Anti-4-1BB*CCR8 antibodies and uses thereof

Abstract

The present disclosure relates to antibodies or antigen-binding fragments thereof comprising a first antigen-binding domain that specifically binds to 4-1BB and a second antigen-binding domain that specifically binds to CCR8.

Description

[0001] Priority claim

[0002] This application claims priority to CN202311271892.4 filed on September 26, 2023. The entire contents of the foregoing are incorporated herein by reference. Technical Field

[0003] The present invention relates to multispecific anti-4-1BB×CCR8 antibodies (eg, bispecific antibodies or antigen-binding fragments thereof) and antibody-drug conjugates derived therefrom. Background Art

[0005] Bispecific antibodies are artificial proteins that can bind to two different types of antigens or two different epitopes at the same time. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivering payloads to target sites. The approval of Catumaxomab (anti-EpCAM and anti-CD3) and Blinatumomab (anti-CD19 and anti-CD3) has become an important milestone in the development of bispecific antibodies.

[0006] Because bispecific antibodies have a variety of applications, there is a need to continue to develop various bispecific antibody-based therapeutic approaches. Summary of the invention

[0008] The present invention relates to multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to 4-1BB and CC motif chemokine receptor 8 (CCR8).

[0009] The CCR8 of the present invention includes CCR8 derived from mice, rats, hamsters, guinea pigs, dogs, pigs, and primates including monkeys and humans. Human CCR8 is preferred.

[0010] The 4-1BB of the present invention includes 4-1BB derived from mice, rats, hamsters, guinea pigs, dogs, pigs, and primates including monkeys and humans. Human 4-1BB is preferred.

[0011] Thus, provided herein is an antibody or antigen-binding fragment thereof comprising: a) a first antigen-binding domain that specifically binds to 4-1BB; b) a second antigen-binding domain that specifically binds to CC motif chemokine receptor 8 (CCR8).

[0012] In some embodiments, 4-1BB is human 4-1BB, and wherein CCR8 is human CCR8.

[0013] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0014] In some embodiments, the second antigen binding domain is a single chain fragment variable (scFv) domain in which VH2 and VL2 are connected by a first linker.

[0015] In some embodiments, the second antigen binding domain is linked to the N-terminus of the light chain via a second linker.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc region.

[0018] In some embodiments, the C-terminus of the VH1 of the first antigen binding domain is linked to the Fc region, optionally through a CH1 domain.

[0019] In some embodiments, the antibody comprises a first heavy chain of VH1 and a first light chain of VL1; and a second heavy chain of VH2 and a second light chain of VL2.

[0020] In some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0021] In some embodiments, the Fc region is the Fc region of human IgG1 or IgG2.

[0022] In some embodiments, the Fc region is a wild-type Fc, or an Fc with enhanced ADCC activity, or an Fc with enhanced ADCP activity.

[0023] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1. The VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR3, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, 3 amino acid sequence are one of the following:

[0024] (1) The selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 4, 5, and 6, respectively;

[0025] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 24, 25, and 26, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 27, 28, and 29, respectively;

[0026] (3) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 50, 51, and 52, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 54, and 55, respectively; and

[0027] (4) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72 and 73, respectively.

[0028] In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH2 CDR3; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL2 CDR3, wherein the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are one of the following:

[0029] (1) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12, and 13, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15, and 16, respectively;

[0030] (2) the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 36, 37, and 38, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 39, 40, and 41, respectively;

[0031] (3) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58, 59 and 60, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62 and 63, respectively; and

[0032] (4) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 76, 77 and 78, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 79, 80 and 81, respectively.

[0033] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 1, 2 and 3, respectively, the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 5 and 6, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12 and 13, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15 and 16, respectively.

[0034] The present disclosure also relates to antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments thereof described herein.

[0035] The present disclosure also relates to methods of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising any one of the antibodies or antigen-binding fragments thereof described herein.

[0036] In some embodiments, the cancer is a solid tumor or a hematological tumor.

[0037] In some embodiments, the cancer is breast cancer, uterine corpus cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, gastric cancer, non-small cell lung cancer, spleen cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer, sarcoma, a blood cell cancer such as a leukemia or lymphoma, bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, or liver cancer.

[0038] In some embodiments, the cancer is breast cancer, uterine corpus cancer, ovarian cancer, lung cancer, colorectal cancer, renal cancer, and sarcoma.

[0039] In some embodiments, the cancer is colon cancer.

[0040] In some embodiments, the subject is further treated with an effective amount of an anti-OX40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, or an anti-PD-L1 antibody.

[0041] The present disclosure also relates to methods of reducing tumor growth rate comprising contacting tumor cells with an effective amount of a composition comprising any one of the antibodies or antigen-binding fragments thereof described herein.

[0042] The present invention also relates to a method for killing tumor cells, comprising contacting the tumor cells with an effective amount of a composition comprising any one of the antibodies or antigen-binding fragments thereof described herein.

[0043] The present disclosure also relates to methods of increasing an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising any one of the antibodies or antigen-binding fragments thereof described herein.

[0044] The present invention also relates to a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier.

[0045] As used herein, the term "antigen binding domain" refers to one or more protein domains (e.g., formed by amino acids from a single polypeptide or formed by amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that are capable of specifically binding to one or more different antigens (e.g., effector antigens or tumor antigens). In some examples, the antigen binding domain can bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen binding domain can be an antibody or a fragment thereof. An example of an antigen binding domain is an antigen binding domain formed by VH-VL. In some embodiments, the antigen binding domain may include an alternative scaffold. In some embodiments, the antigen binding domain is a VHH. Non-limiting examples of antigen binding domains are described herein. Antigen binding domains are known in the art. In some embodiments, the antigen binding domain can bind to a single antigen (e.g., one of an effector antigen and a tumor antigen).

[0046] As used herein, the term "antibody" refers to an antibody that contains at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In certain embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, double antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0047] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, an antigen binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0048] As used herein, the term "multispecific antibody" is an antibody comprising two or more different antigen-binding domains that specifically bind to two or more different epitopes. Two or more different epitopes can be epitopes on the same antigen (e.g., a single polypeptide present on a cell surface) or on different antigens (e.g., present on the same cell surface or on different cell surfaces). On the one hand, a multispecific antibody binds to two different epitopes (i.e., "bispecific antibodies"). On the one hand, a multispecific antibody binds to three different epitopes (i.e., "trispecific antibodies"). On the one hand, a multispecific antibody binds to four different epitopes (i.e., "tetraspecific antibodies"). On the one hand, a multispecific antibody binds to five different epitopes (i.e., "five specific antibodies"). Each binding specificity can exist in any suitable valence state. Non-limiting examples of multispecific antibodies are described herein.

[0049] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. The epitopes can be located on the same antigen or on different antigens.

[0050] As used herein, the term "anti-4-1BB×CCR8 antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that binds to both 4-1BB and CCR8.

[0051] Unless otherwise defined, all technical and scientific terms used herein are generally the same as those understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. Other features and advantages of the present invention will become apparent from the following detailed description and drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figures 1A-1G is a schematic diagram of the 4-1BB×CCR8 bispecific antibody described herein.

[0053] Figure 2 The tumor model and dosing regimen for anti-tumor study 1 using the CT26 tumor model are shown. Mice were treated with 4-1BB-1×CCR8-1 bispecific antibody (BsAb), CCR8-1 mAb, and 4-1BB-1 mAb, with PBS as a control. st )、Second (2 nd ) and the third (3 rd) doses were 1 mg / kg mAb or 1.33 mg / kg BsAb, and the fourth (4 th ) The dose is 10 mg / kg.

[0054] Figure 3 Shown are mean tumor volume curves from Antitumor Study 1. Each curve represents the mean tumor volume for each treatment group.

[0055] Figures 4A-4D Shown are individual tumor volume curves using the CT26 tumor implantation model in antitumor study 1. Each curve represents the tumor volume of each mouse.

[0056] Figure 5 The dosing schedule using the CT26 tumor inoculation model for anti-tumor study 2 is shown. Implanted mice were treated with 4-1BB-1×CCR8-1 bispecific antibody (1.33 or 4 mg / kg), CCR8-1 mAb (1 or 3 mg / kg), 4-1BB-1 mAb (1 or 3 mg / kg), respectively, with PBS as a control.

[0057] Figure 6A-6B Shown are mean tumor volume curves from Antitumor Study 2. Each curve represents the mean tumor volume for each treatment group. Fig. 6A Vaccinated mice were shown to be treated with 4-1BB-1×CCR8-1 bispecific antibody (1.33 mg / kg), CCR8-1 mAb (1 mg / kg), 4-1BB-1 mAb (1 mg / kg), with PBS as a control. Figure 6B Vaccinated mice were shown to be treated with 4-1BB-1×CCR8-1 bispecific antibody (4 mg / kg), CCR8-1 mAb (1 mg / kg), 4-1BB-1 mAb (1 mg / kg), with PBS as a control.

[0058] Figures 7A-7F Shown are individual tumor volume curves from antitumor study 2. Each curve represents a tumor volume curve obtained with 1 mg / kg CCR8-1 mAb ( Fig. 7A )、1mg / kg 4-1BB-1mAb( Figure 7B )、1.33mg / kg 4-1BB-1×CCR8-1 bispecific antibody ( Figure 7C )、3mg / kg CCR8-1 mAb( Fig.7D )、3mg / kg 4-1BB-1mAb( Fig. 7E ) or 4 mg / kg 4-1BB-1×CCR8-1 bispecific antibody ( Figure 7F ) The tumor volume of each individual mouse treated.

[0059] Figure 8The figure shows the survival rate of mice in anti-tumor study 2. Mice were treated with 4-1BB-1×CCR8-1 bispecific antibody (1.33 mg / kg), CCR8-1 mAb (1 mg / kg), and 4-1BB-1 mAb (1 mg / kg), respectively, with PBS as a control.

[0060] Fig. 9 The anti-tumor study 3 showed the dosing regimen using the CT26 tumor transplant model, and the transplanted mice were treated with 4-1BB-1×CCR8-1 bispecific antibody (1.33 mg / kg), CCR8-1 mAb (1 mg / kg), and 4-1BB-1 mAb (1 mg / kg), respectively, with PBS as a control.

[0061] Fig.10 Shown are mean tumor volume curves in antitumor study 3. Each curve represents the mean tumor volume of each treatment group treated with 4-1BB-2×CCR8-1 bispecific antibody (1.33 mg / kg), CCR8-1 mAb (1 mg / kg), or 4-1BB-2 mAb (1 mg / kg), with PBS as control.

[0062] Figures 11A-11D Shown are individual tumor volume curves from antitumor study 3. Each curve represents the tumor volume of the tumor treated with PBS ( Fig.11A )、1mg / kg CCR8-1 mAb( Fig. 11B )、1mg / kg 4-1BB-2mAb( Fig. 11C )、1.33mg / kg4-1BB-1×CCR8-2 bispecific antibody ( Fig.11D ) The tumor volume of each individual mouse treated.

[0063] Figures 12A-12B The binding effect of 4-1BB×CCR8 bispecific antibody on CHO cells expressing human 4-1BB was shown, and the mean fluorescence intensity (MFI of PE, Fig. 12A ) and the percentage of positive cells bound by each antibody ( Fig. 12B ).

[0064] Figures 13A-13B The binding effect of 4-1BB×CCR8 bispecific antibody on CHO cells expressing human CCR8 was shown, and the mean fluorescence intensity (MFI of PE, Fig.13A ) and the percentage of positive cells bound by each antibody ( Fig. 13B ).

[0065] Fig.14 The binding effect of the 4-1BB×CCR8 bispecific antibody on CHO cells co-expressing human 4-1BB and human CCR8 is shown.

[0066] Figures 15A-15B The 4-1BB×CCR8 bispecific antibody was shown to mediate PBMC killing of CHO cells co-expressing human CCR8 and 4-1BB. DETAILED DESCRIPTION

[0068] A multispecific antibody or its antigen-binding fragment is an artificial protein that can simultaneously bind to two or more different epitopes (e.g., on two different antigens). In some embodiments, the multispecific antibody is a bispecific antibody. A bispecific antibody or its antigen-binding fragment can have two arms. Each arm can have a heavy chain variable region and a light chain variable region to form an antigen-binding domain (or antigen-binding region). The two arms can bind to two different antigens. In some embodiments, additional antigen-binding domains can be added to a monoclonal antibody (e.g., added to the C-terminus or N-terminus of a light chain or heavy chain).

[0069] The present invention relates to multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, which comprise a first antigen-binding domain that specifically binds 4-1BB and a second antigen-binding domain that specifically binds CC motif chemokine receptor 8 (CCR8).

[0070] Anti-4-1BB×CCR8 antibody

[0071] Human 4-1BB (also known as tumor necrosis factor receptor superfamily member 9, TNFRSF59 and CD137) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNF superfamily member 9), expressed on a variety of immune cells (including activated T cells, B cells), dendritic cells (DC), activated natural killer (NK) cells, and non-immune cells, such as activated endothelial cells. When activated by human 4-1BB ligands, human 4-1BB co-stimulates T cells, thereby providing an effective T cell immune response and generating immune memory by inducing multiple signaling cascades.

[0072] 4-1BB is a member of the TNF receptor superfamily and is an activation-induced T cell co-stimulatory molecule. Signaling through 4-1BB upregulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells. The importance of the 4-1BB pathway has been highlighted in many diseases, including cancer.

[0073] Signaling through 4-1BB with agonistic monoclonal antibodies, soluble 4-1BBL, or cell lines expressing 4-1BBL in the presence of anti-T cell receptor antibodies has been shown to cause T cell expansion, cytokine induction, and upregulation of anti-apoptotic genes and to prevent activation-induced cell death. Kinetic studies and expression analysis have shown that although CD4+ and CD8 + The expression level of 4-1BB in T cells is similar, but CD8 + The proliferation potential of T cells is significantly higher than that of CD4 + T cells. Similar effects were observed in vivo. In addition to the effects on T cells, ligation of 4-1BB receptors on DCs and macrophages can also induce these cells to produce IL-12 and IL-8, respectively.

[0074] To date, the role of 4-1BB signaling has been studied in a variety of autoimmune processes, including rheumatoid arthritis, experimental autoimmune encephalomyelitis, and systemic lupus erythematosus. 4-1BB signaling has dual immunomodulatory effects.

[0075] A detailed description of 4-1BB and its functions can be found in, e.g., Vinay et al., Mol Cancer Ther. 2012 May; 11(5): 1062-70; and Bartkowiak and Curran 2015, Frontiers in Oncology, 5: 117.

[0076] CC motif chemokine receptor 8 (CCR8) is a protein found primarily on the surface of a subset of highly immunosuppressive regulatory T cells (Tregs) within tumors. It is a regulator of immune responses. As a chemokine receptor, CCR8 acts as an active mediator in concert with its chemokine CC chemokine ligand 1 (CCL1). CCR8 expression is upregulated on a subset of highly immunosuppressive Tregs in multiple cancer types. Studies have found that CCR8 expression is particularly upregulated on Tregs in the tumor microenvironment (TME), while CCR8 found in blood and normal tissues is not upregulated. + Treg levels are low. Tregs act through multiple mechanisms within the TME, including suppression of antigen presenting cell (APC) function, consumption of interleukin 2 (IL-2), and production of immunosuppressive cytokines and metabolites. High Treg infiltration in the TME is associated with poor prognosis in multiple cancer types.

[0077] Compared with other current Treg depletion approaches, targeting CCR8 may have more specific antitumor activity. Other Treg target molecules are also present on peripheral Treg (in the blood), normal tissues, and effector T cells. Therefore, a highly selective target, such as CCR8, is needed that can specifically eliminate tumor-infiltrating Tregs without affecting Tregs in the blood and normal tissues or damaging effector T cells.

[0078] For more information on CCR8, see Li H-L et al. Clinical and prognostic significance of CC chemokine receptor type 8 protein expression in gastrointestinal stromal tumors. World J Gastroenterol. 2020;26(31):4656-4668; Ohue Y, Nishikawa H. Regulatory T (Treg) cells in cancer: can Treg cells be a new therapeutic target? Cancer Sci. 2019;110(7):2080-2089;

[0079] Cascieri MA, Springer MS. The chemokine / chemokine-receptor family: potential and progress for therapeutic intervention. Curr Opin Chem Biol. 2000;4(4):420-427; Campbell JR et al. Fc-optimized anti-CCR8 antibody depletes regulatory T cells in human tumor models. Cancer Res. Published online March 23, 2021. doi:10.1158 / 0008-5472-CAN-20-3585; Van Damme H et al. Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy. J Immunother Cancer. 2021;9(2):e001749; and Barsheshet Y et al. CCR8+FOXp3+ Treg cells as master drivers of immune regulation. Proc Natl Acad Sci. 2017;114(23):6086-6091, each of which is incorporated herein by reference in its entirety.

[0080] The present invention provides antibodies (e.g., bispecific or multispecific) and antigen-binding fragments thereof that specifically bind to (a) 4-1BB (e.g., human 4-1BB), (b) CCR8 (e.g., human CCR8), or (c) 4-1BB and CCR8 (e.g., human 4-1BB and human CCR8). On the one hand, the present invention provides anti-4-1BB×CCR8 multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to 4-1BB; and a second antigen-binding domain that specifically binds to CCR8.

[0081] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0082] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof or the anti-4-1BB×CCR8 multispecific (eg, bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding domain that binds 4-1BB (eg, human 4-1BB).

[0083] In some embodiments, an antigen binding domain that binds 4-1BB (e.g., human 4-1BB) comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1).

[0084] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof or anti-4-1BB×CCR8 multispecific (eg, bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding domain that binds CCR8 (eg, human CCR8).

[0085] In some embodiments, the antigen binding domain that binds CCR8 (e.g., human CCR8) comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0086] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80%, 90%, or 100% identical to a selected VH1 CDR1. The VH1 CDR2 region comprises an amino acid sequence that is at least 80%, 90% or 100% identical to the selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%, 90% or 100% identical to the selected VH1 CDR3; and the first light chain variable region (VL1) comprises CDRs 1, 2 and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%, 90% or 100% identical to the selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%, 90% or 100% identical to the selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%, 90% or 100% identical to the selected VL1 CDR3, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, 3 amino acid sequence are one of the following:

[0087] (1) The selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 4, 5, and 6, respectively;

[0088] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 24, 25, and 26, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 27, 28, and 29, respectively;

[0089] (3) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 50, 51, and 52, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 54, and 55, respectively; and

[0090] (4) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72 and 73, respectively.

[0091] In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80%, 90%, or 100% identical to a selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80%, 90%, or 100% identical to a selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80%, 90%, or 100% identical to a selected VH2 CDR3; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80%, 90%, or 100% identical to a selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80%, 90%, or 100% identical to a selected VL2 CDR2, and the VL2 The CDR3 region comprises an amino acid sequence that is at least 80%, 90% or 100% identical to the selected VL2 CDR3, wherein the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are one of the following:

[0092] (1) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12, and 13, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15, and 16, respectively;

[0093] (2) the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 36, 37, and 38, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 39, 40, and 41, respectively;

[0094] (3) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58, 59 and 60, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62 and 63, respectively; and

[0095] (4) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 76, 77 and 78, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 79, 80 and 81, respectively.

[0096] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 1, 2 and 3, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 5 and 6, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12 and 13, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15 and 16, respectively.

[0097] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 1, 2 and 3, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 5 and 6, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 36, 37 and 38, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 39, 40 and 41, respectively.

[0098] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 1, 2 and 3, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 5 and 6, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58, 59 and 60, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62 and 63, respectively.

[0099] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 1, 2 and 3, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 5 and 6, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 76, 77 and 78, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 79, 80 and 81, respectively.

[0100] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 25 and 26, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 27, 28 and 29, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12 and 13, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15 and 16, respectively.

[0101] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 25 and 26, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 27, 28 and 29, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 36, 37 and 38, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 39, 40 and 41, respectively.

[0102] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 25 and 26, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 27, 28 and 29, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58, 59 and 60, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62 and 63, respectively.

[0103] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 25 and 26, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 27, 28 and 29, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 76, 77 and 78, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 79, 80 and 81, respectively.

[0104] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 50, 51 and 52, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 54 and 55, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12 and 13, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15 and 16, respectively.

[0105] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 50, 51 and 52, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 54 and 55, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 36, 37 and 38, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 39, 40 and 41, respectively.

[0106] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 50, 51 and 52, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 54 and 55, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58, 59 and 60, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62 and 63, respectively.

[0107] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 50, 51 and 52, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 54 and 55, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 76, 77 and 78, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 79, 80 and 81, respectively.

[0108] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72 and 73, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12 and 13, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15 and 16, respectively.

[0109] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72 and 73, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 36, 37 and 38, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 39, 40 and 41, respectively.

[0110] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72 and 73, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58, 59 and 60, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62 and 63, respectively.

[0111] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72 and 73, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 76, 77 and 78, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 79, 80 and 81, respectively.

[0112] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 7, 30, 56 or 74, and the selected VL sequence is SEQ ID NO: 8, 31, 57 or 75.

[0113] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 17, 42, 64 or 82, and the selected VL sequence is SEQ ID NO: 18, 43, 65 or 83.

[0114] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:7, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:8, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:17, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:18.

[0115] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:7, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:8, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:42, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:43.

[0116] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:7, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:8, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:64, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:65.

[0117] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:7, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:8, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:82, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:83.

[0118] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:30, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:31, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:17, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:18.

[0119] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:30, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:31, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:42, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:43.

[0120] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:30, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:31, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:64, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:65.

[0121] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:30, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:31, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:82, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:83.

[0122] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:56, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:57, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:17, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:18.

[0123] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:56, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:57, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:42, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:43.

[0124] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:56, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:57, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:64, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:65.

[0125] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:56, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:57, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:82, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:83.

[0126] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:74, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:75, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:17, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:18.

[0127] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:74, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:75, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:42, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:43.

[0128] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:74, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:75, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:64, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:65.

[0129] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:74, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:75, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:82, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:83.

[0130] In some embodiments, VH1 comprises a VH1 CDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL1 comprises a VL1 CDR1, VL1 CDR2, and VL1 CDR3 identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 7, 30, 56, or 74, and the selected VL sequence is SEQ ID NO: 8, 31, 57, or 75.

[0131] In some embodiments, VH2 comprises a VH2 CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 17, 42, 64, or 82, and the selected VL sequence is SEQ ID NO: 18, 43, 65, or 83.

[0132] In some embodiments, CDR definitions are based on Kabat, Chothia, IMGT, contact, or AbM definitions.

[0133] In some embodiments, the first antigen binding domain specifically binds human or mouse 4-1BB; and / or the second antigen binding domain specifically binds human or mouse CCR8.

[0134] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain.

[0135] In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region). In some embodiments, the C-terminus of the VH1 of the first antigen-binding domain is connected to the Fc region. In some embodiments, the C-terminus of the VH1 of the first antigen-binding domain is connected to the Fc region, optionally via a CH1 domain.

[0137] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can be any suitable structure. In some embodiments, the second antigen-binding domain is a single-chain fragment variable domain (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0138] In some embodiments, the second antigen binding domain is connected to the N-terminus of the light chain. In some embodiments, the second antigen binding domain is connected to the N-terminus of the first light chain variable domain (VL1). In some embodiments, the C-terminus of the second antigen binding domain (e.g., scFv) is connected to the N-terminus of the first light chain variable domain (VL1). In some embodiments, the second antigen binding domain (e.g., scFv) is connected to the N-terminus of the light chain, optionally connected through a second joint. In some embodiments, the second antigen binding domain is connected to the N-terminus of Fc, optionally connected through a second joint. In some embodiments, the second antigen binding domain (e.g., scFv) is connected to the C-terminus of the light chain, optionally connected through a second joint. In some embodiments, the second antigen binding domain is optionally connected to the C-terminus of Fc through a second joint.

[0139] In some embodiments, the anti-4-1BB×CCR8 antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more anti-4-1BB antigen binding domains and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more anti-CCR8 antigen binding domains.

[0140] In some embodiments, the anti-CCR8 antigen binding domain (eg, scFv) is linked to the N-terminus of the heavy or light chain. In some embodiments, the anti-CCR8 antigen binding domain is linked to the C-terminus of the heavy or light chain.

[0141] In some embodiments, the anti-4-1BB antigen binding domain (e.g., scFv) is connected to the N-terminus of the heavy chain or light chain. In some embodiments, the anti-4-1BB antigen binding domain is connected to the C-terminus of the heavy chain or light chain.

[0142] In some embodiments, the anti-4-1BB×CCR8 antibody comprises (1) a first polypeptide, optionally including scFv, VL and CL from the N-terminus to the C-terminus; (2) a second polypeptide, optionally including VH and CH1 from the N-terminus to the C-terminus; (3) a third polypeptide, optionally including VH and CH1 from the N-terminus to the C-terminus, wherein the third polypeptide is optionally bound to the second polypeptide through one or more disulfide bonds; (4) a fourth polypeptide, optionally including scFv, VL and CL from the N-terminus to the C-terminus. In some embodiments, the second polypeptide also includes CH2 and CH3. In some embodiments, the third polypeptide also includes CH2 and CH3.

[0143] In some embodiments, the anti-4-1BB×CCR8 antibody comprises (1) a first polypeptide, optionally including VL, CL and scFv from the N-terminus to the C-terminus; (2) a second polypeptide, optionally including VH and CH1 from the N-terminus to the C-terminus; (3) a third polypeptide, optionally including VH and CH1 from the N-terminus to the C-terminus, wherein the third polypeptide is optionally bound to the second polypeptide through one or more disulfide bonds; (4) a fourth polypeptide, optionally including VL, CL and scFv from the N-terminus to the C-terminus. In some embodiments, the second polypeptide also includes CH2 and CH3. In some embodiments, the third polypeptide also includes CH2 and CH3.

[0144] In some embodiments, the anti-4-1BB×CCR8 antibody comprises (1) a first polypeptide, optionally including VL and CL from the N-terminus to the C-terminus; (2) a second polypeptide, optionally including scFv, VH and CH1 from the N-terminus to the C-terminus; (3) a third polypeptide, optionally including scFv, VH and CH1 from the N-terminus to the C-terminus, wherein the third polypeptide is optionally bound to the second polypeptide through one or more disulfide bonds; (4) a fourth polypeptide, optionally including VL and CL from the N-terminus to the C-terminus. In some embodiments, the second polypeptide also includes CH2 and CH3. In some embodiments, the third polypeptide also includes CH2 and CH3.

[0145] In some embodiments, the anti-4-1BB×CCR8 antibody comprises (1) a first polypeptide, optionally including VL and CL from the N-terminus to the C-terminus; (2) a second polypeptide, optionally including VH, CH1 and scFv from the N-terminus to the C-terminus; (3) a third polypeptide, optionally including VH, CH1 and scFv from the N-terminus to the C-terminus, wherein the third polypeptide is optionally bound to the second polypeptide through one or more disulfide bonds; (4) a fourth polypeptide, optionally including VL and CL from the N-terminus to the C-terminus. In some embodiments, the second polypeptide also includes CH2 and CH3. In some embodiments, the third polypeptide also includes CH2 and CH3.

[0146] In some embodiments, the VH and VL bind and bind to CCR8, while the scFv binds to 4-1BB.

[0147] In some embodiments, the VH and VL combine and bind to 4-1BB, while the scFv binds to CCR8.

[0148] The various schematic diagrams are as follows Figures 1A-1G shown.

[0149] In some embodiments, the bispecific antibody (e.g., an anti-4-1BB×CCR8 antibody) comprises a first heavy chain sequence of VH1, wherein the first heavy chain comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 250%, 251%, 252%, 253%, 254%, 255%, 256%, 257%, 258%, 259%,

[0150] In some embodiments, the bispecific antibody (e.g., anti-4-1BB×CCR8 antibody) includes a first light chain sequence of VL1, wherein the first light chain comprises an amino acid sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10, 33.

[0151] In some embodiments, the bispecific antibody (e.g., anti-4-1BB×CCR8 antibody) comprises a second heavy chain sequence of VH2, wherein the second heavy chain comprises an amino acid sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 19. In some embodiments, the bispecific antibody (e.g., anti-4-1BB×CCR8 antibody) comprises a second light chain sequence of VL2, wherein the second light chain comprises an amino acid sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 20.

[0152] In some embodiments, the bispecific antibody (e.g., an anti-4-1BB×CCR8 antibody) comprises a heavy chain sequence comprising an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 21, 34, 44, 46, 48, 66 or 84. In some embodiments, the bispecific antibody (e.g., an anti-4-1BB×CCR8 antibody) comprises a light chain sequence comprising an amino acid sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22, 35, 45, 47, 49, 67 or 85.

[0153] The linkers described herein can be any suitable linkers known in the art. In some embodiments, the linkers comprise an amino acid sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23 or 86.

[0154] The present invention provides anti-4-1BB×CCR8 antibodies and modified antibodies thereof, including, for example, chimeric antibodies, humanized antibodies and human antibodies.

[0155] Anti-4-1BB×CCR8 antibodies or their antigen-binding fragments can also have various forms. Many different forms of bispecific antibodies or their antigen-binding fragments are known in the art and are described in, for example, Suurs, et al. "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges," Pharmacology & therapeutics (2019), which is incorporated herein by reference in its entirety.

[0156] In some embodiments, the anti-4-1BB×CCR8 antibody is a BiTE, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HSA, or tandem-scFv. In some embodiments, the anti-4-1BB×CCR8 antibody is VHH-scAb, VHH-Fab, double scFab, F(ab')2, double antibody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Trio, Minibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body or scFv1-PEG-scFv2.

[0157] In some embodiments, the anti-4-1BB×CCR8 antibody can be a TrioMab. In a TrioMab, the two heavy chains are from different species, wherein different sequences restrict heavy chain-light chain pairing.

[0158] In some embodiments, the anti-4-1BB×CCR8 antibody has two different heavy chains and two different light chains. Heterodimerization of the heavy chains can be based on knobs-in-holes or some other heavy chain pairing technology.

[0159] In some embodiments, CrossMAb technology can be used to produce bispecific anti-4-1BB×CCR8 antibodies. CrossMAb technology can be used to enforce correct light chain association in bispecific heterodimeric IgG antibodies, and the technology allows the preparation of various bispecific antibody formats, including double (1+1), three (2+1) and four (2+2) valence bispecific antibodies, and antibodies based on non-Fc tandem antigen binding fragments (Fab). These forms can be derived from any existing antibody pairs using domain crossovers without identifying common light chains, post-translational processing / in vitro chemical assembly or introducing a set of mutations forcing correct light chain association. The method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-and multispecific antibodies." MAbs. Vol. 8. No. 6. Taylor & Francis, 2016, which is incorporated herein by reference as a whole. In some embodiments, the CH1 in the heavy chain and the CL domain in the light chain are exchanged.

[0160] Anti-4-1BB×CCR8 antibodies can be bispecific antibodies. The naturally occurring Fab exchange mechanism in IgG4 antibodies is mimicked in a controlled form of IgG1 antibodies, a mechanism known as controlled Fab exchange. This format ensures specific pairing between heavy and light chains.

[0161] In dual variable domain antibodies (DVD-Ig), additional VH and variable light (VL) domains are added to each N-terminus to achieve bispecific targeting. This format is similar to IgG-scFv, but the added binding domains are individually bound to each N-terminus, rather than scFv bound to each heavy chain N-terminus.

[0162] In scFv-IgG, two scFvs are attached to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites and is therefore also called tetravalent. scFv-IgG does not have the problem of pairing heavy and light chains.

[0163] In some embodiments, the anti-4-1BB×CCR8 antibody may have an IgG-IgG format. Two intact IgG antibodies are coupled by chemically linking the C-termini of the heavy chains.

[0164] The anti-4-1BB×CCR8 antibody can also have a Fab-scFv-Fc format. In the Fab-scFv-Fc format, the light chain, the heavy chain, and the third chain containing the Fc region and the scFv are assembled. It can ensure efficient preparation and purification.

[0165] In some embodiments, the anti-4-1BB×CCR8 antibody can be TF. The three Fab fragments are connected by disulfide bridges. The TF configuration does not have an Fc region.

[0166] ADAPTIR has two scFvs attached to each side of the Fc region. It abandons the full IgG as the basis of its construction, but retains the Fc region to extend half-life and facilitate purification.

[0167] Dual affinity redirection (DART) has two peptide chains connecting opposite fragments, namely VLA with VHB and VLB with VHA, and the sulfide bonds at their C-termini fuse them together. In DART, the sulfide bonds can improve the stability of BiTE.

[0168] In DART-Fc, the Fc region is attached to the DART structure. It can be made by assembling three chains, two of which are connected through disulfide bonds, just like DART. One chain contains half of the Fc region, which will dimerize with the third chain to express only the Fc region. Adding the Fc region prolongs the half-life, thereby extending the effective concentration and avoiding continuous IV.

[0169] In a tetravalent DART, four peptide chains are assembled. Basically, two DART molecules have half the Fc region and will dimerize. This form has a bivalent binding to two targets, so it is a tetravalent molecule.

[0170] Tandem diabodies (TandAb) contain two diabodies. Each diabody consists of VHA and VLB fragments and covalently linked VHA and VLB fragments. The two diabodies are connected by a peptide chain. It can improve the stability of the diabody composed of two scFvs. It has two bivalent binding sites.

[0171] scFv-scFv-toxin includes a toxin and two scFvs with a stabilizing linker. It can be used for specific payload delivery.

[0172] In some embodiments, the anti-4-1BB×CCR8 antibody is a bispecific antibody. In some embodiments, the bispecific antibody of the present invention is designed as 1+1 (unit price per target) and has an IgG1 subtype structure. This can reduce the affinity for cells with low expression of 4-1BB and CCR8, and increase the affinity for cells co-expressing 4-1BB and CCR8 to achieve enhanced targeting function.

[0173] In some embodiments, the anti-4-1BB×CCR8 antibody includes a KIH mutation. In some embodiments, the anti-4-1BB×CCR8 antibody comprises a first antigen binding domain that specifically binds to 4-1BB and a second antigen binding domain that specifically binds to CCR8. In some embodiments, the first antigen binding domain comprises one or more heavy chains with knob mutations (knob heavy chains), and the second antigen binding domain comprises one or more heavy chains with hole mutations (hole heavy chains).

[0174] In some embodiments, the anti-4-1BB×CCR8 antibody or antibody fragment thereof comprises a combination of the anti-4-1BB and anti-CCR8 antigen binding domains shown in Table 1.

[0175] The present invention also provides a nucleic acid comprising a polynucleotide encoding an anti-4-1BB×CCR8 antibody. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-4-1BB×CCR8 antibody comprises the CDRs shown in Table 1. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CCR8 and / or 4-1BB.

[0176] Anti-4-1BB×CCR8 antibodies can also be anti-4-1BB×CCR8 antibody variants (including derivatives and conjugates) of anti-4-1BB×CCR8 antibodies or antibody fragments. Additional anti-4-1BB×CCR8 antibodies provided herein are polyclonal, monoclonal, multispecific (multimer, such as bispecific), human antibodies, chimeric antibodies (such as human-mouse chimeras), single-chain antibodies, antibodies made in cells, i.e. antibodies made in cells (intracellular antibodies) and antigen-binding fragments thereof. Anti-4-1BB×CCR8 antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), categories (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses. In some embodiments, anti-4-1BB×CCR8 antibodies or antigen-binding fragments are IgG (e.g., IgG1 Fc region) antibodies or antigen-binding fragments thereof.

[0177] Fragments of anti-4-1BB×CCR8 antibodies are suitable for use in the provided methods as long as they retain the desired affinity and specificity for both CCR8 and 4-1BB. Thus, fragments of anti-4-1BB×CCR8 antibodies will retain the ability to bind to both CCR8 and 4-1BB.

[0178] In some embodiments, the antibodies or antigen-binding fragments thereof have the same light chain variable regions.

[0179] In some embodiments, the multispecific (e.g., bispecific) anti-4-1BB×CCR8 antibodies described herein kill tumor cells and / or reduce tumor size (e.g., through activation of 4-1BB-related signaling pathways and recruitment and elimination) of immune cells expressing CCR8 (e.g., tumor-infiltrating Tregs expressing CCR8) in subjects receiving the antibodies. In some embodiments, by administering the multispecific (e.g., bispecific) anti-4-1BB×CCR8 antibodies described herein, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,

[0180] 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of tumor cells.

[0181] In some embodiments, administration of a multispecific (e.g., bispecific) anti-4-1BB×CCR8 antibody described herein reduces tumor size by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to subjects not administered a multispecific (e.g., bispecific) anti-4-1BB×CCR8 antibody. In some embodiments, administration of a multispecific (e.g., bispecific) anti-4-1BB×CCR8 antibody described herein reduces tumor size by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to a subject administered a monospecific antibody (e.g., a monospecific anti-4-1BB antibody or a monospecific anti-CCR8 antibody).

[0182] In some embodiments, administration of a multispecific (e.g., bispecific) anti-4-1BB×CCR8 antibody as described herein increases the survival rate of a subject to whom the antibody is administered. In some embodiments, the survival rate of a subject to whom a multispecific (e.g., bispecific) anti-4-1BB×CCR8 antibody is not administered is compared, and the survival rate increases by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0183] In some embodiments, the anti-tumor effect of anti-4-1BB×CCR8 antibodies is to enhance tumor immunity and increase survival rate by regulating Treg / Teff cells (eg, in mice or humans).

[0184] Antibodies and their antigen-binding fragments

[0185] In some embodiments, bispecific antibodies (e.g., anti-4-1BB×CCR8 antibodies) include an antigen binding domain derived from an anti-4-1BB antibody and an antigen binding domain derived from an anti-CCR8 antibody. These anti-4-1BB×CCR8 antibodies and antigen binding fragments thereof can have a variety of forms.

[0186] In general, antibodies (also called immunoglobulins) can be composed of two types of polypeptide chains: light chains and heavy chains. The non-limiting anti-4-1BB×CCR8 antibodies of the present invention can be complete tetra-immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the anti-4-1BB×CCR8 antibody can be any isotype, including IgM, IgG, IgE, IgA or IgD or isotypes, including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.

[0187] The hypervariable regions, called complementarity determining regions (CDRs), form loops that constitute the major antigen binding surface of antibodies. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs in the other chain, contribute to the formation of the antigen binding domain.

[0188] Methods for identifying the CDR regions of antibodies by analyzing the amino acid sequence of the antibody are well known, and various definitions of CDRs are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in Martin, "Protein sequence and structure analysis of antibody variabledomains," Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol.275(2):269-94(Jan.1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated herein by reference in its entirety.

[0189] CDR is important for recognizing antigen epitopes. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by the antigen binding domain of an antibody. The minimum amount of an epitope can be about three, four, five, six or seven amino acids, but these amino acids do not need to be located in a continuous linear sequence of the primary structure of the antigen, because the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0190] In some embodiments, the anti-4-1BB×CCR8 antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, with different constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference in its entirety.

[0191] Anti-4-1BB×CCR8 antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, or camelids).Antigen binding domains or antigen binding fragments are parts of antibodies that retain the specific binding activity of intact antibodies, i.e., any part of antibodies that can specifically bind to epitopes on target molecules of intact antibodies. It includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Therefore, in some embodiments, anti-4-1BB×CCR8 antibodies or antigen binding fragments thereof may include, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising a binding domain homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, for example, heavy and / or light chain CDRs of intact antibodies, heavy and / or light chain variable regions of intact antibodies, full-length heavy or light chains of intact antibodies, or single CDRs of heavy or light chains from intact antibodies.

[0192] In some embodiments, the scFv in the anti-4-1BB×CCR8 antibody has two heavy chain variable domains and two light chain variable domains. In some embodiments, the anti-4-1BB×CCR8 scFv has two antigen binding regions (antigen binding regions: A and B), and the two antigen binding regions can bind to their respective target antigens with different affinities.

[0193] In some embodiments, the anti-4-1BB×CCR8 antibody or antigen-binding fragment thereof may comprise one, two or three heavy chain variable region CDRs selected from Table 1.

[0194] In some embodiments, the anti-4-1BB×CCR8 antibodies described herein can be coupled to therapeutic agents. Anti-4-1BB×CCR8 antibody-drug conjugates comprising antibodies or antigen-binding fragments thereof can be covalently or non-covalently bound to therapeutic agents. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, maytansinoid alkaloids such as DM-1 and DM-4, ​​diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is coupled via a linker such as a VC linker. A detailed description of linkers for ADCs can be found in Su, Z. et al. "Antibody–drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0195] In some embodiments, anti-4-1BB×CCR8 antibodies are bispecific antibodies. Bispecific antibodies can be prepared by transforming the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface may include at least a portion of the CH3 domain of an antibody constant domain. In the method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation "cavity" of the same or similar size as the large side chain is created on the interface of the second antibody molecule. This provides a mechanism for increasing heterodimer yields relative to other unwanted end products such as homodimers. The method is described in, for example, WO96 / 27011, the entire contents of which are incorporated herein by reference in their entirety.

[0196] Any anti-4-1BB×CCR8 antibody or antigen-binding fragment thereof described herein can be coupled to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in a solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). The coupling of stabilizing molecules can increase the in vitro half-life of the anti-4-1BB×CCR8 antibody or antigen-binding fragment or prolong the biological activity (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body).

[0197] Antibody characteristics

[0198] Anti-4-1BB×CCR8 antibodies may include anti-4-1BB antigen binding domains and any anti-CCR8 antigen binding domains described herein. In some embodiments, anti-4-1BB×CCR8 antibodies or antigen binding fragments thereof may initiate target cell (e.g., tumor cell, such as CT26 cell) killing, CDC, ADCC or ADCP.

[0199] The present invention provides anti-4-1BB×CCR8 antibodies and antigen-binding fragments thereof that can specifically bind to 4-1BB. In some embodiments, by binding to 4-1BB or activating the interaction of 4-1BB with its ligand, anti-4-1BB×CCR8 antibodies can activate 4-1BB-related signaling pathways, thereby treating cancer. In some embodiments, the antibody can reduce the tumor volume of the subject. Therefore, in some embodiments, the anti-4-1BB×CCR8 antibodies described herein are 4-1BB agonists. In some embodiments, the anti-4-1BB×CCR8 antibodies described herein are 4-1BB antagonists.

[0200] The affinity of the antibody for the antigen can be measured using general techniques, including, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some embodiments, the anti-4-1BB×CCR8 antibody or its antigen-binding fragment can bind to 4-1BB (e.g., human 4-1BB, monkey 4-1BB, mouse 4-1BB, and / or chimeric 4-1BB) with a dissociation rate (koff) of less than 0.1s -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s-1, greater than 0.001 s-1, -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0201] In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0202] In some embodiments, the anti-4-1BB×CCR8 antibody or antigen-binding fragment thereof can bind to 4-1BB (e.g., human 4-1BB, monkey 4-1BB, mouse 4-1BB, and / or chimeric 4-1BB) with a KD of less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 - 8 M, less than 1×10 -9 M or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 - 8M, greater than 1×10 -9 M or greater than 1×10 -10 M.

[0203] The anti-4-1BB×CCR8 antibody or its antigen-binding fragment may also include an antigen-binding domain that can specifically bind to CCR8. In some embodiments, the antibodies or their antigen-binding fragments described herein recruit cells expressing CCR8 (e.g., Tregs expressing CCR8) and mediate the consumption of such cells. In some embodiments, the antibody can reduce the tumor volume of the animal. In some embodiments, the antibody can reduce or slow the progression of an autoimmune disease. Therefore, in some embodiments, the anti-4-1BB×CCR8 antibody described herein is a CCR8 agonist. In some embodiments, the anti-4-1BB×CCR8 antibody described herein is a CCR8 antagonist.

[0204] In some embodiments, the anti-4-1BB×CCR8 antibody or antigen-binding fragment thereof can bind to CCR8 (e.g., human CCR8, monkey CCR8, mouse CCR8 and / or chimeric CCR8) at a rate less than that of the 4-1BB×CCR8 antibody or antigen-binding fragment thereof, and the dissociation rate (koff) is less than 0.1 s -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 , or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0205] In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0206] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10-6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M or greater than 1×10 -10 M.

[0207] Since anti-4-1BB×CCR8 antibodies (e.g., bispecific antibodies) bind to both CCR8 and 4-1BB, for cells expressing both CCR8 and 4-1BB, the antibodies have a higher binding affinity to these cells. Avidity can be used to measure the binding affinity of antibodies to these cells. Avidity is the cumulative strength of the multiple affinities of a single non-covalent binding interaction.

[0208] In some embodiments, anti-4-1BB×CCR8 antibodies, antigen-binding fragments or ADCs have a tumor growth inhibition rate or percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, anti-4-1BB×CCR8 antibodies, antigen-binding fragments or ADCs have a tumor growth inhibition percentage TGI (%) of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150%. Detection can be performed, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more days after the start of treatment. As used herein, tumor growth inhibition rate or percentage (TGI%) is calculated using the formula:

[0209] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0210] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

[0211] In some embodiments, the anti-4-1BB×CCR8 bispecific antibody, antigen binding fragment or ADC reduces tumor volume by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to administration of PBS or no administration of the anti-4-1BB×CCR8 antibody, antigen binding fragment or ADC. Tumor volume is measured in mm 3 The following formula can be used to calculate V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L).

[0212] In some embodiments, anti-4-1BB×CCR8 antibodies, antigen binding fragments or ADCs have functional Fc regions. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the Fc region has enhanced ADCC activity. In some embodiments, the Fc region has enhanced ADCP activity.

[0213] In some embodiments, the anti-4-1BB×CCR8 antibody, antigen binding fragment or ADC does not have a functional Fc region. For example, an anti-4-1BB×CCR8 antibody or its antigen binding fragment is a Fab, Fab', F(ab')2 and Fv fragment. In some embodiments, the anti-4-1BB×CCR8 antibody or its antigen binding fragment described herein has an Fc region without effector function. In some embodiments, Fc is human IgG4Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0214] In some embodiments, the antibody or its antigen-binding fragment comprises a fragment crystallizable region (Fc region). In some embodiments, the bispecific anti-4-1BB×CCR8 antibodies described herein may be designed to have an IgG1 subtype structure with a GAALIE mutation (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the anti-4-1BB×CCR8 antibodies described herein may be designed to have an IgG1 Fc region having an alanine (A) at position 236, a leucine (L) at position 330, and a glutamic acid (E) at position 332 in EU numbering.

[0215] Some other modifications may be made to the Fc region. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric fusion protein thus produced may have any increased in vitro and / or in vivo half-life.

[0216] In some embodiments, the IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0217] In some embodiments, the anti-4-1BB×CCR8 antibody variants provided have a carbohydrate structure lacking fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 sugar chain, relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) connected to Asn297 measured by MALDI-TOF mass spectrometry, for example, according to WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the anti-4-1BB×CCR8 antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0218] In some embodiments, the bispecific anti-4-1BB×CCR8 antibodies described herein have a higher endocytosis rate than the corresponding monoclonal antibodies and / or control bispecific antibodies described herein.

[0219] In some embodiments, anti-4-1BB×CCR8 antibodies are bispecific antibodies. Bispecific antibodies can be prepared by transforming the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface may include at least a portion of the CH3 domain of an antibody constant domain. In the method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation "cavity" of the same or similar size as the large side chain is created on the interface of the second antibody molecule. This provides a mechanism for increasing heterodimer yields relative to other unwanted end products such as homodimers. The method is described in, for example, WO96 / 27011, the entire contents of which are incorporated herein by reference in their entirety.

[0220] In some embodiments, anti-4-1BB×CCR8 bispecific antibodies, antigen binding fragments or ADCs promote antibody-dependent cellular phagocytosis (ADCP), wherein anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADCs bind to target cells (e.g., CT26 cells or CHO cells) that co-express 4-1BB and / or CCR8, marking them for destruction by phagocytes (e.g., macrophages, dendritic cells or neutrophils). In ADCP, the Fab region of the antibody binds to an antigen on the surface of a target cell (e.g., CT26 cells or CHO cells) that co-expresses 4-1BB and / or CCR8, and the Fc region interacts with an Fc receptor (e.g., CD64 or CD32) on the surface of the phagocyte. Once involved, this interaction triggers phagocytes (e.g., macrophages, dendritic cells or neutrophils) to engulf and digest target cells (e.g., CT26 cells or CHO cells) that co-express 4-1BB and / or CCR8, thereby removing the labeled cells (or cell killing). ADCP is a key mechanism in the immune response and is actively exploited in therapeutic antibody therapy, particularly in cancer therapy, where antibodies are designed to target and eliminate tumor cells through phagocytosis. ADCP is the primary mode of action for many monoclonal antibodies known in the field of oncology.

[0221] In some embodiments, anti-4-1BB×CCR8 bispecific antibodies, antigen binding fragments or ADCs promote ADCP of target cells (e.g., CHO cells) co-expressing hCCR8 and h4-1BB by effector M0 macrophages. In some embodiments, the ratio of effector cells (e.g., macrophages) to target cells (e.g., CHO cells co-expressing 4-1BB and / or CCR8) or the E:T ratio can be any ratio between 1:10 and 100:1 (e.g., between 1:5 and 50:1, between 1:2 and 25:1, or between 1:1 and 10:1).

[0222] In some embodiments, the knob-into-hole (KIH) technology can be used, which involves modifying the CH3 domain to create a "knob" or "hole" in each heavy chain to promote heterodimerization. Xu, Yiren, et al. "Production of bispecific antibodies in'knobs-into-holes'using a cell-free expression system." MAbs. Vol. 7. No. 1. Taylor & Francis, 2015, the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, one heavy chain has T366W and / or S354C (knob) substitutions (EU numbering), and the other heavy chain has Y349C, T366S, L368A and / or Y407V (hole) substitutions (EU numbering). In some embodiments, one heavy chain has one or more of the following substitutions Y349C and T366W (EU numbering). The other heavy chain may have one or more of the following substitutions E356C, T366S, L368A, and Y407V (EU numbering). In addition, substitutions (-ppcpScp-->-ppcpPcp-) were introduced in the hinge region of two substituted IgGs.

[0223] Recombinant vector

[0224] The present invention also provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide of the present invention (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains the polynucleotide and / or a vector comprising the polynucleotide), and production of an anti-4-1BB×CCR8 antibody polypeptide or fragment thereof by recombinant technology.

[0225] The full-length sequence of human CCR8 is known in the art (eg, NCBI gene ID: 1237, NCBI reference sequence: NM_005201.4). In some embodiments, a His-tagged human CCR8 protein is used as an immunogen.

[0226] The full-length sequence of human 4-1BB is known in the art (eg, GenBank Accession No.: U03397.1). In some embodiments, a His-tagged human 4-1BB protein is used as an immunogen.

[0227] As used herein, "vector" is any construct that can deliver one or more polynucleotides of interest to a host cell when the vector is introduced into a host cell."expression vector" can deliver and express one or more polynucleotides of interest as encoded polypeptides in a host cell introduced into an expression vector.Therefore, in an expression vector, by being operably connected with a regulatory element (such as a promoter, an enhancer and / or a poly A tail) in a vector or in a genome, the polynucleotides of interest are positioned for expression in a vector.Host cell is at or near or flank the integration site of the polynucleotides of interest so that the polynucleotides of interest will be translated in the host cell introducing the expression vector.

[0228] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection and infection and / or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors combined with a cationic condensing agent.

[0229] In some embodiments, a polynucleotide of the invention (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia virus or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-defective virus. In the latter case, viral propagation generally occurs only in complementing viral packaging cells. Suitable systems are described in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; US ​​Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; US Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al. al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be "naked" as described in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that can be efficiently transported into cells.

[0230] For expression, the DNA insert comprising the polynucleotide encoding the polypeptide of the present invention can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as bacteriophage λPL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and retroviral LTR promoters, etc. Other suitable promoters are known to those skilled in the art. The expression construct can also include sites for transcription initiation, termination, and a ribosome binding site for translation in the transcription region. The coding portion of the mature transcript expressed by the construct can include a translation initiation at the start and a termination codon (UAA, UGA or UAG) appropriately located at the end of the polypeptide to be translated.

[0231] As indicated, the expression vector may include at least one selection marker. These markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells, such as Drosophila S2 and Spodoptera litura Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma cells, and HK 293 cells and plant cells. Culture media and conditions suitable for host cells described herein are known in the art.

[0232] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be apparent to the skilled artisan.

[0233] Non-limiting bacterial promoters suitable for use include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters, and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoters of retroviral LTRs (e.g., promoters of Rous sarcoma virus (RSV)), and metallothionein promoters (e.g., mouse metallothionein)-I promoter.

[0234] In Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For detailed descriptions, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153: 516-544 (1997).

[0235] The introduction of the construct into the host cell can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0236] The transcription of the DNA encoding the anti-4-1BB×CCR8 antibody of the present invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10 to 300 bp, which can enhance the transcriptional activity of the promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located at the 100th to 270th base pair on the rear side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer located on the rear side of the replication origin, and the adenovirus enhancer.

[0237] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

[0238] Polypeptides (e.g., anti-4-1BB×CCR8 antibodies) can be expressed in a modified form, such as a fusion protein (e.g., GST-fusion) or with a histidine tag, and include not only secretion signals, but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in host cells during purification or subsequent processing and storage. In addition, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Among other things, adding peptide moieties to polypeptides to cause secretion or excretion, improve stability, and promote purification is a familiar and conventional technique in the art.

[0239] The present invention also provides nucleic acids that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein. The invention relates to an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the amino acid sequences described herein.

[0240] The present invention also provides nucleic acids having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein. The invention relates to an amino acid sequence having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the amino acid sequences described.

[0241] In some embodiments, the present disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0242] In some embodiments, the amino acid sequence (a) comprises the amino acid sequence; (b) consists of the amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0243] In some embodiments, the nucleic acid sequence (a) comprises a nucleic acid sequence; (b) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0244] In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to ignore optimal alignment and non-homologous sequences for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, and considering the number of gaps and the length of each gap, these gaps need to be introduced to achieve optimal alignment of the two sequences. For example, the Blossum 62 scoring matrix (gap penalty of 12, gap extension penalty of 4, frameshift gap penalty of 5) can be used to complete sequence comparison and determination of the percentage identity between the two sequences.

[0245] The present invention provides one or more nucleic acids encoding any polypeptide described herein. In some embodiments, nucleic acids (e.g., cDNA) include polynucleotides encoding heavy chain polypeptides described herein. In some embodiments, nucleic acids include polynucleotides encoding light chain polypeptides described herein. In some embodiments, nucleic acids include polynucleotides encoding scFv polypeptides described herein.

[0246] In some embodiments, the vector may have two nucleic acids described herein, wherein the vectors co-encode and bind to the VL and VH regions of 4-1BB. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids described herein, wherein the pair of vectors co-encode and bind to the VL and VH regions of 4-1BB.

[0247] In some embodiments, the vector comprises two nucleic acids described herein, wherein the vectors jointly encode and bind to the VL and VH regions of CCR8. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids described herein, wherein the pair of vectors jointly encode and bind to the VL and VH regions of CCR8.

[0248] Treatment

[0249] The methods described herein include methods for treating a disorder associated with cancer. Generally, the methods include administering a therapeutically effective amount of an anti-4-1BB×CCR8 antibody described herein to a subject in need or determined to be in need of such treatment.

[0250] As used herein, "treatment" refers to improving at least one symptom of a condition associated with cancer. Cancer usually results in death; therefore, treatment can extend life expectancy or improve survival rate (e.g., extend by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years). Administration of a therapeutically effective amount of an agent described herein for treating a condition associated with cancer will result in a reduction in the number of cancer cells, a reduction in tumor volume, relief of symptoms, and / or an increase in life expectancy or survival rate.

[0251] As used herein, the term "cancer" refers to cells with autonomous growth ability, i.e., abnormal states or conditions characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the histopathological type or invasive stage. The term "tumor" used herein refers to cancer cells, such as cancer cell clusters. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems, such as malignant tumors affecting the lungs, breasts, thyroids, lymphs, gastrointestinal tracts and urogenital tracts, and adenocarcinomas (e.g., colon cancer) including malignant tumors such as most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestine cancers and esophageal cancers. In some embodiments, the reagents described herein are designed to treat or diagnose cancers in subjects. The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer and melanoma. In some embodiments, cancer is renal cancer or melanoma. Exemplary cancers include cancers formed by tissues of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcoma, for example, which includes malignant tumors composed of carcinoma tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or a cancer in which the tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor of mesenchymal origin.

[0252] In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0253] On the one hand, the present disclosure also provides a method for treating cancer in a subject, a method for reducing the rate at which tumor volume increases over time in a subject, a method for reducing the risk of metastasis, or a method for reducing the risk of metastasis. The risk of additional metastasis occurring in a subject. In some embodiments, treatment can stop, slow down, delay or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the quantity, severity and / or duration of one or more symptoms of cancer in a subject.

[0254] In one aspect, the invention is characterized by administering a therapeutically effective amount of an anti-4-1BB×CCR8 antibody described herein to a subject in need thereof, such as a subject suffering from or identified or diagnosed with cancer (e.g., cancer), B-cell lymphoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell lung cancer (NSCLC), mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, or renal cancer. In some embodiments, the cancer is a solid tumor, lung cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer, or brain cancer.

[0255] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human or non-human to which the treatment according to the method of the present invention is provided. The present invention contemplates veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., people under 18 years of age). In addition to humans, patients also include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other domestic, farm and zoo animals.

[0256] In some embodiments, the compositions and methods described herein can be used to treat patients at risk for cancer. Patients suffering from cancer can be identified using various methods known in the art.

[0257] As used herein, "effective amount" refers to an amount or dosage sufficient to achieve a beneficial or desired result, including stopping, slowing, delaying or inhibiting the progression of a disease such as cancer. The effective amount will vary depending on, for example, anti-4-1BB×CCR8 antibodies, anti-4-1BB×CCR8 antigen-binding fragments, polynucleotides encoding anti-4-1BB×CCR8 antibodies to be administered, vectors containing the polynucleotides and / or compositions thereof, the severity of symptoms and the age and weight of the subject for which the administration route is directed, and therefore administration can be determined on an individual basis.

[0258] An effective amount may be administered once or multiple times. For example, an effective amount of an anti-4-1BB×CCR8 antibody or an anti-4-1BB×CCR8 antigen binding fragment is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow down and / or delay the progression of a patient's autoimmune disease or cancer, or an amount sufficient to improve, stop, stabilize, reverse, slow down and / or delay the proliferation of in vitro cells (e.g., biopsy cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)). As understood in the art, the effective amount of an anti-4-1BB×CCR8 antibody, an anti-4-1BB×CCR8 antigen binding fragment or an anti-4-1BB×CCR8 antibody-drug conjugate may vary, depending on the patient's medical history and other factors, such as the type (and / or dosage) of the drug used.

[0259] The effective amount and schedule for administering the anti-4-1BB×CCR8 antibodies, anti-4-1BB×CCR8 antigen-binding fragments, anti-4-1BB×CCR8 antibody encoding polynucleotides and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. It will be appreciated by those skilled in the art that the dose that must be administered will vary depending on, for example, the mammal to which the anti-4-1BB×CCR8 antibodies, anti-4-1BB×CCR8 antigen-binding fragments, anti-4-1BB×CCR8 antibody encoding polynucleotides and / or compositions disclosed herein will be administered, the route of administration, the specific type of agent or composition disclosed herein used, and other drugs administered to the mammal.

[0260] The effective amount of anti-4-1BB×CCR8 antibody is typically daily dosed at 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose may be less than 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or 0.1 mg / kg. In some embodiments, the dose may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dosage is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg or 0.1 mg / kg.

[0261] In some embodiments, the anti-4-1BB×CCR8 bispecific antibodies described herein kill tumor cells and / or reduce tumor size in subjects receiving the anti-4-1BB×CCR8 bispecific antibodies (e.g., by activating 4-1BB-related signaling pathways or recruiting and depleting immune cells expressing CCR8, such as tumor-infiltrating Tregs expressing CCR8). In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of tumor cells are killed by administering the anti-4-1BB×CCR8 bispecific antibodies described herein.

[0262] In some embodiments, the anti-tumor effect of the anti-4-1BB×CCR8 bispecific described herein is to enhance tumor immunity and improve survival by modulating Treg / Teff cells (e.g., in mice or humans).

[0263] In some embodiments, administration of a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and 4-1BB can reduce the tumor volume (tumor size) of a subject. In some embodiments, the tumor volume of a subject receiving an anti-4-1BB×CCR8 bispecific antibody described herein is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the tumor size of a subject not administered an anti-4-1BB×CCR8 bispecific antibody. In some embodiments, the tumor size of a subject receiving an anti-4-1BB antibody and an anti-CCR8 antibody described herein is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the tumor size when anti-4-1BB antibody or anti-CCR8 antibody is administered alone.

[0264] In some embodiments, administration of the anti-4-1BB×CCR8 bispecific antibody described herein increases the survival rate of subjects receiving the antibody. In some embodiments, the survival rate is increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the survival rate of subjects not administered anti-4-1BB×CCR8 bispecific antibody or administered anti-4-1BB antibody alone or administered anti-CCR8 antibody alone.

[0265] In certain embodiments, one or more other therapeutic agents may be administered to a subject.Other therapeutic agents may include one or more selected from B-Raf inhibitors, 4-1BB inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, CCR8 inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinases (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors.In certain embodiments, other therapeutic agents are indoleamine 2,3-dioxygenase-1) (IDO1) inhibitors (e.g., epacadostat).

[0266] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of 4-1BB inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, activated Hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0267] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trabanib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzatalem.

[0268] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from trabectedin, albumin-bound paclitaxel, tebralib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzatoline.

[0269] In some embodiments, the additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0270] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0271] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-TIM3 antibody, or an anti-GITR antibody.

[0272] Pharmaceutical compositions and routes of administration

[0273] Also provided herein is a pharmaceutical composition containing at least one (e.g., one, two, three, or four) anti-4-1BB×CCR8 antibody (e.g., bispecific antibody), anti-4-1BB×CCR8 antigen-binding fragment, or anti-4-1BB×CCR8 antibody-drug conjugate described herein. The pharmaceutical composition can be formulated in any manner known in the art.

[0274] The pharmaceutical composition is prepared to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and isotonic agents, such as sugars (e.g., dextrose), polyols (e.g., mannitol or sorbitol), salts (e.g., sodium chloride) or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition may be formulated and packaged in ampoules, disposable syringes or multi-dose vials. When necessary, for example, in the preparation of injectables, proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant.

[0275] Compositions containing one or more of any of the anti-4-1BB×CCR8 antibodies or anti-4-1BB×CCR8 antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage).

[0276] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population): the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. If a drug exhibits adverse side effects, care should be taken to minimize the potential for harm (i.e., reduce adverse side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0277] The data obtained from cell culture experiments and animal studies can be used to formulate the appropriate dose of any given agent for a subject (e.g., a human). The therapeutically effective amount of an antibody or antigen binding fragment or antibody-drug conjugate (e.g., anti-4-1BB×CCR8 antibody, anti-4-1BB×CCR8 antigen binding fragment or anti-4-1BB×CCR8 antibody-drug conjugate) is a therapeutically effective amount of a subject (e.g., a human subject determined to have cancer) or a subject determined to be at risk of disease (e.g., a subject who previously had cancer but is now cured) to reduce the severity, frequency and / or duration of one or more symptoms of the subject's (e.g., human) disease. A healthcare professional or veterinary professional can determine the effectiveness and dosage of any antibody or antigen binding fragment or antibody-drug conjugate described herein (e.g., anti-4-1BB×CCR8 antibody, anti-4-1BB×CCR8 antigen binding fragment or anti-4-1BB×CCR8 antibody-drug conjugate) using methods known in the art and by observing one or more disease symptoms of a subject (e.g., a human). Certain factors may influence the dosage and duration required to effectively treat a subject (e.g., the severity of the disease or condition, previous treatments, the general health and / or age of the subject, and the presence of other medical conditions).

[0278] Exemplary dosages include milligram or microgram amounts of any antibody or antigen-binding fragment, or antibody-drug conjugate (e.g., anti-4-1BB×CCR8 antibody, anti-4-1BB×CCR8 antigen-binding fragment, or anti-4-1BB×CCR8 antibody-drug conjugate). The 4-1BB×CCR8 antibody-drug conjugate described herein is contained per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 1 mg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg or about 0.1 mg / kg to about 0.5 mg / kg). Although these dosages cover a wide range, those of ordinary skill in the art will understand that the efficacy of therapeutic agents varies, and the effective amount can be determined by methods known in the art. Typically, a relatively low dose is first administered, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still in the development phase) can then gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, time, mode of administration, route of administration, excretion rate and in vivo half-life of the therapeutic agent.

[0279] The drug of the antibody, antigen binding fragment or antibody-drug conjugate described herein (e.g., anti-4-1BB×CCR8 antibody, anti-4-1BB×CCR8 antigen binding fragment or anti-4-1BB×CCR8 antibody-drug conjugate) can be administered once or multiple times during the treatment process. For example, the antibody, antigen binding fragment or antibody-drug conjugate described herein (e.g., anti-4-1BB×CCR8 antibody, anti-4-1BB×CCR8 antigen binding fragment or anti-4-1BB×CCR8 antibody-drug conjugate) can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In some embodiments, the antibody, antigen binding fragment or antibody-drug conjugate (e.g., anti-4-1BB×CCR8 antibody, anti-4-1BB×CCR8 antigen binding fragment or anti-4-1BB×CCR8 antibody-drug conjugate) is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

[0280] The pharmaceutical composition can be included in a container, package or dispenser together with instructions for administration. The present invention also provides methods for making antibodies or antigen-binding fragments, or antibody-drug conjugates (e.g., anti-4-1BB×CCR8 antibodies, anti-4-1BB×CCR8 antigen-binding fragments, or anti-4-1BB×CCR8 antibody-drug conjugates) for various uses described herein. Example

[0281] The present invention will be further described by the following examples, but these examples are not intended to limit the scope of the invention described in the claims.

[0282] Example 1: Molecular Construction

[0283] To prepare anti-CCR8 monoclonal antibody (CCR8 mAb), the VH region was linked to wild-type mIgG2a Fc, and the VL region was linked to mouse lambda light chain constant region. To prepare anti-4-1BB monoclonal antibody (4-1BB mAb), the VH region was linked to wild-type mIgG2a Fc, and the VL region was linked to mouse kappa light chain constant region.

[0284] To prepare 4-1BB-1×CCR8-1 bispecific antibody (BsAb) and 4-1BB-2×CCR8-1 bispecific antibody ( Figure 1A-1B ), the scFv (single-chain fragment variable region VH-(G4S)4-VL) of the anti-CCR8 antibody was connected to the N-terminus of the anti-4-1BB light chain (LC) through a G4S linker (SEQ ID NO: 86), and the 4-1BB VH was connected to the wild-type mIgG2a Fc.

[0285] like Figure 1C-1G As shown, three 4-1BB-2×CCR8-2 bispecific antibodies were prepared ( Figure 1C-1E ), 1 4-1BB-3×CCR8-3 bispecific antibody ( Figure 1F ) and a 4-1BB-4×CCR8-4 bispecific antibody ( Figure 1G ). All of these antibodies contain wild-type hIgG1 Fc. The three 4-1BB-2×CCR8-2 bispecific antibodies include 4-1BB-2×CCR8-2(LC1)BsAb( Figure 1C )、4-1BB-2×CCR8-2(LC2)BsAb( Figure 1D ) and 4-1BB-2×CCR8-2(HC)BsAb( Figure 1E ).

[0286] The fragments were synthesized (IDT) and assembled (NEB) as described above. HiFi DNA Assembly) into pcDNA3.4 or pcDNA3.4 carrying the required fragment. Transform the assembled plasmid into competent Escherichia coli ( 5-alpha). The clones with correct sequences confirmed by sequencing were further cultured with lysogeny broth (LB medium) containing carbenicillin (100 μg / ml) for plasmid amplification and then plasmid purification (QIAGEN Plasmid Plus Kits). The plasmids were eluted in nuclease-free H2O (Sigma) and stored at -80°C.

[0287] Example 2: Transfection and purification of antibodies

[0288] All antibody light and heavy chain coding sequences were generated by direct DNA synthesis and cloned into mammalian expression vectors. The cloned sequences were verified by Sanger sequencing.

[0289] Chinese hamster ovary (CHO) cells were grown in CHOgro medium (Mirus) supplemented with 4 mM L-glutamine and 0.33% Poloxamer-188 and diluted to 4 × 10 in fresh cell culture medium for transfection. 6 cells / ml. By lipofectamine transfection, 4×10 6 The cells contained 1 μg of plasmid DNA. The transfected cells were kept in a shaking incubator at 32°C, 5% CO2 and 70% humidity at 125 rpm.

[0290] Protein expression titer was measured every 5 days. Cell supernatant was collected by centrifugation 10-14 days after transfection and filtered through a 0.22 μm PES membrane filter. The expressed antibody was purified by Protein A chromatography ( AF-rProtein AHC-650M resin, Tosoh), and anion exchange resin (TOYOPEARL NH2-750F, Tosoh) was used to remove aggregates.

[0291] Purified antibodies were obtained by Software-controlled HPLC (Dionex The antibody purity and aggregation were determined using a size exclusion column TSKgel UP-SW3000, 2 μm, 4.6 mm ID×15 cm (Tosoh).

[0292] All purified antibodies had a monomer purity of 97% or greater, were sterilized through 0.22 μm PES membrane filters, and used for binding and functional assays.

[0293] Example 3: Anti-tumor effect of 4-1BB×CCR8 bispecific antibody in CT26 colon cancer model (Study 1)

[0294] BalB / c mice were used to verify the antitumor effect of 4-1BB×CCR8 bispecific antibody on CT26 colon cancer model. Female BalbC mice aged 6-9 years from the Weitong Lihua Laboratory Research Model and Service Center (Guangdong, China) were used in the experiment. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC). Figure 2 As shown, on day 0, CT26 colon cancer cells (5×10 5 The cells were subcutaneously injected into the right posterior abdomen of mice in 0.1 ml PBS for tumor growth. When the average tumor size reached about 80-120 mm on day 7, 3 At 14:00, mice were randomized and assigned to treatment groups. On days 7, 10, 14, and 18, mice were treated with 4-1BB-1×CCR8-1 bispecific antibody, CCR8-1 mAb, and 4-1BB-1 mAb. The first, second, and third doses were 1 (mAb) and 1.33 (BsAb) mg / kg, respectively, while the fourth dose was 10 mg / kg for both mAb and BsAb. Control mice were treated with phosphate-buffered saline (PBS). The first dose was intravenous, and the second, third, and fourth doses were intraperitoneal. Tumors were measured three times a week in two dimensions using calipers, and the volume was expressed in cubic millimeters using the following formula: V = (L×W×W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). Graphing and statistical analysis were performed using the GraphPad Prism program. Tumor growth was analyzed using two-way analysis of variance and Dunnett's multiple comparison test, and survival percentage data were analyzed using Kaplan-Meyer survival analysis.

[0295] In the comparison of the mean tumor volume between the single-agent mAb administration group and the 4-1BB-1×CCR8-1 bispecific antibody administration group (e.g., single-agent CCR8-1 mAb or single-agent 4-1BB-1 mAb administration group), the mean tumor volume in the 4-1BB-1×CCR8-1 bispecific antibody administration group was significantly smaller than that in the single-agent mAb administration group ( Figure 3 ).

[0296] The study also compared complete tumor remission rates. At the end of the study on day 28, treatment with the 4-1BB-1 × CCR8-1 bispecific antibody (2 of 6 tumor-free mice) resulted in a greater number of tumor-free mice (95% CI, 2.3–3.7) compared with treatment with either CCR8-1 mAb alone (1 of 6 tumor-free mice) or 4-1BB-1 mAb alone (0 of 6 tumor-free mice). Figures 4A-4D ).

[0297] These results indicate that the 4-1BB×CCR8 bispecific antibody has a superior effect in inhibiting tumor growth compared with other single mAbs in this CT26 mouse model. The 4-1BB-1×CCR8-1 bispecific antibody induced a stronger anti-tumor effect in the form of tumor volume inhibition and tumor-free mice compared with either monoclonal anti-CCR8 or anti-4-1BB antibodies alone.

[0298] These results suggest that Treg / Teff cells can be modulated in humans to enhance antitumor immunity and improve survival, as demonstrated by the antitumor effects of anti-4-1BB×CCR8 bispecific antibody administered to mice.

[0299] As observed in the clinical development of Urelumab, the main problem of 4-1BB antibody treatment is dose-limiting hepatotoxicity. The most common adverse events were increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). After treatment with the 4-1BB×CCR8 bispecific antibody, blood samples of CT26 cell-implanted mice were collected to measure ALT and AST. No significant increase in ALT and AST was observed, suggesting that the risk of hepatotoxicity caused by 4-1BB×CCR8 bispecific antibodies is generally small.

[0300] Example 4: Anti-tumor effect of 4-1BB×CCR8 bispecific antibody in CT26 colon cancer model (Study 2)

[0301] BalB / c mice were used to verify the antitumor effect of 4-1BB×CCR8 bispecific antibody on CT26 colon cancer model. Female BalbC mice aged 6-9 years from the Weitong Lihua Laboratory Research Model and Service Center (Guangdong, China) were used in the experiment. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC). Figure 5 As shown, on day 0, CT26 colon cancer cells (5×10 5 The cells were subcutaneously injected into the right posterior abdomen of mice in 0.1 ml PBS for tumor growth. When the average tumor size reached about 80-120 mm on day 7, 3At 14:00 p.m., mice were randomized and assigned to treatment groups. On days 7, 10, and 14, mice were treated with 4-1BB-1×CCR8-1 bispecific antibody, CCR8-1 mAb, or 4-1BB-1 mAb. The doses of monoclonal antibody were 1 mg / kg and 3 mg / kg, and the doses of bispecific antibody were 1.33 mg / kg and 4 mg / kg. Control mice were treated with phosphate-buffered saline (PBS). The first dose was intravenous, and the second and third doses were intraperitoneal. Tumors were measured three times a week in two dimensions using calipers, and the volume was expressed in cubic millimeters using the following formula: V=(L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). GraphPad Prism program was used for plotting and statistical analysis. Two-way ANOVA and Dunnett's multiple comparison test were used to analyze tumor growth, and Kaplan-Meyer survival analysis was used to analyze survival percentage data.

[0302] In the comparison of the mean tumor volume between the monoclonal antibody group and the 4-1BB-1×CCR8-1 bispecific antibody group, on days 17, 19, and 23, the mean tumor volume in the 4-1BB-1×CCR8-1 bispecific antibody group was significantly smaller than that in the monoclonal antibody group ( Figure 6A-6B ).

[0303] At the end of the study on day 49, the complete response rates were compared. For either the 1 mg / kg single mAb or 1.33 mg / kg bispecific antibody dose, treatment with the 4-1BB-1 × CCR8-1 antibody (2 out of 6 tumor-free mice) resulted in a higher number of tumor-free mice than treatment with either the CCR8-1 mAb alone (0 out of 6 tumor-free mice) or the 4-1BB-1 mAb alone (0 out of 6 tumor-free mice). Figures 7A-7C For either the 3 mg / kg single mAb or 4 mg / kg bispecific antibody dose, treatment with the 4-1BB-1 × CCR8-1 antibody (3 out of 6 tumor-free mice) resulted in a higher number of tumor-free mice than treatment with either the CCR8-1 mAb alone (0 out of 6 tumor-free mice) or the 4-1BB-1 mAb alone (0 out of 6 tumor-free mice). Figure 7D-7F ). In addition, the 4-1BB-1×CCR8-1 bispecific antibody administration group required a shorter tumor regression time compared with either monoclonal CCR8-1 or 4-1BB-1 mAb alone.

[0304] The study also calculated the percentage of survival during the treatment period. Compared with treatment with CCR8-1 or 4-1BB-1 monoclonal antibodies alone, treatment with 4-1BB-1×CCR8-1 bispecific antibody increased the overall percentage of survival of mice ( Figure 8 ).

[0305] The above results prove that in the CT26 mouse model, the time required for tumor regression in the 4-1BB×CCR8 bispecific antibody-administered group was short, and it showed a strong regression effect and high survival rate compared with treatment with monoclonal CCR8 or 4-1BB mAb alone.

[0306] As observed in the clinical development of Urelumab, the main problem of 4-1BB antibody treatment is dose-limiting hepatotoxicity. The most common adverse events were increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). After treatment with the 4-1BB×CCR8 bispecific antibody, blood samples of CT26 cell-implanted mice were collected to measure ALT and AST. No significant increase in ALT and AST was observed, suggesting that the risk of hepatotoxicity caused by 4-1BB×CCR8 bispecific antibodies is generally small.

[0307] Example 5: Anti-tumor effect of 4-1BB×CCR8 bispecific antibody in CT26 colon cancer model (Study 3)

[0308] BalB / c mice were used to verify the antitumor effect of 4-1BB×CCR8 bispecific antibody on CT26 colon cancer model. Female BalbC mice aged 6-9 years from the Weitong Lihua Laboratory Research Model and Service Center (Guangdong, China) were used in the experiment. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC). Fig. 9 As shown, on day 0, CT26 colon cancer cells (5×10 5 The cells were subcutaneously injected into the right posterior abdomen of mice in 0.1 ml PBS for tumor growth. When the average tumor size reached about 80-120 mm on day 7, 3 At 14 days, mice were randomized and assigned to treatment groups. On days 7, 10, and 14, mice were treated with 4-1BB-2×CCR8-1 bispecific antibody, CCR8-1 mAb, or 4-1BB-2mAb. The dose of monoclonal antibody was 1 mg / kg, and the dose of bispecific antibody was 1.33 mg / kg. Control mice were treated with phosphate-buffered saline (PBS). The first dose was intravenous, and the second and third doses were intraperitoneal. Tumors were measured three times a week in two dimensions using calipers, and the volume was expressed in cubic millimeters using the following formula: V = (L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). GraphPad Prism program was used for plotting and statistical analysis. Tumor growth was analyzed using two-way analysis of variance and Dunnett's multiple comparison test, and survival percentage data were analyzed using Kaplan-Meyer survival analysis.

[0309] In the comparison of the average tumor volume between the monoclonal antibody administration group and the 4-1BB-2×CCR8-1 bispecific antibody administration group, the average tumor volume of the 4-1BB-2×CCR8-1 bispecific antibody administration group was significantly smaller than that of each monoclonal antibody administration group after day 30 ( Fig.10 ).

[0310] At the end of the study on day 49, the complete response rates were compared. Treatment with the 4-1BB-2 × CCR8-1 bispecific antibody resulted in a higher number of tumor-free mice than treatment with CCR8-1 mAb alone (no tumor-free mice out of 6) or 4-1BB-2 mAb alone (no tumor-free mice out of 6) for either the 1 mg / kg single mAb or 1.33 mg / kg bispecific antibody doses. Figures 11A-11D ).

[0311] The above results prove that in the CT26 mouse model, the time required for tumor regression in the 4-1BB×CCR8 bispecific antibody-administered group was short, and it showed a strong regression effect and high survival rate compared with treatment with monoclonal CCR8 or 4-1BB mAb alone.

[0312] As observed in the clinical development of Urelumab, the main problem of 4-1BB antibody treatment is dose-limiting hepatotoxicity. The most common adverse events were increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). After treatment with the 4-1BB×CCR8 bispecific antibody, blood samples of CT26 cell-implanted mice were collected to measure ALT and AST. No significant increase in ALT and AST was observed, suggesting that the risk of hepatotoxicity caused by 4-1BB×CCR8 bispecific antibodies is generally small.

[0313] Example 6. Binding of 4-1BB×CCR8 bispecific antibody to CHO cells expressing human 4-1BB

[0314] The ability of the 4-1BB×CCR8 bispecific antibody to bind to CHO cells expressing human 4-1BB was evaluated. Approximately 50,000 CHO cells expressing human 4-1BB were incubated on ice for 30 minutes with serially diluted antibodies (4-1BB-2×CCR8-2(LC1)bsAb, 4-1BB-2×CCR8-2(LC2)bsAb and 4-1BB-2×CCR8-2(HC)bsAb) in FACS buffer (PBS+0.5% BSA). After incubation, the cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined, and graphs were plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). Figures 12A-12B As shown, all three 4-1BB×CCR8 bispecific antibodies bound well to CHO cells expressing human 4-1BB.

[0315] Example 7. Binding of 4-1BB×CCR8 bispecific antibody to CHO cells expressing human CCR8

[0316] The ability of the 4-1BB×CCR8 bispecific antibody to bind to CHO cells expressing human CCR8 was evaluated. Approximately 50,000 CHO cells expressing human CCR8 were incubated on ice for 30 minutes with serially diluted antibodies (4-1BB-2×CCR8-2(LC1)bsAb, 4-1BB-2×CCR8-2(LC2)bsAb and 4-1BB-2×CCR8-2(HC)bsAb) in FACS buffer (PBS+0.5% BSA). After incubation, the cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined, and graphs were plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). Figures 13A-13B As shown, the three 4-1BB×CCR8 bispecific antibodies all bound well to CHO cells expressing human CCR8.

[0317] Example 8. Binding of 4-1BB×CCR8 bispecific antibody to CHO cells expressing human CCR8 and 4-1BB

[0318] The ability of the 4-1BB×CCR8 bispecific antibody to bind to CHO cells expressing human CCR8 and 4-1BB was evaluated. Approximately 50,000 CHO cells expressing human CCR8 and 4-1BB were incubated on ice for 30 minutes with serially diluted antibodies (4-1BB-2×CCR8-2(LC1)bsAb, 4-1BB-2×CCR8-2(LC2)bsAb and 4-1BB-2×CCR8-2(HC)bsAb) in FACS buffer (PBS+0.5% BSA). After incubation, the cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined, and graphs were plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). Fig.14 As shown, all three 4-1BB×CCR8 bispecific antibodies bound well to CHO cells expressing human CCR8 and 4-1BB.

[0319] Example 9. 4-1BB×CCR8 bispecific antibody mediates PBMC killing of CHO cells co-expressing human CCR8 and 4-1BB

[0320] The killing efficacy of effector PBMC cells against target CHO cells co-expressing hCCR8 and h4-1BB in the presence of different forms of 4-1BB×CCR8 bispecific antibodies was evaluated. PBMCs were isolated from healthy human blood samples of human donors #16 and #17 by the ficoll method. CHO cells were labeled with CFSE (ThermoFisher, Cat#: C34554). Approximately 100,000 PBMCs were incubated with 10,000 CFSE+CHO cells in the presence of serially diluted antibodies (4-1BB-2×CCR8-2(LC1) BsAb, 4-1BB-2×CCR8-2(LC2) BsAb, and 4-1BB-2×CCR8-2(HC) BsAb) in complete RPMI medium (supplemented with 10% heat-inactivated FBS and 5% penicillin / streptomycin) at 37°C for 24 hours to maintain an effector-target ratio (E:T) of 10:1. After incubation, cells were washed twice with FACS buffer (buffer used in fluorescence activated cell sorting) and stained with 50 μl Live dead Near-IR (diluted 1:2000 in PBS) for 10 min at room temperature and then washed twice. Cells were suspended in 100 μl FACS buffer and stained with Flow cytometry was used for analysis. CHO cells were divided into CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume under all treatment conditions. The percentage of target cell killing was calculated as follows: (absolute number of CFSE+CHO cells in the non-treated group - absolute number of CFSE+Live dead Near-IR-CHO cells in the treated group) / absolute number of CFSE+CHO cells in the non-treated group × 100. Figures 15A-15B As shown, three different forms of 4-1BB×CCR8 bispecific antibodies can effectively enhance the cell killing effect on CHO cells co-expressing hCCR8 and h4-1BB.

[0321] Example 10. Other Exemplary 4-1BB×CCR8 BsAbs

[0322] Several other 4-1BB×CCR8 bispecific antibodies (4-1BB-3×CCR8-3 bsAb, 4-1BB-4×CCR8-4 bsAb) were also tested, and each bispecific antibody showed higher antitumor effects, lower hepatotoxicity, and lower E:T ratios compared with CCR8 mAb and 4-1BB mAb alone.

[0323] These results suggest that Treg / Teff cells can be modulated to enhance tumor immunity and improve survival in humans, as demonstrated by the antitumor effects of anti-4-1BB × CCR8 bispecific antibody administered to mice.

[0324] Table 1: Some amino acid sequences described in the present invention

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

Claims

1. An antibody or an antigen-binding fragment thereof, comprising: a) a first antigen binding domain that specifically binds to 4-1BB; and b) A second antigen binding domain that specifically binds to the CC motif chemokine receptor 8 (CCR8).

2. The antibody or antigen-binding fragment thereof of claim 1, wherein 4-1BB is human 4-1BB, and wherein CCR8 is human CCR8.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the second antigen-binding domain is a single-chain fragment variable (scFv) domain, wherein VH2 and VL2 are connected by a first linker.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the second antigen-binding domain is connected to the N-terminus of the light chain via a second linker.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising an Fc region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the C-terminus of the VH1 of the first antigen-binding domain is linked to the Fc region, optionally via the CH1 domain. 9 . The antibody or antigen-binding fragment thereof according to claim 3 , wherein the antibody comprises a first heavy chain of VH1 and a first light chain of VL1; and a second heavy chain of VH2 and a second light chain of VL2.

10. The antibody or antigen-binding fragment thereof according to any one of claims 7 to 9, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4. The antibody or antigen-binding fragment thereof according to claim 10 , wherein the Fc region is the Fc region of human IgG1 or IgG2. 12 . The antibody or antigen-binding fragment thereof according to claim 11 , wherein the Fc region is a wild-type Fc, or an Fc with enhanced ADCC activity, or an Fc with enhanced ADCP activity.

13. The antibody or antigen-binding fragment thereof according to any one of claims 3 to 12, wherein a first heavy chain variable region (VH1) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; in, The selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, 3 amino acid sequence are one of the following: (1) The selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 4, 5, and 6, respectively; (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 24, 25, and 26, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 27, 28, and 29, respectively; (3) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 50, 51, and 52, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 53, 54, and 55, respectively; (4) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72 and 73, respectively.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein a second heavy chain variable region (VH2) comprising CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and a second light chain variable region (VL2) comprising CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, in, The selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are one of the following: (1) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 11, 12, and 13, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 14, 15, and 16, respectively; (2) the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 36, 37, and 38, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 39, 40, and 41, respectively; (3) the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58, 59, and 60, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 61, 62, and 63, respectively; (4) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 76, 77 and 78, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 79, 80 and 81, respectively.

15. The antibody or antigen-binding fragment thereof according to claim 13 or 14, wherein The selected VH1 CDRs 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL1 CDRs 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 4, 5, and 6, respectively; the selected VH2 CDRs 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 11 12 and 13, respectively, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 14, 15, and 16, respectively.

16. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

17. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-16.

18. The method of claim 17, wherein the cancer is a solid tumor or a hematological tumor.

19. The method of claim 18, wherein the cancer is breast cancer, uterine corpus cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, gastric cancer, non-small cell lung cancer, spleen cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer, sarcoma, a blood cell cancer such as a leukemia or lymphoma, bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, or liver cancer.

20. The method of claim 19, wherein the cancer is breast cancer, uterine corpus cancer, ovarian cancer, lung cancer, colorectal cancer, renal cancer, and sarcoma.

21. The method of claim 18, wherein the cancer is colon cancer.

22. The method of any one of claims 17-21, wherein the subject is further treated with an effective amount of an anti-OX40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, or an anti-PD-L1 antibody.

23. A method for reducing tumor growth rate, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-16.

24. A method for killing tumor cells, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-16.

25. A method for increasing an immune response in a subject, the method comprising An effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-16 is administered to a subject.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, and a pharmaceutically acceptable carrier.

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