Antibodies for T-cell activation

Abstract

This invention provides an anti-CD137 antibody that exhibits potent cross-linking-dependent agonist activity and has the potential to avoid the hepatotoxicity observed in clinical trials. [Solution] The present invention provides an antibody comprising an antigen-binding region that binds to CD137. It also provides a bispecific antibody comprising a first antigen-binding region that binds to CD137 and a second antigen-binding region that binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen. Furthermore, it provides a pharmaceutical composition containing the antibody and a method for treating cancer.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Serial No. 62 / 866,699, filed Jun. 26, 2019, and U.S. Serial No. 62 / 953,302, filed Dec. 24, 2019, each of which is hereby incorporated by reference in its entirety.

[0002] Incorporation of Sequence Listing The contents of the accompanying sequence listing are hereby incorporated by reference into this application. The text file of the accompanying sequence listing is named AP1100_2WO_Sequence_Listing.txt, was created on Jun. 12, 2020, and is 66 kb. This file is accessible using Microsoft Word on a computer using the Windows operating system.

[0003] Field of the Invention The present invention generally relates to antibodies and antigen - binding fragments thereof, and more particularly to antibodies and antigen - binding fragments that enhance T - cell function.

Background Art

[0004] Background Information The immune regulatory mechanisms of the adaptive immune system have become an attractive area in cancer immunotherapy due to their few side effects and long - term protection from cancer recurrence. To fully activate T cells, generally two signals are required: an antigen - specific signal from the T - cell receptor (TCR) and a signal from a co - stimulatory molecule such as CD28, for example. In the past decade, additional co - stimulatory molecules as well as co - inhibitory molecules that can positively or negatively regulate TCR signaling have been discovered on T cells.

[0005] In 1989, CD137 (4-1BB), a co-stimulatory molecule belonging to the TNF receptor superfamily, was cloned from activated T cells (Kwon & Weissman, 1989). The 4-1BB:4-1BBL pathway appears to amplify existing co-stimulatory signals, but in the presence of strong TCR signaling, 4-1BB involvement enables the induction of CD28-independent IL-2 production. CD137 signaling has been demonstrated to enhance TCR signaling, induce cytokine synthesis and T cell proliferation, and inhibit activation-induced apoptosis. CD137 stimulation in T cells induces the NF-κB and PI3K / ERK signaling pathways, which are involved in preventing T cell activation-induced apoptosis and inducing T cell proliferation, respectively. Both CD4 and CD8 T cells respond to CD137 stimulation, resulting in enhanced proliferation and effector function, while CD8 T cells preferentially respond to CD137 signaling by inducing greater cytokine production. CD137 is expressed in multiple hematopoietic cell lines, including regulatory T cells, B cells, natural killer cells (NKs), mononuclear cells, and dendritic cells (DCs), in addition to activated T cells. In DCs, CD137 stimulation increases the secretion of IL-6 and IL-12, and more importantly, enhances the DCs' ability to stimulate T cell proliferation in response to alloantigens and trace antigens. In NKs, CD137 stimulation promotes proliferation and IFN-γ production, but not cytolytic activity. Nevertheless, CD137-stimulated NK cells play a helper role in promoting the proliferation of activated T cells.

[0006] Clinically, the anti-CD137 agonist antibody urelumab (BMS-663513) showed partial remission and some stabilization of the disease. However, most trials resulted in fatal hepatotoxicity. In trials of another anti-CD137 antibody, utomilumab (PF-05082566), objective response rates, including full and partial responses, were achieved at 3.8% in solid tumor patients and 13.3% in Merkel cell carcinoma patients, without causing hepatotoxicity. Urelumab and utomilumab exhibit distinct characteristics. Regarding agonist activity, urelumab is strong and crosslink-independent, while utomilumab is weak and crosslink-dependent. The crystal structures of urelumab and utomilumab with CD137 reveal distinct binding epitopes that influence the CD137-CD137L interaction. The recognition of distinct epitopes and the inhibition of CD137-CD137L binding can result in distinct potencies and toxicity for these two anti-CD137 antibodies. However, ligand binding does not determine CD137-mediated toxicity, as anti-4-1BB monoclonal antibodies (mAbs) 3H3 and 2A exhibit similar hepatotoxicity despite having opposing effects on CD137L binding, respectively. Recently, it has been shown that a manipulated Fc region of a weak agonist antibody preferentially binds to FcγRIIB (with a low A / I FcγR binding ratio), resulting in potent agonist activity comparable to urelumab without inducing hepatotoxicity. Agonist anti-CD137 antibodies have been shown to possess anticancer activity by enhancing T-cell toxicity in a CD40-dependent manner. Furthermore, antigen expression is required for anti-CD137 antibodies to degenerate poorly established antigenic tumors. Furthermore, combining anti-PD-1 and anti-CD137 antibodies demonstrated enhanced antitumor activity in a mouse tumor model, mediated by enhanced T-cell effector function and tumor infiltration, compared to monotherapy with each antibody. In addition to anticancer therapy, anti-CD137 agonist antibodies also showed, depending on the timing of treatment, improvement of experimental autoimmune encephalomyelitis and enhancement of antiviral immunity.

[0007] First, PD-1 was isolated from T cells undergoing apoptosis. Next, its ligand, PD-L1, was identified, and it was demonstrated that the interaction between PD-1 and PD-L1 inhibits T cell activation. PD-1 is not expressed on quiescent T cells but is induced by activation. Sustained PD-1 expression is observed on exhausted T cells in chronic infections and cancer. Under normal conditions, the PD-1 / PD-L1 pathway is important for maintaining peripheral tolerance to prevent autoimmunity. However, in cancer, the self-protective function of PD-L1 is hijacked, and PD-L1 is expressed by various cancer cell types to evade immune system surveillance. Antibodies targeting PD-1 / PD-L1 block inhibitory signaling and restore the anti-cancer activity of T cells. The PD-1 / PD-L1 pathway has been considered a dominant-negative regulator of the effector function of anti-tumor T cells. Clinically, blocking this pathway has achieved high objective response rates ranging from 35% to 87% in some cancer types, such as Hodgkin lymphoma, Merkel cell carcinoma, and melanoma. In other cancer types, such as NSCLC, head and neck cancer, and renal cell carcinoma, lower objective response rates ranging from 15% to 25% have been achieved.

[0008] The combination of anti-PD-1 and anti-CD137 antibodies demonstrated enhanced antitumor activity in a mouse tumor model, compared to monotherapy with each antibody, by enhancing T-cell effector function and tumor infiltration. Clinically, the combination of utomirumab (0.45-5.0 mg / kg) and pembrolizumab showed synergistic antitumor effects in patients with advanced solid tumors without dose-limiting toxicity.

[0009] Based on the immunomodulatory properties of CD137, anti-human 4-1BB agonist antibodies may be used to treat cancer, autoimmune diseases, and infections. However, the use of anti-human 4-1BB agonist antibodies is limited due to hepatotoxicity. Furthermore, no effective therapies combining the blocking of immunosuppressive or tumor signaling pathways with the activation of the 4-1BB:4-1BBL pathway without hepatotoxicity have been described. In other words, there is a need for effective involvement of the 4-1BB:4-1BBL pathway, either combined with the removal of inhibition of immune activation or with the inhibition of tumor cell signaling. [Overview of the Initiative]

[0010] This invention is based on the fundamental discovery that it is possible to generate anti-CD137 antibodies that have potent crosslinking-dependent agonist activity and may avoid the hepatotoxicity observed in clinical trials. This invention is further based on the discovery that bispecific antibodies targeting PD-L1 and CD137 possess special activity that enhances T-cell effector function and inhibits tumor growth in vivo better than monotherapy or combination therapy with each antibody. The bispecific antibody may have a unique anti-CD137 single-chain variable fragment (scFv) that activates T cells, for example, through crosslinking via the other arm of the bispecific antibody that binds to PD-L1. The bispecific antibody may also be directed to non-PD-L1 expressing tumors by replacing the anti-PD-L1 arm with other tumor-specific binders, such as anti-Her2 or anti-tumor-specific glycans, as provided herein. Bispecific antibodies that induce target-dependent T-cell activation can avoid hepatotoxicity while maintaining the antitumor capability of anti-CD137 monoclonal antibodies.

[0011] In some embodiments, the present invention provides the following 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 provided herein comprise a heavy chain variable (V H ) region having an amino acid sequence with at least 80% sequence identity to 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 having an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 18. In another aspect, an antibody or antigen-binding fragment having a V H region having an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a V L region having an amino acid sequence with at least 80% identity to SEQ ID NO: 2 comprises (a) the CDR-H1, CDR-H2, and CDR-H3 of V H , wherein CDR-H1 comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 3, CDR-H2 comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 4, and CDR-H3 comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 5, and the CDR-H1, CDR-H2, and CDR-H3 of V H , and (b) the CDR-L1, CDR-L2, and CDR-L3 of V L , wherein CDR-L1 comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 6, CDR-L2 comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 7, and CDR-L3 comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 8, and the CDR-L1, CDR-L2, and CDR-L3 of V L .

[0012] In another aspect, a V H region having an amino acid sequence with at least 80% identity to SEQ ID NO: 9 and a VL An antibody or its antigen-binding fragment having a region is (a)V H CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 11, CDR-H2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 12, and CDR-H3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 13, V H (b)V L CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 14, CDR-L2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 15, and CDR-L3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 16, V L This includes CDR-L1, CDR-L2, and CDR-L3.

[0013] In yet another embodiment, V includes an amino acid sequence having at least 80% identity with SEQ ID NO: 17. H The region and V containing an amino acid sequence that has at least 80% identity with SEQ ID NO: 18. L An antibody or its antigen-binding fragment having a region is (a)V H CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 19, CDR-H2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 20, and CDR-H3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 21, V H (b)V L CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 22, CDR-L2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 23, and CDR-L3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 24, V LThis includes CDR-L1, CDR-L2, and CDR-L3.

[0014] In one embodiment, the antibody or antigen-binding fragment provided herein comprises an Fc domain. In another embodiment, the Fc domain is an IgG, IgE, IgM, IgD, IgA, or IgY domain. In yet another embodiment, the IgG domain is an IgG1, IgG2, IgG3, or IgG4 domain. In yet another embodiment, the IgG1 domain comprises the amino acid sequence of Sequence ID No. 26. In one embodiment, IgG1 comprises a point mutation that modifies or reduces antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) compared to wild-type IgG1. Exemplary point mutations include the K297A and K322A mutations. In yet another embodiment, the IgG4 domain comprises the amino acid sequence of Sequence ID No. 25. In yet another embodiment, the antigen fragment provided herein comprises scFv, F(ab)2, or Fab.

[0015] In one embodiment, the disclosure also provides a pharmaceutical composition comprising one of the antibodies or antigen-binding fragments thereof provided herein. In one embodiment, the antibody or antigen-binding fragment of the pharmaceutical composition provided herein comprises a pharmaceutically acceptable carrier conjugated to the C-terminus of one or more polypeptides of the antibody or antigen-binding fragment. In another embodiment, the pharmaceutical composition provided herein comprises a bispecific antibody.

[0016] In one embodiment, the Disclosure also provides a method for treating cancer, comprising administering to a subject in need of such treatment an effective amount of an antibody or its antigen-binding fragment or an effective amount of a bispecific antibody provided herein. In one embodiment, the cancer is selected from 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, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.

[0017] In one embodiment, this specification 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 embodiment, the bispecific antibody provided herein comprises (i) an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 17. H (ii) a region and a V containing an amino acid sequence having at least 80% identity with the N-terminal sequence of approximately 100-120 amino acids of a sequence selected from SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 18. L The molecule includes a region, and the second antigen-binding region binds to an immune checkpoint molecule, an immune-stimulating molecule, or a tumor antigen.

[0018] In one embodiment, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 31 or SEQ ID NO: 32. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 30. In one embodiment, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 36. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 35. In one embodiment, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 38. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 37. In one embodiment, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 40. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 39.

[0019] In one embodiment, the bispecific antibody provided herein is (a) V containing the amino acid sequence of SEQ ID NO: 9 H The region and the amino acid sequence of approximately 100-120 amino acids from the N-terminal sequence of SEQ ID NO: 10 are included in V L (b) V having a region or containing the amino acid sequence of SEQ ID NO: 17 H The region and the amino acid sequence of approximately 100-120 amino acids from the N-terminal sequence of SEQ ID NO: 18 are included in V L The device includes a first antigen-binding region having a region. In another embodiment, 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 yet another embodiment, the second antigen-binding region binds to PD-L1, Her2, or a glycan.

[0020] In one embodiment, the present invention discloses that an anti-PD-L1#6-CD137#54 bispecific antibody (bsAb) can act as a platform for target-dependent T cell activation through cross-linking-dependent agonist activity of anti-CD137#54 single-chain antibodies.

[0021] In another embodiment, a bispecific antibody that binds to CD137 and PD-L1 is modified to bind to CD137 and other targets expressed in tumors, including immunomodulatory molecules and tumor-specific markers, such as Her2 or tumor-specific glycans.

[0022] In one embodiment, the first and second antigen-binding regions of the bispecific antibody provided herein comprise an Fc domain, a Fab fragment, a single-stranded variable fragment (scFv), or any combination thereof. In yet another embodiment, the scFv comprises (i) the amino acid sequence of SEQ ID NO: 9. H The region and the amino acid sequence of approximately 100-120 amino acids from the N-terminal sequence of SEQ ID NO: 10 are included in V L (ii) V containing the region or the amino acid sequence of SEQ ID NO: 17 H The region and the amino acid sequence of approximately 100-120 amino acids from the N-terminal sequence of SEQ ID NO: 18 are included in V L Includes the region. In a further embodiment, the bispecific antibody provided herein is a V of scFv. H Region and V L A linker is included between the regions. In a further embodiment, scFv includes an amino acid sequence, SEQ ID NO: 33 or SEQ ID NO: 34.

[0023] In one embodiment, the bispecific antibody provided herein comprises an Fc domain. In another embodiment, the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain. In yet another embodiment, the Fc domain is an IgG domain. In yet another embodiment, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.

[0024] In one embodiment, scFv binds to the C-terminus of the Fc domain. In another embodiment, the bispecific antibody provided herein includes a linker between the Fab domain and the scFv domain. In a further embodiment, the Fab fragment binds to the N-terminus of the Fc domain. In yet another embodiment, Fab includes a PD-L1 binding site, a Her2 binding site, or a glycan binding site, and scFv includes a CD137 binding site.

[0025] In one embodiment, the present invention provides a therapeutic agent and an antibody-drug conjugate comprising any bispecific antibody or antigen-binding fragment thereof provided herein. In one embodiment, the therapeutic agent is covalently bound to the antibody or antigen-binding fragment via a linker.

[0026] In one embodiment, this specification provides a pharmaceutical composition comprising any bispecific antibody provided herein and at least one pharmaceutically acceptable carrier.

[0027] In one embodiment, the present invention provides isolated amino acid sequences described in SEQ ID NOs: 1 to 26. In another embodiment, the present invention provides isolated amino acid sequences described in SEQ ID NOs: 30 to 40.

[0028] In one embodiment, the Disclosure also provides an isolated nucleic acid sequence encoding an antibody, its antigen-binding fragment, or the bispecific antibody of the present invention. In another embodiment, the present invention provides an isolated nucleic acid encoding any one of SEQ ID NOs: 1 to 26. In yet another embodiment, the present invention provides an isolated nucleic acid sequence encoding any one of SEQ ID NOs: 30 to 40. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 shows the screening of phage clones directed towards CD137 by direct ELISA. [Figure 2-1]Figure 2 shows the binding of phage clones directed to CD137 on HEK-293F cells overexpressing CD137, as determined by flow cytometry. [Figure 2-2] See the explanation in Figure 2-1. [Figure 2-3] See the explanation in Figure 2-1. [Figure 3] Figures 3A and 3B show the integrity and purity of anti-CD137 antibody leads purified by one-step Protein G using PAGE. Results for two batches are shown (upper and lower figures). [Figure 4] Figure 4 shows the binding of anti-CD137 antibody leads to activated Jurkat cells, as determined by flow cytometry. [Figure 5] Figure 5 shows the binding activity (EC50) of anti-CD137 antibody leads to recombinant human CD137, as determined by ELISA. [Figure 6] Figure 6 shows the protein aggregation of clones 31 and 54 of the highly concentrated anti-CD137 antibody by SEC-HPLC. [Figure 7-1] Figure 7 shows cytokine production by T cells in the presence of agonist activity of the anti-CD137 antibody lead. [Figure 7-2] See the explanation in Figure 7-1. [Figure 8-1] Figure 8 shows the dose-dependent induction of human T cell cytokine production by anti-CD137 antibody lead clones in primary human T cells. [Figure 8-2] See the explanation in Figure 8-1. [Figure 9] Figure 9 shows that treatment in combination with an anti-CD137 antibody enhances anti-PD-L1 antibody-mediated IFN-γ production by T cells in a mixed lymphocyte response. [Figure 10] Figure 10 shows the clear effect of the anti-CD137 antibody clone on the CD137-CD137L interaction. [Figure 11] Figure 11 shows the pharmacokinetic profiles of clones #31 and #54 of the anti-CD137 antibody in vivo. [Figure 12] Figure 12 shows the various cross-linking requirements for agonist activity of anti-CD137 antibody clones compared to utomirumab (CD137 ref) and urerumab (CD137 ref2). [Figure 13] Figure 13 shows the symmetrical format of the anti-PD-L1-CD137 bispecific antibody (bsAb). [Figure 14] Figure 14 shows the purity and integrity of anti-PD-L1-CD137 bsAb purified with protein G, as determined by SDS-PAGE. Achieving a purity of over 90% is possible with a single-step protein G chromatography procedure. [Figure 15] Figure 15 shows the purity and integrity of anti-PD-L1-CD137 bsAb purified with protein A using μCE-SDS. [Figure 16-1] Figure 16 shows that the anti-PD-L1-CD137 bsAb recognizes both CD137 and PD-L1 simultaneously, as observed by biosensor analysis using ForteBio®. [Figure 16-2] See the explanation in Figure 16-1. [Figure 17] Figure 17 shows that anti-PD-L1#6-CD137#54 bsAb induces synergistic T cell activation in mixed lymphocyte responses compared to monotherapy, combination therapy, or treatment with anti-PD-L1#6-CD137#31 bsAb. [Figure 18] Figures 18A and 18B show that the antigen-specific recall response of memory CD4(A) and memory CD8(B) T cells is significantly enhanced by anti-PD-L1#6-CD137#54 bsAb. [Figure 19] Figure 19 shows that target-dependent T cell activation is induced by anti-PD-L1#6-CD137#31 bsAb or anti-PD-L1#6-CD137#54 bsAb during co-culture of T cells with HEK-293 cells overexpressing PD-L1. [Figure 20A]Figures 20A to 20C show that anti-PD-L1#6-CD137#54 bsAb induces IFN-γ production by T cells (left panel in Figures 20A, 20C, and 20B) and cytotoxicity of cancer cells (right panel in Figure 20B) when co-cultured with PD-L1-positive cancer cells. (A) NCI-H1975, non-small cell lung cancer cells; (B) PC-3, prostate cancer cells; (C) MDA-MB-231, breast cancer cells. [Figure 20B-1] See the explanation in Figure 20A. [Figure 20B-2] See the explanation in Figure 20A. [Figure 20C] See the explanation in Figure 20A. [Figure 21] Figures 21A and 21B show that trastuzumab (Tra)-CD137#54 bsAb or anti-Her2#3-7-CD137#54 bsAb induces IFN-γ production in CD8 T cells when co-cultured with Her2-positive cancer cells. (A) SKBR-3, breast cancer cells; (B) MDA-MD-361, breast cancer cells. [Figure 22A] Figures 22A and 22B show that anti-glycan-CD137#54 bsAb induces IFN-γ production by CD8 T cells (left panel of Figure 22A and Figure 22B) and cytotoxicity of cancer cells (right panel of Figure 22A) when co-cultured with glycan-positive cancer cells. (A) MCF-7, breast cancer cells; (B) NCI-N87, gastric cancer cells. [Figure 22B] See the explanation in Figure 22A. [Figure 23] Figure 23 shows that anti-PD-L1#6-CD137#54 bsAb induces internalization of CD137 expressed in HEK293 cells. [Figure 24A] Figures 24A and 24B show that anti-PD-L1#6-CD137 bsAb rescued T-cell proliferation (A) and cytokine production (B) in the presence of Treg cells. [Figure 24B] See the explanation in Figure 24A. [Figure 25]Figures 25A to 25C show that anti-PD-L1#6-CD137#54 bsAb results in greater tumor growth inhibition compared to combination therapy with anti-PD-L1#6 antibody and anti-CD137#54 antibody in humanized mice xenografted with PD-L1-positive tumor cells from (A) NCI-H292, (B) NCI-H1975, and (C) BxPC-3. [Figure 26-1] Figure 26 shows that PD-L1#6-CD137#54 bsAb, Her2#3-7-CD137#54 bsAb, and glycan-CD137#54 bsAb do not induce significant cytokine release in human PBMCs. [Figure 26-2] See the explanation in Figure 26-1. [Figure 27] Figures 27A and 27B show the PK parameters of anti-PD-L1#6-CD137#54 bsAb in monkeys, in the form of (A) graphs and (B) tables. [Modes for carrying out the invention]

[0030] Detailed description of the invention Before describing the compositions and methods, it should be understood that the present invention is not limited to specific compositions, methods, and experimental conditions described, and therefore the compositions, methods, and conditions may vary. It should also be understood that the scope of the present invention is limited only by the appended claims, and that the technical terms used herein are for the purpose of describing specific embodiments and are not intended to limit them.

[0031] In this specification, in some embodiments, antibodies that bind to CD137 and their antigen-binding fragments are provided. In this specification, amino sequences of antibodies that bind to CD137 are also provided. As used herein, the term “antibody” refers to an immunoglobulin molecule having the ability to specifically bind to an antigen. Unless otherwise specified by context, the term “antibody” includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, and anti-idiotype antibodies. In one embodiment, the antibodies provided herein include monoclonal antibodies. The antibodies provided herein include any isotype and class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). As used herein, “antigen-binding fragment” means 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 fragments of scFv, Fab, or F(ab)2. As used herein, “antigen-binding region” means, for example, a portion of an antibody or immunoglobulin molecule that binds to an antigen or protein by contact with the antigen or protein. Antigen-binding regions are generally heavy-chain variable (V H ) region and light chain variable (V L The antigen-binding region generally includes one or more antigen-binding sites or paratopes.

[0032] The antibodies provided herein include 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 in between. H The antibody provided herein also includes 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 in between. L Includes the region.

[0033] Generally, “sequence identity” or “sequence homology” can be used interchangeably, but they refer to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, or the amino acid sequence of a polypeptide, and comparing these sequences with a second nucleotide or amino acid sequence. As used herein, the terms “percent sequence identity” or “percent identity” refer to the percentage of amino acid residues or nucleotides in a sequence that match those in a reference sequence, after aligning the sequences, introducing gaps, achieving the maximum percentage sequence identity if necessary, and without considering any conservative substitutions as part of the sequence identity. In other words, two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity," also known as "percent homology." The percentage identity with respect 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 two perfectly aligned sequences, obtained by dividing by the length of the reference sequence and multiplying by 100. Percent identity can also be determined by comparing sequence information using advanced BLAST computer programs, including version 2.2.9, which are available, for example, from the National Institutes of Health.The BLAST program is based on the alignment method described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and is discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). In short, the BLAST program determines identity as the number of aligned symbols (i.e., nucleotides or amino acids) equal to the total number of symbols in the shorter of the two sequences. This program can be used to determine percentage identity over the entire length of the sequences being compared. For example, the blastp program provides default parameters to optimize searches on short query sequences. This program also allows the use of SEG filters on mask-off segments of the query sequence, as determined by the SEG program in Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired range of sequence identity is approximately 80% to 100% and integer values ​​in between. Percent identity between the reference sequence and the claimed sequence may 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 suggests 100% identity across the length of the reference sequence.Further programs and methods for comparing sequences and / or evaluating sequence identity include the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner, optionally configured with default settings, available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner, optionally configured with default settings, available at www.ebi.ac.uk / Tools / psa / emboss_water / ), 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, Wis). In some embodiments, the reference to percent sequence identity refers to sequence identity as measured using BLAST (Basic Local Alignment Search Tool). In other embodiments, ClustalW is used for multiple sequence alignments. Optimal alignment can be evaluated using any preferred parameters of the selected algorithm, including default parameters.

[0034] In one embodiment, the antibody or its antigen-binding fragment contains an amino acid sequence having at least about 80% identity with respect to SEQ ID NO: 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. HA region and a V containing an amino acid sequence having at least approximately 80% identity with respect to number 2, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between. L It has a region. Sequence ID 1 provides an amino acid sequence containing the variable region of the heavy chain of anti-CD137 antibody clone #15. Sequence ID 2 provides an amino acid sequence containing the variable region of the light chain of anti-CD137 clone #15.

[0035] The antigen-binding region of an antibody or its antigen-binding fragment generally includes a complementarity-determining region (CDR). The "complementarity-determining region (CDR)" is V H and V L This refers to the hypervariable region. The CDR contains the target protein or antigen-binding site of the antibody that gives it specificity regarding binding to a protein or antigen. H and V L Generally, it includes three consecutively numbered CDRs. As used herein, CDR-H1, CDR-H2, and CDR-H3 are numbered from the N-terminus of the heavy chain polypeptide, the heavy chain variable region (V H This refers to three consecutively arranged CDRs. In this specification, CDR-L1, CDR-L2, and CDR-L3 are numbered from the N-terminus of the light chain polypeptide, and are the light chain variable regions (V L This refers to three consecutive CDRs.

[0036] In one embodiment, V contains an amino acid sequence having at least about 80% identity with SEQ ID NO: 1. H The region and V containing an amino acid sequence that has at least approximately 80% identity with SEQ ID NO: 2. LThe antigen-binding region of an antibody or its antigen-binding fragment having a region is CDR-H1 containing an amino acid sequence having at least approximately 80% identity with respect to SEQ ID NO: at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between, and with respect to SEQ ID NO: at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, and less than The CDR-H2 includes an amino acid sequence having at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between; and the CDR-H3 includes an amino acid sequence with respect to sequence number 5 having at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between. In another embodiment, V includes an amino acid sequence having at least about 80% identity with SEQ ID NO: 1. H The region and V containing an amino acid sequence that has at least approximately 80% identity with SEQ ID NO: 2. LThe antigen-binding region of an antibody or its antigen-binding fragment having a region CDR-L1 containing an amino acid sequence having at least about 80% identity with respect to SEQ ID NO 6, 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, and with respect to SEQ ID NO 7, 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, CDR-L2 includes an amino acid sequence having at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between; and CDR-L3 includes an amino acid sequence with respect to sequence number 8 having at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between.

[0037] In some embodiments, the antibody or its antigen-binding fragment contains an amino acid sequence having at least about 80% identity with respect to SEQ ID NO: 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. H A region and a V containing an amino acid sequence having at least approximately 80% identity with respect to number 10, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between. L The sequence includes the variable region. Sequence ID 9 provides an amino acid sequence containing the variable region of the heavy chain of anti-CD137 antibody clone #31. Sequence ID 10 provides an amino acid sequence containing the variable region of the light chain of anti-CD137 clone #31.

[0038] In one embodiment, V contains an amino acid sequence having at least about 80% identity with SEQ ID NO: 9. H The region and V containing an amino acid sequence that has at least approximately 80% identity with SEQ ID NO: 10. LThe antigen-binding region of an antibody or its antigen-binding fragment containing the region includes CDR-H1 containing an amino acid sequence having at least approximately 80% identity with respect to SEQ ID NO: at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between, and with respect to SEQ ID NO: at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, and less than The CDR-H2 includes an amino acid sequence having at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between; and the CDR-H3 includes an amino acid sequence with respect to sequence number 13 having at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between. In another embodiment, V includes an amino acid sequence having at least about 80% identity with SEQ ID NO: 9. H The region and V containing an amino acid sequence that has at least approximately 80% identity with SEQ ID NO: 10. LThe antigen-binding region of an antibody or its antigen-binding fragment containing the region includes CDR-L1 containing an amino acid sequence having at least approximately 80% identity with respect to SEQ ID NO: at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between, and with respect to SEQ ID NO: at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, and less than The CDR-L2 includes an amino acid sequence having at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between; and the CDR-L3 includes an amino acid sequence with respect to sequence number 16 having at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between.

[0039] In some embodiments, the antibody or its antigen-binding fragment contains an amino acid sequence having at least about 80% identity with respect to SEQ ID NO: 17, 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. H A region and a V containing an amino acid sequence having at least approximately 80% identity with respect to sequence number 18, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between. L It has a region. Sequence ID 17 provides an amino acid sequence containing the variable region of the heavy chain of anti-CD137 antibody clone #54. Sequence ID 18 provides an amino acid sequence containing the variable region of the light chain of anti-CD137 clone #54.

[0040] In one embodiment, V contains an amino acid sequence that has at least about 80% identity with SEQ ID NO: 17. H The region and V containing an amino acid sequence that has at least approximately 80% identity with SEQ ID NO: 18. LThe antigen-binding region of an antibody or its antigen-binding fragment containing the region includes CDR-H1 containing an amino acid sequence having at least approximately 80% identity with respect to SEQ ID NO: at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between, and with respect to SEQ ID NO: at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, and less than The CDR-H2 includes an amino acid sequence having at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between; and the CDR-H3 includes an amino acid sequence with respect to sequence number 21 having at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between. In another embodiment, V includes an amino acid sequence having at least about 80% identity with SEQ ID NO: 17. H The region and V containing an amino acid sequence that has at least approximately 80% identity with SEQ ID NO: 18. LThe antigen-binding region of an antibody or its antigen-binding fragment containing the region includes CDR-L1 containing an amino acid sequence having at least approximately 80% identity with respect to SEQ ID NO: at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between, and with respect to SEQ ID NO: at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, and less than The CDR-L2 includes an amino acid sequence having at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between; and the CDR-L3 includes an amino acid sequence with respect to sequence number 24 having at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, at least approximately 99% identity, at least approximately 99.5% identity, at least approximately 99.9% identity, and any number or range in between.

[0041] The antibodies or antigen-binding fragments provided herein further comprise an Fc domain. As used herein, the term Fc domain refers to a region of the antibody including at least a hinge region, a CH2 domain, and a CH3 domain, unless otherwise specified by context. The terms Fc domain and Fc region may be used interchangeably unless otherwise specified by context. In some embodiments, the Fc domain is an IgG domain, IgE domain, IgM domain, and an IgD domain, IgA domain, or IgY domain. Fc domains from any species with any sequence may be used, including humans, apes, monkeys, mice, rabbits, goats, sheep, guinea pigs, horses, and others. In some embodiments, the Fc domain is engineered, i.e., a non-natural or recombinant Fc domain produced, for example, using molecular biology techniques. In some embodiments, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain. In one embodiment, the IgG4 domain comprises the amino acid sequence of SEQ ID NO: 25. In another embodiment, the IgG1 domain contains the amino acid sequence of SEQ ID NO: 26. In one embodiment, the Fc domain is human.

[0042] In some embodiments, this specification also provides pharmaceutical compositions comprising one of the antibodies or antigen-binding fragments thereof provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is bound to the C-terminus of one or more polypeptides of the antibody or antigen-binding fragment. Any suitable means for binding the pharmaceutically acceptable carrier can be used, including, for example, covalent bonding and the use of a linker.

[0043] In some embodiments, this specification provides isolated amino acid sequences described in SEQ ID NOs: 1 to 26. In some embodiments, this specification also provides isolated nucleic acid sequences encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 26.

[0044] In some embodiments, methods for treating cancer in a subject are provided herein. In some embodiments, the method for treating cancer comprises administering to a subject a certain amount of either a CD137-conjugated antibody or an antigen-binding fragment thereof, which is effective in treating cancer, as provided herein. In some embodiments, 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, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.

[0045] As used herein, the terms “treat,” “cure,” “therapy,” and “therapeutic” mean obtaining a desired pharmacological and / or physiological effect, including, but not limited to, mitigating, delaying, or slowing progression, reducing an effect or symptom, preventing onset, inhibiting or improving the onset of a disease or disorder, or obtaining a beneficial or desired outcome with respect to a disease, disorder or condition, such as a therapeutic and / or preventive effect. As used herein, “treatment” includes all treatments of diseases in mammals, particularly humans, and also includes (a) preventing the onset of a disease in a subject, including subjects who are predisposed to the disease or at risk of developing the disease but have not yet been diagnosed as such, (b) inhibiting the disease, i.e., stopping its onset, and (c) reducing the disease, i.e., regressing the disease. Therapeutic effects include the eradication or recovery of the underlying disorder being treated. Therapeutic effects are also achieved by the eradication or recovery of one or more physiological symptoms associated with the underlying disorder, so that improvement is observed in the subject even though the subject may still suffer from the underlying disorder. In some embodiments, for a preventive effect, a treatment or therapeutic composition is administered to a subject at risk of developing a particular disease, or to a subject in which one or more of the physiological symptoms of a disease have been reported, even if a diagnosis of the disease has not yet been made. The methods of this disclosure may be used in any mammal or other animal. In some embodiments, the treatment results in a reduction or cessation of symptoms. Preventive effects include delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof.

[0046] As used herein, the term “subject” refers to any individual or patient on whom the method disclosed herein is performed. The term “subject” may be used interchangeably with the terms “individual” or “patient.” The subject may be a human, but may also be an animal that would be recognized by those skilled in the art. That is, other animals, including, for example, rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, livestock including cattle, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas), are included within the definition of subject.

[0047] As used herein, the terms “effective dose” or “therapeutic dose” refer to the amount of the antibody, its antigen-binding fragment, or other composition described herein that is sufficient to produce an intended application, not limited to the treatment of a disease, as defined herein. The therapeutic dose may vary depending on the intended therapeutic application (e.g., in vivo) or the patient and disease state being treated, for example, the patient’s weight and age, the severity of the disease state, the form of administration, etc., but can be readily determined by those skilled in the art. The term also applies to the dose that induces a particular response in target cells. The exact dose will vary depending on the specific antibody, its antigen-binding fragment, or other selected composition, the following administration regimens, whether administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the delivery system within the body that carries it.

[0048] In some embodiments, the antibodies or antigen-binding fragments of the present invention are used as monotherapy or in combination with other therapeutic agents, such as radiotherapy agents and cytotoxic chemotherapeutic agents, and other immunomodulatory agents, such as vaccines, interleukins, cytokines, and chemokines, and with biologics as combination therapy. 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. Examples of biologics include CAR T-cell therapy, tumor-infiltrating lymphocyte (TIL) therapy, and monoclonal antibodies, such as alemtuzumab (CAMPATH), trastuzumab (HERCEPTIN), ibritumomab tiuxetan (ZEVALIN), brentuximab vedotin (ADCETRIS), trastuzumab emtansine (ado-trastuzumab emtansine) (KADCYLA), blinatumomab (BLINCYTO), bevacizumab (AVASTIN), and cetuximab (ERBITUX). Antibodies also include checkpoint inhibitors such as PD-1 inhibitors, e.g., pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), and cemiprimab (LIBTAYO); PD-L1 inhibitors, e.g., atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (IMFINZI); and CTLA-4 inhibitors, e.g., ipilimumab (YERVOY); as well as other checkpoint inhibitors such as anti-B7-H3 antibody (MGA271), anti--KIR antibody (lirilumab), and anti-LAG3 antibody (BMS-986016).

[0049] In some embodiments, the present invention further provides the expression, purification, and characterization of anti-CD137 agonist antibodies, as detailed in the following examples. The expression construct for the antibodies provided herein may include a signal sequence. Any suitable signal sequence, such as the sequence of SEQ ID NO: 27, may be used. In some embodiments, T cells treated with an anti-PD-L1 antibody together with the anti-CD137 antibody provided herein showed a further increase in T cell effector function. Without being limited by theory, this indicates that combination therapy or therapy with bispecific antibodies targeting both CD137 and PD-L1 can overcome the lower response rates observed with monotherapy using each antibody alone in clinical trials. In addition to the anti-PD-L1 antibody, a second antibody may be used for combination therapy targeting another immunoenhancing antigen, such as CD40 or CTLA-4, or for therapy with a bispecific antibody targeting CD137 and a second antigen, such as PD-L1, CD40, or CTLA-4.

[0050] For example, bispecific molecules such as bispecific antibodies (bsAbs) provide a means of simultaneously targeting multiple epitopes on the same or different molecular targets with a single therapeutic agent. Without being limited by theory, bispecific molecules as cancer therapeutics have the potential to result in novel or multiple strong activities, lower article costs, and facilitate the development of new therapeutic regimens compared to, for example, a mixture of two monoclonal antibodies (mAbs).

[0051] Accordingly, this specification also provides the expression, purification, and characterization of bifunctional proteins, including bispecific antibodies. As used herein, the term “bifunctional protein” refers to a protein having at least two functions. A non-limiting example of a bifunctional protein is a bispecific antibody capable of binding to two antigens. The bispecific antibodies provided herein may include, for example, an isolated functional scFv fragment that binds to CD137 and fuses with the C-terminus of the Fc domain of an anti-PD-L1 antibody. In some embodiments, the C-terminal scFv that binds to CD137 in the fusion construct provided herein fuses with the Fc domain of an antibody that binds to another immunomodulatory molecule, such as CD40 or CTLA-4. In other embodiments, the C-terminal scFv can bind to an immunomodulatory molecule, such as CD40 or CTLA-4.

[0052] In some embodiments, this specification provides a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region. Generally, the first and second antigen-binding regions specifically bind to different antigens or targets. In some embodiments, the first and second antigen-binding regions bind to different epitopes in the same antigen or target.

[0053] In one embodiment, the bispecific antibody provided herein includes a first antigen-binding region that binds to CD137. The first antigen-binding region includes 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 respect to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 17. HV includes an amino acid sequence with 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, for approximately 100-120 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. L This includes a region. In some embodiments, the second antigen-binding region of the bispecific antibody provided herein binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen.

[0054] V H Sequences or V described in Sequence ID 1, Sequence ID 9 and Sequence ID 17 that include the region L Any number of amino acids in the sequences described in SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 18, which include the region, may be present in the bispecific antibody. H Area or V L The N-terminal or C-terminal sequences of sequences provided herein, having a region, may be included in a bispecific antibody. In one embodiment, about 100-105 amino acids, about 100-110 amino acids, about 100-115 amino acids, about 100-120 amino acids, about 100-125 amino acids, and any number or range in between, of the N-terminal or C-terminal sequences 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, are included in the bispecific antibody provided herein. In another embodiment, the bispecific antibody contains the sequence of SEQ ID NO: 1, SEQ ID NO: 9, or SEQ ID NO: 17. In yet another embodiment, the bispecific antibody contains about 100-120 amino acids of the N-terminal sequence of SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 18. In yet another embodiment, the bispecific antibody contains 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.

[0055] In one embodiment, the first antigen-binding region of the bispecific antibody provided herein has the amino acid sequence of SEQ ID NO: 9. H V has a region and an amino acid sequence of approximately 100-120 amino acids, which is the N-terminal sequence of sequence number 10. L The region includes. In another embodiment, the first antigen-binding region of the bispecific antibody provided herein has the amino acid sequence of SEQ ID NO: 17. H V has a region and an amino acid sequence of approximately 100-120 amino acids, which is the N-terminal sequence of sequence number 18. L Includes the region.

[0056] In some embodiments, the second antigen-binding domain of the bispecific antibody provided herein binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen. As used herein, the term "immune checkpoint molecule" refers to any molecule that inhibits or negatively modulates an immune response. In one embodiment, binding of the second antigen-binding domain to an immune checkpoint molecule inhibits the immune checkpoint molecule. Examples of immune checkpoint molecules include PD-L1, PD-1, CTLA-4, and LAG3. As used herein, the term "immunostimulatory molecule" refers to any molecule that induces, enhances, or positively modulates an immune response. Examples of immunostimulatory molecules include CD28, CD40, CD137, CD27, and ICOS. In some embodiments, binding of the second antigen-binding domain to an immunostimulatory molecule activates the immunostimulatory molecule, resulting in, for example, increased signaling and increased immune activation. As used herein, the term "tumor antigen" refers to any antigen present on the surface of tumor cells or expressed in tumor cells. Examples of tumor antigens include products of mutated oncogenes, products of mutated tumor suppressor genes, products of mutated genes other than oncogenes or tumor suppressor factors, tumor antigens produced by oncogenic viruses, altered cell surface glycolipids and glycoproteins, carcinoembryonic antigens, and others. Tumor antigens also include immunomodulatory molecules such as immune checkpoint inhibitors and immunostimulatory molecules. Therefore, in some embodiments, the tumor antigen, or the second antigen-binding region of the bispecific antibody provided herein, is associated with the function of an immunomodulatory molecule. In one embodiment, the binding of the second antigen-binding region to the tumor antigen results in directing immune cells, such as T cells, towards tumor cells.

[0057] Any combination of the first and second antigen-binding regions may be included in the bispecific antibodies provided herein, for example, the first and second antigen-binding regions which bind to any immune checkpoint molecule, any immunostimulatory molecule, or any tumor antigen. Thus, in some embodiments, the second antigen-binding region of the bispecific antibodies provided herein binds to any immune checkpoint molecule, any immunostimulatory molecule, or any tumor antigen. In some embodiments, the first and second antigen-binding regions bind to the same molecule. For example, the first and second antigen-binding regions may bind to the same or different epitopes of the same molecule. In other embodiments, the first and second antigen-binding regions bind to different molecules.

[0058] In some embodiments, the second antigen-binding domain 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 may be exclusively expressed in cancer cells, such as GloboH. In one embodiment, the second antigen-binding domain binds to PD-L1. In another embodiment, the second antigen-binding domain binds to Her2. In yet another embodiment, the second antigen-binding domain binds to a glycan. In a further embodiment, the glycan is GloboH. By expanding the repertoire of bispecific antibodies that bind to CD137 and antigens expressed on tumors in addition to or other than PD-L1, it becomes possible to direct bispecific antibodies to, for example, cancer types that do not express PD-L1.

[0059] In one embodiment, a bispecific antibody having a first antigen-binding domain that binds to CD137 and a second antigen-binding domain that binds to PD-L1 binds to both CD137 and PD-L1 simultaneously (Figure 16). By designing a bispecific antibody capable of binding to both CD137 and PD-L1, without being limited by theory, the risk of hepatotoxicity and associated mortality observed in clinical trials with anti-CD137 antibodies such as urerumab can be reduced by limiting anti-CD137 binding activity to tumor sites expressing PD-L1. Furthermore, it is thought that simultaneous binding to CD137 and PD-L1 can enhance T cell activation as a result of cross-linking (see also Example 10 below). In another embodiment, a bispecific antibody having a first antigen-binding domain that binds to CD137 and a second antigen-binding domain that binds to PD-L1 induces stronger CD137 uptake compared to a reference antibody such as utomirumab and urerumab (Figure 23).

[0060] In some embodiments, the first antigen-binding region and the second antigen-binding region include scFv, F(ab)2, Fab, or any combination thereof. In one embodiment, the first antigen-binding region includes scFv and the second antigen-binding region includes Fab. In another embodiment, the scFv contained in the bispecific antibody provided herein binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen. In yet another embodiment, the scFv contained in the bispecific antibody provided herein binds to CD137. In some embodiments, the Fab contained in the bispecific antibody provided herein binds to an immune checkpoint molecule, an immunostimulatory molecule, or a tumor antigen. In one embodiment, the Fab contained in the bispecific antibody provided herein binds to PD-L1. In another embodiment, the scFv of the bispecific antibody provided herein contains the amino acid sequence of SEQ ID NO: 9. H The region and the amino acid sequence of approximately 100-120 amino acids from the N-terminal sequence of SEQ ID NO: 10 are included in V LThe region includes. In yet another embodiment, the scFv of the bispecific antibody provided herein includes the amino acid sequence of SEQ ID NO: 17. H The region and the amino acid sequence of approximately 100-120 amino acids from the N-terminal sequence of SEQ ID NO: 18 are included in V L Includes the region.

[0061] In one embodiment, the bispecific antibody provided herein comprises a first antigen-binding region that binds to CD137 and a second antigen-binding region that binds to Her2. The bispecific antibody that binds to CD137 and Her2 includes a CD137-binding scFv fused to the C-terminus of the Fc domain of an antibody that binds to Her2, such as trastuzumab (heavy chain SEQ ID NO: 36) or anti-Her2#3-7 (heavy chain SEQ ID NO: 38). In one embodiment, the bispecific antibody that binds to CD137 and Her2 further comprises the light chain of SEQ ID NO: 35 (trastuzumab) or SEQ ID NO: 37 (anti-Her2#3-7). In another embodiment, the bispecific antibody provided herein comprises a first antigen-binding region that binds to CD137 and a second antigen-binding region that binds to a tumor-specific glycan. A bispecific antibody that binds to CD137 and tumor-specific glycans includes a CD137-binding scFv (heavy chain, SEQ ID NO: 40) fused to the C-terminus of the Fc domain of the antibody that binds to the tumor-specific glycan, such as the anti-glycan provided herein. In one embodiment, the bispecific antibody that binds to CD137 and tumor-specific glycans further includes the light chain (anti-glycan) of SEQ ID NO: 39. In some embodiments, the bispecific antibody that binds to CD137 and Her2 or CD137 and tumor-specific glycans further includes a linker that binds the anti-CD137 scFv to the Fc domain. Any linker can be used, such as a GS linker (SEQ ID NO: 28), a G4S linker (SEQ ID NO: 29), or a complex thereof. In one embodiment, the linker is a G4S linker.

[0062] Therefore, the present invention provides a platform for target-dependent T cell activation. In one embodiment, the agonist activity of anti-CD137 scFv is induced by binding to tumor-specific antigens such as PD-L1, as shown in Figures 19 and 20. 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.

[0063] In some embodiments, the bispecific antibodies provided herein are V of scFv H Region and V L Linkers are included between the regions. Any linker can be used. For example, a linker can contain any amino acid sequence. A linker can be of 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. A linker can also contain a complex of amino acid sequences. Any number of amino acid sequence complexes can be included in a linker. Exemplary linker sequences are provided in SEQ ID NOs. 28 and SEQ ID NOs. 29. In some embodiments, scFv contains the amino acid sequence of SEQ ID NOs. 33 or SEQ ID NOs. 34.

[0064] In some embodiments, the bispecific antibodies provided herein further comprise an Fc domain. In some embodiments, the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain. Fc domains from any species with any sequence are available, including humans, apes (apes), monkeys, mice, rabbits, goats, sheep, guinea pigs, horses, and others. In some embodiments, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain. In one embodiment, the IgG4 domain comprises the amino acid sequence of SEQ ID NO: 25. In another embodiment, the IgG1 domain comprises the amino acid sequence of SEQ ID NO: 26. In one embodiment, the Fc domain is human. Generally, human Fc domains are not immunogenic in humans and are therefore suitable for use in human therapeutics.

[0065] In some embodiments, the scFv of the bispecific antibody provided herein is bound to the C-terminus of the Fc domain. In one embodiment, the linker is located between the Fc domain and the scFv. In another embodiment, the linker binds the scFv to the Fc domain. Any linker can be used, such as the G4S linker provided herein.

[0066] In some embodiments, the Fab of the bispecific antibody provided herein binds to the N-terminus of the Fc domain. In one embodiment, the Fab binds directly to the N-terminus of the Fc domain via a peptide bond. In another embodiment, the Fab domain binds to the N-terminus of the Fc domain via a linker.

[0067] In some embodiments, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 31 or SEQ ID NO: 32. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 30. In one embodiment, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 36. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 35. In one embodiment, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 38. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 37. In one embodiment, the bispecific antibody provided herein includes the heavy chain sequence of SEQ ID NO: 40. In another embodiment, the bispecific antibody provided herein further includes the light chain sequence of SEQ ID NO: 39. For example, 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 others may contain one or more G linkers (SEQ ID NO: 28), one or more G4S linkers (SEQ ID NO: 29), or any complex of G linkers or G4S linkers, but any other suitable linker may be used.

[0068] In some embodiments, this specification provides isolated amino acid sequences described in SEQ ID NOs. 30-40. In some embodiments, this specification also provides isolated nucleic acid sequences encoding any one of the amino sequences of SEQ ID NOs. 30-40.

[0069] In some embodiments, this specification provides antibody-drug conjugates. The antibody-drug conjugates provided herein may include any antibody or its antigen-binding fragment provided herein. For example, any antibody or its antigen-binding fragment that specifically binds to CD137 may be included in the antibody-drug conjugate. Any bispecific antibody or its antigen-binding fragment provided herein may also be included in the antibody-drug conjugate. In some embodiments, the antibody-drug conjugates provided herein include therapeutic agents. Any therapeutic agent, including small molecules, may be included in the antibody-drug conjugate provided herein. In some embodiments, the therapeutic agent has cytotoxic activity. Any chemotherapeutic agent having cytotoxic activity may be included in the antibody-drug conjugate. Examples of chemotherapeutic drugs include actinomycin, all-trans retinoic acid, anti-estrogens, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, eposilon, etoposide, fluorouracil, and gemcitabine. Examples include, but are not limited to, hydroxyurea, idarubicin, imatinib, irinotecan, mechloretamine, mercaptopurine, methotrexate, mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, taxol, taxotere, tamoxifen, teniposide, thioguanine, topotecan, barrubicin, vemurafenib, vinblastine, vincristine, and vindesine.

[0070] In some embodiments, the antibody-drug conjugates provided herein are used in the treatment of cancer. For example, the antibody in the antibody-drug conjugate binds to an antigen on a tumor cell, thereby directing a small molecule with cytotoxic activity or other therapeutic agent contained in the antibody-drug conjugate towards the tumor cell. Once the antibody-drug conjugate binds to the tumor cell, the small molecule or other therapeutic agent is taken up and released from the tumor cell.

[0071] In some embodiments, the therapeutic agent contained in the antibody-drug molecule provided herein is covalently bound to the antibody or its antigen-binding fragment provided herein, or to the bispecific antibody or its antigen-binding fragment provided herein. A linker may be used to covalently bind the therapeutic agent to the antibody or its antigen-binding fragment, or to the bispecific antibody or its antigen-binding fragment. Any suitable linker may be used to covalently bind the therapeutic agent to the antibody or its antigen-binding fragment, or to the bispecific antibody or its antigen-binding fragment provided herein. In some embodiments, the linker contained in the antibody-drug conjugate provided herein is stable outside the target cell, including in the bloodstream, and is cleaved inside the target cell to release the therapeutic agent. A therapeutic agent having cytotoxic activity can, for example, induce target cell death upon release. Therefore, in some embodiments, the therapeutic agent is selectively directed to tumor cells. Selective targeting of the therapeutic agent to tumor cells generally results in reduced cytotoxicity and increased tolerability, for example, compared to non-tumor cells.

[0072] In this specification, in some embodiments, pharmaceutical compositions comprising bispecific antibodies provided herein are provided. Any bispecific antibody provided herein may be included in a pharmaceutical composition. In one embodiment, the bispecific antibody included in a pharmaceutical composition provided herein binds to CD137, PD-L1, or both CD137 and PD-L1. Antibody-drug conjugates provided herein may also be included in a pharmaceutical composition. In some embodiments, the pharmaceutical composition is used for the treatment of cancer. Any cancer can be treated using the pharmaceutical composition provided herein. 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, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, and leukemia.

[0073] In some embodiments, methods for treating cancer in a subject are provided herein. Methods for treating cancer include administering to a subject a certain amount of any bispecific antibody or antigen-binding fragment thereof provided herein that is effective in treating cancer. In some embodiments, 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, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia. [Examples]

[0074] Example 1 This embodiment describes antibody production from an OmniMab library.

[0075] To generate therapeutic antibodies against CD137, selection was performed using an OmniMab phagemid library. This phagemid library was constructed by AP Biosciences Inc. (APBio Inc.) from peripheral blood mononuclear cells collected from over 100 healthy donors. Pre-coated CD137-Fc recombinant protein was incubated with the supernatant containing rescued phages for 1 hour and washed three times with PBS containing 0.1% Tween-20. Binding phages were detected with HRP-labeled anti-M13 antibody (Roche), and a TMB substrate was used for signal generation. OD450 readings were recorded.

[0076] The first panning was performed using Hyperphage (M13K07ΔρIII, Progen, Heidelberg, Germany). Solid-phase and cell panning for CD137 were used for the selection and isolation of CD137-specific binders from the OmniMab library. Solid-phase panning was performed using recombinant human CD137-ECD-Fc (APBio Inc.), which was used for selection in the first panning. HEK293 cells expressing CD137 were used for the second and third enrichments. After the third panning, specific CD137 binders were screened and isolated by direct ELISA and FACS (Figures 1 and 2). For FACS analysis, 293F cells stably expressing CD137 were stained with anti-CD137 phage supernatant (50 ul / well) and their CD137 binding activity was examined. 293F cells stably expressing CD137 were also incubated on ice for 1 hour with 2.5 ug / ml anti-CD137 antibody (Ab) as a control. These cells were washed three times with 1×PBS and then incubated on ice for 1 hour with anti-M13 antibody (Progen). Again, the cells were washed three times with 1×PBS and incubated on ice for another 1 hour with anti-mouse IgG-Alexa 488 (Invitrogen Inc.). After staining, the cells were washed three times with 1×PBS, resuspended in 1×PBS, and analyzed using FACS Calibur (BD Biosciences, Inc.) and FlowJo (TreeStar, LLC). FACS analysis of 293F cell clone 13 stably expressing CD137 is shown in Figure 2. Positive binders were isolated and sent for sequencing to confirm heavy chain sequence and diversity. As shown in Figures 1 and 2, several clones that specifically recognized the CD137 antigen were isolated compared to the negative control.

[0077] These results indicate that phage clones obtained after three CD137-specific enrichments specifically recognize CD137.

[0078] Example 2 This example describes the subcloning, expression, and purification of a protein that specifically binds to CD137 in IgG form.

[0079] To rapidly screen for candidates with functional properties in T cell activation, the heavy and light chains of positive CD137 or PD-L1 binders identified by ELISA were amplified, digested, and subcloned into IgG expression vectors carrying the IgG4 constant region (SEQ ID NO: 25), produced by APBio. After sequence validation, plasmids were prepared for antibody expression using 293fectin transfection reagent (Invitrogen) and transfected into HEK293 cells. Four days after culturing, antibodies secreted into serum-free medium were affinity-purified from the culture supernatant by protein G chromatography. The purified antibodies were concentrated and subsequently dialyzed in PBS buffer. The final concentration of the dialyzed protein was determined using a NanoDrop2000 spectrophotometer, and its purity and integrity were determined by SDS-PAGE, with or without a reducing agent.

[0080] Figure 3 shows representative PAGE gel analyses of the first batch (Figure 3, top) and second batch (Figure 3, bottom) of purified anti-CD137 antibody leads. Culture supernatants from mammalian cells collected 4 days after transfection were purified using protein G chromatography (Thermo Fisher). The purified proteins were analyzed under reducing and non-reducing conditions before being loaded onto gels (3 μg / lane). The results showed that under non-reducing conditions, both proteins had a molecular weight of approximately 145 kDa, while under reducing conditions, the heavy and light chains had molecular weights of approximately 55 kDa and 25 kDa, respectively. A purity of over 90% was achieved with a single step of protein G chromatography.

[0081] These results demonstrate that the integrity of various purified antibody leads is normal in HEK293 cells.

[0082] Example 3 This example illustrates the binding of an anti-CD137 antibody to Jurkat cells.

[0083] Purified anti-CD137 antibody leads were also applied to Jurcut cells that had induced CD137, and their binding activity was determined by FACS. Jurcut cells were treated with PMA (10 ng / ml) and ionomycin (1 μg / ml) to induce CD137 expression for 2 days. Stimulated cells were incubated on ice for 1 hour with anti-CD137 (0.5 μg / ml) and reference (ref) antibody (0.5 μg / ml) as positive controls, left unstained, or incubated with OX40 reference (ref) antibody as a negative control. Cells were washed three times with 1×PBS and then incubated on ice for another 1 hour with Alexa-488-labeled goat anti-human IgG (H+L) (Invitrogen Inc.). After staining, the cells were washed three times with 1×PBS, resuspended in 1×PBS, and then analyzed using FACS Calibur (BD Biosciences, Inc.) and FlowJo (TreeStar, LLC). Among the CD137 antibody reads, some reads exhibited binding activity comparable to the reference antibody, as shown in Figure 4.

[0084] These results, as seen in flow cytometry, demonstrate the binding of anti-CD137 antibody leads to activated Jurcut cells.

[0085] Example 4 This example describes the determination of anti-CD137 antibody binding activity by ELISA.

[0086] To perform a direct ligand binding assay of an anti-CD137 antibody that binds to CD137, pre-coated wells were prepared using recombinant CD137 / Fc (100 ng / ml). Briefly, purified human CD137-IgG4 Fc (APBio) was dialyzed in phosphate-buffered saline (PBS) to 1 mg / ml, and then diluted in PBS to a final concentration of 1 μg / ml. Nunc-Immuno Maxisorp 96-well plates were pre-coated with 0.1 ml of recombinant CD137 protein per well, leaving empty wells for nonspecifically binding controls, and incubated overnight at 4°C. After removing the recombinant CD137 protein solution, 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 milk powder in PBS) was added to all wells, and the plates were incubated at room temperature for 1 hour. The samples were washed three times with 0.4 ml of washing buffer, excluding the blocking buffer.

[0087] Pre-coated wells were incubated with serial dilutions of purified anti-CD137 antibody. Serial dilutions of CD137 antibody were prepared in PBS, and 0.1 ml was added to each well. The plate was incubated at room temperature for 1 hour. After removing the antibody solution, the plate was washed three times with 0.4 ml of wash buffer. HRP-labeled goat anti-human IgG, F(ab')2 specific F(ab')2 antibody (Jackson Immunoresearch #109-036-097) was diluted 1:2000 in PBS and added at a rate of 0.1 ml per well. The plate was incubated at room temperature for 1 hour and washed three times with 0.4 ml of wash buffer per well. The plate was colored with 0.1 ml of TMB reagent (Invitrogen) and incubated at room temperature for 1-5 minutes. The reaction was stopped by adding 0.05 ml of 1N HCl, and the absorbance at 450 nm was read using Bio-Tek Spectra. The OD450 readings were plotted against the concentration of anti-CD137, and the 50% effective concentration (EC2) of the anti-CD137 antibody that binds to CD137 / Fc was determined. 50 The value was calculated. EC 50The values ​​were calculated using GraphPad Prism (GraphPad Software, San Diego, California). EC values ​​were calculated for clones 31 and 54 of the anti-CD137 antibody. 50 The value showed binding activity comparable to that of the reference antibody. EC calculated for anti-CD137 specific antibody leads. 50 The value showed good binding activity compared to the reference antibody (Figure 5).

[0088] Before color development, anti-PD-L1 antibodies were detected using HRP-labeled anti-human IgG1 Fab antibodies, and the OD450 readings were plotted against the anti-PD-L1 concentration.

[0089] Example 5 This example describes the evaluation of protein aggregation of highly concentrated anti-CD137 antibody leads using SEC-HPLC.

[0090] SEC-HPLC was performed using a Waters Alliance Separation Module 2695 equipped with a Waters 2996 Photodiode Array detector. For SEC separation, a homogeneous solvent, 25 mM sodium phosphate, 200 mM NaCl, pH 6.8, was used as the mobile phase buffer. Samples were loaded onto an XBridge Protein BEH SEC column (Waters, catalog number #176007640). The flow rate was 0.4 mL / min, and the sample injection volume was 10 μL. Peaks were detected by absorbance at 280 nm. Before injection into the SEC column, all samples were filtered through a 0.22 μm filter (Millipore, catalog number #SLGP003RB) to remove any precipitated protein material. Data were analyzed using Empower 2 software. As shown in Figure 6, the main peak percentages of clones 31 and 54 of the highly concentrated anti-CD137 antibody exceeded 90%, and there was no apparent protein aggregation.

[0091] These results indicate that high concentrations of anti-CD137 antibody do not result in aggregation.

[0092] Example 6 This example describes the agonist activity of the anti-CD137 antibody.

[0093] Purified antibody leads were functionally screened for their ability to enhance human CD3+ cell activation, as seen in enhanced cytokine production, proliferation, and induction of human CD3+ T cell proliferation. Anti-CD3 antibody (1 μg / ml, OKT3, BioLegend, catalog no. 317304), anti-CD137 antibody leads, or isotype antibodies (1, 3, and 10 μg / ml) were coated onto Maxisorp 96-well plates. Human CD3+ T cells were isolated from the peripheral blood of healthy adult volunteers using RosetteSep® Human T Cell Enrichment Cocktail (STEMCELL, catalog no. 15061). Isolated CD3+ T cells were labeled with CFSE (CellTrace® CFSE cell proliferation kit, Life Technologies, catalog number C34554) and seeded in wells pre-coated with RPMI1640 medium (containing 10% fetal bovine serum, 2.5 mM L-glutamine, and 1 × penicillin / streptomycin) (1 × 10^5 cells per well). After 3 days, cell proliferation was analyzed by flow cytometry, and cytokine production of IL-2 and INF-γ was analyzed by ELISA. As shown in Figures 7 and 8, anti-CD137 antibody leads #15, #31, and #54 showed dose-dependent and donor-dependent agonist activity that enhanced CD3+ T cell activation in at least two of the four donors tested. Clones #31 and #54 of the anti-CD137 group exhibited comparable or higher agonist activity compared to the reference antibody (utomirumab; Chin et al., 2018; antibody sequence; also see U.S. Patent No. 8,337,850), as evidenced by enhanced T cell activation. Therefore, clones #31 and #54 were selected for the construction of bispecific antibodies, as described below.

[0094] Example 7 This embodiment describes combination therapy with anti-PD-L1 and anti-CD137 antibodies in a mixed lymphocyte reaction.

[0095] Mononuclear cells were isolated from the peripheral blood of healthy donors using RosetteSep® Human Monocyte Enrichment Cocktail (catalog number 15068) and cultured for 6 days in RPMI1640 differentiation medium containing human GM-CSF and IL-4 (1000 U / ml each, R&D). Dendritic cell (DC) differentiation was verified by flow cytometry based on the expression of DC-SIGN, CD14, CD80, or CD83. Differentiated DCs were used as antigen-presenting cells (APCs) in mixed lymphocyte reactions (MLR). Allogeneic CD4+ T cells were isolated from human peripheral blood using RosetteSep® Human CD4+ T Cell Enrichment Cocktail (catalog number 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 for 3–5 days in the presence of antibody leads (0.4, 2, and 10 μg / ml). CD4+ T cell proliferation was analyzed by flow cytometry, and cytokine production of IL-2 and IFN-γ in the culture medium was detected by ELISA. IL-2 and IFN-γ production was significantly increased in the presence of anti-PD-L1 antibody in the MLR compared to isotype control antibodies. Interestingly, anti-CD137 antibodies, such as clone #31, enhanced anti-PD-L1 antibody-mediated IFN-γ production in the MLR in two distinct donor pairs, as shown in Figure 9.

[0096] Example 8 This example describes the effect of an anti-CD137 antibody on the CD137-CD137L interaction.

[0097] HEK-293F / CD137 cells were incubated on ice for 30 minutes with isotype control and anti-CD137 antibody (50 μg / ml), washed twice with PBS / 2% FBS (PBS2), and then incubated on ice for 20 minutes with His-labeled 4-1BBL (0.5 μg / ml, Acro BIOSYSTEMS). After two washes with PBS2, the presentation of anti-CD137 antibody and 4-1BBL on HEK-293F / CD137 cells was detected using anti-human Fc-A488 (Jackson ImmunoResearch) and anti-His antibody-APC (Biolegend), respectively, followed by analysis using a Calibur flow cytometer (BD). Almost all cells were A488 positive, except for those incubated with the isotype control. The mean fluorescence intensity (MFI) of the APC channel was calculated using FlowJo (TreeStar, LLC), and the values ​​are shown as a histogram (Figure 10). As shown in Figure 10, reference antibody 1 (ref1) and clone #54 effectively blocked the CD137-CD137L interaction, while reference antibody 2 (ref2), clone #15, and clone #31 were ineffective or inefficient in blocking the CD137-CD137L interaction.

[0098] Example 9 This embodiment describes the pharmacokinetics of an anti-CD137 antibody lead in vivo.

[0099] Antibodies were administered to SCID-beige mice by intravenous bolus injection at a dose of 5 mg per kg of body weight. Peripheral blood was collected at the time shown in the figure after injection. Antibody plasma concentrations were detected by ELISA as described below. Wells pre-coated with CD137-human Fc (1 μg / mL) were incubated with gradually increasing concentrations of purified anti-CD137 IgG4 antibody to create a standard curve for calculating antibody concentrations in plasma (using fresh preparations in blocking solution). Samples collected at various time points were added to CD137-human Fc wells pre-coated for detection. After washing with 0.1% Tween-20 in PBS before color development, the bound antibodies were detected using HRP-labeled anti-human Fab antibody (0.4 μg / mL). Antibody plasma concentrations were calculated by interpolation. PK parameters were calculated using PKSolver software (Zhang, Huo, Zhou, & Xie, 2010). The antibody showed good results for approximately 176 hours. 1 / 2 This resulted in an AUC of approximately 7800 ug / ml*h (Figure 11).

[0100] Example 10 This example describes the crosslinking-dependent agonist activity of an anti-CD137 antibody lead.

[0101] Stable clones of CD137 reporter cells were generated by transfection of HEK293 cells with NF-κB-mediated luciferase and full-length CD137, followed by selection with hygromycin and G418, respectively. For agonist activity assays, the reporter cells were incubated with anti-CD137 antibodies (10, 2, and 0.4 μg / ml), either alone or crosslinked with goat anti-human IgG (5 μg / ml, Jackson ImmunoResearch, catalog no. 109-006-008), for 5 hours. Luciferase activity was detected using the ONE-Glo® luciferase activity assay system (Promega, catalog no. E6120). Consistent with previous reports, utomirumab (CD137 ref, Figure 12) exhibited crosslinking-dependent agonist activity, while urerumab (CD137 ref2, Figure 12) showed crosslinking-independent agonist activity that can cause the severe hepatotoxicity observed in clinical trials. Compared to utomirumab, CD137#54 showed greater agonist activity upon crosslinking, while its agonist activity without crosslinking was moderate, similar to that of utomirumab (Figure 12). Without being limited by theory, this property of CD137#54 could induce target-dependent T cell activation when contained in bispecific antibodies with tumor-specific binders.

[0102] In summary, these results demonstrate the distinct cross-linking dependence of anti-CD137#15, anti-CD137#31, and anti-CD137#54 agonist activity.

[0103] Example 11 This embodiment describes the construction, expression, and purification of an anti-PD-L1-CD137 bispecific antibody.

[0104] Clone 6 of the anti-PD-L1 antibody was used in ADCC-free IgG form, and the anti-CD137 antibody was used in scFv format, and both were fused to the C-terminus of the antibody Fc region of clone 6 of anti-PD-L1. The bispecific antibody construct containing the Fc region of the anti-PD-L1 antibody fused with CD137 scFv is schematically shown in Figure 10 and in Table 1 (sequence) below. A short, flexible peptide linker (GGGGS) 2 (SEQ ID NO: 29) was placed between the heavy chain C-terminus of the anti-PD-L1 antibody's Fc region (SEQ ID NO: 25 or SEQ ID NO: 26) and the N-terminal module of the anti-CD137 scFv to ensure accurate folding and minimize steric hindrance. The amino acid sequences of the anti-PD-L1-CD137 scFv heavy chain are shown in SEQ ID NOs: 31 and 32. The antibody-Fc fusion protein construct was expressed using the Gibco ExpiCHO expression system and purified from the cell culture supernatant of transfected cells via a single-step protein G chromatography process.

[0105] The antibody fused to anti-CD137 scFv may include, in addition to the bispecific anti-PD-L1 antibody Fc fused to anti-CD137 scFv as described above, anti-inhibitory immune checkpoint antibodies such as anti-PD-1, anti-CTLA-4, anti-LAG3, and others, or immunostimulatory antibodies such as anti-CD28, anti-CD40, anti-CD137, anti-CD27, anti-ICOS, and others. For each bispecific antibody, it is possible to generate a bispecific antibody by placing a certain linker between the antibody Fc domain and anti-CD137 scFv.

[0106] The purity of the bispecific antibody exceeded 90% (Figures 14 and 15). This purity was achieved in a single-step purification process and was consistent with the purified fusion protein having an accurate molecular weight (Mw = 220 kDa). Figure 14 shows a representative PAGE gel analysis of the purified anti-PD-L1-CD137 bispecific antibody (bsAb). Culture supernatant from mammalian cells collected 4 days after transfection was purified using protein G chromatography (Thermo Fisher). The purified protein was analyzed under reducing and non-reducing conditions before being loaded onto the gel (3 μg / lane). The results showed that under non-reducing conditions, both proteins had a molecular weight of approximately 220 kDa, and under reducing conditions, the heavy chain -CD137 scFv and light chain had molecular weights of approximately 85 kDa and 25 kDa, respectively. Figure 15 shows the purity and integrity of anti-PD-L1 #6-CD137 #54 bsAb purified by one step of Protein A using μCE-SDS.

[0107] Example 12 This embodiment describes antigen recognition by an anti-PD-L1-CD137 bispecific antibody.

[0108] The binding activity of the anti-PD-L1-CD137 bispecific antibody was determined by ForteBio® (Menlo Park, California) biosensor analysis. His-labeled CD137 (ACROBiosystems) was loaded at 5 μg / mL in DPBS containing 0.02% Tween-20 and 0.1% BSA onto a HIS1K (anti-Penta-HIS) biosensor (catalog no. 18-5120) for 5 minutes. The sensor was then exposed to the antibody as shown at 100 nM using the same buffer for 5 minutes, followed by binding with a second antigen (PD-L1 fused to the mouse Fc domain) at 100 nM for another 5 minutes. The binding chart shown in Figure 16 was then created using Octet Data Acquisition and Analysis software as described by the manufacturer. Compared to the control antibody, both bispecific antibodies (anti-PD-L1#6-CD137#31 and anti-PD-L1#6-CD137#54) were able to first recognize CD137 and then similarly recognize PD-L1, demonstrating that bispecific antibodies can simultaneously target PD-L1 and CD137.

[0109] In summary, these results indicate that the anti-PD-L1-CD137 bispecific antibody simultaneously recognizes PD-L1 and CD137, as determined by ForteBio® biosensor analysis.

[0110] Example 13 This embodiment describes the enhancement of T cell activation by anti-PD-L1 antibody and anti-PD-L1-CD137 scFv bispecific antibody (bsAb) in a mixed allogeneic lymphocyte reaction.

[0111] Mononuclear cells were isolated from the peripheral blood of healthy donors using RosetteSep® Human Monocyte Enrichment Cocktail (catalog no. 15068) and cultured for 6 days in RPMI1640 differentiation medium containing human GM-CSF and IL-4 (1000 U / ml each, R&D). Allogeneic CD4+ T cells were isolated from human peripheral blood using RosetteSep® Human CD4+ T Cell Enrichment Cocktail (catalog no. 15062). The purity of the CD4+ T cells was approximately 95% based on CD3 and CD4 expression. CFSE-labeled CD4+ T cells were co-cultured with DCs for 3-5 days in the presence of antibody leads (1, 3, and 10 μg / ml). CD4+ T cell proliferation was analyzed by flow cytometry, and cytokine production of IL-2 and IFN-γ in the culture medium was detected by ELISA. Compared to monotherapy and combination therapy with anti-PD-L1 and anti-CD137 antibodies, anti-PD-L1#6-CD137#54 significantly enhanced T cell activation (Figure 17, showing results for two donor pairs).

[0112] These results indicate that anti-PD-L1#6-CD137#54 bsAb induced more potent T cell activation in mixed lymphocyte responses compared to treatment with anti-PD-L1 and anti-CD137 antibodies alone or in combination, and compared to treatment with anti-PD-L1#6-CD137#31 bsAb.

[0113] Example 14 This example describes the enhancement of antigen-specific T cell activation by an anti-PD-L1-CD137 scFv bispecific antibody lead.

[0114] Human memory CD4 T cells and CD8 T cells were isolated using the EasySep® Human Memory CD4+ T Cell Enrichment Kit (STEMCELL, catalog no. 19157) and the Human CD8+ T Cell Isolation Kit (STEMCELL, catalog no. 17953), respectively. Memory CD4-T cells were co-cultured with autologous immature DCs and stimulated with CEFX Ultra SuperStim Pool MHC-II subset (1 ug / ml, JPT) for 7 days in the presence of antibodies (0.4, 2, and 10 μg / ml). TLR-DCs were generated for co-culture with CD8-T cells. 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), poly(I:C) (10 μg / ml, Sigma), and R848 (5 μg / ml, Sigma) to the differentiation medium. Co-cultures of CD8 T cells and autologous TLR-DCs were stimulated for 7 days with CEFX Ultra SuperStim Pool (1 μg / ml, JPT) in the presence of antibodies (0.4, 2, and 10 μg / ml). Similar to the results observed in the MLR of Example 12, anti-PD-L1#6-CD137#54 bsAb enhanced the recall response of memory CD4 T cells (Figure 18, A) and CD8 T cells (Figure 18, B) compared to monotherapy with anti-PD-L1 or anti-CD137 monoclonal antibodies or combination therapy with anti-PD-L1 and anti-CD137 monoclonal antibodies (Figures 18A-18B).

[0115] In summary, the results shown in Figure 17 (Example 13) and Figure 18 (This Example) demonstrate that anti-PD-L1#6-CD137#54 bsAb significantly enhanced T cell activation that was more robust than that observed with treatment using either anti-PD-L1 or anti-CD137 monoclonal antibodies alone or in combination. Furthermore, the T cell activation observed with anti-PD-L1#6-CD137#54 bsAb was antigen-dependent, as seen in recall response assays in CD4 and CD8 T cells. Without being limited by theory, it is suggested that the greater T cell activation observed with anti-PD-L1#6-CD137#54 bsAb compared to anti-PD-L1#6-CD137#31 bsAb allows the anti-CD137#54 arm of the bispecific antibody to bind to the intrinsic CD137 epitope without steric hindrance, resulting in the binding of the anti-PD-L1 arm of the bispecific antibody to PD-L1.

[0116] Example 15 This example describes target-dependent T cell activation induced by an anti-PD-L1-CD137 bispecific antibody.

[0117] Human T cells were isolated using RosetteSep® Human T Cell Enrichment Cocktail (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 cells or parental HEK293 cells under antibody treatment as shown (Figure 19). Compared to monotherapy or combination therapy with anti-PD-L1 and anti-CD137 monoclonal antibodies, the anti-PD-L1-CD137 bispecific antibody significantly enhanced T cell activation when co-cultured with PD-L1 overexpressing cells, as shown in Figure 19, but not significantly enhanced when co-cultured with PD-L1-negative parental cells.

[0118] In summary, these results indicate that, in the presence of plate-bound anti-CD3 (OKT3), target-dependent T cell activation was induced only by anti-PD-L1#6-CD137 bsAb when co-cultured with PD-L1 overexpressing HEK-293 cells, rather than with parental HEK293 cells.

[0119] Example 16 This example describes tumor antigen-dependent T cell activation induced by an anti-tumor antigen-specific CD137#54 bispecific antibody.

[0120] 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 the presence of anti-CD3 (OKT3) coated polybeads in a 1:1 ratio. After 3 days, T cell activation was analyzed based on IFN-γ production measured by ELISA, and the cytotoxicity of tumor cells was detected using the CytoTox 96® cytotoxicity assay (Promega, catalog number #G1780).

[0121] Compared to monotherapy or combination therapy with anti-PD-L1#6 antibody and anti-CD137#54 antibody, more robust IFN-γ production was induced by anti-PD-L1#6-CD137#54 bsAb (Figure 20). Higher tumor cell cytotoxicity of CD8 T cells was observed by co-culture with PC-3 cells (Figure 20B). In addition to PD-L1-positive tumors, Her2-positive (SKBR-3 and MDA-MB-361) tumor cells and glycan-positive (MCF-7 and NCI-N87) tumor cells targeted with anti-Her2 (trastuzumab or #3-7) and anti-tumor glycan antibodies bound to CD137#54 scFv also resulted in far more potent CD8 T-cell IFN-γ production (Figures 21A-21B and 22A-22B) and tumor cell cytotoxicity (Figure 22A) compared to monotherapy or combination therapy.

[0122] These results indicate that target-dependent T cell activation was specifically induced by tumor-targeting CD137#54 bsAb.

[0123] Example 17 This embodiment describes the induction of CD137 uptake by an anti-PD-L1#6-CD137#54 bispecific antibody.

[0124] To investigate whether CD137 uptake is also induced not only by the anti-PD-L1#6-CD137#54 bispecific antibody but also by the reference antibodies urelumab and utoliumab, an uptake assay was performed using CD137-expressing HEK293 cells (Figure 23). 5 × 10^3 CD137-expressing cells per well were pre-seed in a black 96-well plate containing Dulbecco's Modified Eagle Medium (Invitrogen) with 10% fetal bovine serum (Gibco) and incubated overnight at 37°C and 5% CO2. Antibodies as shown were labeled with pHAb amine-reactive dye (Promega Corp.) according to the manufacturer's protocol and then prepared in medium as 3-fold serial dilutions starting from 100 nM. The medium of the pre-seed cells was then replaced with medium containing the labeled antibody, and the cells were cultured in an incubator for a further 24 hours. After incubation, cells were rinsed and maintained in PBS for fluorescence recording with SpectraMax iD3. EC50 values ​​were calculated using GraphPad Prism. As shown in Figure 23, stronger induction of CD137 uptake was observed with treatment with anti-PD-L1#6-CD137#54 bispecific antibody compared to treatment with reference anti-CD137 antibody. Without being limited by theory, if the bispecific antibody (Ab) binds to T cells only via CD137 binding, this level of CD137 uptake may reduce CD137 activation. Therefore, when a bispecific antibody is administered in vivo, lower toxicity may be observed with the bispecific antibody compared to the reference antibody, and especially compared to urelumab.

[0125] Example 18 This example describes the rescue of T-reg cell-mediated inhibition of T cell proliferation by an anti-PD-L1#6-CD137#54 bispecific antibody.

[0126] The T-reg suppression assay was established using the mixed lymphocyte reaction described in Example 12. T-reg cells were EasySep® Human CD4 + CD127 low CD25 + Regulatory T cells were isolated from peripheral blood using a regulatory T cell isolation kit (STEMCELL, catalog number 18063) and then expanded using Dynabeads® human T-reg expander (Gibco, catalog number 11129D). The expanded T-reg cells significantly suppressed CD4 T cell proliferation and IL-2 production. The suppressive activity of T-reg cells was abolished by the addition of anti-PD-L1#6-CD137#54 bsAb to the culture medium (Figures 24A-24B). Anti-PD-L1#6-CD137#54 bsAb rescued T cell proliferation (Figure 24, A) and cytokine production (Figure 24, B) in the presence of T-reg cells. These results indicate that anti-PD-L1#6-CD137#54 bsAb can rescue T-reg-mediated suppression of T cell activation.

[0127] Example 19 This example describes the inhibition of tumor growth in vivo by an anti-PD-L1#6-CD137#54 bispecific antibody.

[0128] To verify the antitumor activity of anti-PD-L1#6-CD137#54 bsAb, 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 then xenografted subcutaneously into SCID-beige mice to evaluate in vivo anticancer activity. Seven days after tumor inoculation, equimolar doses of mAb (molecular weight 150 kDa, 1 mg / kg) and bsAb (molecular weight 195 kDa, 1.3 mg / kg) were administered intraperitoneally twice a week. Tumor size (mm 3The tumor growth inhibition index (TGI) was measured twice a week and calculated as (length × width × width) / 2. In the NCI-H292 tumor model, the TGI of anti-PD-L1#6-CD137#54 bsAb (TGI: 67.5%) exceeded that of MPDL-3280a (TGI: 44.3%) and the TGI of the PD-L1#6+CD137#54 combination (TGI: -18.77%) (Figure 25, A). Similarly, in the NCI-H1975 tumor model, the TGI of anti-PD-L1#6-CD137#54 bsAb (TGI: 80%) exceeded that of the PD-L1#6+CD137#54 combination (TGI: 67.2%) (Figure 25, B). In addition to lung cancer, anti-PD-L1#6-CD137#54 bsAb also showed greater antitumor activity (TGI of 43% at 1.3 mg / kg) compared to combination therapy (TGI of -9.2% at 10 mg / ml, respectively) in the BxPC-3 pancreatic cancer model (Figure 25, Figure C). In other words, anti-PD-L1#6-CD137#54 bsAb demonstrated antitumor activity in two different cancer models in vivo.

[0129] In summary, these results indicate that in a mouse xenograft tumor model, treatment with anti-PD-L1#6-CD137#54 bsAb results in greater inhibition of tumor growth compared to combination therapy with anti-PD-L1 and anti-CD137 antibodies.

[0130] Example 20 This embodiment describes cytokine release in vitro in the presence of bispecific antibodies.

[0131] Human PBMCs from three donors were incubated for 24 hours at 0.67, 6.67, and 66.67 nM 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 bound to CD137#54 scFv). Cytokines released into the culture medium were detected by multiplex ProcartaPlex immunoassay (ThermoFisher Scientific). OKT3 induced significant cytokine release, while the three bispecific antibodies did not (Figure 26).

[0132] These results indicate that, compared to OKT3, bsAbs of PD-L1#6-CD137#54, Her2#3-7-CD137#54, and glycan-CD137#54 did not induce significantly higher cytokine release than the background when incubated with human PBMCs.

[0133] Example 21 This embodiment describes the pharmacokinetic parameters of the anti-PD-L1#6-CD137#54 bispecific antibody in rhesus monkeys.

[0134] Bispecific anti-PD-L1#6-CD137#54 antibodies (5 and 25 mg per kg of body weight) were administered intravenously as a bolus injection to two groups of rhesus monkeys (one male and one female per group). Peripheral blood samples were collected at 0.5, 6, 24, 48, 72, and 144 hours post-injection. Plasma concentrations of the antibodies were determined by ELISA. Maxi-soap plates (Invitrogen) were coated with CD137-Fc fusion protein (AP Biosciences, 1 μg / mL), and then serially diluted plasma samples and anti-PD-L1#6-CD137#54 bsAb were added as a standard curve. The bound antibodies were detected using TMB substrate with biotinylated PD-L1-Fc fusion protein (AP Biosciences) and HRP-labeled streptavidin. Plasma concentrations of the antibodies were calculated by interpolation. PK parameters were calculated using PKSolver software (Zhang, Huo, Zhou, & Xie, 2010). The t of anti-PD-L1#6-CD137#54 bsAb was calculated. 1 / 2 The response times were approximately 87 and 49 hours in the 25 mg / kg injection group and the 5 mg / kg injection group, respectively (Figures 27A-27B), and no increase in ALT / AST levels was observed during the experiment (not shown).

[0135] array TIFF2026086946000002.tif158149TIFF2026086946000003.tif222149TIFF20260869460 00004.tif228149TIFF2026086946000005.tif229149TIFF2026086946000006.tif211149

[0136] (Table 1) Sequence of defined CDR regions of monoclonal antibodies and single-strand variable fragments (scFv) of bispecific antibodies TIFF2026086946000007.tif137154

[0137] References TIFF2026086946000008.tif45139

[0138] As used herein and in the appended claims, the singular forms "a," "an," and "the" imply the plural form unless otherwise explicitly stated in the context. That is, for example, the phrase "the method" includes one or more methods and / or steps of the type described herein, which would become apparent to a person skilled in the art by reading this disclosure.

[0139] The publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and separately indicated as if each individual publication, patent, or patent application were incorporated by reference.

[0140] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Any methods and substances similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but it will be understood that modifications and variations are included within the spirit and scope of this disclosure.

[0141] Scope: Throughout this disclosure, various aspects of the invention may exist in the form of scope. It should be understood that descriptions in the form of scope are merely for convenience and brevity and should not be interpreted as fixed limitations on the scope of the invention. Therefore, a description of a scope should be considered to specifically disclose not only all possible sub-scopes but also the individual numbers within that scope. For example, a description of a scope such as 1-6 should be considered to have not only the specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, but also the individual numbers within that scope 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 applies regardless of the width of the scope.

[0142] While the present invention has been described with reference to the above embodiments, it should be understood that modifications and variations are included within the spirit and scope of the present invention. Therefore, the present invention is limited only to the following claims.

[0143] Sequence information SEQUENCE LISTING <110> AP BIOSCIENCES, INC. <120> ANTIBODIES FOR T-CELL ACTIVATION <150> US 62 / 866,699 <151> 2019-06-26 <150> US 62 / 953,302 <151> 2019-12-24 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 clone 15 heavy chain <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asp Leu Tyr Gln Leu Leu Phe Pro Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 clone 15 light chain <400> 2 Gln Leu Val Leu Thr Gln Pro Pro Ser Ala Ser Ala Ser Leu Gly Ala 1 5 10 15 Ser Val Thr Leu Thr Cys Thr Leu Ser Ser Gly Tyr Ser Asn Tyr Lys 20 25 30 Val Asp Trp Tyr Gln Gln Arg Pro Gly Lys Gly Pro Arg Phe Val Met 35 40 45 Arg Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Asp Gly Ile Pro 50 55 60 Asp Arg Phe Ser Val Leu Gly Ser Gly Leu Asn Arg Tyr Leu Thr Ile 65 70 75 80 Lys Asn Ile Gln Glu Glu Asp Glu Ser Asp Tyr His Cys Gly Ala Asp 85 90 95 His Gly Ser Gly Ser Asn Leu Phe Trp Val Phe Gly Gly Gly Thr Lys 100 105 110 Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe 115 120 125 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 130 135 140 Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala 145 150 155 160 Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys 165 170 175 Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 180 185 190 Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu 195 200 205 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 220 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H1 of anti-CD137 clone 15 <400> 3 Gly Gly Thr Phe Ser Ser Tyr 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H2 of anti-CD137 clone 15 <400> 4 Ile Pro Ile Leu Gly Ile 1 5 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H3 of anti-CD137 clone 15 <400> 5 Asp Leu Tyr Gln Leu Leu Phe Pro Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L1 of anti-CD137 clone 15 <400> 6 Thr Leu Ser Ser Gly Tyr Ser Asn Tyr Lys Val Asp 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L2 of anti-CD137 clone 15 <400> 7 Val Gly Thr Gly Gly Ile Val Gly Ser Lys Gly Asp 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L3 of anti-CD137 clone 15 <400> 8 Gly Ala Asp His Gly Ser Gly Ser Asn Leu Phe Trp Val 1 5 10 <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 clone 31 heavy chain <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Arg Gly Ala Phe Asp Pro Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 clone 31 light chain <400> 10 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asn Phe Val Ser Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Glu Leu 35 40 45 Met Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ile Thr Arg Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H1 of anti-CD137 clone 31 <400> 11 Gly Tyr Thr Phe Thr Gly Tyr 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H2 of anti-CD137 clone 31 <400> 12 Asn Pro Asn Ser Gly Gly 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H3 of anti-CD137 clone 31 <400> 13 Asp Leu Arg Gly Ala Phe Asp Pro 1 5 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L1 of anti-CD137 clone 31 <400> 14 Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr Asn Phe Val Ser 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L2 of anti-CD137 clone 31 <400> 15 Asp Val Ser Asp Arg Pro Ser 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L3 of anti-CD137 clone 31 <400> 16 Ser Ser Tyr Thr Ser Ser Ile Thr Arg Tyr Val 1 5 10 <210> 17 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 clone 54 heavy chain <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Thr Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 18 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-CD137 clone 54 light chain <400> 18 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr Tyr 85 90 95 Val Phe Gly Thr Gly Thr Lys Val Thr Val Phe Gly Gln Pro Lys Ala 100 105 110 Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 130 135 140 Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val 145 150 155 160 Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser 210 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H1 of anti-CD137 clone 54 <400> 19 Gly Gly Thr Phe Ser Ser Tyr 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H2 of anti-CD137 clone 54 <400> 20 Ile Pro Ile Leu Gly Ile 1 5 <210> 21 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H3 of anti-CD137 clone 54 <400> 21 Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp 1 5 10 15 Ile <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L1 of anti-CD137 clone 54 <400> 22 Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala Ser 1 5 10 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L2 of anti-CD137 clone 54 <400> 23 Gln Asp Ser Lys Arg Pro Ser 1 5 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L3 of anti-CD137 clone 54 <400> 24 Gln Ala Trp Asp Gly Ser Ser Thr Tyr Val 1 5 10 <210> 25 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> constant domain in heavy chain (IgG4) <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 26 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> constant domain in heavy chain (engineered IgG1) <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 27 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> SP1 <400> 27 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 28 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> GS linker <400> 28 Gly Gly Gly Gly Ser 1 5 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> (G4S)2 linker <400> 29 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 30 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-PD-L1#6 Light Chain <400> 30 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Leu Ser Leu 85 90 95 Asn Ala Trp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 31 <211> 700 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-PD-L1 #6-CD137 #31 bsAb Heavy Chain <400> 31 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Gln Ser Ala Leu Thr Gln Pro Ala 450 455 460 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 465 470 475 480 Thr Ser Ser Asp Val Gly Ala Tyr Asn Phe Val Ser Trp Tyr Gln Gln 485 490 495 Arg Pro Gly Lys Ala Pro Glu Leu Met Ile Tyr Asp Val Ser Asp Arg 500 505 510 Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 515 520 525 Ala Ser Leu Thr Ile Ser Gly Leu Gln Thr Glu Asp Glu Ala Asp Tyr 530 535 540 Tyr Cys Ser Ser Tyr Thr Ser Ser Ile Thr Arg Tyr Val Phe Gly Thr 545 550 555 560 Gly Thr Lys Val Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly 565 570 575 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 580 585 590 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 595 600 605 Tyr Thr Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly 610 615 620 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 625 630 635 640 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 645 650 655 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 660 665 670 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Leu Arg Gly Ala Phe Asp Pro 675 680 685 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 690 695 700 <210> 32 <211> 712 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-PD-L1#6-CD137 #54 bsAb Heavy Chain <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser 450 455 460 Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val Lys Val Ser Cys Lys 465 470 475 480 Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln 485 490 495 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Ile Leu 500 505 510 Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr 515 520 525 Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg 530 535 540 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Pro Pro Tyr Tyr Asp 545 550 555 560 Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp Ile Trp Gly Gln Gly 565 570 575 Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu 595 600 605 Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Ser Ile 610 615 620 Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala Ser Trp Tyr Gln 625 630 635 640 Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile Tyr Gln Asp Ser Lys 645 650 655 Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn 660 665 670 Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala Gly Asp Glu Ala Asp 675 680 685 Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr Tyr Val Phe Gly Thr 690 695 700 Gly Thr Lys Val Thr Val Leu Gly 705 710 <210> 33 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CD137#31-scFv <400> 33 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asn Phe Val Ser Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Glu Leu 35 40 45 Met Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ile Thr Arg Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 130 135 140 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 165 170 175 Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly 180 185 190 Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu 195 200 205 Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Asp Leu Arg Gly Ala Phe Asp Pro Trp Gly Gln Gly Thr Thr Val Thr 225 230 235 240 Val Ser Ser Ala <210> 34 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CD137#54-scFv <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Thr Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser 145 150 155 160 Pro Gly Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu 165 170 175 Lys Tyr Ala Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu 180 185 190 Val Ile Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe 195 200 205 Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu 210 215 220 Gln Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser 225 230 235 240 Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 245 250 255 <210> 35 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Trastuzumab Light Chain <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 36 <211> 714 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Trastuzumab-CD137 #54 bsAb Heavy Chain <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val 450 455 460 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val Lys Val Ser 465 470 475 480 Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val 485 490 495 Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Ile Pro 500 505 510 Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr 515 520 525 Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser 530 535 540 Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Pro Pro Tyr 545 550 555 560 Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp Ile Trp Gly 565 570 575 Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly Gly Ser Gly 580 585 590 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr 595 600 605 Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala 610 615 620 Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr Ala Ser Trp 625 630 635 640 Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile Tyr Gln Asp 645 650 655 Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser 660 665 670 Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala Gly Asp Glu 675 680 685 Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr Tyr Val Phe 690 695 700 Gly Thr Gly Thr Lys Val Thr Val Leu Gly 705 710 <210> 37 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-Her2 #3-7 Light Chain <400> 37 Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 Tyr Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr 35 40 45 Leu Ile Tyr Ser Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Val Leu Tyr Met Gly Ser 85 90 95 Gly Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 38 <211> 717 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-Her2 #3-7-CD137 #54 bsAb Heavy Chain <400> 38 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Asp Asn Trp Asn His Gly Pro Tyr Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 450 455 460 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val 465 470 475 480 Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile 485 490 495 Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg 500 505 510 Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly 515 520 525 Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 530 535 540 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 545 550 555 560 Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp 565 570 575 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 595 600 605 Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly 610 615 620 Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr 625 630 635 640 Ala Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile 645 650 655 Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly 660 665 670 Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala 675 680 685 Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr 690 695 700 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 705 710 715 <210> 39 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-Glycan Light Chain <400> 39 Glu Ile Val Leu Thr Gln Ser Pro Ser Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Glu Asp Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Gln Pro Trp Ile Tyr 35 40 45 Gly Thr Ser Asn Lys Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg Arg Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 40 <211> 717 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> Anti-Glycan-CD137 #54 bsAb Heavy Chain <400> 40 Gln Ile Thr Leu Gln Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Tyr Arg Phe 20 25 30 Asp Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Val Arg Gly Leu His Asp Tyr Tyr Tyr Tyr Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 450 455 460 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Thr Val 465 470 475 480 Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile 485 490 495 Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg 500 505 510 Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln Gly 515 520 525 Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 530 535 540 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 545 550 555 560 Pro Pro Tyr Tyr Asp Ser Ser Gly Tyr Tyr Pro Leu Gly Ala Phe Asp 565 570 575 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 595 600 605 Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly 610 615 620 Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Glu Lys Tyr 625 630 635 640 Ala Ser Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Ile Leu Val Ile 645 650 655 Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly 660 665 670 Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala 675 680 685 Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Gly Ser Ser Thr 690 695 700 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 705 710 715 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H1 of anti-Her2#3-7 <400> 41 Gly Tyr Ser Phe Thr Ser Tyr 1 5 <210> 42 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H2 of anti-Her2#3-7 <400> 42 Tyr Pro Gly Asp Ser Asp 1 5 <210> 43 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-H3 of anti-Her2#3-7 <400> 43 Gln Asp Asn Trp Asn His Gly Pro Tyr Asp Ala Phe Asp Ile 1 5 10 <210> 44 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L1 of anti-Her2#3-7 <400> 44 Gly Leu Ser Ser Gly Ser Val Ser Thr Ser Tyr Tyr Pro Ser 1 5 10 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L2 of anti-Her2#3-7 <400> 45 Ser Thr Asn Thr Arg Ser Ser 1 5 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <223> CDR-L3 of anti-Her2#3-7 <400> 46 Val Leu Tyr Met Gly Ser Gly Ile Trp Val 1 5 10

Claims

1. (i) A V containing an amino acid sequence that has at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO:

17. Ｈ Domain and, (ii) A V containing an amino acid sequence that has at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO:

18. Ｌ Domain and An antibody or antigen-binding fragment thereof, comprising the antibody or antigen-binding fragment thereof, which binds to CD137.

2. The aforementioned V Ｈ The region includes an amino acid sequence having at least 80% identity with SEQ ID NO: 1, and the V Ｌ The antibody or antigen-binding fragment according to claim 1, wherein the region comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

2.

3. (a) V Ｈ CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 3, CDR-H2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and CDR-H3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 5, the V Ｈ The CDR-H1, CDR-H2, and CDR-H3, (b) V Ｌ CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 6, CDR-L2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and CDR-L3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 8, the V Ｌ CDR-L1, CDR-L2, and CDR-L3 The antibody or antigen-binding fragment according to claim 2, comprising:

4. The above-mentioned V Ｈ region contains an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and the above-mentioned V Ｌ region contains an amino acid sequence having at least 80% identity with SEQ ID NO: 10, the antibody or antigen-binding fragment according to claim 1.

5. (a) V Ｈ CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 11, CDR-H2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 12, and CDR-H3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 13, the V Ｈ The CDR-H1, CDR-H2, and CDR-H3, (b) V Ｌ CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 14, CDR-L2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 15, and CDR-L3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 16, the V Ｌ CDR-L1, CDR-L2, and CDR-L3 The antibody or antigen-binding fragment according to claim 4, comprising:

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

18.

7. (a) V Ｈ CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 19, CDR-H2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 20, and CDR-H3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 21, the V Ｈ The CDR-H1, CDR-H2, and CDR-H3, (b) V Ｌ CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 22, CDR-L2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 23, and CDR-L3 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 24, the V Ｌ CDR-L1, CDR-L2, and CDR-L3 The antibody or antigen-binding fragment according to claim 6, comprising:

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, and 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.

11. The antibody or antigen-binding fragment according to claim 10, wherein the IgG1 domain comprises the amino acid sequence of SEQ ID NO:

26.

12. The antibody or antigen-binding fragment according to claim 10, wherein the IgG4 domain comprises the amino acid sequence of SEQ ID NO:

25.

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

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable carrier is bound to the C-terminus of one or more polypeptides of the antibody or antigen-binding fragment.

16. A method for treating cancer in the subject, Administering a therapeutically effective amount of the antibody or antigen-binding fragment or pharmaceutical composition described in any one of claims 1 to 15 to the subject, thereby treating the cancer. The method, including the method described above.

17. The method according to claim 16, wherein the cancer is selected from 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, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.

18. The isolated amino acid sequences described in SEQ ID NOs: 1 to 26.

19. An isolated nucleic acid sequence encoding one of sequence numbers 1 through 26.

20. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to CD137.

21. The aforementioned antibody (i) A V containing an amino acid sequence that has at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO:

17. Ｈ Domain and, (ii) A V containing an amino acid sequence having at least 80% identity with the N-terminal sequence of approximately 100 to 120 amino acids of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO:

18. Ｌ Domain and Includes, The second antigen-binding region binds to an immune checkpoint molecule, an immune-stimulating molecule, or a tumor antigen. The bispecific antibody according to claim 20.

22. The first antigen-binding region described above is (a) V containing the amino acid sequence of SEQ ID NO: 9 Ｈ V includes the region and the amino acid sequence of approximately 100 to 120 amino acids from the N-terminal sequence of Sequence ID No.

10. Ｌ Area, or (b) V containing the amino acid sequence of SEQ ID NO: 17 Ｈ V includes the region and the amino acid sequence of approximately 100 to 120 amino acids from the N-terminal sequence of Sequence ID No.

18. Ｌ region A bispecific antibody according to claim 21, comprising:

23. The bispecific antibody according to claim 21, wherein the second antigen-binding region binds to an antigen selected from PD-L1, PD-1, CTLA-4, LAG3, CD28, CD40, CD137, CD27, ICOS, Her2, or glycan.

24. The bispecific antibody according to claim 23, wherein the second antigen-binding region binds to PD-L1, Her2, or glycan.

25. The bispecific antibody according to claim 23, wherein the first antigen-binding region and the second antigen-binding region include scFv, F(ab)2, Fab, or any combination thereof.

26. The bispecific antibody according to claim 25, wherein the first antigen-binding region comprises scFv and the second antigen-binding region comprises Fab.

27. The aforementioned scFv is, (i) V containing the amino acid sequence of SEQ ID NO: 9 Ｈ V includes the region and the amino acid sequence of approximately 100 to 120 amino acids from the N-terminal sequence of Sequence ID No.

10. Ｌ Area, or (ii) V containing the amino acid sequence of SEQ ID NO: 17 Ｈ V contains the region and approximately 100 to 120 amino acids of the N-terminal sequence of Sequence ID No.

18. Ｌ region A bispecific antibody according to claim 26, comprising:

28. The V of the scFv Ｈ region and the V Ｌ The bispecific antibody according to claim 27, further comprising a linker between the region and the region.

29. The bispecific antibody according to claim 28, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO:

34.

30. The bispecific antibody according to claim 26, further comprising an Fc domain.

31. The bispecific antibody according to claim 30, wherein the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain.

32. The bispecific antibody according to claim 31, wherein the Fc domain is an IgG domain.

33. The bispecific antibody according to claim 32, wherein the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.

34. The bispecific antibody according to claim 30, wherein the scFv is linked to the C-terminus of the Fc domain.

35. The bispecific antibody according to claim 30, further comprising a linker between the Fc domain and the scFv.

36. The bispecific antibody according to claim 30, wherein the Fab is linked to the N-terminus of the Fc domain.

37. The bispecific antibody according to claim 20, comprising the heavy chain sequence of SEQ ID NO: 31 or SEQ ID NO:

32.

38. The bispecific antibody according to claim 37, further comprising the light chain sequence of SEQ ID NO:

30.

39. A bispecific antibody according to claim 20, comprising the heavy chain sequence of SEQ ID NO:

36.

40. The bispecific antibody according to claim 39, further comprising the light chain sequence of Sequence ID No.

35.

41. The bispecific antibody according to claim 20, comprising the heavy chain sequence of SEQ ID NO:

38.

42. The bispecific antibody according to claim 41, further comprising the light chain sequence of Sequence ID No.

37.

43. The bispecific antibody according to claim 20, comprising the heavy chain sequence of SEQ ID NO:

40.

44. The bispecific antibody according to claim 43, further comprising the light chain sequence of Sequence ID No.

39.

45. The isolated amino acid sequences described in SEQ ID NOs. 30 to 40.

46. An isolated nucleic acid sequence encoding one of sequence numbers 30 through 40.

47. An antibody-drug conjugate comprising a therapeutic agent and an antibody or antigen-binding fragment according to any one of claims 1 to 13, or a bispecific antibody or its antigen-binding fragment according to any one of claims 20 to 44.

48. The antibody-drug conjugate according to claim 47, wherein the therapeutic agent is covalently bound to the antibody or antigen-binding fragment via a linker.

49. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 20 to 44 and at least one pharmaceutically acceptable carrier.

50. A method for treating cancer in the subject, Administering a therapeutically effective amount of the bispecific antibody or its antigen-binding fragment according to any one of claims 20 to 44 to the subject, thereby treating the cancer. The method, including the method described above.

51. The method according to claim 50, wherein the cancer is selected from 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, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.

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