T cell activation antibodies

Abstract

The name of the invention is T cell activation antibodies. The present invention provides antibodies 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 immune stimulatory molecule or a tumor antigen. The invention provides pharmaceutical compositions comprising the antibodies and methods of treating cancer.

Description

[0001] This application is a divisional application. The application date of the original application is June 24, 2020, the application number is 2020800451461 (PCT / US2020 / 039218), and the name of the invention is “T cell activation antibody”.

[0002] Cross-references to Related Patent Applications

[0003] This application claims priority under Section 119(e) of the United States Patent Act to 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.

[0004] Inclusion of sequence listing

[0005] The material in the accompanying sequence listing is incorporated by reference into this application and becomes a part of this application. The accompanying sequence listing text file is named AP1100_2WO_Sequence_Listing.txt, was created on June 12, 2020, and is 66 kb 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] Immunomodulation of the adaptive immune system has become an attractive area of cancer immunotherapy due to its limited side effects and long-term potential to suppress cancer recurrence. Full T cell activation typically involves two signals: an antigen-specific signal from the T cell receptor (TCR) and a signal from a co-stimulatory molecule, such as CD28. Over 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 participates in strong TCR signaling and 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 demonstrated that CD137 signaling can promote TCR signaling, induce cytokine synthesis and T cell proliferation, and inhibit activation-induced apoptosis. CD137 stimulation on T cells induces the 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 with enhanced proliferation and effector function, but CD8 T cells preferentially respond to CD137 signaling by inducing greater cytokine production. In addition to its expression on activated T cells, CD137 is also expressed on multiple hematopoietic cell lineages, including regulatory T cells, B cells, natural killer cells (NK), monocytes, and dendritic cells (DC). In DCs, CD137 stimulation increases the secretion of IL-6 and IL-12, and more importantly, it enhances the ability of DCs to stimulate T cell proliferation in response to alloantigens and epiantigens. In NKs, 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) has shown partial remission and partial stabilization of the disease. However, fatal hepatotoxicity has led to the termination of most trials. In patients with solid tumors and Merkel cell carcinoma, trials of another anti-CD137 antibody, utomilumab (PF-05082566), achieved objective response rates (including complete and partial responses) of 3.8% and 13.3%, respectively, without causing hepatotoxicity. Urelumab and utomilumab exhibit different properties. The agonist activity of urelumab is strong and independent of cross-linking, while the agonist activity of utomilumab is weak and dependent 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 potencies and toxicities 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 while revealing similar hepatotoxicity profiles. Recently, studies have 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 agonist anti-CD137 antibodies have anticancer activity by enhancing T cell cytotoxicity in a CD40-dependent manner. In addition, anti-CD137 antibodies require antigen presentation to restore established low-antigenicity tumors. Furthermore, the combination of anti-PD-1 and anti-CD137 antibodies demonstrated enhanced anti-tumor activity in mouse tumor models by enhancing T cell effector function and tumor infiltration compared to treatment with either antibody alone. Studies have shown that, in addition to anti-cancer therapy, anti-CD137 agonist antibodies can 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 blocks T cell activation. PD-1 is not expressed on resting T cells but is induced upon activation. PD-1 is persistently 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 suppresses the self-protective function of PD-L1, with various cancer cell types expressing PD-L1 to evade immune surveillance. Antibodies targeting PD-1 / PD-L1 block inhibitory signaling and restore T cell anti-cancer activity. The PD-1 / PD-L1 pathway has long been considered 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 cancer types, such as Hodgkin lymphoma, Merkel cell carcinoma, and melanoma. Other cancer types, 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 has shown enhanced antitumor activity by enhancing T cell effector function and tumor infiltration compared to treatment with each antibody alone. In the clinic, the combination of utolutumab (0.45–5.0 mg / kg) and pembrolizumab has demonstrated synergistic antitumor effects in patients with advanced solid tumors 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 currently no description of an effective treatment method that combines the 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, which have strong cross-linking-dependent agonist activity and may circumvent the liver toxicity that occurred 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) that can activate T cells after cross-linking through the other arm of the bispecific antibody that binds to PD-L1. As described herein, by replacing the anti-PD-L1 arm with 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 antibodies of the present invention comprise a heavy chain variable (V H ) region comprising 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, comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 18. On the other hand, H region (comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 1) and V L The antibody or antigen-binding fragment of the present invention comprises a region (comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 2) comprising (a) V H CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3, wherein CDR-H2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4, wherein CDR-H3 comprises an amino acid sequence that 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, and 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 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 CDR-H1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 11, wherein CDR-H2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 12, and wherein CDR-H3 comprises an amino acid sequence that 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, and 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 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 CDR-H1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 19, wherein CDR-H2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 20, wherein CDR-H3 comprises an amino acid sequence that 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, and wherein the amino acid sequence comprised by CDR-L3 is at least 80% identical to SEQ ID NO: 24.

[0018] In one aspect, the antibody or its antigen-binding fragment of the present invention comprises an Fc domain. In another aspect, the Fc domain is an IgG, IgE, IgM, IgD, IgA or IgY domain. In another aspect, the IgG domain is an IgG1, IgG2, IgG3 or IgG4 domain. In another aspect, the IgG1 domain comprises the amino acid sequence of SEQ ID NO: 26. In certain aspects, compared to 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. In another aspect, the IgG4 domain comprises the amino acid sequence of SEQ ID NO: 25. In another aspect, 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 of the present invention. In one aspect, 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. In another aspect, the pharmaceutical composition of the present invention comprises a bispecific antibody.

[0020] In one embodiment, the present disclosure further provides a method for treating cancer, comprising the steps of 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. In one aspect, 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 approximately 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 present 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 serve 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 antibodies of the present invention comprise 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] In one aspect, the scFv is linked to the C-terminus of the Fc domain. In another aspect, the bispecific antibody of the present invention comprises a linker between the Fab domain and the scFv domain. In another aspect, the Fab fragment is linked to the N-terminus of the Fc domain. In another aspect, 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 of the present invention (including any bispecific antibody) 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 disclosure provides an isolated amino acid sequence encoding any one of SEQ ID NOs: 1-26. In another embodiment, the present disclosure provides an isolated amino acid sequence encoding any one of SEQ ID NOs: 30-40. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Direct screening of phage clones targeting CD137 using ELISA is shown.

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

[0035] Figures 3A-3B Shown is the integrity and purity of anti-CD137 antibody leads purified by one-step protein G purification of polyacrylamide gel electrophoresis (PAGE). 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 was shown by 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 Shown are cytokine production by T cells in the presence of agonistic activity from anti-CD137 antibody leads.

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

[0042] Figure 10 A significant effect of anti-CD137 antibody clones on CD137-CD137L interaction was demonstrated.

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

[0044] Figure 12 Different requirements for cross-linking for agonistic activity of anti-CD137 antibody clones compared to utolutumab (CD137 no. 2) and usrulumab (CD137 no. 3) are shown.

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

[0046] Figure 14 The purity and integrity of anti-PD-L1-CD137 bsAbs purified by Protein G using SDS-PAGE are shown. One-step Protein G chromatography yields >90% purity.

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

[0048] Figure 16 showed that anti-PD-L1-CD137 bsAbs recognized CD137 and PD-L1 simultaneously. Biosensor analysis shown.

[0049] Figure 17 We showed that anti-PD-L1#6-CD137#54bsAb induced synergistic T cell activation in mixed lymphocyte reactions 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] Figure 19 We showed that anti-PD-L1#6-CD137#31 or anti-PD-L1#6-CD137#54 bsAbs induced target-dependent T cell activation when T cells were co-cultured with PD-L1-overexpressing HEK-293 cells.

[0052] Figures 20A-20C It showed that after co-culture with PD-L1 positive cancer cells, anti-PD-L1#6-CD137#54bsAb induced T cells to produce IFN-γ ( Figure 20A 、 Figure 20C and Figure 20B Left) and cancer cell toxicity ( Figure 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 IFN-γ production induced by trastuzumab (Tra)CD137#54 or anti-Her2#3-7-CD137#54 bsAbs 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 It was shown that anti-glycan CD137#54bsAb induced IFN-γ production by CD8 T cells after co-culture with glycan-positive cancer cells. Figure 22A Left picture and Figure 22B ) and cancer cell toxicity ( Figure 22A Right panel). (A) MCF-7, breast cancer cells; (B) NCI-N87, gastric cancer cells.

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

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

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

[0058] Figure 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 should be understood that the present invention is not limited to the specific compositions, methods, and experimental conditions described, as these may vary. It should also be understood that the terminology used herein is intended to describe, and not to limit, the specific embodiments, as the scope of the present invention is limited only by the appended claims.

[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" as used in the present invention refers to an immunoglobulin molecule that has the ability to specifically bind to an antigen. Unless the context clearly indicates otherwise, 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. In one aspect, 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 portion of an immunoglobulin molecule or antibody that has the ability to specifically bind to the same antigen as the immunoglobulin molecule or antibody. Exemplary antigen-binding fragments include scFv, Fab, or F(ab)2 fragments. As used herein, the term "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 variable heavy chain (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 therebetween, to the sequence of SEQ ID NO: 1; SEQ ID NO: 9; or SEQ ID NO: 17. The antibody of the present invention further 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 to the sequence of SEQ ID NO: 2; SEQ ID NO: 10; or SEQ ID NO: 18, and any number or range therebetween.

[0063] In general, "sequence identity" or "sequence homology" are used interchangeably to refer to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, sequence identity determination techniques include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence or amino acid sequence of a polypeptide encoded thereby, and comparing such sequences with a second nucleotide or amino acid sequence. As used herein, the terms "percent (%) sequence identity" or "percent (%) identity" (also including "homology") refer to the percentage of amino acid residues or nucleotides in a sequence that are identical to the amino acid residues or nucleotides in a reference sequence, after aligning the sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity, without taking into account any conservative substitutions that are part of the sequence identity. Thus, by determining the "percent identity" (also referred to as "percent homology") of two or more sequences (polynucleotides or amino acids), two or more sequences can be compared. The percent identity to 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 and multiplied by 100. For example, the 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 of the United States of America 87:2264-2268 (1990), as described by Altschul et al. in J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proceedings of the National Academy of Sciences of the United States of America 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 of the two sequences. This program can be used to determine percent identity over the entire length of the compared sequences. The default parameters are optimized for searches using short query sequences, such as those performed using the blastp program. This program also allows for the use of SEG filters to screen query sequence segments as determined by the SEG program of Wootton and Federhen, Computers and Chemistry, 17:149-163 (1993). The desired degree of sequence identity ranges from approximately 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%. Generally, 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-Wunsch algorithm (e.g., see EMBOSS Needle aligner available 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 available 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 chosen algorithm (including the 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 the amino acid sequence comprising the heavy chain variable region of anti-CD137 antibody clone #15. SEQ ID NO: 2 provides the 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 consecutive CDRs of the light chain variable region (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 (numbered starting 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, 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, 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: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 a CDR-H3 comprising an amino acid sequence identical to SEQ ID NO:4. 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 therebetween. In another 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-L1 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, 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: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 a CDR-L3 comprising an amino acid sequence identical to SEQ ID NO:7. 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 the amino acid sequence comprising the heavy chain variable region of anti-CD137 antibody clone #31. SEQ ID NO: 10 provides the 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, 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, 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 a CDR-H3 comprising an amino acid sequence identical to SEQ ID NO:12. 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 therebetween. In another 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-L1 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, 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:15. 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 the amino acid sequence comprising the heavy chain variable region of anti-CD137 antibody clone #54. SEQ ID NO: 18 provides the 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 of the present invention 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 a CDR-H3 comprising an amino acid sequence identical to SEQ ID NO:20. 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 therebetween. In another 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 of the present invention comprises a CDR-L1 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, 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:23. 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 antibodies or antigen-binding fragments thereof of the present invention further comprise an Fc domain. Unless the context clearly indicates otherwise, the term Fc domain as used herein refers to an antibody region comprising at least a hinge region, a CH2 domain, and a CH3 domain. Unless the context clearly indicates 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. Fc domains of any sequence and any species can be used, including humans, apes, monkeys, mice, rabbits, goats, sheep, guinea pigs, horses, and the like. In some aspects, the Fc domain employs an engineered Fc domain, i.e., a non-naturally occurring or recombinant Fc domain generated, for example, using molecular biology techniques. In some aspects, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain. In one aspect, 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 pharmaceutical compositions comprising any of the antibodies or antigen-binding fragments 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 by any suitable means, including, for example, covalent coupling and the use of a linker.

[0073] In some embodiments, the present invention provides isolated amino acid sequences as shown in SEQ ID NOs: 1-26. In some embodiments, the present invention also provides isolated nucleic acid sequences encoding any one of the amino acid sequences of SEQ ID NOs: 1-26.

[0074] In certain embodiments, the present invention provides a subject cancer treatment method. In some aspects, cancer treatment method includes administering to the subject a certain amount of the present invention (can effectively treat cancer) any antibody or its antigen binding fragment that is 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] As used herein, the terms "treat," "treatment," "therapy," "therapeutic," and the like 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 with respect to a disease, disorder, or medical condition, such as therapeutic benefit and / or prophylactic benefit. "Treatment" as used herein includes treating a disease in mammals, particularly humans, and includes: (a) preventing a subject from developing the disease, including a subject susceptible to or at risk of developing the disease but not yet diagnosed with the disease; (b) inhibiting the disease, i.e., stopping 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 be suffering from the underlying disorder, a therapeutic benefit may be achieved by eradicating or improving one or more physiological symptoms associated with the underlying disorder, such that an improvement in the subject's condition is observed. In some aspects, although a diagnosis may not yet be made for such a disease, to obtain a preventative benefit, treatment is provided or a therapeutic composition is administered to a subject who is likely to develop a particular disease or to a subject who reports one or more physiological symptoms of a disease. The methods of the present disclosure can be used in any mammal or other animal. In some aspects, treatment can alleviate or stop symptoms. A preventative effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing down, stopping, or reversing the progression of a disease or condition, or any combination thereof.

[0076] The term "subject" as used in the present invention refers to any individual or patient using the methods disclosed herein. The term "subject" can be used interchangeably with the term "individual" or "patient." Those skilled in the art will appreciate 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 included in the definition of subject.

[0077] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an antibody, antigen-binding fragment thereof, or other composition described herein that is sufficient to achieve the intended use, including but not limited to the treatment of diseases as defined herein. A 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 route of administration, etc.), and can be readily determined by one of ordinary skill in the art. The term also applies to a dose that will induce a specific response in the target cell. The specific dosage will vary depending on the specific antibody, antigen-binding fragment thereof, or other composition selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be administered, and the physical delivery system used to carry the drug.

[0078] In some aspects, as combined therapy, the antibody of the present invention or its antigen-binding fragment is as monotherapy or is combined with other therapeutic agents (such as radiotherapy, cytotoxic chemotherapy) and other immunomodulators (such as 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 imfinzia (IMFINZI), CTLA-4 inhibitors such as ipilimumab (YERVOY), and other checkpoint inhibitors such as anti-B7-H3 antibody (MGA271), anti-KIR antibody (lirilumab), and anti-LAG3 antibody (BMS-986016).

[0079] As described in the examples below, in some embodiments, the present invention further provides 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 can 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 show that T cell effector function is further enhanced. Without being limited by theory, this situation shows that combined therapy or treatment with bispecific antibodies targeting CD137 and PD-L1 can overcome the lower 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) can be used, or a bispecific antibody targeting CD137 and a second antigen (such as PD-L1, CD40 or CTLA-4) can be used for treatment.

[0080] Bispecific molecules, such as bispecific antibodies (bsAbs), provide a means of simultaneously targeting 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 compared to a mixture of two monoclonal antibodies (mAbs), reduce cost of goods, and facilitate the development of new treatment options.

[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 antibodies of the present invention may comprise an isolated 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 bind to immunomodulatory molecules (such as CD40 or CTLA-4).

[0082] In some embodiments, the present invention provides bispecific antibodies 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 the group consisting of 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 in between, with about 100 to 120 amino acids of the N-terminal sequence of a sequence selected from SEQ ID NO: 2; SEQ ID NO: 10; and SEQ ID NO: 18. 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 in the sequence described in the 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, and any number or range therebetween, 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. 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 present invention comprises a V H region (having 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 having 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" as 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 "immune stimulatory molecule" as used in the present invention refers to any molecule that induces, enhances, 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, increasing signal transduction and immune activation. The term "tumor antigen" as 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, 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 antibodies 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 can 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 that binds 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 ( Figure 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 ( Figure 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 comprising 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 an 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 tumor-specific glycans, 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 multiples thereof. In one aspect, the linker is a G4S linker.

[0092] Accordingly, the present invention provides a target-dependent T cell activation platform. Figure 19 As shown in Figures 21 and 22, the agonist activity of anti-CD137 scFv is induced upon binding 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 present invention further comprise a scFv VH Area and V L The linker between the regions. Any linker can be used. For example, the linker can comprise any amino acid sequence. The linker can have any length, for example, 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 linker can also comprise any multiple of the amino acid sequence. Any number of multiples of the amino acid sequence can be included in a linker. Exemplary linker 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 antibodies of the present invention further comprise an Fc domain. In certain aspects, the Fc domain is an IgG domain, an IgE domain, an IgM domain, an IgD domain, an IgA domain, or an IgY domain. Fc domains 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. In one aspect, 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 human. Generally, human Fc domains are not immunogenic in humans and are therefore suitable for human therapy.

[0095] In some aspects, the scFv of the bispecific antibody of the present invention is coupled to the C-terminus of the Fc domain. In one aspect, a linker is included between the Fc domain and the scFv. In another aspect, the linker connects the scFv to the Fc domain. Any linker can be used, for example, 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 linker.

[0097] In some aspects, the bispecific antibodies of the present invention comprise a heavy chain sequence of SEQ ID NO: 31 or SEQ ID NO: 32. In another aspect, the bispecific antibodies of the present invention further comprise a light chain sequence of SEQ ID NO: 30. In one aspect, the bispecific antibodies of the present invention comprise a heavy chain sequence of SEQ ID NO: 36. In another aspect, the bispecific antibodies of the present invention further comprise a light chain sequence of SEQ ID NO: 35. In one aspect, the bispecific antibodies of the present invention comprise a heavy chain sequence of SEQ ID NO: 38. In another aspect, the bispecific antibodies of the present invention further comprise a light chain sequence of SEQ ID NO: 37. In one aspect, the bispecific antibodies of the present invention comprise a heavy chain sequence of SEQ ID NO: 40. In another aspect, the bispecific antibodies of the present invention further comprise 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 include 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 isolated amino acid sequences as shown in SEQ ID NOs: 30-40. In some embodiments, the present invention also provides isolated nucleic acid sequences encoding any one of the amino acid sequences of SEQ ID NOs: 30-40.

[0099] In some embodiments, the present invention provides antibody-drug conjugates. The antibody-drug conjugates of the present invention may comprise 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 conjugates of the present invention comprise a therapeutic agent. Any therapeutic agent, including small molecules, may be included in the antibody-drug conjugates 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, doxepidermis, 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, valbivac, 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 the tumor cell, it is internalized in the tumor cell and releases the small molecule or other therapeutic agent.

[0101] In some aspects, the therapeutic agent contained in the antibody-drug molecule of the present invention is covalently linked to the antibody of the present invention or its antigen-binding fragment or the bispecific antibody of the present invention or its antigen-binding fragment. A connexon can be used to covalently link a therapeutic agent to an antibody or its antigen-binding fragment, or to a bispecific antibody or its antigen-binding fragment. Any suitable connexon can be used to covalently link a therapeutic agent to an antibody of the present invention or its antigen-binding fragment, or to a bispecific antibody of the present invention or its antigen-binding fragment. In some aspects, the connexon contained in the antibody-drug conjugate of the present invention is relatively stable outside the target cell (including in the circulation) and is cracked 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 a reduction in cytotoxicity to non-tumor cells while increasing 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 the pharmaceutical composition. On the one hand, the bispecific antibody included in the pharmaceutical composition of the present invention binds to CD137 or PD-L1 or binds to 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. The pharmaceutical composition of the present invention can be used to treat any cancer. 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 comprises administering to the subject a certain amount of any of the bispecific antibodies of the present invention (effectively treating cancer) or its antigen-binding fragment. 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 using the OmniMab phage library. The phage library was established by APBiosciences Inc. (APBio Inc.) and selected from peripheral blood mononuclear cells from more than 100 healthy donors. Pre-coated CD137-Fc recombinant protein was incubated with supernatant containing rescued phage for 1 hour and washed three times with PBS containing 0.1% Tween 20. Bound phage was detected by HRP-conjugated 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, direct ELISA and FACS were used to screen and isolate 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 cultured on ice for 1 hour with 2.5ug / ml anti-CD137 antibodies (Abs). The cells were washed three times with 1x PBS and then cultured on ice for 1 hour with anti-M13 antibodies (Progen). The cells were washed three times with 1X PBS again and then cultured on ice for another 1 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 2. 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 with 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 reagent).

[0113] Figure 3 shows representative PAGE gel analysis of the first batch (Figure 3, top) and second batch (Figure 3, bottom) of purified anti-CD137 antibody leads. Mammalian cell culture supernatants collected 4 days after transfection were purified using Protein G chromatography (Thermo Fisher Scientific). 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 145 kDa, while under reducing conditions, the molecular weights of the heavy and light chains were ~55 kDa and ~25 kDa, respectively. One-step Protein G chromatography yielded a purity exceeding 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 antibodies to Jurkat cells.

[0117] Purified anti-CD137 antibody leads are also applied to Jurkat cells induced by CD137 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 to culture stimulated cells 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 are several leads with comparable binding activity to the reference antibody, e.g. Figure 4 shown.

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

[0119] Example 4

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

[0121] For direct ligand binding assays of anti-CD137 antibodies binding to CD137, pre-coated membrane wells were prepared using recombinant CD137 / Fc (100 ng / ml). Briefly, purified human CD137-IgG4 Fc (APBio) was dialyzed against phosphate-buffered saline (PBS), adjusted to 1 mg / ml, and then diluted with PBS to a final concentration of 1 μg / ml. Each well of a Nunc-Immuno Maxisorp 96-well plate was pre-coated with 0.1 ml of recombinant CD137 protein, leaving an empty well for a nonspecific binding control, and incubated overnight at 4°C. The CD137 recombinant protein solution was removed, and the 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 dry milk in PBS) was added to all wells and incubated at room temperature for 1 hour. The blocking buffer was removed, and the plates were washed three times with 0.4 ml of wash buffer.

[0122] The pre-coated wells were cultured with serial dilutions of purified anti-CD137 antibodies. Serial dilutions of CD137 antibodies were prepared in PBS, with 0.1 ml serial dilutions added to each well. The culture plates were incubated at room temperature for 1 hour. The antibody solution was removed and the culture plates were washed three times with 0.4 ml wash buffer. RP-coupled goat anti-human IgG, F(ab')2 specific F(ab')2 antibodies (Jackson Immunoresearch#109-036-097) were diluted with PBS at 1:2000 and added in 0.1 ml / well. The culture plates were incubated at room temperature for 1 hour and washed three times with 0.4 ml wash buffer / well. The culture plates were developed with 0.1 ml TMB reagent (Invitrogen) and incubated at room temperature for 1 to 5 minutes. 0.05 ml 1N HCl was added to stop the reaction and absorbance was read at 450 nm on a Bio-Tek Spectra. OD450 readings were plotted against anti-CD137 concentrations, and the 50% effective concentration (EC) of anti-CD137 antibody binding to CD137 / Fc was calculated. 50 EC values were 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 antibodies were 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 demonstrates 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 SEC separation was performed using an isocratic 25 mM sodium phosphate, 200 mM NaCl, pH 6.8 mobile phase buffer. The flow rate was 0.4 mL / min and the injection volume was 10 μL. Peaks were detected by absorbance at 280 nm. All samples were filtered using a 0.22 μm filter (Millipore, catalog #SLGP003RB) prior to injection onto the SEC column 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 antibodies 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 to determine their ability to enhance human CD3+ cell activation, such as enhancing cytokine production, proliferation, and inducing human CD3+ T cell proliferation. Anti-CD3 antibody (1 μg / ml, OKT3, BioLegend catalog number 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 (1×10 cells per well) using RPMI1640 medium (containing 10% fetal bovine serum, 2.5 mM L-glutamine, and 1× 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. Compared to a reference antibody, anti-CD137 clones #31 and #54 exhibited comparable or greater agonistic activity in enhancing T cell activation (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 bispecific antibody construction, 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 (Cat. 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). Dendritic cell (DC) differentiation was verified by flow cytometry based on the expression of DC-SIGN, CD14, CD80, or CD83. The differentiated DCs were used as antigen-presenting cells (APCs) in mixed lymphocyte reactions (MLRs). 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 were significantly increased in the presence of anti-PD-L1 antibodies in the MLR compared to isotype control antibodies. Interestingly, for example, anti-CD137 antibodies (e.g., clone #31) further promoted anti-PD-L1 antibody-mediated IFN-γ production in MLRs with two different donor pairs, e.g., Figure 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 with isotype control and anti-CD137 antibodies (50 μg / ml) for 30 minutes, 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 histograms ( Figure 10 ).like Figure 10 As shown, reference antibody 1 (ref1) and clone #54 effectively blocked the CD137-CD137L interaction, whereas reference antibody 2 (ref2), clone #15 and clone #31 were less efficient or less effective in blocking the 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 kilogram of 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. 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, 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( Figure 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 the reporter cells and cultured for 5 hours. ONE-Glo TM Luciferase activity was detected using the luciferase assay system (Promega, catalog number E6120). Figure 12 ) showed cross-linking-dependent agonist activity, whereas urerulumab (CD137 ref2, Figure 12 ) exhibited cross-linking-independent agonist activity, leading to the severe hepatotoxicity observed in clinical trials. Compared to utolumab, CD137#54 exhibited greater agonist activity upon cross-linking, whereas in the absence of cross-linking, the agonist activity was modest, similar to that of utolumab ( Figure 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 demonstrate distinct cross-linking dependencies for 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] Anti-PD-L1 antibody clone 6 was used in the form of IgG without ADCC, and anti-CD137 antibody was used in the form of scFv and fused to the C-terminus of the Fc region of anti-PD-L1 clone 6 antibody. The construction of a bispecific antibody comprising the Fc region of anti-PD-L1 antibody fused to CD137 scFv is shown in Table 1 below (sequence), and the schematic diagram is shown in Figure 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 sequences of the anti-PD-L1-CD137 scFv heavy chain are 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, antibodies 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% ( Figure 14 and Figure 15 In a one-step purification process, a purity greater than 90% was achieved, which is consistent with a purified fusion protein with the correct molecular weight (Mw = 220 kD). Figure 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 Scientific). 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. Figure 15 The purity and integrity of the protein A-purified anti-PD-L1#6-CD137#54 bsAb using μCE-SDS are shown.

[0149] Example 12

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

[0151] pass The binding activity of the anti-PD-L1-CD137 bispecific antibody was determined by biosensor analysis (Menlo Park, CA). His-tagged CD137 (ACROBiosystems) was loaded onto HIS1K (anti-Penta-HIS) biosensors (Cat. 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 a mouse Fc domain) for 5 minutes. The data were then compiled using Octet data acquisition and analysis software as described by the manufacturer. Figure 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, demonstrating that bispecific antibodies can simultaneously target PD-L1 and CD137.

[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 antibodies and anti-PD-L1-CD137scFv bispecific antibodies (bsAbs) significantly enhance T cell activation in an allogeneic mixed lymphocyte reaction.

[0155] Using RosetteSep TM Human Monocyte Enrichment Fluid (Cat. 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 leads (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 ( Figure 17 , showing the results for two donor pairs).

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

[0157] Example 14

[0158] This example demonstrates the promotion of antigen-specific T cell activation via 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 (1 ug / ml, JPT) in the presence of antibodies (0.4, 2, and 10 μg / ml) 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 (10 ng / ml, PeproTech), TNF-α (10 ng / ml, PeproTech), IFN-γ (5000 IU / ml, PeproTech), PGE2 (250 ng / ml, Sigma), polyI:C (10 μg / ml, Sigma), and R848 (5 μg / 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 response 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 18A -B).

[0160] In summary, Figure 17The results shown in (Example 13) and Figure 18 (this Example) show that anti-PD-L1#6-CD137#54bsAb significantly enhanced T cell activation, which was more robust than the T cell activation seen when treated with anti-PD-L1 and anti-CD137 monoclonal antibodies alone or in combination. In addition, based on the 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 had 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 treatment of the indicated antibodies ( Figure 19 ).like Figure 19 As shown, compared with monotherapy or combination therapy with 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 demonstrate that target-dependent T-cell activation is induced exclusively by the 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 demonstrates 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 determined by ELISA and assayed by CytoTox. Tumor cytotoxicity was detected using a 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). In co-culture with PC-3 cells, CD8 T cells were observed to have higher tumor cytotoxicity ( Figure 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 #54scFv also produced stronger IFN-γ production compared with either single-agent or combination therapy ( Figure 21A -B and Figure 22A -B) and CD8 T cell tumor cytotoxicity ( Figure 22A ).

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

[0170] Example 17

[0171] This example demonstrates 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 utolutumab, internalization assays were performed using CD137-expressing HEK293 cells ( Figure 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 under 5% CO2. The indicated antibodies were labeled with pHAb amine reactive dye (Promega Corp.) according to the manufacturer's protocol and then prepared by 3-fold serial dilution in culture medium starting from 100 nM. The culture medium containing the labeled antibody was then replaced with the culture 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. Figure 23 As shown, enhanced induction of CD137 internalization was observed with 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 binds to T cells solely through CD137 engagement, the increased level of CD137 internalization could result in reduced CD137 activation. Accordingly, upon in vivo administration of the bispecific antibody, the bispecific antibody exhibited lower toxicity compared to the reference antibody, particularly urerulumab.

[0173] Example 18

[0174] This example demonstrates 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 established 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#54 bsAb to the culture. Figure 24A -B). In the presence of Treg cells, anti-PD-L1#6-CD137#54bsAb rescued both T cell proliferation (Figure 24, Panel A) and cytokine production (Figure 24, Panel B). These results indicate that anti-PD-L1#6-CD137#54bsAb can rescue Treg-mediated suppression of T cell activation.

[0176] Example 19

[0177] This example demonstrates 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 pre-mixed with human PBMCs and xenografted subcutaneously into SCID-beige mice to evaluate the in vivo anti-cancer 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) was measured twice a week. 3 ), calculated as (length × width × width) / 2. In the NCI-H292 tumor model, the tumor growth inhibition index (TGI) of the anti-PD-L1#6-CD137#54 bsAb (TGI: 67.5%) was greater than that of MPDL-3280a (TGI: 44.3%) and the PD-L1#6 + CD137#54 combination (TGI: -18.77%) ( Figure 25 , Panel A). Similarly, in the NCI-H1975 tumor model, the TGI of the anti-PD-L1#6-CD137#54 bsAb (TGI: 80%) was greater than that of the PD-L1#6 + CD137#54 combination (TGI: 67.2%) ( Figure 25 , Panel 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 1.3 mg / kg was 43%) compared to the combination therapy (TGI of each 10 mg / 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 than combined treatment with anti-PD-L1 and anti-CD137 antibodies.

[0180] Example 20

[0181] This example demonstrates 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 hours. Cytokine release into the culture medium was measured using a multiplex ProcartaPlex Immunoassay (Thermo Fisher Scientific). OKT3 induced deep cytokine release, while the three bispecific antibodies did not induce cytokine release ( Figure 26 ).

[0183] These results indicate that, compared with OKT3, PD-L1#6-CD137#54, Her2#3-7-CD137#54, and glycan-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 macaques (one male and one female per group) were administered a bispecific anti-PD-L1#6-CD137#54 antibody (5 and 25 mg / kg body weight) via intravenous bolus. Peripheral blood was collected at 0.5, 6, 24, 48, 72, and 144 hours after injection. Plasma concentrations of the antibody were determined by ELISA. CD137-Fc fusion protein (AP Biosciences, 1 μg / mL) was coated on MaxiSorp plates (Invitrogen), and serially diluted plasma samples and the anti-PD-L1#6-CD137#54 bsAb were used as a standard curve. Bound antibody was 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 ( Figure 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] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT

[0238] YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD

[0239] GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK

[0240] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS

[0241] LSLSLGK

[0242] SEQ ID NO 26: Constant domain in the heavy chain (engineered IgG1)

[0243] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

[0244] YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW

[0245] YVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE

[0246] MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

[0247] QKSLSLSPG

[0248] SEQ ID NO 27: SP1

[0249] METDTLLLWVLLLWVPGSTG

[0250] SEQ ID NO 28: GS linker

[0251] GGGGS

[0252] SEQ ID NO 29: (G4S)2 linker

[0253] GGGGSGGGGS

[0254] SEQ ID NO 30: Anti-PD-L1 #6 light chain

[0255] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQ

[0256] SEDEADYYCATWDLSLNAWVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV

[0257] KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0258] SEQ ID NO 31: Anti-PD-L1 #6-CD137 #31 bsAb heavy chain

[0259] QVQLVQSGAEVKKPGSSVKVSCKASGGTFRRYSISWVRQAPGQGLEWMGGIIPVFGAAKYAQKFQGRVTITADEFTSTAY

[0260] MELSSLTSEDTAVYYCALSGDSDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

[0261] SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

[0262] VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY

[0263] KCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

[0264] SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGQSALTQPASVSGSPGQSITISCTG

[0265] TSSDVGAYNFVSWYQQRPGKAPELMIYDVSDRPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCSSYTSSITRYVFGT

[0266] GTKVTVLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGT

[0267] NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRGAFDPWGQGTTVTVSSA

[0268] SEQ ID NO 32: Anti-PD-L1#6-CD-137#54 bsAb heavy chain

[0269] QVQLVQSGAEVKKPGSSVKVSCKASGGTFRRYSISWVRQAPGQGLEWMGGIIPVFGAAKYAQKFQGRVTITADEFTSTAY

[0270] MELSSLTSEDTAVYYCALSGDSDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN

[0271] SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

[0272] VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY

[0273] KCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

[0274] SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTVKVSCK

[0275] ASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPYYD

[0276] SSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKYASWYQ

[0277] QKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLGSEQ ID NO 33:CD137#31-scFv

[0278] QSALTQPASVSGSPGQSITISCTGTSSDVGAYNFVSWYQQRPGKAPELMIYDVSDRPSGVSNRFSGSKSGNTASLTISGL

[0279] QTEDEADYYCSSYTSSITRYVFGTGTKVTVLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYM

[0280] HWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRGAFDPWGQGTTVT

[0281] VSSA

[0282] SEQ ID NO 34: CD137#54 - scFv

[0283] QVQLVQSGAEVKKPGSTVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAY

[0284] MELSSLRSEDTAVYYCASPPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVS

[0285] PGQTASITCSGDKLGEKYASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGS

[0286] STYVFGTGTKVTVLG

[0287] SEQ ID NO 35: Trastuzumab light chain

[0288] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP

[0289] EDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

[0290] ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0291] SEQ ID NO 36: Trastuzumab CD137#54 bsAb heavy chain

[0292] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY

[0293] LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

[0294] WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0295] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGK

[0296] EYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0297] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTVKVS

[0298] CKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPPY

[0299] YDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKYASW

[0300] YQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLGSEQ ID NO 37: Anti-Her2 #3-7 light chain

[0301] QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGA

[0302] QADDESDYYCVLYMGSGIWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK

[0303] AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSEQ ID NO 38: Anti-Her2#3-7-CD137#54bsAb heavy chain

[0304] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAY

[0305] LQWSSLKASDTAMYYCARQDNWNHGPYDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

[0306] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

[0307] LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWL

[0308] NGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0309] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTV

[0310] KVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAS

[0311] PPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKY

[0312] ASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLGSEQ ID NO 39: Anti-glycan light chain

[0313] EIVLTQSPSTLSLSPGERATLSCQASEDVSYMHWYQQKPGQAPQPWIYGTSNKASGVPSRFSGSGSGTDFTLTISSLQPE

[0314] DVATYYCQQWSRRPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

[0315] SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0316] SEQ ID NO 40: Anti-glycan CD137 #54bsAb heavy chain

[0317] QITLQESGPTLVKPTQTLTLTCTFSGFSLYRFDMGVGWIRQPPGQGLEWLAHIWWDDDKYYNPALKSRLTISKDTSKNQV

[0318] VLTMTNMDPVDTATYYCARVRGLHDYYYYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

[0319] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

[0320] LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWL

[0321] NGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0322] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGSTV

[0323] KVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAS

[0324] PPYYDSSGYYPLGAFDIWGQGTMVTVSSAGGGGSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDKLGEKY

[0325] ASWYQQKAGQSPILVIYQDSKRPSGIPERFSGSNSGNTATLTISGLQAGDEADYYCQAWDGSSTYVFGTGTKVTVLGSEQ ID NO 41: CDR-H1 of anti-Her2#3-7

[0326] GYSFTSY

[0327] SEQ ID NO 42: CDR-H2 of anti-Her2#3-7

[0328] YPGDSD

[0329] SEQ ID NO 43: CDR-H3 of anti-Her2#3-7

[0330] QDNWNHGPYDAFDI

[0331] SEQ ID NO 44: CDR-L1 of anti-Her2#3-7

[0332] GLSSGSVSTSYYPS

[0333] SEQ ID NO 45: CDR-L2 of anti-Her2#3-7

[0334] STNTRSS

[0335] SEQ ID NO 46: CDR-L3 of anti-Her2#3-7

[0336] VLYMGSGIWV

[0337] Table 1. Sequences of defined CDR regions of monoclonal antibodies and bispecific antibody single-chain variable fragments (scFv).

[0338]

[0339] References

[0340] Kwon, BS and Weissman, SM (1989). cDNA sequences of two inducible T cell genes. Proceedings of the National Academy of Sciences of the United States of America, 86(6), 1963-1967.

[0341] Zhang, Y., Huo, M., Zhou, J., and Xie, S. (2010). PKSolver: An add-in 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

[0342] 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

[0343] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents 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 become apparent to those skilled in the art upon reading this disclosure and the like.

[0344] 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.

[0345] Unless otherwise specified, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. In the practice or testing of the present invention, although any methods and materials similar or equivalent to those described in the present invention can be used, it is understood that modifications and variations are within the spirit and scope of the present disclosure.

[0346] Range: Throughout this disclosure, various aspects of the invention may be presented in the form of ranges. It should be understood that descriptions in range format are for convenience and brevity only and should not be construed as inflexible limitations on the scope of the invention. Accordingly, range descriptions should be considered to have specifically disclosed all possible subranges and individual numerical values within such ranges. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges 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 individual numbers within such ranges, 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.

[0347] Although the present invention has been described with reference to the above examples, it will 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.

Claims

1. An antibody or antigen-binding fragment thereof, comprising: (i) A V H A 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% identity to a sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 10; and SEQ ID NO: 18, wherein The antibody or antigen-binding fragment binds to CD137.

2. The antibody or antigen-binding fragment according to claim 1, wherein V H The amino acid sequence contained in the region has at least 80% identity with SEQ ID NO: 1, wherein V L The region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

2.

3. The antibody or antigen-binding fragment according to claim 2, wherein The antibody or antigen-binding fragment comprises: (a)V H CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3, wherein CDR-H2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4, and wherein CDR-H3 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 5; 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:6, wherein the amino acid sequence comprised by CDR-L2 is at least 80% identical to SEQ ID NO:7, and wherein the amino acid sequence comprised by CDR-L3 is at least 80% identical to SEQ ID NO:

8.

4. The antibody or antigen-binding fragment according to claim 1, wherein The V H The amino acid sequence contained in the region has at least 80% identity with SEQ ID NO: 9, wherein the V L The region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

10.

5. The antibody or antigen-binding fragment according to claim 4, wherein The antibody or antigen-binding fragment comprises: (a)V H CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 11, wherein CDR-H2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 12, and wherein CDR-H3 comprises an amino acid sequence that 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, and wherein the amino acid sequence comprised by CDR-L3 is at least 80% identical to SEQ ID NO:

16.

6. The antibody or antigen-binding fragment according to claim 1, wherein The V H The region comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 17, wherein the V L The region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

18.

7. The antibody or antigen-binding fragment according to claim 6, wherein The antibody or antigen-binding fragment comprises: (a)V H CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 19, wherein CDR-H2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 20, and wherein CDR-H3 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 21; 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: 22, wherein the amino acid sequence comprised by CDR-L2 is at least 80% identical to SEQ ID NO: 23, and wherein the amino acid sequence comprised by CDR-L3 is at least 80% identical to SEQ ID NO:

24.

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

9. The antibody or antigen-binding fragment according to claim 8, wherein The Fc domain is an IgG domain, an IgE domain, an IgM domain, an IgD domain, an IgA domain or an IgY domain.

10. The antibody or antigen-binding fragment according to claim 9, wherein The IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain or an IgG4 domain.

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