T cell activating antibodies

Abstract

The present invention provides an antibody comprising an antigen binding region that binds to CD137. The present invention also provides a bispecific antibody comprising a first antigen binding region that binds to CD137 and a second antigen binding region that binds to an immune checkpoint molecule, an immunostimulatory molecule or a tumor antigen. The present invention provides a pharmaceutical composition comprising the antibody and a method for treating cancer.

Description

[0001] Cross-references to related patent applications

[0002] This application claims priority under Section 119(e) of the U.S. Patent Application No. 62 / 866,699 filed on June 26, 2019 and U.S. Patent Application No. 62 / 953,302 filed on December 24, 2019, which are incorporated herein by reference in their entirety and become a part of the present invention.

[0003] Inclusion of sequence listing

[0004] The materials in the attached sequence listing are incorporated by reference into this application and become a part of this application. The attached sequence listing text file is named AP1100_2WO_Sequence_Listing.txt, created on June 12, 2020, and is 66kb in size. This file can be accessed using Microsoft Word on a computer using the Windows operating system. Background Art Technical Field

[0006] The present invention generally relates to antibodies and antigen-binding fragments thereof, and more particularly to antibodies and antigen-binding fragments thereof for enhancing T cell function.

[0007] Background Information

[0008] Adaptive immune system immunomodulation has become an attractive area of ​​cancer immunotherapy due to its fewer side effects and long-term suppression of cancer recurrence. Full activation of T cells usually involves two signals, one from the T cell receptor (TCR) antigen-specific signal and the other from the co-stimulatory molecule (such as CD28). In the past decade, additional co-stimulatory and co-inhibitory molecules have been discovered on T cells that can positively or negatively regulate TCR signaling.

[0009] In 1989, CD137 (4-1BB), a co-stimulatory molecule belonging to the TNF receptor superfamily, was cloned from activated T cells (Kwon and Weissman, 1989). Although 4-1BB is involved in strong TCR signaling that can induce IL-2 production in a CD28-independent manner, the 4-1BB:4-1BBL pathway appears to amplify existing co-stimulatory signals. Studies have shown that CD137 signaling can promote TCR signaling, induce cytokine synthesis and T cell proliferation, and inhibit activation-induced apoptosis. CD137 stimulation on T cells can induce NF-κB and PI3K / ERK signaling pathways (responsible for preventing T cell activation-induced apoptosis and inducing T cell proliferation, respectively). Both CD4 and CD8 T cells respond to CD137 stimulation, thereby enhancing expansion and effector function, while CD8 T cells preferentially respond to CD137 signaling by inducing greater cytokine production. In addition to expression on activated T cells, CD137 is expressed on multiple hematopoietic cell lineages, including regulatory T cells, B cells, natural killer cells (NK), monocytes, and dendritic cells (DC). In DC, CD137 stimulation increases the secretion of IL-6 and IL-12, and more importantly, it enhances the ability of DC to stimulate T cell proliferation in response to alloantigens and epiantigens. In NK, CD137 stimulation promotes proliferation and IFN-γ production, but does not promote cytolytic activity. However, CD137-stimulated NK cells have an auxiliary role in promoting the expansion of activated T cells.

[0010] Clinically, the anti-CD137 agonist antibody Urelumab (BMS-663513) showed partial remission and partial stabilization of the disease. However, fatal hepatotoxicity caused most trials to be terminated. In patients with solid tumors and Merkel cell carcinoma, trials of another anti-CD137 antibody Utomilumab (PF-05082566) achieved objective response rates of 3.8% and 13.3%, respectively (including complete response and partial response), without causing hepatotoxicity. Urelumab and Utomilumab exhibit different properties. The agonist activity of Urelumab is strong and does not depend on cross-linking, while the agonist activity of Utomilumab is weak and depends on cross-linking. For CD137, the crystal structures of Urelumab and Utomilumab reveal different binding epitopes that affect the CD137-CD137L interaction. Different epitope recognition and blockade of CD137-CD137L binding may result in different potency and toxicity of these two anti-CD137 antibodies. However, ligand binding cannot determine CD137-mediated toxicity, as the anti-4-1BB monoclonal antibodies (mAbs) 3H3 and 2A have opposite effects on CD137L binding but reveal similar hepatotoxicity profiles. Recently, it was shown that the engineered Fc region of a weak agonist antibody preferentially binds to FcγRIIB (low A / I FcγR binding ratio) and produces potent agonist activity comparable to that of urerulumab without inducing hepatotoxicity. Studies have shown that agonistic anti-CD137 antibodies have anticancer activity by enhancing T cell cytotoxicity in a CD40-dependent manner. In addition, anti-CD137 antibodies require antigen expression to restore established low-antigenic tumors. Moreover, the combination of anti-PD-1 and anti-CD137 antibodies showed enhanced antitumor activity in a mouse tumor model by enhancing T cell effector function and tumor infiltration compared with treatment with each antibody alone. In addition to anticancer therapy, the study suggests that anti-CD137 agonist antibodies may also improve experimental autoimmune encephalomyelitis and enhance antiviral immunity, depending on the timing of treatment.

[0011] PD-1 was first isolated from apoptotic T cells. Its ligand PD-L1 was subsequently identified, and studies have shown that the interaction between PD-1 and PD-L1 can block T cell activation. PD-1 is not expressed on resting T cells, but is induced when activated. PD-1 is continuously expressed on exhausted T cells in chronic infection and cancer. Under normal circumstances, the PD-1 / PD-L1 pathway is important for maintaining peripheral immune tolerance and preventing autoimmunity. However, cancer inhibits the self-protection function of PD-L1 and expresses PD-L1 through various types of cancer cells to avoid immune system surveillance. Antibodies targeting PD-1 / PD-L1 block inhibitory signaling and restore the anti-cancer activity of T cells. The PD-1 / PD-L1 pathway has been regarded as a dominant negative regulator of anti-tumor T cell effector function. Clinically, blockade of this pathway has achieved high objective response rates of 35% to 87% in certain types of cancer (such as Hodgkin lymphoma, Merkel cell carcinoma, and melanoma). Other types of cancer, such as NSCLC, head and neck cancer, and renal cell carcinoma, achieved lower objective response rates of 15% to 25%.

[0012] In mouse tumor models, the combination of anti-PD-1 and anti-CD137 antibodies showed enhanced antitumor activity by enhancing T cell effector function and tumor infiltration compared with treatment with each antibody alone. In the clinic, the combination of utolumab (0.45–5.0 mg / kg) and pembrolizumab in patients with advanced solid tumors showed synergistic antitumor effects without dose-limiting toxicities.

[0013] Based on the immunomodulatory effect of CD137, anti-human 4-1BB agonist antibodies can be used to treat cancer, autoimmune diseases and infectious diseases. However, due to hepatotoxicity, the use of anti-human 4-1BB agonist antibodies is limited. In addition, there is no description of effective treatment methods that combine activation of the 4-1BB:4-1BBL pathway (without hepatotoxicity) with blocking immunosuppression or tumor signaling pathways. Therefore, effective participation of the 4-1BB:4-1BBL pathway is required, combined with the removal of immune activation inhibition or inhibition of tumor cell signaling. Summary of the invention

[0014] The present invention is based on the groundbreaking discovery that anti-CD137 antibodies can be generated, and anti-CD137 antibodies have strong cross-linking-dependent agonist activity, which may circumvent the liver toxicity that occurs in clinical trials. The present invention is further based on such findings that bispecific antibodies targeting PD-L1 and CD137 are very active, can enhance T cell effector function, and inhibit tumor growth in vivo, and their effects are better than various antibody monotherapy or combination therapy. For example, the bispecific antibody may have a unique anti-CD137 single-chain variable fragment (scFv), which can activate T cells after cross-linking through the other arm of the bispecific antibody bound to PD-L1. As described herein, by changing the anti-PD-L1 arm to other tumor-specific binders (such as anti-Her2 or anti-tumor-specific glycans), bispecific antibodies can also target non-PD-L1 expressing tumors. While maintaining the anti-tumor effect of anti-CD137 monoclonal antibodies, bispecific antibodies that induce target-dependent T cell activation can avoid liver toxicity.

[0015] In some embodiments, the present invention provides three agonist antibodies or antigen-binding fragments thereof: anti-CD137 antibody clone 15 (CD137#15), anti-CD137 antibody clone 31 (CD137#31) and anti-CD137 antibody clone 54 (CD137#54). In one aspect, the anti-CD137 antibody of the present invention comprises a heavy chain variable (V H ) region, which comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 17; and a light chain variable (V L ) region, which comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 18. On the other hand, having V H A region (comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1) and V L The antibody or antigen-binding fragment of the present invention comprises (a) V H CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence comprised by CDR-H1 is at least 80% identical to SEQ ID NO:3, wherein the amino acid sequence comprised by CDR-H2 is at least 80% identical to SEQ ID NO:4, wherein the amino acid sequence comprised by CDR-H3 is at least 80% identical to SEQ ID NO:5; and (b) V LCDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence comprised by CDR-L1 is at least 80% identical to SEQ ID NO:6, wherein the amino acid sequence comprised by CDR-L2 is at least 80% identical to SEQ ID NO:7, wherein the amino acid sequence comprised by CDR-L3 is at least 80% identical to SEQ ID NO:8.

[0016] On the other hand, with V H A region (comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 9) and V L The antibody or antigen-binding fragment thereof comprises (a) V H CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence comprised by CDR-H1 is at least 80% identical to SEQ ID NO: 11, wherein the amino acid sequence comprised by CDR-H2 is at least 80% identical to SEQ ID NO: 12, wherein the amino acid sequence comprised by CDR-H3 is at least 80% identical to SEQ ID NO: 13; and (b) V L CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence comprised by CDR-L1 is at least 80% identical to SEQ ID NO:14, wherein the amino acid sequence comprised by CDR-L2 is at least 80% identical to SEQ ID NO:15, wherein the amino acid sequence comprised by CDR-L3 is at least 80% identical to SEQ ID NO:16.

[0017] On the other hand, with V H A region (comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 17) and V L The antibody or antigen-binding fragment thereof comprises (a) V H CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence comprised by CDR-H1 is at least 80% identical to SEQ ID NO: 19, wherein the amino acid sequence comprised by CDR-H2 is at least 80% identical to SEQ ID NO: 20, wherein the amino acid sequence comprised by CDR-H3 is at least 80% identical to SEQ ID NO: 21; and (b) V LCDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence comprised by CDR-L1 is at least 80% identical to SEQ ID NO:22, wherein the amino acid sequence comprised by CDR-L2 is at least 80% identical to SEQ ID NO:23, wherein the amino acid sequence comprised by CDR-L3 is at least 80% identical to SEQ ID NO:24.

[0018] On the one hand, the antibody or its antigen-binding fragment of the present invention comprises an Fc domain. On the other hand, the Fc domain is an IgG, IgE, IgM, IgD, IgA or IgY domain. On the other hand, the IgG domain is an IgG1, IgG2, IgG3 or IgG4 domain. On the other hand, the IgG1 domain comprises the amino acid sequence of SEQ ID NO:26. In some aspects, compared with wild-type IgG1, IgG1 comprises a point mutation that modifies or reduces antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Typical point mutations include K297A and K322A mutations. On the other hand, the IgG4 domain comprises the amino acid sequence of SEQ ID NO:25. On the other hand, the antigen fragment of the present invention includes scFv, F(ab)2 or Fab.

[0019] In one embodiment, the present disclosure further provides a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof of the present invention. On the one hand, the antibody or antigen-binding fragment of the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier coupled to one or more polypeptide C-termini of the antibody or antigen-binding fragment thereof. On the other hand, the pharmaceutical composition of the present invention comprises a bispecific antibody.

[0020] In one embodiment, the disclosure further provides a method for treating cancer, the method comprising the following steps: administering an effective amount of an antibody of the present invention or its antigen-binding fragment, or an effective amount of a bispecific antibody of the present invention to a subject in need thereof. On the one hand, the cancer includes prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer or leukemia.

[0021] In one embodiment, the present invention provides a bispecific antibody comprising a first antigen binding region and a second antigen binding region, wherein the first antigen binding region binds to CD137. In one aspect, the bispecific antibody of the present invention comprises (i) a V HA region comprising an amino acid sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 9; and SEQ ID NO: 17; and (ii) a V L A region comprising an amino acid sequence having at least 80% sequence identity with an N-terminal sequence of about 100 to 120 amino acids of a sequence selected from SEQ ID NO: 2; SEQ ID NO: 10; and SEQ ID NO: 18, wherein the second antigen binding region binds to an immune checkpoint molecule, an immunostimulatory molecule or a tumor antigen.

[0022] In one aspect, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 31 or SEQ ID NO: 32. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 30. In one aspect, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 36. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 35. In one aspect, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 38. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 37. In one aspect, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 40. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 39.

[0023] In one aspect, the bispecific antibody of the invention comprises a first antigen-binding region having (a) a V H region (comprising the amino acid sequence of SEQ ID NO: 9), and a V L region (an amino acid sequence of about 100 to 120 amino acids comprising the N-terminal sequence of SEQ ID NO: 10); or (b) a V H region (comprising the amino acid sequence of SEQ ID NO: 17), and a V L In another aspect, the second antigen-binding region binds to an antigen selected from PD-L1, PD-1, CTLA-4, LAG3, CD28, CD40, CD137, CD27, ICOS, Her2, or a glycan. In another aspect, the second antigen-binding region binds to PD-L1, Her2, or a glycan.

[0024] On the one hand, the present invention discloses that an anti-PD-L1#6-CD137#54 bispecific antibody (bsAb) can be used as a platform to fully exert the activation effect of target-dependent T cells through the cross-linking-dependent agonist activity of the anti-CD137#54 single chain.

[0025] On the other hand, bispecific antibodies that bind to CD137 and PD-L1 are modified to bind to CD137 and other targets expressed on tumors, including immunomodulatory molecules and tumor-specific markers such as Her2 or tumor-specific glycans.

[0026] In one aspect, the first antigen-binding region and the second antigen-binding region of the bispecific antibody of the present invention comprise an Fc domain, a Fab fragment, a single-chain variable fragment (scFv), or any combination thereof. In another aspect, the scFv comprises (i) a V H region (comprising the amino acid sequence of SEQ ID NO: 9), and a V L region (an amino acid sequence of about 100 to 120 amino acids comprising the N-terminal sequence of SEQ ID NO: 10); or (ii) a V H region (comprising the amino acid sequence of SEQ ID NO: 17), and a V L region (an amino acid sequence of about 100 to 120 amino acids comprising the N-terminal sequence of SEQ ID NO: 18). On the other hand, the bispecific antibody of the present invention comprises a scFv V H Area and V L In another aspect, the scFv comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0027] In one aspect, the bispecific antibody of the present invention comprises an Fc domain. In another aspect, the Fc domain is an IgG domain, an IgE domain, an IgM domain, an IgD domain, an IgA domain or an IgY domain. In another aspect, the Fc domain is an IgG domain. In another aspect, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain or an IgG4 domain.

[0028] On the one hand, the scFv is connected to the C-terminus of the Fc domain. On the other hand, the bispecific antibody of the present invention comprises a linker between the Fab domain and the scFv domain. On the other hand, the Fab fragment is connected to the N-terminus of the Fc domain. On the other hand, the Fab comprises a PD-L1 binding site, a Her2 binding site or a glycan binding site, and the scFv comprises a CD137 binding site.

[0029] In one embodiment, the present invention provides an antibody-drug conjugate comprising a therapeutic agent and an antibody (including any bispecific antibody) of the present invention or an antigen-binding fragment thereof. In one aspect, the therapeutic agent is covalently linked to the antibody or antigen-binding fragment via a linker.

[0030] In one embodiment, the present invention provides a pharmaceutical composition comprising any bispecific antibody of the present invention, and at least one pharmaceutically acceptable carrier.

[0031] In one embodiment, the present invention provides an isolated amino acid sequence as shown in SEQ ID NO: 1-26. In another embodiment, the present invention provides an isolated amino acid sequence as shown in SEQ ID NO: 30-40.

[0032] In one embodiment, the present disclosure further provides an isolated amino acid sequence encoding the antibody, antigen-binding fragment thereof, or bispecific antibody of the present invention. In another embodiment, the present invention provides an isolated amino acid sequence encoding any one of SEQ ID NOs: 1-26. In another embodiment, the present invention provides an isolated amino acid sequence encoding any one of SEQ ID NOs: 30-40. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The direct screening of phage clones targeting CD137 by ELISA is shown.

[0034] Figure 2 Shown is the binding of phage clones targeting CD137 on CD137 overexpressing HEK-293F cells using flow cytometry.

[0035] Figure 3A-3B The integrity and purity of the anti-CD137 antibody lead by one-step protein G purification by polyacrylamide gel electrophoresis (PAGE) are shown. Results for two batches (upper and lower panels) are shown.

[0036] Figure 4 Shown is the binding of anti-CD137 antibody leads on activated Jurkat cells by flow cytometry.

[0037] Figure 5 The binding activity of anti-CD137 antibody leads to recombinant human CD137 using ELISA (EC 50 ).

[0038] Figure 6 Protein aggregation of high concentration anti-CD137 antibodies clone 31 and clone 54 using SEC-HPLC is shown.

[0039] Figure 7 Cytokine production by T cells in the presence of agonistic activity of anti-CD137 antibody leads is shown.

[0040] Figure 8 Shown is the dose-dependent induction of human T cell cytokine production by anti-CD137 antibody lead clones in primary human T cells.

[0041] Fig. 9 It was shown that combination therapy with anti-CD137 antibody promoted anti-PD-L1 antibody-mediated IFN-γ production by T cells in mixed lymphocyte reactions.

[0042] Fig.10 The significant effects of anti-CD137 antibody clones on CD137-CD137L interaction were shown.

[0043] Fig.11 Shown are the in vivo pharmacokinetic profiles of anti-CD137 antibody clones #31 and #54.

[0044] Fig.12 Different requirements for cross-linking for agonistic activity of anti-CD137 antibody clones compared to utolumab (CD137 number 2) and usrulumab (CD137 number 3) are shown.

[0045] Fig.13 Symmetrical formats of anti-PD-L1-CD137 bispecific antibodies (bsAbs) are shown.

[0046] Fig.14 The purity and integrity of protein G-purified anti-PD-L1-CD137 bsAbs by SDS-PAGE are shown. One-step protein G chromatography yields more than 90%.

[0047] Fig.15 The purity and integrity of protein A-purified anti-PD-L1-CD137 bsAbs using the μCE-SDS method are shown.

[0048] Fig.16 It was shown that anti-PD-L1-CD137 bsAbs recognize both CD137 and PD-L1. Biosensor analysis as shown.

[0049] Fig.17 It was shown that in mixed lymphocyte reactions, anti-PD-L1#6-CD137#54bsAb induced synergistic T cell activation compared with monotherapy, combination therapy, or anti-PD-L1#6-CD137#31bsAb treatment.

[0050] Figures 18A-18BIt was shown that anti-PD-L1#6-CD137#54bsAb could significantly enhance the antigen-specific recall responses of memory CD4 (A) and memory CD8 (B) T cells.

[0051] Fig.19 It was shown that anti-PD-L1#6-CD137#31 or anti-PD-L1#6-CD137#54 bsAbs could induce target-dependent T cell activation while co-culturing T cells with PD-L1-overexpressing HEK-293 cells.

[0052] Figures 20A-20C The results showed that anti-PD-L1#6-CD137#54bsAb induced T cells to produce IFN-γ ( Fig. 20A , Fig. 20C and Fig. 20B Left) and cancer cell toxicity ( Fig. 20B Right panel). (A) NCI-H1975, non-small cell lung cancer cells; (B) PC-3, prostate cancer cells; (C) MDA-MB-231, breast cancer cells.

[0053] Figures 21A-21B Figure 2 shows that trastuzumab (Tra)CD137#54 or anti-Her2#3-7-CD137#54bsAbs induced IFN-γ production in CD8 T cells after co-culture with Her2-positive cancer cells. (A) SKBR-3, breast cancer cells; (B) MDA-MD-361, breast cancer cells.

[0054] Figures 22A-22B The results showed that anti-glycan CD137#54bsAb induced IFN-γ production by CD8 T cells after co-culture with glycan-positive cancer cells. Fig.22A Left picture and Fig. 22B ) and cancer cell toxicity ( Fig.22A Right panel). (A) MCF-7, breast cancer cells; (B) NCI-N87, gastric cancer cells.

[0055] Fig.23 Anti-PD-L1#6-CD137#54 bsAb is shown to induce internalization of CD137 expressed on HEK293 cells.

[0056] Figures 24A-24B Anti-PD-L1#6-CD137 bsAb rescues T cell proliferation (A) and cytokine production (B) in the presence of Treg cells.

[0057] Figures 25A-25CShown that in humanized mice transplanted with PD-L1-positive (A) NCI-H292, (B) NCI-H1975, and (C) BxPC-3 tumor cells, anti-PD-L1#6-CD137#54 bsAb produced greater tumor growth inhibition than combined treatment with anti-PD-L1#6 and anti-CD137#54 antibodies.

[0058] Fig.26 It was shown that PD-L1#6-CD137#54, Her2#3-7-CD137#54 and glycan-CD137#54 bsAbs did not induce significant cytokine release in human PBMCs.

[0059] Figures 27A-27B PK parameters of anti-PD-L1#6-CD137#54bsAb in monkeys are shown in (A) graphical and (B) tabular form. DETAILED DESCRIPTION

[0060] Before describing the present compositions and methods, it is to be understood that, since the specific compositions, methods and experimental conditions described may vary, the present invention is not limited to such specific compositions, methods and experimental conditions. It is also to be understood that, since the scope of the present invention is limited only to the scope of the appended claims, the terms used in the present invention are intended only to describe, but not to limit, specific embodiments.

[0061] In some embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to CD137. The present invention also provides antibody amino acid sequences that bind to CD137. The term "antibody" used in the present invention refers to an immunoglobulin molecule that has the ability to specifically bind to an antigen. Unless otherwise clearly stated in the context, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, and anti-idiotypic antibodies. On the one hand, the antibodies of the present invention include monoclonal antibodies. The antibodies of the present invention include any isotype and class (such as IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The "antigen-binding fragment" used in the present invention refers to a fragment or part of an immunoglobulin molecule or antibody that has the ability to specifically bind to the same antigen as an immunoglobulin molecule or antibody. Exemplary antigen-binding fragments include scFv, Fab, or F(ab)2 fragments. As used herein, "antigen binding region" refers to the portion of an antibody or immunoglobulin molecule that binds to an antigen or protein, for example, by contacting the antigen or protein. The antigen binding region generally includes the heavy chain variable (V H ) region and light chain variable (V LThe antigen binding region generally comprises one or more antigen binding sites or fill sites.

[0062] The antibody of the present invention has a V H A region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range between the two with the sequence of SEQ ID NO:1; SEQ ID NO:9; or SEQ ID NO:17. The antibody of the present invention also comprises a V L A region comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range in between, to the sequence of SEQ ID NO:2; SEQ ID NO:10; or SEQ ID NO:18.

[0063] In general, "sequence identity" or "sequence homology" are used interchangeably and refer to the exact correspondence of nucleotides to nucleotides or amino acids to amino acids of two polynucleotide or polypeptide sequences, respectively. In general, sequence identity determination techniques include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby or the amino acid sequence of a polypeptide, and comparing such sequences with a second nucleotide or amino acid sequence. The terms "percent (%) sequence identity" or "percent (%) identity" (also including "homology") used in the present invention refer to the percentage of amino acid residues or nucleotides in a sequence that is identical to the amino acid residues or nucleotides in a reference sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Therefore, by determining the "percent identity" (also called "percent homology") of two or more sequences (polynucleotides or amino acids), two or more sequences can be compared. The percent identity with a reference sequence (e.g., a nucleic acid or amino acid sequence) (which may be a sequence within a longer molecule (e.g., a polynucleotide or polypeptide)) can be calculated as the number of exact matches between the two best aligned sequences divided by the length of the reference sequence multiplied by 100. For example, the advanced BLAST computer program (including version 2.2.9) provided by the National Institutes of Health can also be used to compare sequence information and determine the percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proceedings of the National Academy of Sciences 87:2264-2268 (1990), as described by Altschul et al. in J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). In short, the BLAST program defines identity as the number of identical alignment symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. This program can be used to determine the percent identity over the entire length of the compared sequences. The default parameters are used to optimize searches using short query sequences, such as with the blastp program. This program also allows the use of SEG filters to mask query sequence fragments determined by the SEG program of Wootton and Federhen, Computers and Chemistry, 17:149-163 (1993). The desired degree of sequence identity ranges from about 80% to 100%, with integer values ​​in between.The percent identity between the reference sequence and the sequence to be protected can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or at least 99.9%. In general, an exact match represents 100% identity over the length of the reference sequence. Additional programs and methods for comparing sequences and / or assessing sequence identity include the Needleman-Wungsch algorithm (e.g., see EMBOSS Needle aligner provided at www.ebi.ac.uk / Tools / psa / emboss_needle / , which can optionally be used with default settings), the Smith-Waterman algorithm (e.g., see EMBOSS Water aligner provided at www.ebi.ac.uk / Tools / psa / emboss_water / , which can optionally be used with default settings), the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin). In some aspects, references to sequence identity refer to sequence identity measured using BLAST (Basic Local Alignment Search Tool). In other aspects, ClustalW is used for multiple sequence alignment. The best alignment can be assessed using any suitable parameters of the selected algorithm (including default parameters).

[0064] In one aspect, the antibody or antigen-binding fragment thereof has a V H A region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 1; and a V LA region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween. SEQ ID NO: 1 provides an amino acid sequence comprising the heavy chain variable region of anti-CD137 antibody clone #15. SEQ ID NO: 2 provides an amino acid sequence comprising the light chain variable region of anti-CD137 clone #15.

[0065] The antigen binding region of the antibody or antigen binding fragment thereof generally comprises a complementarity determining region (CDR). H and V L The CDRs include the target protein or antigen binding site of the antibody that confers protein or antigen binding specificity. H and V L Usually includes three CDRs numbered in sequence. CDR-H1, CDR-H2 and CDR-H3 used in the present invention refer to the heavy chain variable region (V H ) are three consecutively arranged CDRs (numbered from the N-terminus of the heavy chain polypeptide). The CDR-L1, CDR-L2 and CDR-L3 used in the present invention refer to the light chain variable region (V L ) are three consecutively arranged CDRs of the light chain polypeptide (numbering starts from the N-terminus of the light chain polypeptide).

[0066] In one aspect, the antibody or antigen-binding fragment thereof (having a V H A region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 1, and a V LThe antigen binding region comprises a CDR-H1 comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:2, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, and comprises a CDR-H2 comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, to SEQ ID NO:3. NO:4 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity and any number or range therebetween, and comprises CDR-H3, which comprises an amino acid sequence identical to SEQ ID NO:5 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range between the two. On the other hand, the antibody or antigen-binding fragment thereof (having a V H A region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 1, and a V LThe antigen binding region comprises a CDR-L1 comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:2, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, and comprises a CDR-L2 comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, to SEQ ID NO:6. NO:7 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity and any number or range therebetween, and comprises CDR-L3, which comprises an amino acid sequence identical to SEQ ID NO:8 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between.

[0067] In some aspects, the antibody or antigen-binding fragment thereof comprises a V H A region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:9, and a V LA region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween. SEQ ID NO: 9 provides an amino acid sequence comprising the heavy chain variable region of anti-CD137 antibody clone #31. SEQ ID NO: 10 provides an amino acid sequence comprising the light chain variable region of anti-CD137 clone #31.

[0068] In one aspect, the antibody or antigen-binding fragment thereof (comprising a V H A region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 9, and a V LThe antigen binding region comprises a CDR-H1 comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO: 10, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, and a CDR-H2 comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, to SEQ ID NO: 11. NO:12 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity and any number or range therebetween, and comprises CDR-H3, which comprises an amino acid sequence identical to SEQ ID NO:13 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range between the two. On the other hand, the antibody or antigen-binding fragment thereof (comprising a V H A region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:9, and a V LThe antigen binding region comprises a CDR-L1 comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO: 10, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, and a CDR-L2 comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, to SEQ ID NO: 14. NO:15 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity and any number or range therebetween, and comprises a CDR-L3 comprising an amino acid sequence identical to SEQ ID NO:16 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between.

[0069] In some aspects, the antibody or antigen-binding fragment thereof has a V H A region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 17, and a V LA region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween. SEQ ID NO: 17 provides an amino acid sequence comprising the heavy chain variable region of anti-CD137 antibody clone #54. SEQ ID NO: 18 provides an amino acid sequence comprising the light chain variable region of anti-CD137 clone #54.

[0070] In one aspect, the antibody or antigen-binding fragment thereof (comprising a V H A region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 17, and a V LThe antigen binding region comprises a CDR-H1 comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, to SEQ ID NO:19, and a CDR-H2 comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, to SEQ ID NO:19. NO:20 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity and any number or range therebetween, and comprises CDR-H3, which comprises an amino acid sequence identical to SEQ ID NO:21 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range between the two. On the other hand, the antibody or antigen-binding fragment thereof (comprising a V H A region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 17, and a V LThe antigen binding region comprises a CDR-L1 comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO: 18, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, and a CDR-L2 comprising an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.9% identical, and any number or range therebetween, to SEQ ID NO: 22. NO:23 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity and any number or range therebetween, and comprises a CDR-L3 comprising an amino acid sequence identical to SEQ ID NO:24 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between.

[0071] The antibody or its antigen-binding fragment of the present invention further comprises an Fc domain. Unless the context clearly states otherwise, the term Fc domain used in the present invention refers to an antibody region, which at least comprises a hinge region, a CH2 domain and a CH3 domain. Unless the context clearly states otherwise, the terms Fc domain and Fc region are used interchangeably. In some aspects, the Fc domain is an IgG domain, an IgE domain, an IgM domain, an IgD domain, an IgA domain or an IgY domain. Any sequence and any species of Fc domain can be used, including humans, apes, monkeys, mice, rabbits, goats, sheep, guinea pigs, horses, etc. In some aspects, the Fc domain uses an engineered Fc domain, that is, a non-naturally occurring or recombinant Fc domain generated using molecular biology techniques. In some aspects, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain or an IgG4 domain. On the one hand, the IgG4 domain comprises the amino acid sequence of SEQ ID NO:25. In another aspect, the IgG1 domain comprises the amino acid sequence of SEQ ID NO: 26. In one aspect, the Fc domain is a human Fc domain.

[0072] In some embodiments, the present invention also provides a pharmaceutical composition comprising any antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier is coupled to the C-terminus of one or more polypeptides of the antibody or antigen-binding fragment. For example, the pharmaceutically acceptable carrier can be coupled in any appropriate manner, such as covalent coupling and the use of a linker.

[0073] In some embodiments, the present invention provides an isolated amino acid sequence as shown in SEQ ID NO: 1-26. In some embodiments, the present invention also provides an isolated nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NO: 1-26.

[0074] In some embodiments, the present invention provides a method for treating cancer in a subject. In some aspects, the method for treating cancer includes administering to a subject a certain amount of the present invention (can effectively treat cancer) any antibody or its antigen binding fragment combined with CD137. In some aspects, the cancer is prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer or leukemia.

[0075] The terms "treat", "treatment", "therapy", "therapeutic" and the like as used herein refer to achieving a desired pharmacological and / or physiological effect, including but not limited to alleviating, delaying or slowing progression, alleviating effects or symptoms, preventing onset, inhibiting and ameliorating onset, obtaining beneficial or desired results regarding a disease, disorder or medical condition, such as therapeutic benefit and / or preventive benefit. "Treatment" as used herein includes treating a disease in a mammal, especially a human, and includes: (a) preventing a subject from developing the disease, including a subject susceptible to the disease or at risk of developing the disease but not yet diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., remission of the disease. Therapeutic benefit includes eradication or improvement of the underlying disorder being treated. Moreover, although the subject may still suffer from the underlying disorder, therapeutic benefit can be achieved by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing an improvement in the subject's condition. In some aspects, although a diagnosis may not yet be made for such diseases, in order to obtain a preventive benefit, treatment is provided or a therapeutic composition is administered to a subject who is likely to develop a particular disease or to report one or more physiological symptoms of a disease. The disclosed methods can be used in any mammal or other animal. In some aspects, treatment can alleviate or stop symptoms. The preventive effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of a disease or condition symptom, slowing down, stopping or reversing the progression of a disease or condition, or any combination thereof.

[0076] The term "subject" used in the present invention refers to any individual or patient using the disclosed method of the present invention. The term "subject" can be used interchangeably with the term "individual" or "patient". It will be understood by those skilled in the art that although the subject may be an animal, the subject may be a human. Therefore, other animals (including mammals, such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals (including cattle, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans and gorillas)) are all included in the definition of subject.

[0077] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of an antibody, an antigen binding fragment thereof, or other composition described herein sufficient to achieve the intended use, including but not limited to the treatment of diseases defined herein. The therapeutically effective amount can vary depending on the intended therapeutic use (e.g., in vivo) or the patient being treated and the condition (e.g., the patient's weight and age, the severity of the condition, the mode of administration, etc.), and a person of ordinary skill in the art can readily determine the therapeutically effective amount. The term also applies to a dose that will induce a specific response in a target cell. The specific dose will vary depending on the specific antibody, antigen binding fragment thereof, or other composition selected, the dosing regimen to be followed, whether it is co-administered with other compounds, the time of administration, the administration tissue, and the physical delivery system for carrying the drug.

[0078] In some aspects, as a combined therapy, the antibody or its antigen-binding fragment of the present invention is used as a monotherapy or in combination with other therapeutic agents (e.g., radiotherapy, cytotoxic chemotherapy) and other immunomodulators (e.g., vaccines, interleukins, cytokines, chemokines and biological products). Exemplary interleukins for immunotherapy include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21 and IL-23. Exemplary cytokines for immunotherapy include interferon, TNF-α, TGF-β, G-CSF and GM-CSF. Exemplary chemokines for immunotherapy include CCL3, CCL26 and CXCL7. Exemplary biologics include CAR T cell therapy, tumor infiltrating lymphocyte (TIL) therapy, and monoclonal antibodies, such as alemtuzumab (CAMPATH), trastuzumab (HERCEPTIN), ibritumomab tiuxetan (ZEVALIN), brentuximab (ADCETRIS), ado-trastuzumab emtansine (KADCYLA), blinatumomab (BLINCYTO), bevacizumab (AVASTIN), and cetuximab (ERBITUX). For example, antibodies also include checkpoint inhibitors, which include PD-1 inhibitors such as pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), and cemiplimab (LIBTAYO), PD-L1 inhibitors such as atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (IMFINZI), CTLA-4 inhibitors such as ipilimumab (YERVOY), and other checkpoint inhibitors such as anti-B7-H3 antibodies (MGA271), anti-KIR antibodies (lirilumab), and anti-LAG3 antibodies (BMS-986016).

[0079] As described in the following examples, in some embodiments, the present invention further provides the expression, purification and characterization of anti-CD137 agonist antibodies. A signal sequence may be included in the expression construct of the antibody of the present invention. Any suitable signal sequence may be used, such as the sequence of SEQ ID NO:27. In some aspects, T cells treated with anti-PD-L1 antibodies and anti-CD137 antibodies of the present invention indicate that T cell effector function is further enhanced. In the absence of theoretical limitations, this situation indicates that the combined therapy or treatment using bispecific antibodies targeting CD137 and PD-L1 can overcome the low response rate of monotherapy when each antibody is used alone in clinical trials. For example, in addition to anti-PD-L1 antibodies, a second antibody for combined therapy targeting other immune enhancing antigens (such as CD40 or CTLA-4) may be used, or a bispecific antibody targeting CD137 and a second antigen (such as PD-L1, CD40 or CTLA-4) may be used for treatment.

[0080] Bispecific molecules, such as bispecific antibodies (bsAbs), provide a means to simultaneously target multiple epitopes on the same or different molecular targets using a single therapeutic agent. For example, without being limited by theory, bispecific molecules as cancer therapeutics have the potential to confer novel or more potent activities, reduce cost of goods, and facilitate the development of new treatment options compared to a mixture of two monoclonal antibodies (mAbs).

[0081] Therefore, the present invention also provides the expression, purification and characterization of bifunctional proteins including bispecific antibodies. The term "bifunctional protein" used in the present invention refers to a protein having at least two functions. Non-limiting examples of bifunctional proteins include bispecific antibodies that can bind to two antigens. For example, the bispecific antibody of the present invention may include a separated functional scFv fragment that binds to CD137 and is fused to the C-terminus of the Fc domain of an anti-PD-L1 antibody. In some aspects, the C-terminal positioning scFv that binds to CD137 in the fusion construct of the present invention is fused to the Fc domain of an antibody that binds to other immunomodulatory molecules (such as CD40 or CTLA-4). For example, in other aspects, the C-terminal positioning scFv can be combined with an immunomodulatory molecule (such as CD40 or CTLA-4).

[0082] In some embodiments, the present invention provides a bispecific antibody comprising a first antigen binding region and a second antigen binding region. Generally, the first antigen binding region and the second antigen binding region specifically bind to different antigens or targets. In some aspects, the first antigen binding region and the second antigen binding region bind to different epitopes in the same antigen or target.

[0083] In one embodiment, the bispecific antibody of the present invention comprises a first antigen binding region that binds to CD137. The first antigen binding region comprises a V H A region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to a sequence selected from SEQ ID NO: 1; SEQ ID NO: 9 and SEQ ID NO: 17, and a V L A region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range between the two. In some aspects, the second antigen binding region of the bispecific antibody of the present invention binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen.

[0084] SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 17 (including V H region) or SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 18 (comprising V L Any number of amino acids of the sequence described in the V region) can be included in the bispecific antibody. H Area or V LIn one aspect, the bispecific antibody of the present invention comprises about 100 to 105 amino acids, about 100 to 110 amino acids, about 100 to 115 amino acids, about 100 to 120 amino acids, about 100 to 125 amino acids of the N-terminal or C-terminal sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17 or SEQ ID NO:18, and any number or range therebetween. In another aspect, the bispecific antibody comprises the sequence of SEQ ID NO:1, SEQ ID NO:9 or SEQ ID NO:17. In another aspect, the bispecific antibody comprises about 100 to 120 amino acids of the N-terminal sequence of SEQ ID NO:2, SEQ ID NO:10 or SEQ ID NO:18. In another aspect, the bispecific antibody comprises about 112 amino acids of the N-terminal sequence of SEQ ID NO:10 or about 108 amino acids of the N-terminal sequence of SEQ ID NO:18.

[0085] In one aspect, the first antigen-binding region of the bispecific antibody of the invention comprises a V H region (having the amino acid sequence of SEQ ID NO: 9), and a V L region (having an amino acid sequence of about 100 to 120 amino acids of the N-terminal sequence of SEQ ID NO: 10). On the other hand, the first antigen-binding region of the bispecific antibody of the present invention comprises a V H region (comprising the amino acid sequence of SEQ ID NO: 17), and a V L region (an amino acid sequence of about 100 to 120 amino acids comprising the N-terminal sequence of SEQ ID NO: 18).

[0086] In some aspects, the second antigen binding region of the bispecific antibody of the present invention binds to an immune checkpoint molecule, an immunostimulatory molecule or a tumor antigen. The term "immune checkpoint molecule" used in the present invention refers to any molecule that inhibits or negatively regulates an immune response. On the one hand, the binding of the second antigen binding region to the immune checkpoint molecule inhibits the immune checkpoint molecule. Exemplary immune checkpoint molecules include PD-L1, PD-1, CTLA-4 and LAG3. The term "immunostimulatory molecule" used in the present invention refers to any molecule that induces, strengthens or negatively regulates an immune response. Exemplary immunostimulatory molecules include CD28, CD40, CD137, CD27 and ICOS. For example, in some aspects, the binding of the second antigen binding region to the immunostimulatory molecule activates the immunostimulatory molecule, increases signal transduction and immune activation. The term "tumor antigen" used in the present invention refers to any antigen present on the surface of a tumor cell or expressed by a tumor cell. Exemplary tumor antigens include products of mutant oncogenes, products or mutant tumor suppressor genes, products of mutant genes other than oncogenes or tumor suppressor genes, tumor antigens produced by oncogenic viruses, altered cell surface glycolipids and glycoproteins, tumor-fetal antigens, etc. Tumor antigens also include immunomodulatory molecules, such as immune checkpoint inhibitors and immunostimulatory molecules. Accordingly, in some aspects, the tumor antigen binding of the second antigen binding region of the bispecific antibody of the present invention is combined with the function of an immunomodulatory molecule. For example, on the one hand, the combination of the second antigen binding region and the tumor antigen targets immune cells (such as T cells) to tumor cells.

[0087] Any combination of the first and second antigen binding regions may be included in the bispecific antibodies of the present invention, including, for example, the first and second antigen binding regions that bind to any immune checkpoint molecule, any immunostimulatory molecule, or any tumor antigen. Accordingly, in some aspects, the second antigen binding region of the bispecific antibody of the present invention binds to any immune checkpoint molecule, any immunostimulatory molecule, or any tumor antigen. In some aspects, the first and second antigen binding regions bind to the same molecule. For example, the first and second antigen binding regions may bind to the same or different epitopes of the same molecule. In other aspects, the first and second antigen binding regions bind to different molecules.

[0088] In some aspects, the second antigen binding region binds to an antigen selected from PD-L1, PD-1, CTLA-4, LAG3, CD28, CD40, CD137, CD27, ICOS, human epidermal growth factor receptor 2 (Her2) or a glycan. Exemplary glycans include N-glycans, O-glycans and glycosphingolipids. Glycans can be expressed only in cancer cells, such as GloboH. On the one hand, the second antigen binding region binds to PD-L1. On the other hand, the second antigen binding region binds to Her2. On the other hand, the second antigen binding region binds to a glycan. On the other hand, the glycan is GloboH. For example, expanding the bispecific antibody library bound to CD137 and antigens expressed on tumors other than PD-L1 can target bispecific antibodies to cancer types that do not express PD-L1.

[0089] In one aspect, a bispecific antibody having a first antigen-binding region that binds to CD137 and a second antigen-binding region that binds to PD-L1 simultaneously binds to CD137 and PD-L1 ( Fig.16 ). Without being limited by theory, by designing bispecific antibodies that can bind to both CD137 and PD-L1, the anti-CD137 binding activity can be restricted to tumor sites expressing PD-L1, which can reduce the risk of hepatotoxicity and its associated mortality seen in clinical trials of anti-CD137 antibodies such as ustulumab. In addition, studies have shown that simultaneous binding to CD137 and PD-L1 can enhance T cell activation due to cross-linking (see also Example 10 below). On the other hand, bispecific antibodies having a first antigen binding region that binds to CD137 and a second antigen binding region that binds to PD-L1 can induce stronger CD137 internalization compared to reference antibodies such as ustulumab and ustulumab ( Fig.23 ).

[0090] In some aspects, the first antigen binding region and the second antigen binding region comprise scFv, F(ab)2, Fab, or any combination thereof. On the one hand, the first antigen binding region comprises scFv and the second antigen binding region comprises Fab. On the other hand, the scFv contained in the bispecific antibody of the present invention binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen. On the other hand, the scFv contained in the bispecific antibody of the present invention binds to CD137. In some aspects, the Fab contained in the bispecific antibody of the present invention binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen. On the one hand, the Fab contained in the bispecific antibody of the present invention binds to PD-L1. On the other hand, the scFv of the bispecific antibody of the present invention comprises a V H region (comprising the amino acid sequence of SEQ ID NO: 9), and a V Lregion (an amino acid sequence of about 100 to 120 amino acids of the N-terminal sequence of SEQ ID NO: 10). On the other hand, the scFv of the bispecific antibody of the present invention comprises a V H region (comprising the amino acid sequence of SEQ ID NO: 17), and a V L region (an amino acid sequence of about 100 to 120 amino acids comprising the N-terminal sequence of SEQ ID NO: 18).

[0091] On the one hand, the bispecific antibody of the present invention comprises a first antigen binding region bound to CD137 and a second antigen binding region bound to Her2. The bispecific antibody bound to CD137 and Her2 comprises a scFv bound to CD137, and the scFv is fused to the C-terminus of the Fc domain of the antibody bound to Her2, such as trastuzumab (heavy chain SEQ ID NO: 36) or anti-Her2#3-7 (heavy chain SEQ ID NO: 38). In certain aspects, the bispecific antibody bound to CD137 and Her2 further comprises a light chain of SEQ ID NO: 35 (trastuzumab) or SEQ ID NO: 37 (anti-Her2#3-7). On the other hand, the bispecific antibody of the present invention comprises a first antigen binding region bound to CD137 and a second antigen binding region bound to tumor-specific glycans. The bispecific antibody that binds to CD137 and tumor-specific glycans comprises a scFv that binds to CD137, and the scFv is fused to the C-terminus of the Fc domain of the antibody that binds to the tumor-specific glycan, such as the anti-glycan (heavy chain SEQ ID NO: 40) of the present invention. In certain aspects, the bispecific antibody that binds to CD137 and tumor-specific glycans further comprises a light chain of SEQ ID NO: 39 (anti-glycan). In some aspects, the bispecific antibody that binds to CD137 and Her2 or CD137 and tumor-specific glycans further comprises a linker that connects the anti-CD137 scFv to the Fc domain. Any linker can be used, such as a GS linker (SEQ ID NO: 28), a G4S linker (SEQ ID NO: 29) or a multiple thereof. In one aspect, the linker is a G4S linker.

[0092] Accordingly, the present invention provides a target-dependent T cell activation platform. Fig.19 As shown in Figures 21 and 22, the agonist activity of anti-CD137 scFv is induced when it binds to tumor-specific antigens such as PD-L1. The anti-CD137 agonist activity can also be activated by binding to other tumor-specific antigens such as Her2 and tumor-specific glycans, as shown in Figures 21 and 22.

[0093] In some aspects, the bispecific antibodies of the invention further comprise a scFv VH Area and V L The connexon between the regions. Any connexon can be used. For example, the connexon can comprise any amino acid sequence. The connexon can have any length, such as 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids or more amino acids. The connexon can also comprise any multiple of amino acid sequences. Any number of multiples of amino acid sequences can be included in one connexon. Exemplary connexon sequences are provided in SEQ ID NO: 28 and SEQ ID NO: 29. In some aspects, the scFv comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0094] In some aspects, the bispecific antibody of the present invention further comprises an Fc domain. In some aspects, the Fc domain is an IgG domain, an IgE domain, an IgM domain, an IgD domain, an IgA domain or an IgY domain. The Fc domain of any sequence and any species can be used, including humans, apes, monkeys, mice, rabbits, goats, sheep, guinea pigs, horses, etc. In some aspects, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain or an IgG4 domain. On the one hand, the IgG4 domain comprises the amino acid sequence of SEQ ID NO:25. On the other hand, the IgG1 domain comprises the amino acid sequence of SEQ ID NO:26. On the one hand, the Fc domain is human. Generally, the human Fc domain is not immunogenic in humans and is therefore suitable for human treatment.

[0095] In some aspects, the scFv of the bispecific antibody of the present invention is coupled to the C-terminus of the Fc domain. On the one hand, a linker is included between the Fc domain and the scFv. On the other hand, the linker connects the scFv to the Fc domain. Any linker can be used, such as the G4S linker of the present invention.

[0096] In some aspects, the Fab of the bispecific antibody of the present invention is connected to the N-terminus of the Fc domain. On the one hand, the Fab is directly connected to the N-terminus of the Fc domain via a peptide bond. On the other hand, the Fab domain is connected to the N-terminus of the Fc domain via a connector.

[0097] In some aspects, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 31 or SEQ ID NO: 32. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 30. In one aspect, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 36. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 35. In one aspect, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 38. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 37. In one aspect, the bispecific antibody of the present invention comprises a heavy chain sequence of SEQ ID NO: 40. In another aspect, the bispecific antibody of the present invention further comprises a light chain sequence of SEQ ID NO: 39. Although any other suitable linker may be used, the heavy chain sequences (such as SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40 and other sequences) may contain one or more G linkers (SEQ ID NO:28), one or more G4S linkers (SEQ ID NO:29), or any multiple of G linkers or G4S linkers.

[0098] In some embodiments, the present invention provides an isolated amino acid sequence as shown in SEQ ID NO: 30-40. In some embodiments, the present invention also provides an isolated nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NO: 30-40.

[0099] In some embodiments, the present invention provides antibody-drug conjugates. The antibody-drug conjugates of the present invention may include any antibody or antigen-binding fragment thereof of the present invention. For example, any antibody or antigen-binding fragment thereof that specifically binds to CD137 may be included in the antibody-drug conjugate. Any bispecific antibody or antigen-binding fragment thereof of the present invention may also be included in the antibody-drug conjugate. In some aspects, the antibody-drug conjugate of the present invention comprises a therapeutic agent. Any therapeutic agent, including small molecules, may be included in the antibody-drug conjugate of the present invention. In some aspects, the therapeutic agent has cytotoxic activity. Any chemotherapeutic agent with cytotoxic activity may be included in the antibody-drug conjugate. Exemplary chemotherapeutic agents include, but are not limited to, dactinomycin, all-trans retinoic acid, antiestrogens, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorpheniramine, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, docetaxel, doxorubicin, doxorubicin, epirubicin, etomycin, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, methotrexate, mercaptopurine, methotrexate, mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, paclitaxel, taxotere, tamoxifen, teniposide, thioguanine, topotecan, vabicin, vemurafenib, vinblastine, vincristine, and vindesine.

[0100] In some aspects, the antibody-drug conjugates of the present invention are used to treat cancer. For example, the antibody contained in the antibody-drug conjugate binds to an antigen on a tumor cell, thereby targeting a small molecule with cytotoxic activity or other therapeutic agent contained in the antibody-drug conjugate to the tumor cell. When the antibody-drug conjugate binds to a tumor cell, the small molecule or other therapeutic agent is internalized and released in the tumor cell.

[0101] In some aspects, the therapeutic agent contained in the antibody-drug molecule of the present invention is covalently linked to the antibody or its antigen-binding fragment of the present invention or the bispecific antibody or its antigen-binding fragment of the present invention. The linker can be used to covalently link the therapeutic agent to the antibody or its antigen-binding fragment, or to covalently link the bispecific antibody or its antigen-binding fragment. Any suitable linker can be used to covalently link the therapeutic agent to the antibody or its antigen-binding fragment of the present invention, or to covalently link the bispecific antibody or its antigen-binding fragment of the present invention. In some aspects, the linker contained in the antibody-drug conjugate of the present invention is more stable outside the target cell (including in the circulation) and is cleaved in the target cell to release the therapeutic agent. For example, a therapeutic agent with cytotoxic activity can induce target cell death when released. Accordingly, in some aspects, the therapeutic agent selectively targets tumor cells. For example, the selective targeting of a therapeutic agent to tumor cells generally results in reduced cytotoxicity to non-tumor cells, while increased tolerance.

[0102] In some embodiments, the present invention provides a pharmaceutical composition comprising a bispecific antibody of the present invention. Any bispecific antibody of the present invention may be included in a pharmaceutical composition. On the one hand, the bispecific antibody contained in the pharmaceutical composition of the present invention is combined with CD137 or PD-L1 or combined with CD137 and PD-L1 simultaneously. The antibody-drug conjugate of the present invention may also be included in the pharmaceutical composition. In some aspects, the pharmaceutical composition is used for cancer treatment. Any cancer can be treated with the pharmaceutical composition of the present invention. Exemplary cancers include prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer and leukemia.

[0103] In some embodiments, the present invention provides a method for treating cancer in a subject. The method for treating cancer includes administering to a subject a certain amount of any bispecific antibody or antigen-binding fragment thereof of the present invention (effective for treating cancer). In some aspects, the cancer is prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer or leukemia.

[0104] Example

[0105] Example 1

[0106] This example illustrates the generation of antibodies from the OmniMab library.

[0107] To generate anti-CD137 therapeutic antibodies, selection was performed through the OmniMab phage library. The phage library was established by APBiosciences Inc. (APBio Inc.) and peripheral blood mononuclear cells from more than 100 healthy donors were selected. The pre-coated CD137-Fc recombinant protein was cultured with the supernatant containing the rescued phage for 1 hour and washed three times with PBS containing 0.1% Tween 20. The bound phage was detected by HRP-coupled anti-M13 antibody (Roche) and signal development was performed using TMB substrate. OD450 readings were recorded.

[0108] The first round of elution was performed using Hyperphage (M13K07ΔρIII, Progen, Heidelberg, Germany). Solid phase elution and anti-CD137 cell elution were used to select and isolate CD137-specific binders from the OmniMab library. Solid phase elution was performed using the recombinant human CD137-ECD-Fc (APBio Inc.) used in the first round of selection. CD137-expressing HEK293 cells were used for the second and third rounds of enrichment. After three rounds of elution, specific CD137 binders ( Figure 1 and Figure 2 ). For FACS analysis, 293F cells stably expressing CD137 were stained with anti-CD137 phage supernatant (50ul / well) to detect CD137 binding activity. As a control, 293F cells stably expressing CD137 were also incubated on ice for 1 hour with 2.5ug / ml anti-CD137 antibodies (Abs). The cells were washed three times with 1x PBS and then incubated on ice for 1 hour with anti-M13 antibodies (Progen). The cells were washed three times with 1X PBS again and then incubated on ice for another hour with anti-mouse IgG-Alexa488 (Invitrogen Inc.). After staining, the cells were washed three times with 1x PBS and resuspended in 1x PBS before analysis by FACSCalibur (BD Biosciences, Inc.) and FlowJo (TreeStar, LLC). FACS analysis of 293F cell clone 13 stably expressing CD137 is shown in Figure 1. Figure 2 As shown. Positive binders were isolated and sent for sequencing to confirm the sequence and diversity of the heavy chain. Figure 1 and Figure 2 As shown, several clones that specifically recognized the CD137 antigen were isolated compared to the negative control.

[0109] These results indicate that the phage clones obtained after three rounds of CD137-specific enrichment can specifically recognize CD137.

[0110] Example 2

[0111] This example demonstrates the subcloning, expression, and purification of a CD137-specific binding protein in IgG format.

[0112] To rapidly screen candidates with T cell activation function, the heavy and light chains of positive CD137 or PD-L1 binders identified by ELISA were amplified, digested, and subcloned into an IgG expression vector carrying an IgG4 constant region generated by APBio (SEQ ID NO.25). After sequence verification, plasmids were prepared and transfected into HEK293 cells using 293fectin transfection reagent (Invitrogen) for antibody expression. After 4 days of culture, antibodies secreted into serum-free medium were affinity purified from the culture supernatant using protein G chromatography. The purified antibodies were concentrated and then dialyzed in PBS buffer. The final concentration of the dialyzed protein was determined using a NanoDrop2000 spectrophotometer, and the purity and integrity were determined using SDS-PAGE (with or without reducing agents).

[0113] Figure 3 shows a representative PAGE gel analysis of the first batch (Figure 3, top) and the second batch (Figure 3, bottom) of purified anti-CD137 antibody leads. Protein G chromatography (Thermo Fisher) was used to purify the mammalian cell culture supernatant collected 4 days after transfection. The purified proteins were analyzed under reducing or non-reducing conditions before loading on the gel (3μg / lane). The results showed that under non-reducing conditions, the molecular weight of the two proteins was about 145kDa, while under reducing conditions, the molecular weights of the heavy chain and light chain were ~55kDa and ~25kDa, respectively. One-step protein G chromatography can obtain a purity of more than 90%.

[0114] These results indicate that the integrity of the various purified antibody leads is normal in HEK293 cells.

[0115] Example 3

[0116] This example demonstrates the binding of anti-CD137 antibody to Jurkat cells.

[0117] Purified anti-CD137 antibody leads were also applied to CD137-induced Jurkat cells to determine binding activity by FACS. Jurkat cells were treated with PMA (10 ng / ml) and ionomycin (1 μg / ml) to induce CD137 expression and continued for 2 days. Anti-CD137 (0.5 μg / ml) and reference (ref) Ab (0.5 μg / ml) were used as positive controls, and stimulated cells were cultured on ice for 1 hour without staining, or OX40 reference (ref) Ab was used as a negative control for culture. Cells were washed three times with 1x PBS and then cultured on ice for another 1 hour with Alexa-488-conjugated goat anti-human IgG (H+L) (Invitrogen Inc.). After staining, cells were washed three times with 1x PBS and resuspended in 1x PBS before analysis by FACS Calibur (BD Biosciences, Inc.) and FlowJo (TreeStar, LLC). Among the CD137 antibody leads, there were several leads that had comparable binding activity to the reference antibody, e.g. Figure 4 shown.

[0118] These results indicate that binding of anti-CD137 antibodies can lead to activation of Jurkat cells as shown by flow cytometry.

[0119] Example 4

[0120] This example illustrates the use of ELISA to measure the binding activity of anti-CD137 antibodies.

[0121] For direct ligand binding analysis of anti-CD137 antibody binding to CD137, pre-coated membrane wells were prepared using recombinant CD137 / Fc (100 ng / ml). Briefly, purified human CD137-IgG4Fc (APBio) was dialyzed in phosphate-buffered saline (PBS), adjusted to 1 mg / ml, and then diluted with PBS to a final concentration of 1 μg / ml. Nunc-Immuno Maxisorp 96-well plates were pre-coated with 0.1 ml of recombinant CD137 protein per well, leaving empty wells for non-specific binding controls and incubated overnight at 4°C. The CD137 recombinant protein solution was removed and the culture plates were washed three times with 0.4 ml of wash buffer (0.1% Tween 20 in PBS). 0.4 ml of blocking buffer (5% low-fat milk powder in PBS) was added to all wells and incubated for 1 hour at room temperature. The blocking buffer was removed and washed three times with 0.4 ml of wash buffer.

[0122] The pre-coated membrane wells were cultured with serial dilutions of purified anti-CD137 antibodies. Serial dilutions of CD137 antibodies were prepared in PBS, and 0.1 ml serial dilutions were added to each well. The culture plate was cultured at room temperature for 1 hour. The antibody solution was removed and the culture plate was washed three times with 0.4 ml washing buffer. RP-coupled goat anti-human IgG, F(ab')2 specific F(ab')2 antibody (Jackson Immunoresearch#109-036-097) was diluted with PBS at 1:2000 and added in an amount of 0.1 ml / well. The culture plate was cultured at room temperature for 1 hour and washed three times in an amount of 0.4 ml washing buffer / well. The culture plate was developed with 0.1 ml TMB reagent (Invitrogen) and cultured at room temperature for 1 to 5 minutes. 0.05 ml 1N HCl was added to stop the reaction and the absorbance was read at 450 nm on the Bio-Tek Spectra. The OD450 readings were plotted against the anti-CD137 concentrations and the 50% effective concentration (EC) of anti-CD137 antibody binding to CD137 / Fc was calculated. 50 ) values. EC was calculated using GraphPad Prism (GraphPad Software, San Diego, CA). 50 EC values ​​of anti-CD137 antibodies clone 31 and clone 54 50 The calculated values ​​showed comparable binding activity to the reference antibody. The EC values ​​of the anti-CD137 specific antibody leads were 50 The calculated values ​​showed good binding activity ( Figure 5 ).

[0123] Before development, anti-PD-L1 antibody was detected with HRP-conjugated anti-human IgG1 Fab antibody, and OD450 readings were plotted against anti-PD-L1 concentration.

[0124] Example 5

[0125] This example illustrates the evaluation of protein aggregation induced by high concentrations of anti-CD137 antibody by SEC-HPLC.

[0126] SEC-HPLC was performed using a Waters Alliance separation module 2695 and a Waters 2996 photodiode array detector. The samples were loaded onto an XBridge protein BEH SEC column (Waters, catalog #176007640) and an isocratic 25 mM sodium phosphate, 200 mM NaCl, pH 6.8 was used as the mobile phase buffer for SEC separation. The flow rate was 0.4 mL / min and the injection volume was 10 μL. Peaks were detected by absorbance at 280 nm. Prior to injection onto the SEC column, all samples were filtered using a 0.22 μm filter (Millipore, catalog #SLGP003RB) to remove any precipitated protein material. Data analysis was performed using Empower 2 software. Figure 6 As shown, the main peak percentage of high-concentration anti-CD137 antibody clone 31 and clone 54 was greater than 90%, with no obvious protein aggregates.

[0127] These results indicate that high concentrations of anti-CD137 antibodies do not produce aggregates.

[0128] Example 6

[0129] This example illustrates the agonistic activity of anti-CD137 antibodies.

[0130] Purified antibody leads were functionally screened for their ability to enhance human CD3+ cell activation, such as enhancing cytokine production, proliferation, and inducing proliferation of human CD3+ T cells. Anti-CD3 antibody (1 μg / ml, OKT3, BioLegend Cat. No. 317304), anti-CD137 antibody leads or isotype antibodies (1, 3, and 10 μg / ml) were coated onto Maxisorp 96-well plates. TM Human CD3+ T cells were isolated from the peripheral blood of healthy adult volunteers using human T cell enrichment solution (STEMCELL catalog number 15061). TM The isolated CD3+ T cells were labeled with CFSE cell proliferation kit (Life Technologies, catalog number C34554) and seeded in pre-coated wells (1x10 cells per well) using RPMI1640 medium (containing 10% fetal bovine serum, 2.5 mM L-glutamine, 1x penicillin / streptomycin). 5 After 3 days, cell proliferation was analyzed by flow cytometry, and cytokine production of IL-2 and INF-γ was analyzed by ELISA. Figure 7 and Figure 8As shown, anti-CD137 antibody leads #15, #31, and #54 exhibited agonistic activity, enhancing CD3+ T cell activation in at least two of the four donors tested in a dose- and donor-dependent manner. Anti-CD137 clones #31 and #54 exhibited comparable or higher agonistic activity in enhancing T cell activation compared to the reference antibody (Utolumab; Chin et al., 2018; antibody sequences are available at www.kegg.jp / dbget-bin / www_bget?dr:D10997; see also U.S. Patent No. 8,337,850). Therefore, clones #31 and #54 were selected for the construction of bispecific antibodies as described below.

[0131] Example 7

[0132] This example illustrates the use of combination therapy with anti-PD-L1 and anti-CD137 antibodies in a mixed lymphocyte reaction.

[0133] Using RosetteSep TM Human Monocyte Enrichment Fluid (Catalog No. 15068) was used to isolate monocytes from the peripheral blood of healthy donors and cultured for 6 days in RPMI1640 differentiation medium containing human GM-CSF and IL-4 (1000 U / ml each, R&D). Dendritic cell (DC) differentiation was verified by flow cytometry by expression of DC-SIGN, CD14, CD80, or CD83. The differentiated DCs were used as antigen presenting cells (APCs) in mixed lymphocyte reactions (MLR). TM Allogeneic CD4+ T cells were isolated from human peripheral blood using human CD4+ T cell enrichment fluid (Catalog No. 15062). The purity of CD4+ T cells was approximately 95% based on CD3 and CD4 expression. CFSE-labeled CD4+ T cells were co-cultured with DCs in the presence of antibody leads (0.4, 2, and 10 μg / ml) for 3 and 5 days. CD4+ T cell proliferation was analyzed by flow cytometry, and cytokine production of IL-2 and INF-γ in the culture medium was analyzed by ELISA. IL-2 and INF-γ production was significantly increased in the presence of anti-PD-L1 antibodies in the MLR compared to isotype control antibodies. Interestingly, for example, anti-CD137 antibodies (such as clone #31) further promoted anti-PD-L1 antibody-mediated IFN-γ production in MLRs with two different donor pairs, such as Fig. 9 shown.

[0134] Example 8

[0135] This example illustrates the effect of anti-CD137 antibodies on the CD137-CD137L interaction.

[0136] HEK-293F / CD137 cells were cultured on ice for 30 minutes with isotype control and anti-CD137 antibodies (50 μg / ml), washed twice with PBS / 2% FBS (PBS2), and cultured on ice with His-tagged 4-1BBL (0.5 μg / ml, Acro BIOSYSTEMS) for 20 minutes. After washing twice with PBS2, anti-human Fc-A488 (Jackson ImmunoResearch) and anti-His antibody APC (Biolegend) were used to detect the presence of anti-CD137 antibodies and 4-1BBL on HEK-293F / CD137 cells, respectively, and then analyzed using a Calibur flow cytometer (BD). Except for the isotype control culture, almost all cells were A488 positive. The mean fluorescence intensity (MFI) of the APC channel was calculated using FlowJo (TreeStar, LLC), and the values ​​were displayed as a histogram ( Fig.10 ).like Fig.10 As shown, reference antibody 1 (ref1) and clone #54 effectively blocked CD137-CD137L interaction, while reference antibody 2 (ref2), clone #15 and clone #31 were less efficient or less effective in blocking CD137-CD137L interaction.

[0137] Example 9

[0138] This example illustrates the in vivo pharmacokinetics of an anti-CD137 antibody lead.

[0139] The antibody was administered to SCID-beige mice by intravenous bolus injection at a dose of 5 mg per kg body weight. After the injection was completed, peripheral blood was collected at the specified time points. The antibody plasma concentration was detected by ELISA as described below. The CD137 human Fc (1 μg / mL) pre-coated wells were co-incubated with titrated concentrations of purified anti-CD137 IgG4 antibodies, a standard curve was prepared, and the antibody concentration in plasma was calculated (using a fresh preparation in blocking solution). During the detection, the samples collected at different time points were also applied to the pre-coated CD137 human Fc wells. After washing with 0.1% Tween 20 in PBS, the bound antibody was detected using HRP-conjugated anti-human Fab antibody (0.4 μg / mL), and then the color was developed. The antibody plasma concentration was calculated by interpolation. PK parameters were calculated using PKSolver software (Zhang, Huo, Zhou, and Xie, 2010). The antibody showed good t 1 / 2 (about 176 hours), AUC is about 7800ug / ml*h( Fig.11 ).

[0140] Example 10

[0141] This example illustrates the cross-linking-dependent agonistic activity of anti-CD137 antibody leads.

[0142] Stable CD137 reporter cell clones were generated by transfecting HEK293 cells with NF-κB-driven luciferase and full-length CD137, followed by selection with hygromycin and G418, respectively. For agonist activity assays, anti-CD137 antibodies (10, 2, and 0.4 μg / ml) alone or cross-linked with goat anti-human IgG (5 μg / ml, Jackson ImmunoResearch, catalog number 109-006-008) were added to reporter cells and cultured for 5 hours. ONE-Glo TM Luciferase activity was detected using the luciferase assay system (Promega, catalog number E6120). Fig.12 ) showed cross-linking-dependent agonist activity, while urerulumab (CD137 ref2, Fig.12 ) showed cross-link-independent agonist activity, leading to the severe hepatotoxicity observed in clinical trials. CD137#54 showed greater agonist activity when cross-linked compared to Urolumab, whereas in the absence of cross-linking, the agonist activity was modest and similar to that of Urolumab ( Fig.12 Without being limited by theory, such features of CD137#54 may induce target-dependent T cell activation when included in a bispecific antibody containing a tumor-specific binder.

[0143] Taken together, these results show different cross-linking dependencies of the agonist activities of anti-CD137#15, anti-CD137#31, and anti-CD137#54.

[0144] Example 11

[0145] This example demonstrates the construction, expression, and purification of an anti-PD-L1-CD137 bispecific antibody.

[0146] The anti-PD-L1 antibody clone 6 was used in the form of IgG without ADCC, and the anti-CD137 antibody was used in the form of scFv and fused to the C-terminus of the Fc region of the anti-PD-L1 clone 6 antibody. The construction of the bispecific antibody comprising the Fc region of the anti-PD-L1 antibody fused to the CD137 scFv is shown in Table 1 below (sequence), and the schematic diagram is shown in Fig.10A short flexible peptide linker (GGGGS) 2 (SEQ ID NO: 29) was placed between the C-terminus of the anti-PD-L1 antibody heavy chain in the Fc region (SEQ ID NO: 25 or SEQ ID NO: 26) and the N-terminal module of the anti-CD137 scFv to ensure proper folding and minimize steric hindrance. The amino acid sequence of the anti-PD-L1-CD137 scFv heavy chain is shown in SEQ ID NO: 31 and SEQ ID NO: 32. The antibody Fc fusion protein construct was expressed using the Gibco ExpiCHO expression system, and the cell culture supernatant of the transfected cells was purified by one-step protein G chromatography.

[0147] In addition to the bispecific anti-PD-L1 antibody Fc fused to the above-mentioned anti-CD137 scFv, the antibody fused to the anti-CD137 scFv may include anti-inhibitory immune checkpoint antibodies, such as anti-PD-1, anti-CTLA-4, anti-LAG3, etc., or immunostimulatory antibodies, such as anti-CD28, anti-CD40, anti-CD137, anti-CD27, anti-ICOS, etc. For bispecific antibodies, a linker is placed between the antibody Fc domain and the anti-CD137 scFv to generate a bispecific antibody.

[0148] The purity of the bispecific antibody was greater than 90% ( Fig.14 and Fig.15 In a one-step purification process, a purity of greater than 90% was achieved, which is consistent with a purified fusion protein with the correct molecular weight (Mw = 220 kD). Fig.14 Representative PAGE gel analysis of purified anti-PD-L1-CD137 bispecific antibodies (bsAbs) is shown. Mammalian cell culture supernatants collected 4 days after transfection were purified using protein G chromatography (Thermo Fisher). Purified proteins were analyzed under reducing or non-reducing conditions before loading on the gel (3 μg / lane). The results showed that under non-reducing conditions, the molecular weight of both proteins was approximately 220 kDa, while under reducing conditions, the molecular weights of the heavy chain CD137 scFv and light chain were ∼85 kDa and ∼25 kDa, respectively. Fig.15 The purity and integrity of the protein A purified anti-PD-L1#6-CD137#54 bsAb using the μCE-SDS method are shown.

[0149] Example 12

[0150] This example illustrates antigen recognition by an anti-PD-L1-CD137 bispecific antibody.

[0151] pass (Menlo Park, CA) Biosensor analysis to determine the binding activity of anti-PD-L1-CD137 bispecific antibodies. His-tagged CD137 (ACROBiosystems) was loaded onto HIS1K (anti-Penta-HIS) biosensors (Catalog No. 18-5120) at a concentration of 5 μg / mL in DPBS containing 0.02% Tween 20 and 0.1% BSA for 5 minutes. The sensors were then exposed to the indicated antibodies at 100 nM for 5 minutes using the same buffer, followed by association with 100 nM of the second antigen (PD-L1 fused to mouse Fc domain) for 5 minutes. The data were then compiled using Octet Data Acquisition and Analysis Software as described by the manufacturer. Fig.16 Binding diagram shown. Compared with the control antibody, both bispecific antibodies (anti-PD-L1#6-CD137#31 and anti-PD-L1#6-CD137#54) can first recognize CD137 and then recognize PD-L1, proving that bispecific antibodies can target PD-L1 and CD137 simultaneously.

[0152] Taken together, these results indicate that As determined by biosensor analysis, the anti-PD-L1-CD137 bispecific antibody can simultaneously recognize PD-L1 and CD137.

[0153] Example 13

[0154] This example demonstrates that anti-PD-L1 antibody and anti-PD-L1-CD137scFv bispecific antibody (bsAb) can significantly enhance T cell activation in an allogeneic mixed lymphocyte reaction.

[0155] Using RosetteSep TM Human monocyte enrichment solution (Catalog No. 15068) was used to isolate monocytes from the peripheral blood of healthy donors and cultured for 6 days in RPMI1640 differentiation medium containing human GM-CSF and IL-4 (1000 U / ml each, research and development). TM Allogeneic CD4+ T cells were isolated from human peripheral blood using Human CD4+ T Cell Enrichment Fluid (Catalog No. 15062). The purity of CD4+ T cells was approximately 95% based on CD3 and CD4 expression. CFSE-labeled CD4+ T cells were co-cultured with DCs in the presence of antibody guides (1, 3, and 10 μg / ml) for 3 and 5 days. CD4+ T cell proliferation was analyzed by flow cytometry, and cytokine production of IL-2 and INF-γ in the culture medium was analyzed by ELISA. Anti-PD-L1#6-CD137#54 significantly enhanced T cell activation compared to monotherapy and combination therapy with anti-PD-L1 and anti-CD137 antibodies ( Fig.17 , showing the results for two donor pairs).

[0156] These results suggest that anti-PD-L1#6-CD137#54bsAb induced more potent T cell activation in mixed lymphocyte reactions compared with anti-PDL-1 and anti-CD137 antibody monotherapy or combination therapy and with anti-PD-L1#6-CD137#31bsAb treatment.

[0157] Example 14

[0158] This example demonstrates the promotion of antigen-specific T cell activation by anti-PD-L1-CD137 scFv bispecific antibody leads.

[0159] Use EasySep separately TM Human memory CD4+ T cell enrichment kit (STEMCELL, catalog number 19157) and human CD8+ T cell isolation kit (STEMCELL, catalog number 17953) were used to isolate human memory CD4 and CD8 T cells. Co-culture of memory CD4-T cells and autologous immature DCs was stimulated with CEFX Ultra SuperStim Pool MHC-II subset (1ug / ml, JPT) in the presence of antibodies (0.4, 2, and 10μg / ml) and continued for 7 days. When co-cultured with CD8-T cells, TLR-DCs generated as immature DCs were matured for 24 hours by adding IL-1β (10ng / ml, PeproTech), TNF-α (10ng / ml, PeproTech), IFN-γ (5000IU / ml, PeproTech), PGE2 (250ng / ml, Sigma), poly I:C (10μg / ml, Sigma), and R848 (5μg / ml, Sigma) to the differentiation medium. Co-cultures of CD8 T cells and TLR-DCs were stimulated with CEFX Ultra SuperStim Pool (1 μg / ml, JPT) in the presence of antibodies (0.4, 2, and 10 μg / ml) for 7 days. Similar to the results observed in Example 12 MLR, anti-PD-L1#6-CD137#54 bsAb promoted the recall responses of memory CD4 T cells (Figure 18, Panel A) and CD8 T cells (Figure 18, Panel B) compared to monotherapy with anti-PD-L1 or anti-CD137 mAbs or combination therapy with anti-PD-L1 and anti-CD137 mAbs (Figure 18, Panel A). Fig.18A -B).

[0160] In summary, Fig.17The results shown in (Example 13) and Figure 18 (this Example) indicate that anti-PD-L1#6-CD137#54bsAb significantly enhanced T cell activation, which was more robust than that seen when anti-PD-L1 and anti-CD137 monoclonal antibodies were used alone or in combination. In addition, based on recall response analysis using CD4 and CD8 T cells, T cell activation was found to be antigen-dependent after treatment with anti-PD-L1#6-CD137#54bsAb. Without being limited by theory, the anti-PD-L1#6-CD137#54bsAb has greater T cell activation potency than the anti-PD-L1#6-CD137#31bsAb, indicating that the anti-CD137#54 arm of the bispecific antibody can bind to a unique CD137 epitope without steric hindrance due to PD-L1 binding to the anti-PD-L1 arm of the bispecific antibody.

[0161] Example 15

[0162] This example illustrates target-dependent T cell activation induced by an anti-PD-L1-CD137 bispecific antibody.

[0163] Using RosetteSep TM Human T cells were isolated from human T cell enrichment buffer (STEMCELL catalog number 15061). Purified T cells were activated with plate-bound anti-CD3 (OKT3, 1 μg / ml) and co-cultured with PD-L1 overexpressing or parental HEK293 cells under the indicated antibody treatment ( Fig.19 ).like Fig.19 As shown, compared with monotherapy or combination therapy of anti-PD-L1 and anti-CD137 monoclonal antibodies, the anti-PD-L1-CD137 bispecific antibody significantly promoted T cell activation when co-cultured with PD-L1-overexpressing but not PD-L1-negative parental cells.

[0164] Taken together, these results indicate that target-dependent T cell activation was induced exclusively by anti-PD-L1#6-CD137 bsAb when cocultured with PD-L1-overexpressing HEK-293 cells (but not parental HEK293 cells) in the presence of plate-bound anti-CD3 (OKT3).

[0165] Example 16

[0166] This example illustrates tumor antigen-dependent T cell activation induced by the anti-tumor antigen-specific CD137#54 bispecific antibody.

[0167] Human CD8-T cells were isolated by positive selection as described above (Example 15). Purified CD8-T cells were co-cultured with PD-L1 positive tumor cells (NCI-H1975, PC-3 and MDA-MD-231) in a 1:1 ratio in the presence of anti-CD3 (OKT3) coated polystyrene microspheres. After 3 days, T cell activation was determined based on IFN-γ production as measured by ELISA and by CytoTox Tumor cell toxicity was detected by cytotoxicity assay (Promega, catalog number G1780).

[0168] Compared with monotherapy or combination therapy using anti-PD-L1#6 and anti-CD137#54 antibodies, IFN-γ production induced by anti-PD-L1#6-CD137#54bsAb was more robust (Figure 20). During co-culture with PC-3 cells, CD8 T cells were observed to have higher tumor cytotoxicity ( Fig. 20B In addition to PD-L1-positive tumors, Her2-positive (SKBR-3 and MDA-MB-361) and glycan-positive (MCF-7 and NCI-N87) tumor cells targeted by anti-Her2 (trastuzumab or #3-7) and anti-tumor glycan antibodies conjugated to CD137 #54 scFv also generated stronger IFN-γ production compared with either monotherapy or combination therapy ( Fig.21A -B and Fig.22A -B) and CD8 T cell tumor cytotoxicity ( Fig.22A ).

[0169] These results indicate that the tumor-targeting CD137#54bsAb specifically induced target-dependent T cell activation.

[0170] Example 17

[0171] This example illustrates the induction of CD137 internalization by the anti-PD-L1#6-CD137#54 bispecific antibody.

[0172] To test whether CD137 internalization is also induced by the anti-PD-L1#6-CD137#54 bispecific antibody and the reference antibodies ustulumab and utolumab, internalization analysis was performed using CD137-expressing HEK293 cells ( Fig.23 ). Place 5x10 3CD137-expressing cells were pre-seeded in black 96-well plates in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Gibco) and cultured overnight at 37°C, 5% CO2. The indicated antibodies were labeled with pHAb amine reactive dye (Promega Corp.) according to the manufacturer's protocol and prepared by 3-fold serial dilutions in culture medium starting from 100 nM. The medium containing the labeled antibody was then replaced with the medium in which the cells were pre-seeded, and the cells were cultured in the incubator for another 24 hours. After incubation, the cells were rinsed and stored in PBS, and fluorescence was recorded using SpectraMax iD3. EC50 values ​​were calculated using GraphPad Prism. Fig.23 As shown, a stronger induction of CD137 internalization was observed in the anti-PD-L1#6-CD137#54 bispecific antibody treatment compared to the reference anti-CD137 antibody treatment. Without being limited by theory, if the bispecific Ab only binds to T cells through CD137 engagement, the internalization level of CD137 can reduce CD137 activation. Accordingly, after in vivo administration of the bispecific antibody, the toxicity of the bispecific antibody was lower than that of the reference antibody, especially compared to Urecitabine.

[0173] Example 18

[0174] This example illustrates the rescue of Treg cell-mediated suppression of T cell proliferation by the anti-PD-L1#6-CD137#54 bispecific antibody.

[0175] A Treg suppression assay was set up using a mixed lymphocyte reaction as described in Example 12. Using EasySep TM Human CD4 + CD127 low CD25 + Treg cells were isolated from peripheral blood using the Regulatory T Cell Isolation Kit (STEMCELL, catalog number 18063). Treg cells were expanded using Human Treg Expander (Gibco, catalog number 11129D). The expanded Treg cells significantly suppressed CD4 T cell proliferation and IL-2 production. The suppressive activity of Treg cells was eliminated by adding anti-PD-L1#6-CD137#54bsAb to the culture ( Fig.24A -B). In the presence of Treg cells, anti-PD-L1#6-CD137#54bsAb rescued both T cell proliferation (Figure 24, Figure A) and cytokine production (Figure 24, Figure B). The above results indicate that anti-PD-L1#6-CD137#54bsAb can rescue Treg-mediated inhibition of T cell activation.

[0176] Example 19

[0177] This example illustrates the inhibition of tumor growth by the anti-PD-L1#6-CD137#54 bispecific antibody in vivo.

[0178] To validate the anti-tumor activity of anti-PD-L1#6-CD137#54bsAb (which does not cross-react with mouse PD-L1 and mouse CD137), human tumor cells (NCI-H292, NCI-H1975, and BxPC-3) were premixed with human PBMCs and xenografted subcutaneously into SCID-beige mice to evaluate the in vivo anticancer activity. Seven days after tumor inoculation, equimolar amounts of mAb (MW 150 kDa, 1 mg / kg) and bsAb (MW 195 kDa, 1.3 mg / kg) were injected intraperitoneally twice a week. Tumor size (mm 3 ), calculated as (length × width × width) / 2. In the NCI-H292 tumor model, the tumor growth inhibition index (TGI) of anti-PD-L1#6-CD137#54bsAb (TGI: 67.5%) was greater than that of MPDL-3280a (TGI: 44.3%) and the combination of PD-L1#6+CD137#54 (TGI: -18.77%) (Figure 25, Figure A). Similarly, in the NCI-H1975 tumor model, the TGI of anti-PD-L1#6-CD137#54bsAb (TGI: 80%) was greater than that of the combination of PD-L1#6+CD137#54 (TGI: 67.2%) (Figure 25, Figure B). In addition to lung cancer, in the BxPC-3 pancreatic cancer model, anti-PD-L1#6-CD137#54bsAb also showed more potent anti-tumor activity (TGI 1.3mg / kg was 43%) compared to the combination therapy (TGI each was 10mg / ml: -9.2%) (Figure 25, Figure C). Therefore, in two different types of in vivo tumor models, anti-PD-L1#6-CD137#54bsAb showed anti-tumor activity.

[0179] In summary, the above results indicate that in a mouse xenograft tumor model, anti-PD-L1#6-CD137#54bsAb treatment has a stronger inhibitory effect on tumor growth compared with the combined treatment of anti-PD-L1 and anti-CD137 antibodies.

[0180] Example 20

[0181] This example illustrates cytokine release in the presence of a bispecific antibody in vitro.

[0182] Human PBMCs from three donors were incubated with isotype, anti-CD3 antibody (OKT3, as a positive control) and three bispecific antibodies (anti-PD-L1#6, anti-Her2#3-7 and anti-glycan coupled to CD137#54scFv) at 0.67, 6.67 and 66.67 nM for 24 h. Cytokine release into the culture medium was measured using multiplex ProcartaPlex Immunoassay (Thermo Fisher Scientific). OKT3 induced deep cytokine release, while the three bispecific antibodies did not induce cytokine release ( Fig.26 ).

[0183] These results indicate that, in contrast to OKT3, PD-L1#6-CD137#54, Her2#3-7-CD137#54, and syndecan-CD137#54 bsAbs do not induce significant cytokine release when cultured with human PBMCs.

[0184] Example 21

[0185] This example illustrates the pharmacokinetic parameters of the anti-PD-L1#6-CD137#54 bispecific antibody in rhesus monkeys.

[0186] Two groups of rhesus monkeys (one male and one female in each group) were administered with bispecific anti-PD-L1#6-CD137#54 antibody (5 and 25 mg / kg body weight) by intravenous bolus. Peripheral blood was collected at several time points, 0.5, 6, 24, 48, 72, and 144 hours after injection. Plasma concentrations of antibodies were determined by ELISA. CD137-Fc fusion protein (AP Biosciences, 1 μg / mL) was coated on MaxiSorp plates (Invitrogen), and then serially diluted plasma samples and anti-PD-L1#6-CD137#54 bsAb were used as standard curves. Bound antibodies were detected by biotinylated PD-L1-Fc fusion protein (AP Biosciences) and HRP-conjugated streptavidin using TMB substrate. Antibody plasma concentrations were calculated by interpolation. PK parameters were calculated using PKSolver software (Zhang, Huo, Zhou, & Xie, 2010). In the 25 mg / kg and 5 mg / kg injection groups, the t 1 / 2 About 87 hours and 49 hours respectively ( Fig.27A -B), no increase in ALT / AST levels was observed during the experiment (not shown).

[0187] sequence

[0188] SEQ ID NO 1: Anti-CD137 Clone 15 Heavy Chain

[0189] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASDLYQLLFPYYYGMDVWGQGTTVTVSS

[0190] SEQ ID NO 2: Anti-CD137 Clone 15 Light Chain

[0191] QLVLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQRPGKGPRFVMRVGTGGIVGSKGDGIPDRFSVLGSGLNRYLTIKNIQEEDESDYHCGADHGSGSNLFWVFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0192] SEQ ID NO 3: CDR-H1 of anti-CD137 clone 15

[0193] GGTFSSY

[0194] SEQ ID NO 4: CDR-H2 of anti-CD137 clone 15

[0195] IPILGI

[0196] SEQ ID NO 5: CDR-H3 of anti-CD137 clone 15

[0197] DLYQLLFPYYYGMDV

[0198] SEQ ID NO 6: CDR-L1 of anti-CD137 clone 15

[0199] TLSSGYSNYKVD

[0200] SEQ ID NO 7: CDR-L2 of anti-CD137 clone 15

[0201] VGTGGIVGSKGD

[0202] SEQ ID NO 8: CDR-L3 of anti-CD137 clone 15

[0203] GADHGSGSNLFWV

[0204] SEQ ID NO 9: Anti-CD137 Clone 31 Heavy Chain

[0205] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRGAFDPWGQGTTVTVSS

[0206] SEQ ID NO 10: Anti-CD137 Clone 31 Light Chain

[0207] QSALTQPASVSGSPGQSITISCTGTSSDVGAYNFVSWYQQRPGKAPELMIYDVSDRPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCSSYTSSITRYVFGTGTKV TVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0208] SEQ ID NO 11: CDR-H1 of anti-CD137 clone 31

[0209] GYTFTGY

[0210] SEQ ID NO 12: CDR-H2 of anti-CD137 clone 31

[0211] NPNSGG

[0212] SEQ ID NO 13: CDR-H3 of anti-CD137 clone 31

[0213] DLRGAFDP

[0214] SEQ ID NO 14: CDR-L1 of anti-CD137 clone 31

[0215] TGTSSDVGAYNFVS

[0216] SEQ ID NO 15: CDR-L2 of anti-CD137 clone 31

[0217] DVSDRPS

[0218] SEQ ID NO 16: CDR-L3 of anti-CD137 clone 31

[0219] SSYTSSITRYV

[0220] SEQ ID NO 17: Anti-CD137 Clone 54 Heavy Chain

[0221] QVQLVQSGAEVKKPGSTVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPYYDSSGYYPLGAFDIWGQGTMVTVSS

[0222] SEQ ID NO 18: Anti-CD137 Clone 54 Light Chain

[0223] SYELTQPPSVSVSPGQTASITCSGDKLGEKYASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTV FGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0224] SEQ ID NO 19: CDR-H1 of anti-CD137 clone 54

[0225] GGTFSSY

[0226] SEQ ID NO 20: CDR-H2 of anti-CD137 clone 54

[0227] IPILGI

[0228] SEQ ID NO 21: CDR-H3 of anti-CD137 clone 54

[0229] PPYYDSSGYYPLGAFDI

[0230] SEQ ID NO 22: CDR-L1 of anti-CD137 clone 54

[0231] SGDKLGEKYAS

[0232] SEQ ID NO 23: CDR-L2 of anti-CD137 clone 54

[0233] QDSKRPS

[0234] SEQ ID NO 24: CDR-L3 of anti-CD137 clone 54

[0235] QAWDGSSTYV

[0236] SEQ ID NO 25: Constant domain in the heavy chain (IgG4)

[0237] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0238] SEQ ID NO 26: Constant domain in the heavy chain (engineered IgG1)

[0239] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0240] SEQ ID NO 27: SP1

[0241] METDTLLLWVLLLWVPGSTG

[0242] SEQ ID NO 28: GS linker

[0243] GGGGS

[0244] SEQ ID NO 29: (G4S)2 linker

[0245] GGGGSGGGGS

[0246] SEQ ID NO 30: Anti-PD-L1 #6 light chain

[0247] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDLSLNAWVVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0248] SEQ ID NO 31: Anti-PD-L1#6-CD137#31bsAb heavy chain

[0249] QVQLVQSGAEVKKPGSSVKVSCKASGGTFRRYSISWVRQAPGQGLEWMGGIIPVFGAAKYAQKFQGRVTITADEFTSTAYMELSSLTSEDTAVYYCALSGDSDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGQSALTQPASVSGSPGQSITISCTGTSSDVGAYNFVSWYQQRPGKAPELMIYDVSDRPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCSSYTSSITRYVFGTGTKVTVLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRGAFDPWGQGTTVTVSSA

[0250] SEQ ID NO 32: Anti-PD-L1#6-CD137#54 bsAb heavy chain

[0251] QVQLVQSGAEVKKPGSSVKVSCKASGGTFRRYSISWVRQAPGQGLEWMGGIIPVFGAAKYAQKFQGRVTITADEFTSTAYMELSSLTSEDTAVYYCALSGDSDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKYASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLG

[0252] SEQ ID NO 33:CD137#31-scFv

[0253] QSALTQPASVSGSPGQSITISCTGTSSDVGAYNFVSWYQQRPGKAPELMIYDVSDRPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCSSYTSSITRYVFGTGTKVTVLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRGAFDPWGQGTTVTVSSA

[0254] SEQ ID NO 34: CD137#54-scFv

[0255] QVQLVQSGAEVKKPGSTVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKYASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLG

[0256] SEQ ID NO 35: Trastuzumab light chain

[0257] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0258] SEQ ID NO 36: Trastuzumab CD137#54 bsAb heavy chain

[0259] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKYASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLG

[0260] SEQ ID NO 37: Anti-Her2 #3-7 light chain

[0261] QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCVLYMGSGIWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0262] SEQ ID NO 38: Anti-Her2#3-7-CD137#54 bsAb heavy chain

[0263] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQDNWNHGPYDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKYASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLG

[0264] SEQ ID NO 39: Anti-glycan light chain

[0265] EIVLTQSPSTLSLSPGERATLSCQASEDVSYMHWYQQKPGQAPQPWIYGTSNKASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQWSRRPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0266] SEQ ID NO 40: Anti-glycan CD137 #54bsAb heavy chain

[0267] QITLQESGPTLVKPTQTLTLTCTFSGFSLYRFDMGVGWIRQPPGQGLEWLAHIWWDDDKYYNPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARVRGLHDYYYYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKYASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLG

[0268] SEQ ID NO 41: CDR-H1 of anti-Her2 #3-7

[0269] GYSFTSY

[0270] SEQ ID NO 42: CDR-H2 of anti-Her2 #3-7

[0271] YPGDSD

[0272] SEQ ID NO 43: CDR-H3 of anti-Her2#3-7

[0273] QDNWNHGPYDAFDI

[0274] SEQ ID NO 44: CDR-L1 of anti-Her2#3-7

[0275] GLSSGSVSTSYYPS

[0276] SEQ ID NO 45: CDR-L2 of anti-Her2#3-7

[0277] STNTRSS

[0278] SEQ ID NO 46: CDR-L3 of anti-Her2#3-7

[0279] VLYMGSGIWV

[0280] Table 1. Sequences of defined CDR regions of monoclonal antibodies and bispecific antibody single chain variable fragments (scFv).

[0281]

[0282] References

[0283] Kwon, BS and Weissman, SM (1989). cDNA sequences of two inducible T cell genes. Proceedings of the National Academy of Sciences, 86(6), 1963-1967.

[0284] Zhang, Y., Huo, M., Zhou, J., and Xie, S. (2010). PKSolver: an add-on for Microsoft Excel for pharmacokinetic and pharmacodynamic data analysis. Computer Methods and Programs in Biomedicine, 99(3), 306-314. doi:10.1016 / j.cmpb.2010.01.007

[0285] Chin, SM, et al. (2018). Structure of the 4-1BB / 4-1BBL complex and the unique binding and functional properties of utolumab and usrulumab. Nature Communications 9:4679. DOI: 10.1038 / s41467-018-07136-7

[0286] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein that will be apparent to those skilled in the art upon reading this disclosure and the like.

[0287] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0288] Unless otherwise specified, all scientific and technological terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. In the practice or inspection of the present invention, although any method and material similar or equivalent to the method and material described in the present invention can be adopted, it is understood that modifications and variations are included in the spirit and scope of the present disclosure.

[0289] Range: In the present disclosure, various aspects of the invention may be presented in the form of ranges. It should be understood that the description in range form is only for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the range description should be considered to have specifically disclosed all possible sub-ranges and each numerical value within this range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within this range, such as 1, 2, 2.1, 2.2, 2.7, 3, 4, 5, 5.5, 5.75, 5.8, 5.85, 5.9, 5.95, 5.99 and 6. This requirement applies regardless of the breadth of the range.

[0290] Although the present invention has been described with reference to the above examples, it should be understood that modifications and variations are encompassed within the spirit and scope of the present invention. Accordingly, the present invention is limited only by the following claims. Sequence Listing <110> Yuan Xiang Biotech Co., Ltd. He Zhenghong You Zhongzhe Xu Jingxuan Huang Bolin <120> T cell activating antibodies <130> AP1100-2WO <150> US 62 / 866,699 <151> 2019-06-26 <150> US 62 / 953,302 <151> 2019-12-24 <160> 46 <170> PatentIn Version 3.5 <210> 1 <211> 124 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> Anti-CD137 Clone 15 Heavy Chain <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asp Leu Tyr Gln Leu Leu Phe Pro Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 Clone 15 Light Chain <400> 2 Gln Leu Val Leu Thr Gln Pro Pro Ser Ala Ser Ala Ser Leu Gly Ala 1 5 10 15 Ser Val Thr Leu Thr Cys Thr Leu Ser Ser Gly Tyr Ser Asn Tyr Lys 20 25 30 Val Asp Trp Tyr Gln Gln Arg Pro Gly Lys Gly Pro Arg Phe Val Met 35 40 45 Arg Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Asp Gly Ile Pro 50 55 60 Asp Arg Phe Ser Val Leu Gly Ser Gly Leu Asn Arg Tyr Leu Thr Ile 65 70 75 80 Lys Asn Ile Gln Glu Glu Asp Glu Ser Asp Tyr His Cys Gly Ala Asp 85 90 95 His Gly Ser Gly Ser Asn Leu Phe Trp Val Phe Gly Gly Gly Thr Lys 100 105 110 Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe 115 120 125 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 130 135 140 Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala 145 150 155 160 Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys 165 170 175 Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 180 185 190 Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu 195 200 205 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 220 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H1 of anti-CD137 clone 15 <400> 3 Gly Gly Thr Phe Ser Ser Tyr 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H2 of anti-CD137 clone 15 <400> 4 Ile Pro Ile Leu Gly Ile 1 5 <210> 5 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H3 of anti-CD137 clone 15 <400> 5 Asp Leu Tyr Gln Leu Leu Phe Pro Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 6 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L1 of anti-CD137 clone 15 <400> 6 Thr Leu Ser Ser Gly Tyr Ser Asn Tyr Lys Val Asp 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L2 of anti-CD137 clone 15 <400> 7 Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Asp 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L3 of anti-CD137 clone 15 <400> 8 Gly Ala Asp His Gly Ser Gly Ser Asn Leu Phe Trp Val 1 5 10 <210> 9 <211> 117 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> Anti-CD137 Clone 31 Heavy Chain <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Arg Gly Ala Phe Asp Pro Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti - CD137 Clone 31 Light Chain <400> 10 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asn Phe Val Ser Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Glu Leu 35 40 45 Met Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ile Thr Arg Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H1 of anti-CD137 clone 31 <400> 11 Gly Tyr Thr Phe Thr Gly Tyr 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H2 of anti-CD137 clone 31 <400> 12 Asn Pro Asn Ser Gly Gly 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H3 of anti-CD137 clone 31 <400> 13 Asp Leu Arg Gly Ala Phe Asp Pro 1 5 <210> 14 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L1 of anti-CD137 clone 31 <400> 14 Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr Asn Phe Val Ser 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L2 of anti-CD137 clone 31 <400> 15 Asp Val Ser Asp Arg Pro Ser 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L3 of anti-CD137 clone 31 <400> 16 Ser Ser Tyr Thr Ser Ser Ile Thr Arg Tyr Val 1 5 10 <210> 17 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 Clone 54 Heavy Chain <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Thr Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 18 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 Clone 54 Light Chain <400> 18 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr Tyr 85 90 95 Val Phe Gly Thr Gly Thr Lys Val Thr Val Phe Gly Gln Pro Lys Ala 100 105 110 Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 130 135 140 Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val 145 150 155 160 Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser 210 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H1 of anti-CD137 clone 54 <400> 19 Gly Gly Thr Phe Ser Ser Tyr 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H2 of anti-CD137 clone 54 <400> 20 Ile Pro Ile Leu Gly Ile 1 5 <210> twenty one <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H3 of anti-CD137 clone 54 <400> twenty one Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp 1 5 10 15 Ile <210> twenty two <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L1 of anti-CD137 clone 54 <400> twenty two Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala Ser 1 5 10 <210> twenty three <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L2 of anti-CD137 clone 54 <400> twenty three Gln Asp Ser Lys Arg Pro Ser 1 5 <210> twenty four <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L3 of anti-CD137 clone 54 <400> twenty four Gln Ala Trp Asp Gly Ser Ser Thr Tyr Val 1 5 10 <210> 25 <211> 327 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> Heavy chain constant region (IgG4) <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 26 <211> 329 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> Heavy chain constant region (engineered IgG1) <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 27 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> SP1 <400> 27 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 28 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> GS linker <400> 28 Gly Gly Gly Gly Ser 1 5 <210> 29 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> (G4S)2 linker <400> 29 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 30 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Synthesis <220> <221> misc_feature <223> Anti-PD-L1 #6 light chain <400> 30 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Leu Ser Leu 85 90 95 Asn Ala Trp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 31 <211> 700 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-PD-L1 #6-CD137 #31 bsAb Heavy Chain <400> 31 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Gln Ser Ala Leu Thr Gln Pro Ala 450 455 460 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 465 470 475 480 Thr Ser Ser Asp Val Gly Ala Tyr Asn Phe Val Ser Trp Tyr Gln Gln 485 490 495 Arg Pro Gly Lys Ala Pro Glu Leu Met Ile Tyr Asp Val Ser Asp Arg 500 505 510 Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 515 520 525 Ala Ser Leu Thr Ile Ser Gly Leu Gln Thr Glu Asp Glu Ala Asp Tyr 530 535 540 Tyr Cys Ser Ser Tyr Thr Ser Ser Ile Thr Arg Tyr Val Phe Gly Thr 545 550 555 560 Gly Thr Lys Val Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly 565 570 575 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 580 585 590 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 595 600 605 Tyr Thr Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly 610 615 620 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 625 630 635 640 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 645 650 655 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 660 665 670 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Leu Arg Gly Ala Phe Asp Pro 675 680 685 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 690 695 700 <210> 32 <211> 712 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-PD-L1#6-CD137 #54 bsAb Heavy Chain <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser 450 455 460 Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val Lys Val Ser Cys Lys 465 470 475 480 Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln 485 490 495 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Ile Leu 500 505 510 Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr 515 520 525 Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg 530 535 540 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Pro Pro Tyr Tyr Asp 545 550 555 560 Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp Ile Trp Gly Gln Gly 565 570 575 Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu 595 600 605 Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Ser Ile 610 615 620 Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala Ser Trp Tyr Gln 625 630 635 640 Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile Tyr Gln Asp Ser Lys 645 650 655 Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn 660 665 670 Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala Gly Asp Glu Ala Asp 675 680 685 Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr Tyr Val Phe Gly Thr 690 695 700 Gly Thr Lys Val Thr Val Leu Gly 705 710 <210> 33 <211> 244 <212> PRT <213> Artificial sequence <220> <223> Synthetic <220> <221> misc_feature <223> CD137#31-scFv <400> 33 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asn Phe Val Ser Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Glu Leu 35 40 45 Met Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ile Thr Arg Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 130 135 140 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 165 170 175 Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly 180 185 190 Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu 195 200 205 Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Asp Leu Arg Gly Ala Phe Asp Pro Trp Gly Gln Gly Thr Thr Val Thr 225 230 235 240 Val Ser Ser Ala <210> 34 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CD137#54-scFv <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Thr Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser 145 150 155 160 Pro Gly Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu 165 170 175 Lys Tyr Ala Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu 180 185 190 Val Ile Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe 195 200 205 Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu 210 215 220 Gln Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser 225 230 235 240 Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 245 250 255 <210> 35 <211> 214 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> Trastuzumab light chain <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 36 <211> 714 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Trastuzumab CD137 #54 bsAb Heavy Chain <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val 450 455 460 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val Lys Val Ser 465 470 475 480 Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val 485 490 495 Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Ile Pro 500 505 510 Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr 515 520 525 Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser 530 535 540 Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Pro Pro Tyr 545 550 555 560 Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp Ile Trp Gly 565 570 575 Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly Gly Ser Gly 580 585 590 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr 595 600 605 Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala 610 615 620 Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala Ser Trp 625 630 635 640 Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile Tyr Gln Asp 645 650 655 Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser 660 665 670 Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala Gly Asp Glu 675 680 685 Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr Tyr Val Phe 690 695 700 Gly Thr Gly Thr Lys Val Thr Val Leu Gly 705 710 <210> 37 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthesis <220> <221> misc_feature <223> Anti-Her2 #3-7 light chain <400> 37 Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 Tyr Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr 35 40 45 Leu Ile Tyr Ser Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Val Leu Tyr Met Gly Ser 85 90 95 Gly Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 38 <211> 717 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-Her2 #3-7-CD137 #54 bsAb Heavy Chain <400> 38 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Asp Asn Trp Asn His Gly Pro Tyr Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 450 455 460 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val 465 470 475 480 Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile 485 490 495 Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg 500 505 510 Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly 515 520 525 Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 530 535 540 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 545 550 555 560 Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp 565 570 575 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 595 600 605 Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly 610 615 620 Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr 625 630 635 640 Ala Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile 645 650 655 Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly 660 665 670 Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala 675 680 685 Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr 690 695 700 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 705 710 715 <210> 39 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-glycan light chain <400> 39 Glu Ile Val Leu Thr Gln Ser Pro Ser Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Glu Asp Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Gln Pro Trp Ile Tyr 35 40 45 Gly Thr Ser Asn Lys Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg Arg Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 40 <211> 717 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-glycan CD137 #54 bsAb heavy chain <400> 40 Gln Ile Thr Leu Gln Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Tyr Arg Phe 20 25 30 Asp Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Val Arg Gly Leu His Asp Tyr Tyr Tyr Tyr Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 450 455 460 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val 465 470 475 480 Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile 485 490 495 Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg 500 505 510 Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly 515 520 525 Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 530 535 540 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 545 550 555 560 Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp 565 570 575 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 595 600 605 Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly 610 615 620 Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr 625 630 635 640 Ala Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile 645 650 655 Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly 660 665 670 Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala 675 680 685 Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr 690 695 700 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 705 710 715 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H1 against Her2#3-7 <400> 41 Gly Tyr Ser Phe Thr Ser Tyr 1 5 <210> 42 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H2 of anti-Her2#3-7 <400> 42 Tyr Pro Gly Asp Ser Asp 1 5 <210> 43 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-H3 of anti-Her2#3-7 <400> 43 Gln Asp Asn Trp Asn His Gly Pro Tyr Asp Ala Phe Asp Ile 1 5 10 <210> 44 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L1 of anti-Her2#3-7 <400> 44 Gly Leu Ser Ser Gly Ser Val Ser Thr Ser Tyr Tyr Pro Ser 1 5 10 <210> 45 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L2 of anti-Her2#3-7 <400> 45 Ser Thr Asn Thr Arg Ser Ser 1 5 <210> 46 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> misc_feature <223> CDR-L3 of anti-Her2#3-7 <400> 46 Val Leu Tyr Met Gly Ser Gly Ile Trp Val 1 5 10

Claims

1. An antibody or an antigen-binding fragment thereof, comprising: (a)V H Area, the V H The area contains V H CDR-H1, V H CDR-H2 and V H CDR-H3, where: V H CDR-H1 consists of the amino acid sequence of SEQ ID NO: 19, V H CDR-H2 consists of the amino acid sequence of SEQ ID NO: 20, and V H CDR-H3 consists of the amino acid sequence of SEQ ID NO: 21; and (b)V L Area, the V L The area contains V L CDR-L1, V L CDR-L2 and V L CDR-L3, where: V L CDR-L1 consists of the amino acid sequence of SEQ ID NO: 22, V L CDR-L2 consists of the amino acid sequence of SEQ ID NO: 23, and V L CDR-L3 consists of the amino acid sequence of SEQ ID NO:24, Wherein, the antibody or antigen-binding fragment thereof binds to CD137.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: (i) V H The region comprises the amino acid sequence of SEQ ID NO: 17; and (ii) V L The region comprises the amino acid sequence of SEQ ID NO:

18.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody comprises an Fc domain.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein The Fc domain is an IgG domain, an IgE domain, an IgM domain, an IgD domain, an IgA domain or an IgY domain.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain or an IgG4 domain.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The IgG1 domain comprises the amino acid sequence of SEQ ID NO:

26.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein The IgG4 domain comprises the amino acid sequence of SEQ ID NO:

25.

8. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The antigen-binding fragment comprises scFv, F(ab)2 or Fab.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, wherein The pharmaceutically acceptable carrier is coupled to the C-terminus of one or more polypeptides of the antibody or antigen-binding fragment thereof.

11. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein: The first antigen binding region binds to CD137, Wherein the first antigen binding region comprises: (i)V H A region comprising the amino acid sequence of SEQ ID NO: 17; and (ii) V L A region comprising an amino acid sequence of an N-terminal sequence of 100 to 120 amino acids of SEQ ID NO: 18; and Wherein, the second antigen binding region binds to an immune checkpoint molecule, an immunostimulatory molecule or a tumor antigen.

12. The bispecific antibody according to claim 11, wherein The second antigen binding region binds to an antigen selected from PD-L1, PD-1, CTLA-4, LAG3, CD28, CD40, CD137, CD27, ICOS, Her2 or glycan.

13. The bispecific antibody according to claim 12, wherein: The second antigen binding region binds to PD-L1, Her2 or glycan.

14. The bispecific antibody according to claim 12, wherein: The first antigen binding region and the second antigen binding region comprise scFv, F(ab)2, Fab or any combination thereof.

15. The bispecific antibody according to claim 14, wherein The first antigen binding region comprises a scFv, and wherein the second antigen binding region comprises a Fab.

16. The bispecific antibody according to claim 15, wherein The scFv comprises: V H Area, the V H The region comprises the amino acid sequence of SEQ ID NO: 17, and V L Area, the V L The region comprises an amino acid sequence of 100 to 120 amino acids of the N-terminal sequence of SEQ ID NO:

18.

17. The bispecific antibody according to claim 16, further comprising a V of scFv H Area and V L Connectors between regions.

18. The bispecific antibody according to claim 17, wherein The scFv comprises the amino acid sequence of SEQ ID NO:

34. The bispecific antibody according to claim 15 , further comprising an Fc domain.

20. The bispecific antibody according to claim 19, wherein The Fc domain is an IgG domain, an IgE domain, an IgM domain, an IgD domain, an IgA domain or an IgY domain.

21. The bispecific antibody according to claim 20, wherein The Fc domain is an IgG domain.

22. The bispecific antibody according to claim 21, wherein The IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain or an IgG4 domain.

23. The bispecific antibody according to claim 19, wherein The scFv is linked to the C-terminus of the Fc domain.

24. The bispecific antibody of claim 19, further comprising a linker between the Fc domain and the scFv.

25. The bispecific antibody according to claim 19, wherein The Fab is linked to the N-terminus of the Fc domain.

26. The bispecific antibody according to claim 11, wherein The antibody comprises the sequence of SEQ ID NO:

32.

27. The bispecific antibody according to claim 26, further comprising the sequence of SEQ ID NO:

30.

28. The bispecific antibody according to claim 11, wherein The antibody comprises the sequence of SEQ ID NO:

36.

29. The bispecific antibody according to claim 28, further comprising the sequence of SEQ ID NO:

35.

30. The bispecific antibody according to claim 11, wherein The antibody comprises the sequence of SEQ ID NO:

38. The bispecific antibody according to claim 30 , further comprising the sequence of SEQ ID NO:

37.

32. The bispecific antibody according to claim 11, wherein The antibody comprises the sequence of SEQ ID NO:

40.

33. The bispecific antibody according to claim 32, further comprising the sequence of SEQ ID NO:

39.

34. An antibody comprising any one of the amino acid sequences shown in SEQ ID NOs: 32, 34, 36, 38 and 40.

35. An isolated nucleic acid encoding the antibody of claim 34.

36. An antibody-drug conjugate comprising a therapeutic agent and the antibody or antigen-binding fragment thereof of any one of claims 1-8, or the bispecific antibody or antigen-binding fragment thereof of any one of claims 11-33.

37. The antibody-drug conjugate according to claim 36, wherein The therapeutic agent is covalently linked to the antibody or antigen-binding fragment thereof via a linker.

38. A pharmaceutical composition comprising the bispecific antibody of any one of claims 11-33, and at least one pharmaceutically acceptable carrier.

39. Use of a bispecific antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating cancer in a subject, wherein the cancer comprises PD-L1-positive cancer cells, wherein the bispecific antibody comprises a first antigen-binding region and a second antigen-binding region, wherein, The first antigen binding region binds to CD137, Wherein the first antigen binding region comprises: (i)V H A region comprising the amino acid sequence of SEQ ID NO: 17; and (ii) V L A region comprising an amino acid sequence of an N-terminal sequence of 100 to 120 amino acids of SEQ ID NO: 18; and Wherein, the second antigen binding region binds to PD-L1.

40. The use according to claim 39, wherein The cancer is selected from prostate cancer, lung cancer, lymphoma, breast cancer, head and neck cancer, ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer or hematopoietic cancer.

41. The use according to claim 39, wherein The cancer is selected from melanoma, renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), or leukemia.

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