as background levels of binding and does not specifically bind in significant amounts to other antigens present in the sample.

[0214] As used herein, "antigen-binding domain" refers to the portion of an antibody that specifically binds to an antigen. In some embodiments, it comprises at least six CDRs and specifically binds to an epitope (or three CDRs for single-domain antibodies). The "antigen-binding domain" of a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen-binding domain that specifically binds to a first epitope and a second antigen-binding domain that specifically binds to a second epitope. Multispecific antibodies can be bispecific, trispecific, tetraspecific, etc., with an antigen-binding domain directed to each particular epitope. Multispecific antibodies can be multivalent (e.g., a bispecific tetravalent antibody) comprising multiple antigen-binding domains, for example, two, three, four, or more antigen-binding domains that specifically bind to a first epitope and two, three, four, or more antigen-binding domains that specifically bind to a second epitope.

[0215] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0216] The terms "humanized" or "humanized antibody" refer to forms of antibodies that contain sequences from non-human (e.g., mouse, rabbit, llama, etc.) and human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies comprise substantially all of at least one, and typically 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 FR regions are those of a human immunoglobulin sequence. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). Where necessary to distinguish humanized antibodies from rodent parent antibodies, the name of the antibody clone is prefixed with "hum," "hu," "Hu," or "h." Humanized forms of rodent / camelid antibodies generally contain the same CDR sequences of the rodent parent antibody, but may contain certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0217] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest amino acid sequence identity, as determined with a reference variable region amino acid sequence or subsequence, compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence can also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that comprise variable regions. Sequence identity can be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0218] By "2+2 format" is meant a bispecific antibody that targets two different antigens or two different epitopes, such an antibody comprising two first antigen-binding domains that specifically bind to a first antigen or first epitope, and two second antigen-binding domains that specifically bind to a second antigen or second epitope.

[0219] “Equilibrium dissociation constant (K DThe term "dissociation rate constant (kd, time -1 ) is the association rate constant (ka, time -1 , M -l ) The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than or equal to 10 -8 Less than m, e.g., about 10 -9 Less than M or 10 -10 M or less, and in some embodiments, about 10 -11 Under M, 10 -12 Less than M or 10 -13 It is less than M.

[0220] The terms "cancer" or "tumor" as used herein have the broadest meaning understood in the art and refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer is not limited to any particular type or location.

[0221] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" refers to the substitution of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to GPC3 or CD137. In particular, common conservative changes of amino acids are known in the art.

[0222] As used herein, the term "knobs-into-holes" technology refers to amino acids that together direct the pairing of two polypeptides, either in vitro or in vivo, by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide at the interface where they interact. For example, knobs-into-holes can be used to direct the pairing of two polypeptides, such as the Fc:Fc binding interface of an antibody, C L :C H I interface or V H / V LKnobs-into-holes have been introduced into the interface (see, e.g., US 2011 / 0287009, US 2007 / 0178552, WO 96 / 027011, WO 98 / 050431, and Zhu et al., Protein Science. 1997;6:781-788). In some embodiments, the knobs-into-holes ensure the correct pairing of two different heavy chains during the production of multispecific antibodies. For example, an antibody having knobs-into-hole amino acids in the Fc region of a multispecific antibody may further comprise a single variable domain linked to each Fc region, or may further comprise a different heavy chain variable domain paired with a similar or different light chain variable domain. The knobs-into-holes technology can also be used with VH or VL regions to ensure correct pairing.

[0223] As used herein, the term "knob," in the context of "knob-into-hole" technology, refers to an amino acid change that introduces a protuberance (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0224] As used herein, the term "hole," in the context of "knob-into-hole," refers to an amino acid change that introduces a socket or cavity (hole) in a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0225] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 1990;215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as starting points for searches to find longer HSPs containing them. Word hits are extended outward along each end of each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted if the cumulative alignment score falls by an amount X from the maximum achieved value, if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and both strands are compared.For amino acid sequences, the BLAST program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix of 50 (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA. 1989;89:10915) alignment (B), an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0226] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0227] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 1988;4:11-17, which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), which has been incorporated into the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0228] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0229] The term "operably linked," in the context of nucleic acids, refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcriptional sequence are physically contiguous to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous to or located in close proximity to the coding sequence whose transcription they enhance.

[0230] As used herein, the term "pharmaceutically acceptable excipient" includes all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0231] As used herein, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case will be apparent to one of ordinary skill in the art and can also be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined components for effective treatment of the disease, disorder, or condition.

[0232] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in this disclosure. Such administration encompasses the co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also encompasses co-administration in multiple or separate containers (e.g., capsules, powders, and liquids) for each active ingredient. The powders and / or liquids may be reconstituted or diluted to the desired dosage prior to administration. Furthermore, such administration also encompasses the use of various therapeutic agents in a sequential manner, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effects of the drug combination in treating the conditions or disorders described herein.

[0233] As used herein, the term "in combination with" means that the antibody herein is administered to a subject simultaneously with, immediately before, or immediately after the administration of an additional therapeutic agent. In certain embodiments, the antibody herein is administered as a combination with an additional therapeutic agent.

[0234] equivalent While the present invention has been described with reference to its detailed description, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0235] It is understood that one, some, any, or all of the features of the various embodiments described herein may be combined to form additional embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. [Example]

[0236] Example 1. Generation of single-chain anti-huCD137 VHH antibodies CD137 recombinant protein for phage campaigns and binding assays To discover VHH antibodies against human CD137, several recombinant proteins were designed, expressed, and subjected to phage panning and screening. The cDNA coding region of full-length human CD137 (huCD137) was ordered based on the CD137 GenBank sequence (accession number: NM_001561.4, the gene is available from Sinobio, catalog number HG10041-M, and is referred to herein as SEQ ID NO: 36). The human CD137 ligand (TNFSF9) was ordered based on the CD137 ligand GenBank sequence (accession number: NM_003811.3, the gene is available from Sinobio, catalog number HG15693-G). Briefly, the coding region of the extracellular domain (ECD) consisting of amino acids (AA) 24-183 of huCD137 (SEQ ID NO: 35) and the coding region of the ECD consisting of amino acids 71-254 of huCD137 ligand (SEQ ID NO: 37) were amplified by PCR. The coding region of mIgG2a Fc (SEQ ID NO: 39) was PCR-amplified and then conjugated with the ECD of human CD137 or the ECD of human CD137 ligand by overlap PCR to create mIgG2a Fc fusion proteins. The PCR products were then cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad, CA, USA) to obtain two recombinant mIgG2a Fc fusion protein expression plasmids: human CD137 ECD-mIgG2a and human CD137 ligand ECD-mIgG2a. Alternatively, the coding region of the ECD consisting of AA24-183 of huCD137 (SEQ ID NO: 35) was also cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad, CA, USA) fused with a 6xHis tag at the C-terminus to obtain human CD137-ECD-his. For the production of recombinant fusion proteins, the plasmids were transiently transfected into a HEK293-based mammalian cell expression system (developed in-house) and cultured for 5–7 days in a CO2 incubator equipped with a rotating shaker. The supernatant containing the recombinant protein was collected and removed by centrifugation.Recombinant proteins were purified using a Protein A column (catalog no. 17127901, GE Life Sciences) or Ni-NTA agarose (catalog no. R90115, Invitrogen). All recombinant proteins were dialyzed against phosphate-buffered saline (PBS) and stored in small aliquots at −80°C in a freezer.

[0237] Llama immunization and phage library construction One llama was immunized with human CD137 ECD-mIgG2a. Two weeks after the fourth immunization, llama PBMCs were collected for RNA extraction using standard techniques (Chomczynski, et al., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Analytical. Biochem. 1987;162(1):156-159).

[0238] A phage library was constructed by reverse transcription and splice overlap extension PCR. The PCR products were double-digested with NcoI / NotI and ligated into the phagemid vector pCANTAB-5E. The repertoire was then transformed into Escherichia coli TG1 bacteria and verified by DNA Sanger sequencing of random clones (more than 96 clones analyzed). After a rescue step using KM13 helper phage, phages were purified directly from the culture supernatant by two precipitations with PEG / NaCl. After transformation into E. coli bacteria, phages with sizes >10 7 A library of

[0239] Phage display panning and screening Phage display selection was performed by phage display using standard protocols (Silacci et al., (2005) Proteomics, 5, 2340-50; Zhao et al., (2014) PLoS One, 9, e111339). Briefly, 10 μg / ml of immobilized human CD137 ECD-mIgG2a in immunotubes (catalog 470319, ThermoFisher) was used in rounds 1 and 2. Hut78 / huCD137 cells were used for round 3 selection. Immunotubes were blocked with 5% milk powder (w / v) in PBS supplemented with 1% Tween 20 (MPBST) for 1 hour. After washing with PBST (PBS buffer supplemented with 0.05% Tween 20), 5 × 10 IgG from each sublibrary were selected. 12 (Round 1) or 5 x 10 11 Phages (round 2) were depleted with human CD40 ECD-mIgG2a in MPBST for 1 hour and then incubated with antigen for 1 hour. In the third and fourth rounds of selection, cell panning was performed using Hut78 / huCD137 cells along with HEK293 (ATCC, CRL-1573) cells as depletion cells. After washing with PBST, bound phages were eluted with 100 mM triethylamine (Sigma-Aldrich). The eluted phages were used to infect mid-logarithmic phase E. coli TG1 bacteria, which were plated on TYE agar plates supplemented with 2% glucose and 100 μg / ml ampicillin. After three rounds of selection, individual clones were selected, and phage-containing supernatants were prepared using standard protocols. Anti-huCD137 VHH antibodies were screened using phage ELISA.

[0240] For phage ELISA, Maxisorp immunoplates were coated with antigen and blocked with 5% milk powder (w / v) in PBS buffer. Phage supernatants were blocked with MPBST for 30 min and added to the wells of the ELISA plate for 1 h. After washing with PBST, bound phages were detected using HRP-conjugated anti-M13 antibody (GE Healthcare) and 3,3',5,5'-tetramethylbenzidine substrate (catalog: 00-4201-56, eBioscience, USA). CD137-expressing cells (10 5 Cells (0.01% / well) were incubated with ELISA-positive phage supernatants and subsequently bound with Alexa Fluro-647-labeled anti-M13 antibody (GE Healthcare). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA).

[0241] Expression and purification of Fc-fused VHH antibodies Next, we constructed anti-huCD137 VHH antibodies in a human Fc-fused VHH antibody format (VHH-Fc) using an expression vector developed in-house. The VHH domain antibody was fused to the N-terminus of human Fc via a G4S linker (SEQ ID NO: 62). We used an Fc-null version of human IgG1 (an inactive Fc lacking FcγR binding, SEQ ID NO: 19). Expression and preparation of the Fc-fused VHH antibodies were achieved by transfection into 293G cells and purification using a Protein A column (Cat. No. 17543802, GE Life Sciences). The purified antibody was concentrated to 0.5-5 mg / mL in PBS, aliquoted, and stored in a -80°C freezer.

[0242] Example 2. Characterization of purified anti-huCD137 VHH antibodies For antigen ELISA, Maxisorp immunoplates were coated with antigen and blocked with 3% BSA (w / v) in PBS buffer (blocking buffer). Monoclonal VHH antibodies were blocked with blocking buffer for 30 minutes and added to the wells of the ELISA plate for 1 hour. After washing with PBST, bound antibodies were detected using an HRP-conjugated anti-human IgG antibody (Sigma, A0170) and 3,3',5,5'-tetramethylbenzidine substrate (catalog: 00-4201-56, eBioscience, USA). Ligand competition was also applied to the ELISA assay. The results of ELISA analysis and ligand competition for one representative clone, BGA-9612, are shown in Figure 1. The results showed that BGA-9612 (SEQ ID NOs: 1-5) could bind to human CD137 with good affinity, but did not show binding to mouse CD137. BGA-9612 binding to human CD137 may be reduced by competition with the huCD137 ligand.

[0243] Example 3. Humanization of anti-human CD137 VHH BGA-9612 For the humanization of BGA-9612, human germline IgG genes were searched for sequences sharing high homology with the BGA-9612 variable region cDNA sequence by comparing with the human immunoglobulin gene databases on the IMGT and NCBI websites. Human IGVH genes, which are frequently present in the human antibody repertoire (Glanville et al., 2009 PNAS 106:20216-20221) and share high homology with BGA-9612, were selected as templates for humanization.

[0244] Humanization was performed using the CDR grafting method (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and the humanized VHH derived from BGA-9612 was engineered into an Fc-VHH format using an expression vector developed in-house. The humanized VHH from BGA-9612 was fused to the C-terminus of the Fc using a G4S linker (SEQ ID NO: 62) in an Fc-VHH format using an expression vector developed in-house containing a human IgG1 Fc variant (SEQ ID NO: 19) with an adaptable subcloning site. Expression and preparation of the humanized VHH derived from BGA-9612 was achieved by transfection of the construct into ExpiCHO™ cells and purification using a Protein A column. The purified antibody was concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80°C in a freezer.

[0245] Framework Swapping In the first round of humanization, mutations from Camelidae to human amino acid residues in the framework regions were guided by the simulated 3D structure. Structurally important Camelidae framework residues for maintaining the canonical structure of the CDRs, including amino acid residues R27, F37, E44, R45, Q46, Y47, G49, V78, I94, and Q103 (Kabat numbering), were retained in the first round of humanization. Specifically, the CDRs of BGA-9612 VHH (SEQ ID NOS: 1-3) were grafted into the framework of the human germline variable region gene IGVH3-23 while retaining some Camelidae framework residues, resulting in BGA-6582 (SEQ ID NOS: 8-9). The binding affinities of BGA-9612 and BGA-6582 by SPR are shown in Table 2.

[0246] In these examples, Kabat numbering and definitions were used for CDR and VH / VL sequences, and EU numbering was used for Fc sequences. [Table 2]

[0247] Based on BGA-6582, several single mutations were made to convert the retained camelid residues in the framework regions to the corresponding human germline residues and a combination of single mutations, as shown in Table 3. All humanized mutations were made using primers containing mutations at specific positions and a site-directed mutagenesis kit (catalog no. FM111-02, TransGen, Beijing, China). The desired mutations were confirmed by sequence analysis. These further humanized VHHs from BGA-6582 were tested in SPR binding assays, as shown in Table 3. BGA-3726 (SEQ ID NOS: 1-3 and 6-7), which contains amino acid Q46E based on BGA-6582, was selected for further manipulation. Amino acid residues F37, Y47, G49, and I94 in the framework regions are important for binding to CD137 (Kabat numbering). [Table 3]

[0248] Improved biophysical properties Humanized VHH BGA-3726 did not exhibit better overall biophysical properties (e.g., Tm or Tagg) than camelid VHH BGA-9612 (data not shown). Therefore, BGA-3726 was further engineered by introducing mutations into the CDR and framework regions to improve its biophysical properties for therapeutic use in humans.

[0249] Amino acid N73 in framework region 3 (FR3) of BGA-3726 was identified as a hotspot for deamination. To mitigate the risk of post-translational modifications (PTMs), the subsequent amino acid S74 was mutated to alanine (a back mutation to a camelid amino acid residue).

[0250] In summary, based on BGA-3726, the following engineered versions of humanized VHHs were derived from the above mutation process: (1) BGA-3544 (Kabat numbering) (SEQ ID NOS: 1, 10, 3, and 11-12), which contains amino acids N64K and N65G in HCDR2 and L5V, S82bN, A84P, and L108Q in the framework; (2) BGA-70, which contains L5V, S82bN, A84P, and L108Q in the framework; 31 (Kabat numbering) (SEQ ID NOS: 1-3, and 15-16), (3) BGA-9502 (Kabat numbering) (SEQ ID NOS: 13-14) containing amino acids N64K and N65G in HCDR2 and L5V, S74A, S82bN, A84P, and L108Q in the framework, and (4) BGA-2524 (Kabat numbering) (SEQ ID NOS: 17-18) containing L5V, S74A, S82bN, A84P, and L108Q in the framework.

[0251] For affinity measurements, the antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The results of the SPR binding profiles of the anti-CD137 antibodies are summarized in Table 4. BGA-3544 and BGA-7031 have similar binding affinities with dissociation constants of 17.6 nM and 11.7 nM, respectively, which are comparable to that of BGA-9612 (15.2 nM). BGA-6582 also has a binding affinity comparable to that of BGA-9612. [Table 4]

[0252] The biophysical properties of the chimeric and humanized Fc-VHHs were tested. The biophysical properties tested included melting temperature by DSC, aggregation temperature by SLS266, hydrophobicity by HIC-HPLC, and self-association tendency by AC-SINS (see detailed description below). BGA-9612 demonstrated optimal thermal stability by Tm and Tagg, and good colloidal stability by AC-SINS. As shown in Table 5, the humanized VHHs BGA-3544 and BGA-7031 exhibited overall biophysical properties comparable to those of BGA-9612 (chimera). Furthermore, BGA-3544 and BGA-7031 showed improved Tm compared to BGA-9612 (chimera). [Table 5]

[0253] Melting temperatures (Tm) were measured using a high-throughput MicroCal™ VP-Capillary DSC (Malvern Instruments, Northampton, MA). Thermograms of each protein (350 μL at 0.5 mg / mL) were acquired from 20°C to 100°C using a scan rate of 60°C / hr. A buffer-alone thermogram was subtracted from each protein sample. Results indicate the transition temperature (Tm) midpoint and calorimetric enthalpy (ΔH) values ​​for the samples.

[0254] The aggregation temperature Tagg (°C) represents the colloidal stability of the sample and was obtained by monitoring the onset of aggregation with an SLS266 using UNCLE™ (Unchained Lab, Pleasanton, CA). The sample was loaded into a Uni and the temperature was increased from 15°C to 95°C. Back-reflecting optics cannot detect near-ultraviolet light scattering by protein aggregates, and therefore only unscattered light reaches the detector. Therefore, the reduction in back-reflected light is a direct measure of aggregation in the sample.

[0255] To determine the hydrophobicity of a given VHH using HPLC e2695 (Waters Corporation, Milford, MA), protein samples were diluted with mobile phase A (50 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and then filtered before loading onto a MAbPac™ HIC-10 column (Thermo Fisher Scientific, Waltham, MA) equilibrated with mobile phase A. Samples were eluted using a reverse gradient from mobile phase A to mobile phase B (50 mM sodium phosphate, pH 7.0). Following elution, the A280 nm fractions were recorded as a function of time, and the data were then exported and analyzed using Empower™ software. The retention time of each sample was compared to a reference. Retention time is characteristic of the hydrophobicity of the VHH, with longer elution times correlating with a higher degree of hydrophobicity.

[0256] The AC-SINS assay measures protein self-interactions by capturing VHHs on the surface of gold colloids, which exhibit surface resonance vibrations at frequencies in the visible light spectrum. As the immobilized antibodies self-interact, the colloids aggregate, shifting their vibrational frequency and absorbing at longer wavelengths. Gold nanoparticles were incubated with an 80 / 20 (v / v) mixture of capture / non-capture antibodies. The coated gold nanoparticles were then 10x concentrated in PBS. Samples were prediluted to a concentration of 50 μg / ml in PBS. 10 μL of 10x concentrated AuNPs were incubated with 100 μL of sample in a 384-well plate in the dark at room temperature for 2 hours. The absorbance spectrum of each well was then read from 510 to 570 nm using a BMG ClarioStar™ (BMG Labtech, Offenburg, Germany). The red-shift in the maximum absorption peak and its intensity indicates the self-interaction tendency of the VHH sample tested.

[0257] Example 4. Binding activity of humanized VHHs to native CD137 To assess the binding activity of anti-CD137 antibodies to native CD137 on live cells, HuT78 cells were engineered to overexpress human CD137. Live HuT78 / CD137 cells were seeded into 96-well plates and incubated with serial dilutions of anti-CD137 antibodies. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC of dose-dependent binding to human native CD137. 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism™. The data are shown in Figure 2 and Table 6. The humanized VHHs retained subnanomolar binding affinity to native CD137. [Table 6]

[0258] Example 5. Anti-GPC3xCD137 multispecific antibody Agonistic anti-huCD137 antibodies have demonstrated toxicity in clinical settings, which may indicate that systemic FcγR crosslinking is not ideal for CD137 activation. Our goal was to achieve potent tumor-specific CD137 stimulation for a wide range of cancers without systemic CD137 activation. To overcome the dependency on FcγR crosslinking, we generated an anti-GPC3xCD137 multispecific antibody containing the following features, as shown in Figure 3. This specific construct included a 2:2 modular IgG fusion-like multispecific antibody format, a bivalent F(ab')2 fragment that binds to human GPC3, a VH domain fragment with a C-terminal fusion to the CH3 that binds huCD137, and a null version of the Fc of huIgG1 (IgG1mf Fc, SEQ ID NO: 53) that lacks FcγR binding but retains FcRn binding. The amino acid and DNA sequences of the GPC3 antibody are shown in Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

[0259] To further increase the in vivo half-life of the GPC3xCD137 multispecific antibody, YTE mutations (M252Y / S254T / T256E, EU numbering) located in CH2 of the IgG1mf Fc region were introduced into IgG1mf Fc to generate IgG1mf Fc-YTE (SEQ ID NO: 20).

[0260] The following GPC3xCD137 polyspecific antibodies were generated:

[0261] BE-933: The GPC3 antibody (SEQ ID NOs: 41-50) was combined with the CD137 VHH BGA-9612 (SEQ ID NOs: 1-5) to yield construct BE-933 (SEQ ID NOs: 23-24 for VL, SEQ ID NOs: 27-28 for VH).

[0262] BE-774: The YTE mutations in Fc were introduced into BE-933 to generate BE-774 (SEQ ID NOs: 23-24 for VL, SEQ ID NOs: 25-26 for VH).

[0263] BE-653: The GPC3 antibody (SEQ ID NOs: 41-50) was combined with the CD137 VHH BGA-7031 (SEQ ID NOs: 1-3 and 15-16) to yield construct BE-653 (SEQ ID NOs: 23-24 for VL, SEQ ID NOs: 29-30 for VH).

[0264] BE-915: The GPC3 antibody (SEQ ID NOs: 41-50) was combined with the CD137 VHH BGA-2524 (SEQ ID NOs: 1-3 and 17-18) and YTE mutations in the Fc to yield construct BE-915 (SEQ ID NOs: 23-24 for VL, SEQ ID NOs: 21-22 for VH).

[0265] BE-647: The GPC3 antibody (SEQ ID NOs: 41-50) was combined with the CD137 VHH BGA-9502 (SEQ ID NOs: 1, 10, 3, 13-14) to yield construct BE-647 (SEQ ID NOs: 23-24 for VL, SEQ ID NOs: 31-32 for VH).

[0266] BE-621: The GPC3 antibody (SEQ ID NOs: 41-50) was combined with the CD137 VHH BGA-9502 (SEQ ID NOs: 1, 10, 3, 13-14) as well as YTE mutations in the Fc to yield construct BE-621 (SEQ ID NOs: 23-24 for VL, SEQ ID NOs: 33-34 for VH).

[0267] Example 6. Target binding activity of anti-GPC3xCD137 bispecific antibodies BE-774 was characterized for its binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences). Briefly, anti-kappa antibody was immobilized on an activated CM5 biosensor chip (catalog: BR100839, GE Life Sciences). BE-774 was flowed over the chip surface and captured by the anti-kappa antibody. Next, serial dilutions (6.0 nM to 2150 nM) of human CD137 ECD-mIgG2a or huGPC3-His were flowed over the chip surface, and the change in surface plasmon resonance signal was analyzed to determine the association rate (k) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k onThe EC2 value was calculated as . The results demonstrated that BE-774 exhibited binding to huCD137 and huGPC3, as shown in Table 8 below. To evaluate the binding activity of the BE-774 bispecific antibody to native huCD137 on live cells, Hut78 cells were transfected to overexpress human CD137. Live Hut78 / huCD137-expressing cells were seeded into 96-well plates and incubated with serial dilutions of BE-774. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC2 value for dose-dependent binding to human native CD137 50 The EC50 values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism™. As shown in Figure 4A, BE-774 specifically bound to native CD137 on live cells in a dose-responsive manner with an EC50 of 0.7 nM. To evaluate binding to huGPC3 on live cells, HepG2 cells expressing huGPC3 were seeded into 96-well plates and incubated with serial dilutions of BE-774. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC50 values ​​for dose-dependent binding to human native GPC3 were calculated. 50 The EC50 values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism™. As shown in Figure 4B, BE-774 demonstrated specific binding to native GPC3 on live cells in a dose-responsive manner with an EC50 of 0.9 nM. [Table 8]

[0268] Example 7. Anti-GPC3xCD137 induces activation of T cells co-cultured with GPC3-positive tumor cells The functional activity of the anti-GPC3xCD137 bispecific antibody was evaluated in in vitro coculture experiments using human peripheral blood mononuclear cells (PBMCs) and an OS8-expressing hepatocellular carcinoma (HCC) cell line. OS8 is a single-chain variable fragment (scFv) of the anti-human CD3 antibody OKT3 fused to the C-terminal domain (113–220 aa) of mouse CD8α, including the hinge, transmembrane, and cytoplasmic domains. When expressed on target cells, OS8 was able to provide signal 1 for T cell activation (Figure 5A). Highly GPC3-expressing HepG2 cells were selected to evaluate the functional activity of the GPC3xCD137 bispecific antibody, while SK-HEP-1 (GPC3-negative) was used as a negative control cell line.

[0269] Frozen human PBMCs (AllCells) were thawed in RPMI 1640 medium and incubated overnight at 37°C. OS8-expressing target cells were seeded into 384-well plates and allowed to adhere for 16 hours. The following day, PBMCs were added to the 384-well plates at an effector-to-target cell ratio (E:T) of 2:1. Co-cultured cells were then treated with serial dilutions of BE-774 or BE-653 for 48 hours at 37°C. Culture supernatants were collected for subsequent measurement of IFN-γ and IL-2 concentrations using a TR-FRET-based method (Degorce, Francois, et al. Current Chemical Genomics. 2009, 3:22) as described in the manufacturer's manual (Cisbio). These results demonstrated that anti-GPC3xCD137 bispecific antibodies, including BE-774 and BE-653, induced dose-dependent cytokine release in PBMCs cocultured with HepG2 cells, but not in GPC3-negative cells (Figure 5B). PBMCs from two donors were tested, and the results are shown in Figure 5B.

[0270] Example 8. Anti-GPC3xCD137 enhances T cell killing activity against GPC3-positive tumor cells The ability of the anti-GPC3xCD137 bispecific antibody to induce T cell killing activity was evaluated in coculture experiments by impedance measurements using an xCELLigence™ RTCA MP instrument (Agilent Technologies). Frozen human PBMCs (AllCells) were thawed in RPMI 1640 medium and incubated overnight at 37°C. Target cells were seeded into 96-well E-plates (Agilent Technologies) and allowed to adhere for 16 hours. The following day, PBMCs were added to the 96-well E-plates at an effector-to-target cell ratio (E:T) of 5:1. The cocultured cells were then treated with serial dilutions of BE-774 or BE-653 in combination with EpCAM / CD3 bispecific T cell engager (BiTE), which provides signal 1 for T cell activation (Figure 6A). The experiment was continued for 4 days to measure electrode impedance with live, adherent target cells. These results showed that both BE-774 and BE-653 dose-dependently enhanced T cell killing activity against GPC3-expressing HepG2 cells, but not against GPC3-negative SK-OV-3 cells (Fig. 6B).

[0271] Example 9. Pharmacokinetics of BE-774 and BE-933 in cynomolgus monkeys Blood samples were collected from cynomolgus monkeys at 0, 0.00694 (10 min), 0.0417 (1 h), 0.167 (4 h), 0.333 (8 h), 1, 4, 7, 10, 14, 21, and 28 days after intravenous administration of 5 mg / kg BE-774 or BE-933. Serum was then separated by centrifugation (4°C, 3500 × g, 2 min). BE-774 or BE-933 concentrations were measured by an in-house developed ELISA. Briefly, for BE-774 or BE-933 measurements, tagged GPC3 protein (C414, Novoprotein, China) was used as the capture reagent, and biotin-labeled CD137 antigen (41B-H82E6, ACRO, China) was used as the detection reagent. The pharmacokinetic (PK) profiles of BE-774 and BE-933 at a dose level of 5 mg / kg are shown in Figure 7. The PK parameters of BE-774 and BE-933 at a dose level of 5 mg / kg are shown in Table 9. BE-774 with the YTE mutation in Fc showed a significantly improved pharmacokinetic profile compared to BE-933. [Table 9]

[0272] Example 10. Pharmacokinetics of BE-774 and BE-933 in the hFcRn mouse model After intravenous administration of 3 mg / kg of BE-774 or BE-933 in hFcRn mice, blood samples were collected at 0, 0.0833 (2 hours), 1, 3, 7, 10, 14, 21, and 28 days, followed by centrifugation (4°C, 3500 × g, 2 minutes) to separate serum. BE-774 or BE-933 concentrations were measured by an in-house developed ELISA. Briefly, for BE-774 or BE-933 measurement, tagged GPC3 protein (C414, Novoprotein, China) was used as the capture reagent, and biotin-labeled CD137 antigen (41B-H82E6, ACRO, China) was used as the detection reagent. The pharmacokinetic (PK) profiles of BE-774 and BE-933 at the 3 mg / kg dose level are shown in Figure 8. The PK parameters of BE-774 and BE-933 at the 3 mg / kg dose level are shown in Table 10. BE-933 had a favorable pharmacokinetic profile in hFcRn mice, and BE-774, which has the YTE mutation in the Fc, showed a significantly improved pharmacokinetic profile compared to BE-933. [Table 10]

[0273] Example 11. SPR binding of BE-915 to CD137 and GPC3 The binding kinetics of the antibodies were measured using surface plasmon resonance (SPR). SPR was used to measure the on-rate constants (K) of the antibodies to recombinant proteins of CD137 and GPC3. a ) and off-rate constant (K d ) and then measure the affinity constant (K D ) were determined. These results show that BE-915 has good binding affinity to both human CD137 and human GPC3 (Tables 11 and 12).

[0274] To test the binding specificity of BE-915 to CD137 from different species, SPR binding experiments were performed using human CD137 (Cat: 41B-H5256, Acrobio, CHINA) and cynomolgus monkey CD137 ECD (SEQ ID NO: 102) as bait proteins. As shown in Table 11, BE-915 exhibited high binding affinity to human CD137, with a K D is approximately 3.6 nM, which is the same as that for cynomolgus monkey CD137 (K D The binding affinity of the anti-CD137 antibody BGA-2524 used in BE-915 to human CD137 and cynomolgus monkey CD137 is also shown to be high.

[0275] To test the binding specificity of BE-915 to GPC3 from different species, SPR binding studies were performed using human GPC3 (Cat. No. 10088-H08H, Sino Biological, China) and cynomolgus monkey GPC3 (Cat. No. GP3-C5225, Acrobio, China) as bait proteins. As shown in Table 12, BE-915 exhibited binding affinity to human GPC3, with a K D The K for cynomolgus monkey GPC3 was approximately 2.4 nM. D was approximately 0.53 nM. [Table 11] [Table 12]

[0276] Example 12. Binding of BE-915 to native human CD137 and human GPC3 To verify the binding of BE-915 to native human CD137 and human GPC3 expressed on cells, the binding of BE-915 was evaluated using HuT78 cells overexpressing human CD137 (HuT78 / CD137) and HepG2 cells expressing native human GPC3, separately. Fluorescence-activated cell sorting (FACS) showed that BE-915 inhibited the EC , , and , respectively, of human CD137 and human GPC3 in a dose-dependent manner, as shown in Figure 9. 50 demonstrated potent binding activities of 0.69 nM (Figure 9B) and 1.09 nM (Figure 9A). The isotype control huIgG (catalog: 02-7102, Thermo, USA) showed no binding activity to HuT78 / CD137 or HepG2 cells.

[0277] Example 13. Binding specificity of BE-915 to other TNFRSF members To test the binding specificity of BE-915 to other TNFRSF members, ELISA was performed by coating plates with TNFRSF4 / OX40 (catalog: OXO-H5252, Acrobio, CHINA), TNFRSF7 / CD27 (catalog: CD7-H5257, Acrobio, CHINA), TNFRSF14 / HVEM (catalog: CD7-H522b, Acrobio, CHINA), TNFRSF8 / CD30 (catalog: HVM-H5258, Acrobio, CHINA), and human CD137 (catalog: 41B-H5256, Acrobio, CHINA) followed by binding with E-915. As shown in Figure 10, BE-915 specifically binds to human CD137 but not to other TNFRSF members. It has also been shown that the anti-CD137 antibody BGA-2524 used in BE-915 specifically binds to human CD137.

[0278] Example 14. BE-915 competes with human CD137L for binding to human CD137 To accurately evaluate the blocking effect of BE-915 on CD137-CD137L binding at the cellular level, a cell-based blocking assay using HuT78 / CD137 was established. Competitive blocking of CD137L on the interaction between CD137 and BE-915 was measured by detecting the binding of BE-915 (starting at 15 μg / mL, followed by three-fold serial dilutions) to human CD137 expressed on HuT78 in the presence of 10 μg / mL, 1 μg / mL, 0.1 μg / mL, or 0 μg / mL of CD137L (Cat. No. 41L-H52D4, Acrobio, China). Binding signals were detected using the secondary antibody anti-hFc 647 (Cat. No. 109-605-098, Jackson, USA). huIgG (Cat. No. 02-7102, Thermo, USA) was used as an isotype control (Figure 11A). As the CD137L concentration increased, the binding of BE-915 to CD137 decreased (Figure 11A). Competitive blocking of the interaction between CD137 and CD137L by BE-915 was measured by detecting the binding of CD137L (starting at 15 μg / mL, followed by 3-fold serial dilutions) to CD137 expressed on HuT78 in the presence of 1 μg / mL, 0.1 μg / mL, or 0 μg / mL of BE-915. The binding signal was detected using a secondary antibody, anti-his 647 (catalog: A01802, Genscript, China). hu IgG (catalog: 02-7102, Thermo, USA) was used as an isotype control (Figure 11B). As the BE-915 concentration increased, the binding of CD137L to CD137 decreased (Figure 11B). These data demonstrate that BE-915 cross-competes with CD137L for binding to human CD137. They also show that the anti-CD137 antibody BGA-2524 used in BE-915 cross-competes with CD137L for binding to human CD137.

[0279] Example 15. BE-915 induces T cell activation co-cultured with GPC3-positive tumor cells The functional activity of the GPC3 x CD137 bispecific antibody BE-915 was evaluated in an in vitro coculture assay using human peripheral blood mononuclear cells (PBMCs) and OS8-expressing hepatocellular carcinoma (HCC) cell lines (Figure 12A). Based on FACS analysis, three HCC cell lines with high to low GPC3 expression, HepG2, Huh7, and Hep3B (Figure 12B), were selected to evaluate the effect of GPC3 levels on the functional activity of BE-915. SK-HEP-1, which does not express GPC3, was used as a negative control cell line.

[0280] Frozen human PBMCs (OriBiotech) were thawed in RPMI 1640 medium and incubated overnight at 37°C. OS8-expressing target cells were seeded into 384-well plates and allowed to adhere for 16 hours. The following day, PBMCs were added to the 384-well plates at an effector-to-target cell ratio (E:T) of 2:1. Co-cultured cells were then treated with serial dilutions of BE-915 for 48 hours at 37°C. Culture supernatants were collected for subsequent measurement of IFN-γ and IL-2 concentrations using a TR-FRET-based method (Degorce, Francois, et al. Current Chemical Genomics. 2009, 3:22) as described in the manufacturer's manual (Cisbio). Results showed that BE-915 induced dose-dependent cytokine release in PBMCs from two independent donors co-cultured with GPC3-expressing cells, but not in GPC3-negative cells (Figure 12C).

[0281] Example 16. BE-915 enhances PBMC-based cell killing cultured with GPC3-positive tumor cells The killing activity of BE-915-controlled T cells was evaluated in coculture experiments with impedance measurements using an xCELLigence RTCA MP instrument (Agilent Technologies). Frozen human PBMCs (OriBiotech) were thawed in RPMI 1640 medium and incubated overnight at 37°C. Target cells were seeded into 96-well E-plates (Agilent Technologies) and allowed to adhere for 16 hours. The next day, PBMCs were added to the 96-well E-plates at an effector-to-target cell ratio (E:T) of 5:1. The cocultured cells were then treated with serial dilutions of BE-915 combined with EpCAM / CD3 bispecific T cell engager (BiTE), which provides signal 1 for T cell activation (Figure 13A). Based on FACS analysis, three HCC cell lines with high to low GPC3 expression, HepG2, Huh7, and Hep3B (Figure 13B), were selected to evaluate the effect of GPC3 levels on the functional activity of BE-915. SK-OV-3, which does not express GPC3, was used as a negative control cell line.

[0282] The experiment was continued for 4 days to measure electrode impedance with live, adherent target cells. Consistent with the cytokine production assay, BE-915 dose-dependently enhanced T cell killing activity against GPC3-expressing cells, but not against GPC3-negative cells (Figure 13C). PBMCs from two donors were used in this experiment.

[0283] Example 17. Pharmacokinetic profile of BE-915 in cynomolgus monkeys Blood samples were collected from cynomolgus monkeys at 0, 0.167 hours, 1 hour, 4 hours, 8 hours, and 1, 3, 6, 9, 13, 20, and 27 days after intravenous infusion of 5 mg / kg BE-915. Serum was then separated by centrifugation (4°C, 3000 × g, 15 minutes). BE-915 concentrations were measured using an in-house developed ELISA ligand-binding assay. Briefly, for BE-915, tagged GPC3 antigen (C414, Novoprotein, China) was used as the capture reagent, and biotin-labeled CD137 antigen (41B-H82E6, ACRO, China) was used as the detection reagent. The resulting pharmacokinetic profiles and parameters are shown in Figure 14 and Table 13, respectively. In the 5 mg / kg dose group, BE-915 levels were below the lower limit of quantitation (0.0391 μg / mL) on day 13 after dosing. Anti-drug antibodies (ADAs) were detected in serum from day 9 in the 5 mg / kg group, indicating a potential impact on the pharmacokinetic curve. PK parameters were calculated after removing time-point concentration values. The clearance of BE-915 was 10.4 mL / day / kg, with an antibody-like half-life of 3.2 days. [Table 13]

[0284] Example 18. Efficacy of BE-915 monotherapy in the MC38 / hGPC3 model in humanized CD137 knock-in mice The in vivo efficacy of BE-915 was investigated in the MC38 / hGPC3 mouse colorectal cancer model in humanized CD137 knock-in mice. MC38 / hGPC3 cells were subcutaneously implanted into the right flank of recipient mice. Seven days after cell inoculation, mice were randomized into four groups according to tumor volume. BE-915 was administered intraperitoneally on day 1 and once weekly for 18 days. BE-915 (0.1, 0.5, and 3.0 mg / kg, once weekly) effectively inhibited tumor growth. Tumor volume was significantly reduced at the study endpoint (D18). Furthermore, the tumor-free rates for the 0.1, 0.5, and 3.0 mg / kg groups were 0%, 0%, and 10%, respectively, on day 18 (Figure 15 and Table 14). There was no significant effect on animal weight in any treatment group throughout the study. [Table 14]

[0285] Example 19. Efficacy of the combination of BE-915 and anti-PD-1 antibody in the LL / 2 / hGPC3 model in humanized 4-1BB knock-in mice The antitumor activity of the combination of BE-915 and an anti-mouse PD-1 antibody was investigated in a LL / 2 / hGPC3 syngeneic model (lung cancer) in humanized CD137 knock-in mice. LL / 2 / hGPC3 cells were implanted into the mice. Seven days after cell inoculation, the mice were randomized into four groups according to tumor volume. Mice treated with the combination of BE-915 (10.0 mg / kg, once weekly) and an anti-mouse PD-1 antibody (10.0 mg / kg, once weekly) showed synergistic tumor growth inhibition. The tumor growth inhibition rate in the combination group was 74.7% on day 13, which was significantly higher than that in the groups treated with BE-915 (34.1%) or anti-PD-1 (38.0%) alone (Figure 16 and Table 15). No significant effect on animal weight was observed in any of the treatment groups throughout the study. [Table 15]

[0286] Example 20. Biophysical properties of BE-915 The biophysical properties of BE-774 (using camelid CD137 VHH BGA-9612) and BE-915 (using humanized CD137 VHH BGA-2524) were tested. The biophysical properties tested included melting temperature, aggregation temperature, hydrophobicity by HIC-HPLC, and self-association tendency by AC-SINS (see detailed description below). BE-915 demonstrated optimal thermal stability by Tm and Tagg, and good colloidal stability by AC-SINS. As shown in Table 16, BE-915 exhibited overall biophysical properties comparable to those of BE-774. The results also show that the humanized CD137 VHH BGA-2524 used in BE-915 has overall biophysical properties comparable to those of the camelid CD137 VHH BGA-9612 used in BE-774. [Table 16]

[0287] The melting temperature (Tm) and aggregation temperature (Tag) (°C) were determined by UNCLE™ (Unchained Lab, Pleasanton, CA), an instrument that simultaneously measures intrinsic fluorescence and static light scattering. During the measurement, 9 μL of a 1 mg / mL protein sample in PBS buffer was loaded into a cuvette. The sample was held at 20°C for 120 seconds and then heated to 95°C at a rate of 0.3°C / min. Both fluorescence and static light scattering (266 nm) were collected after excitation at 266 nm.

[0288] To determine the hydrophobicity of a given antibody using an HPLC system, 50 μg of sample was diluted at 1 mg / ml with mobile phase A solution (1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0) to achieve a final ammonium sulfate concentration of approximately 1 M before analysis. A MABPac HIC-10 column was used with a linear gradient of mobile phase A and mobile phase B solution (50 mM sodium phosphate, pH 7.0) over 29 minutes at a flow rate of 0.5 mg / min. Peak retention times were monitored at A280 absorbance.

[0289] The AC-SINS assay measures protein self-interactions by capturing antibodies on the surface of gold colloids, which exhibit surface resonant vibrations at frequencies in the visible spectrum. As the immobilized antibodies self-interact, the colloids aggregate, shifting their vibrational frequency and absorbing at longer wavelengths. Gold nanoparticles were incubated with an 80 / 20 (v / v) mixture of capture and non-capture antibodies. The coated gold nanoparticles were then spun down and resuspended in PBS. Samples were diluted to 0.05 mg / ml in conjugation buffer, and 45 μl of each dilution was loaded into a 384-well plate. Five μl of the previously prepared gold nanoparticles were then added to each well of the plate containing the mAb and buffer control. The plate was then covered with an aluminum lid, incubated at room temperature for 2 hours, and quickly spun down at 3000 rpm. The absorbance spectrum of each well was then read from 450 to 650 nm using a plate reader. Each sample spectrum was recorded and analyzed for a red shift in the maximum absorption peak compared to the buffer. This red shift and its intensity indicate the self-interaction tendency of the tested mAb samples.

[0290] The amino acid and DNA sequences of the anti-CD137 VHH and anti-GPC3xCD137 bispecific antibody are shown in Table 17 below. [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] Table 17-6 Table 17-7 Table 17-8 Table 17-9 Table 17-10 Table 17-11 Table 17-12 Table 17-13 Table 17-14 Table 17-15 Table 17-16 Table 17-17 Table 17-18 Table 17-19 [Table 17-20] [Table 17-21] [Table 17-22] [Table 17-23]

[0291] Example 21. Structural and functional CD137 epitope mapping To better understand how the anti-CD137 single-domain antibody arm can have high affinity for CD137 and achieve robust agonism of the CD137 / CD137L interaction, we determined the crystal structure of VHH (BGA-2524) in complex with CD137.

[0292] A. Expression, purification, and crystallization of CD137 and VHH (BGA-2524) Human CD137 ectodomain (CRD1-3, amino acids 24-105 of SEQ ID NO: 35 (human CD137-full length)) partially containing three CRDs was expressed in HEK293G cells. The cDNA encoding CD137 was cloned into the pMAX vector with an N-terminal secretory sequence and a C-terminal TEV cleavage site followed by an Fc tag. The culture supernatant containing the secreted CD137-Fc fusion protein was mixed with Mab Select Sure™ resin (GE Healthcare Life Sciences) for 3 hours at 4°C. The protein was washed with a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, then eluted with 50 mM acetic acid (pH adjusted to 3.5 with 5 M NaOH), and finally neutralized with 1 / 10 CV of 1.0 M Tris-HCl pH 8.0. The eluted protein was mixed with TEV protease (10:1 molar ratio) and dialyzed overnight against buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl) at 4° C. The mixture was loaded onto a Ni-NTA column (Qiagen) and Mab Select Sure™ resin to remove the TEV protease and Fc tag, and the flow-through was then further purified by size-exclusion chromatography in buffer (20 mM Tris pH 8.0, 100 mM NaCl) using a HiLoad 16 / 600 Superdex™ 75 pg column (GE Healthcare Life Sciences).

[0293] The DNA sequence encoding VHH (BGA-2524) was cloned into the PET21a vector with an N-terminal HIS-MBP tag followed by a TEV protease site. Protein expression in Shuffle T7 was induced with 1 mM IPTG at 18°C ​​for 16 hours at an OD600 of 0.6-1.0. Cells were harvested by centrifugation at 7,000 g for 10 minutes. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl) and lysed on ice by sonication. The lysate was then centrifuged at 48,000 g for 30 minutes at 4°C. The supernatant was mixed with Talon resin and batch mixed for 3 hours at 4°C. The resin was washed with lysis buffer containing 5 mM imidazole, and the protein was further eluted in lysis buffer containing 100 mM imidazole. The eluate was mixed with TEV protease (10:1 molar ratio) and dialyzed against buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl) overnight at 4° C. The mixture was loaded onto a Talon column to remove the TEV protease and HIS-MBP tag, and the flow-through was then further purified by size-exclusion chromatography in buffer (20 mM Tris pH 8.0, 100 mM NaCl) using a HiLoad 16 / 600 Superdex™ 75 pg column (GE Healthcare Life Sciences).

[0294] Excess purified CD137 was mixed with purified VHH(BGA-2524) (1.2:1 molar ratio) to generate the CD137 / VHH(BGA-2524) complex. The complex was then further purified by gel filtration in buffer (20 mM Tris pH 8.0, 100 mM NaCl) using a Superdex™ 75 Increase 10 / 300 column (GE Healthcare Life Sciences). The CD137 / VHH(BGA-2524) complex (10 mg / ml) was crystallized in 18% PEG 4000, 0.1 M Tris pH 8.7, 0.2 M LiSO. Crystals cryoprotected with 20% PEG 4000, 0.1 M Tris pH 8.7, 0.2 M LiSO, 10% glycerol were flash-frozen in liquid nitrogen. X-ray diffraction data were collected at beamline BL02U1 of the Shanghai Synchrotron Radiation Facility (Shanghai, China).

[0295] B. Data Collection and Structuring Solutions X-ray diffraction data were collected under cryogenic cooling conditions at 100 Kelvin on beamline BL02U1 at the Shanghai Synchrotron Radiation Facility (Shanghai, China). Diffraction images were processed using integrated data processing software using XDS (Kabsch, W., Xds. Acta Crystallogr D Biol Crystallogr, 2010. 66(Pt 2):pp. 125-32). The structures of human CD137 (PDB: 6MGP) and our own VHH model were used as search models. Initial solutions were found using the molecular replacement program PHASER (McCoy, AJ, et al., Phaser crystallographic software. J Appl Crystallogr, 2007. 40(Pt 4):pp. 658-674). This model was then iteratively constructed manually using the program COOT (Emsley, P. and K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr, 2004. 60(Pt 12 Pt 1): pp. 2126-32) and refined using PHENIX (Adams, P.D., et al., PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr, 2010. 66(Pt 2): pp. 213-21). The final model was refined to acceptable R and R-free values ​​and Ramachandran statistics (calculated by Molprobity). Data processing and refinement statistics are shown in Table 18. [Table 18-1] [Table 18-2]

[0296] C. Structure of VHH (BGA-2524) bound to human CD137 dimer VHH(BGA-2524) complexed with CD137 was crystallized in the P21 space group, with two complexes in the asymmetric unit, diffracting to 1.71 Å. The structure of VHH(BGA-2524) bound to human CD137 shows that VHH(BGA-2524) makes partial steric interfacial contacts with CD137L binding (Figure 17). The buried surface area between VHH(GA-2524) and CD137 is approximately 863 Å. 2The VHH(BGA-2524) interactions are clustered around the CRD2 domain of CD137. VHH(BGA-2524) mainly binds to the CRD2 domain of CD137 via CDR residues on the sides. All CDRs of VHH(BGA-2524) are involved in CD137 dimer binding, with CDR3 potentially contributing the most. CDR1 and CDR3 bind to both monomers of the CD137 dimer, while CDR2 binds only to one monomer of the CD137 dimer. The curved CDR3 loop always covers the hydrophobic patch of the VHH framework, which may result in better biophysical properties (Figure 18). VHH(BGA-2524) CDR1 Tyr32 contacts one monomer of the CD137 dimer at residue Gly98, while CDR1 Asn31 and Ala33 contact the other monomer of the CD137 dimer at residues Ile64 and Gln67. VHH(BGA-2524) CDR2 Trp52, Ser53, Tyr55, and His57 contact only one monomer of the CD137 dimer at residues Asp38, Pro49, Pro50, Asn51, and Ile64. VHH(BGA-2524) CDR3 residues Leu98, Thr104, Thr106, and Tyr109 contact one monomer of the CD137 dimer at residues Ser55, Ala56, Arg75, Glu85, and Ala97, while CDR3 residues Leu98, Lys99, Tyr100, and Pro101 contact another monomer of the CD137 dimer at residues Phe36, Pro49, Thr61, Cys62, Asp63, and Ile64. VHH(BGA-2524) interacts with CD137 using a combination of hydrogen bonds and salt bridges, along with hydrophobic interactions. For example, VHH(BGA-2524) CDR2 residue His57 forms two salt bridges with CD137 residue Asp38. VHH(BGA-2524) CDR3 residue Lys99 forms two salt bridges with CD137 residue Asp63.VHH(BGA-2524) residues Tyr32, Ser53, His57, Leu98, Lys99, Pro101, and Thr106 form one hydrogen bond with CD137 residues Gly98, Asn51, Asp38, Ile64, Asp63, Thr61, and Ser55, respectively (i.e., Tyr32-Gly98, Ser53-Asn51, His57-Asp38, Leu98-Ile64, Lys99-Asp63, Pro101-Thr61, and Thr106-Ser55). VHH(BGA-2524) residue Trp52 forms two hydrogen bonds with CD137 residues Pro50 and Asn51. VHH(BGA-2524) residue Tyr109 forms two hydrogen bonds with CD137 residues Arg75 and Glu85, and VHH(BGA-2524) residue Tyr100 forms two hydrogen bonds with CD137 residue Cys62 (Figure 19).

[0297] Based on the crystal structure of the VHH(BGA-2524) / CD137 complex, we determined the residues of CD137 that VHH(BGA-2524) contacts (i.e., the epitope residues of CD137 that VHH(BGA-2524) binds) and the residues of VHH(BGA-2524) that CD137 contacts (i.e., the paratope residues of VHH(BGA-2524) that CD137 contacts). Tables 19 and 20 below show the residues of CD137 and VHH(BGA-2524) that they contact, assessed using a contact distance stringency of 3.7 Å, the point at which van der Waals (non-polar) interaction forces are at their highest. [Table 19] [Table 20]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human CD137, (1) The antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35); (2) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35); or (3) The antibody or antigen-binding fragment thereof specifically binds to a human CD137 dimer comprising or consisting of a first human CD137 monomer and a second human CD137 monomer, wherein the first human CD137 monomer comprises or consists of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 (SEQ ID NO: 35). and / or the antibody or antigen-binding fragment thereof specifically binds to an epitope on the second human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and / or the antibody or antigen-binding fragment thereof binds to a human CD137 dimer and promotes human CD137 clustering.

2. An antibody or antigen-binding fragment thereof that specifically binds to human CD137, (i) a heavy chain variable region (VH) comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; or (ii) the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO:

3.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or (v) the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:

4.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 4 are inserted, deleted, or substituted.

5. 10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, (i) a heavy chain variable region (VH) comprising SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15; or (v) the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO:

4.

6. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

7. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising a heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type.

8. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

9. 10. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which has reduced or no glycosylation or is hypofucosylated.

10. 10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising an increase in bisecting GlcNac structures.

11. 10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

53.

12. 10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function and / or extended half-life, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO:

20.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of the preceding claims and a pharmaceutically acceptable carrier.

14. A method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in any one of claims 1 to 12, or the pharmaceutical composition described in claim 13.

15. 15. The method of claim 14, wherein the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

16. The method of any one of claims 14 to 15, wherein the antibody or antigen-binding fragment thereof is administered in combination with another therapeutic agent.

17. 17. The method of claim 16, wherein the therapeutic agent is an anti-PD-1 antibody.

18. 18. The method of claim 17, wherein the anti-PD1 antibody is tislelizumab.

19. at least a first antigen-binding domain that specifically binds to a human tumor-associated antigen (TAA); and A multispecific antibody or antigen-binding fragment thereof comprising at least a second antigen-binding domain that specifically binds to human CD137, wherein the second antigen-binding domain comprises: (1) An antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35); (2) an antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35); or (3) The multispecific antibody or antigen-binding fragment thereof, which is an antibody or antigen-binding fragment thereof that specifically binds to a human CD137 dimer comprising or consisting of a first human CD137 monomer and a second human CD137 monomer, and which specifically binds to an epitope on the first human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67, and specifically binds to an epitope on the second human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and / or binds to the human CD137 dimer and promotes human CD137 clustering.

20. 1. A multispecific antibody or antigen-binding fragment thereof, comprising at least a first antigen-binding domain that specifically binds to a human tumor-associated antigen (TAA) and at least a second antigen-binding domain that specifically binds to human CD137, wherein the second antigen-binding domain: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3; or (ii) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO:

3.

21. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 20, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:

4.

22. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 21, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15; or (v) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO:

4.

23. 23. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 22, wherein the TAA is GPC3.

24. A multispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to human glypican 3 (GPC3) and a second antigen-binding domain that specifically binds to human CD137.

25. 25. The multispecific antibody or antigen-binding fragment thereof according to claim 24, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (1) An antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, and Gln67 of human CD137 (SEQ ID NO: 35); (2) an antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98 of human CD137 (SEQ ID NO: 35); or (3) The multispecific antibody or antigen-binding fragment thereof, which is an antibody or antigen-binding fragment thereof that specifically binds to a human CD137 dimer comprising or consisting of a first human CD137 monomer and a second human CD137 monomer, and which specifically binds to an epitope on the first human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Phe36, Asp38, Pro49, Pro50, Asn51, Thr61, Cys62, Asp63, Ile64, Gln67, and specifically binds to an epitope on the second human CD137 monomer (SEQ ID NO: 35) comprising or consisting of amino acid residues Ser55, Ala56, Arg75, Glu85, Ala97, and Gly98, and / or binds to the human CD137 dimer and promotes human CD137 clustering.

26. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 24 to 25, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3; or (ii) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO:

3.

27. 27. The multispecific antibody or antigen-binding fragment thereof of claim 26, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:

4.

28. 28. The multispecific antibody or antigen-binding fragment thereof of claim 27, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 4 have been inserted, deleted, or substituted.

29. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 24 to 28, wherein the second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 17; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15; or (v) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO:

4.

30. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 29, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises: (a) a heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 45, (b) an HCDR2 of SEQ ID NO: 46, and (c) an HCDR3 of SEQ ID NO: 47; and (d) a light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 48, (e) an LCDR2 of SEQ ID NO: 49, and (f) an LCDR3 of SEQ ID NO:

50.

31. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 30, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises:

1. The multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 41, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:

43.

32. 32. The multispecific antibody or antigen-binding fragment thereof of claim 31 , wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in SEQ ID NO: 41 or SEQ ID NO: 43 are inserted, deleted, or substituted.

33. 33. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 32, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 41 and a light chain variable region (VL) comprising SEQ ID NO:

43.

34. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 33, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises: (a) a heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 45, (b) an HCDR2 of SEQ ID NO: 46, and (c) an HCDR3 of SEQ ID NO: 47; and (d) a light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 48, (e) an LCDR2 of SEQ ID NO: 49, and (f) an LCDR3 of SEQ ID NO: 50; The second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3; or (ii) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 10, and (c) an HCDR3 of SEQ ID NO:

3.

35. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 34, wherein the first antigen-binding domain that specifically binds to human GPC3 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 43; The second antigen-binding domain that specifically binds to human CD137 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 4; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 11; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 13; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 15; or (v) the multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO:

17.

36. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 35, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab') ２ The multispecific antibody or antigen-binding fragment thereof,

37. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 36, wherein the first antigen-binding domain that specifically binds to human GPC3 is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a single-domain antibody, a Fab fragment, a Fab' fragment, or a F(ab') ２ It is a fragment, The second antigen-binding domain that specifically binds to human CD137 is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a single-domain antibody, a Fab fragment, a Fab' fragment, or a F(ab') fragment. ２ The multispecific antibody or antigen-binding fragment thereof, which is a fragment thereof.

38. 38. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 37, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody.

39. 39. The multispecific antibody or antigen-binding fragment thereof of claim 38, wherein the bispecific antibody is in a 2+2 format.

40. 40. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 39, comprising a linker according to SEQ ID NO: 60 to SEQ ID NO:

101.

41. 41. The multispecific antibody or antigen-binding fragment thereof of claim 40, wherein the linker is SEQ ID NO:

62.

42. 41. The multispecific antibody or antigen-binding fragment thereof of claim 40, wherein the linker is SEQ ID NO:

67.

43. 43. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 42, comprising a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type, The multispecific antibody or antigen-binding fragment thereof, wherein the heavy chain constant region comprises a CH1 and / or Fc domain.

44. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 43, wherein the multispecific antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

45. 45. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 44, which has reduced or no glycosylation or is hypofucosylated.

46. 46. ​​The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 45, comprising an increase in bisecting GlcNac structures.

47. 47. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 46, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO:

53.

48. 48. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 47, comprising an Fc domain, wherein the Fc domain is an IgG1 Fc with reduced effector function and / or extended half-life, and optionally the Fc domain comprises the amino acid sequence of SEQ ID NO:

20.

49. 49. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 48, comprising an Fc domain, wherein the Fc domain is an IgG4 Fc.

50. 50. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 49, a) the heavy chain variable region (VH) of the first antigen-binding domain that specifically binds to human GPC3, a CH1 domain, the Fc domain, and the heavy chain variable region (VH) of the second antigen-binding domain that specifically binds to human CD137 are arranged in an N-terminal to C-terminal direction in the first polypeptide; Optionally, the C-terminus of the Fc domain is linked to the N-terminus of the heavy chain variable region (VH) of the second antigen-binding domain via a linker; and b) the multispecific antibody or antigen-binding fragment thereof, wherein the light chain variable region (VL) and the first light chain constant region of the first antigen-binding domain that specifically binds to human GPC3 are arranged in an N-terminal to C-terminal direction within the second polypeptide.

51. 51. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 50, (i) a first polypeptide of SEQ ID NO: 25 and a second polypeptide of SEQ ID NO: 23; (ii) a first polypeptide of SEQ ID NO: 21 and a second polypeptide of SEQ ID NO: 23; (iii) a first polypeptide of SEQ ID NO: 33 and a second polypeptide of SEQ ID NO: 23; (iv) a first polypeptide of SEQ ID NO: 27 and a second polypeptide of SEQ ID NO: 23; (v) a first polypeptide of SEQ ID NO: 29 and a second polypeptide of SEQ ID NO: 23, or (vi) the multispecific antibody or antigen-binding fragment thereof, comprising a first polypeptide of SEQ ID NO: 31 and a second polypeptide of SEQ ID NO:

23.

52. A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 51 and a pharmaceutically acceptable carrier.

53. 52. A method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the multispecific antibody or antigen-binding fragment thereof according to any one of claims 19 to 51, or the pharmaceutical composition of claim 52.

54. 54. The method of claim 53, wherein the cancer is an advanced or metastatic solid tumor.

55. 55. The method of any one of claims 53 to 54, wherein the cancer expresses GPC3.

56. 56. The method of any one of claims 53 to 55, wherein the cancer is liver cancer, lung cancer, gastric cancer, germ cell tumor, thyroid cancer, pancreatic cancer, ovarian cancer, skin cancer, kidney cancer, esophageal cancer, atypical teratoid rhabdoid tumor of the brain, or undifferentiated synovial sarcoma.

57. 57. The method of claim 56, wherein the liver cancer is hepatoblastoma or hepatocellular carcinoma (HCC).

58. 57. The method of claim 56, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

59. 59. The method of claim 58, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.

60. 59. The method of claim 58, wherein the non-small cell lung cancer is GPC3+ squamous non-small cell lung cancer.

61. 57. The method of claim 56, wherein the gastric cancer is alpha-fetoprotein+ (AFP+) gastric cancer.

62. 57. The method of claim 56, wherein the kidney cancer is Wilms' tumor.

63. 57. The method of claim 56, wherein the esophageal cancer is esophageal squamous cell carcinoma.

64. 57. The method of claim 56, wherein the esophageal cancer is GPC3+ esophageal squamous cell carcinoma.

65. 57. The method of claim 56, wherein the germ cell tumor is a yolk sac tumor or a non-dysgerminoma.

66. 66. The method of any one of claims 53 to 65, wherein the multispecific antibody or antigen-binding fragment thereof, or the pharmaceutical composition is administered in combination with another therapeutic agent.

67. 67. The method of claim 66, wherein the therapeutic agent is an anti-PD1 or anti-PDL1 antibody.

68. 68. The method of claim 67, wherein the anti-PD1 antibody is tislelizumab.

69. An isolated nucleic acid encoding the antibody, multispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 12 and 19 to 51.

70. A vector comprising the nucleic acid of claim 69.

71. 71. A host cell comprising the nucleic acid of claim 69 or the vector of claim 70.

72. 72. A process for producing a multispecific antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 71 and recovering the antibody or antigen-binding fragment thereof from the culture.

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