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

Abstract

This disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB and an antibody or antigen-binding fragment thereof that specifically binds to CCR8, and methods of uses thereof.

Description

[0001] ANTI-4-1BB AND ANTI-CCR8 ANTIBODIES AND USES THEREOF

[0002] CLAIM OF PRIORITY

[0003] This application claims priority to PCT / CN2023 / 121286, filed on September 26, 2023. The entire contents of the foregoing are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named 55679-0012WO2_ST26_SL.XML.” The XML file, created on September 25, 2024, is 77,404 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This disclosure relates to anti-4- IBB antibodies or antigen-binding fragments thereof, anti-CCR8 antibodies or antigen-binding fragments thereof, and methods of using the antibodies described herein.

[0008] BACKGROUND

[0009] Cancer is currently one of the diseases that have the highest human mortality. According to the World Health Organization statistical data, in 2012, the number of global cancer incidence and death cases reached 14 million and 8.2 million, respectively. In China, the newly diagnosed cancer cases are 3.07 million, and the death toll is 2.2 million. Recent clinical and commercial success of anticancer antibodies has created great interest in antibody-based therapeutics.

[0010] There is a need to develop antibodies for use in various antibody-based therapeutics to treat cancers.

[0011] SUMMARY

[0012] This disclosure relates to antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to 4-1BB. This disclosure also relates to antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to C-C motif chemokine receptor 8 (CCR8). This disclosure also relates to methods of treating a subject in need thereof using the antibodies or antigen-binding fragments thereof that specifically bind to 4-1BB and / or the antibodies or antigen-binding fragments thereof that specifically bind to CCR8.

[0013] Accordingly, provided herein are methods of treating a subject having a tumor, the method comprising administering a therapeutically effective amount of: a) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB to the subject.

[0014] The present disclosure is also related to methods of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of: a) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB to the subject.

[0015] The present disclosure is also related to methods of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of: a) a composition comprising an antibody or antigenbinding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB to the subject.

[0016] In some embodiments, the 4-1BB is human 4-1BB, and wherein the CCR8 is human CCR8.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CCR8 is BMS-986340, LM-108, S-531011, SRF-114, ABBV-514, BAY3375968, CM369, HBM-1022, JTX-1811, PSB-114, ZL-1218, FPA 157, GB2101 orHFB-101110, and wherein the antibody or antigenbinding fragment thereof that specifically binds to 4-1BB is Utomilumab, Urelumab, EU-101, YH-004, ADG-106 or LVGN-6051.

[0018] In some embodiments, the tumor is a solid tumor or a blood tumor.

[0019] In some embodiments, the cancer is a solid tumor.

[0020] In some embodiments, the cancer is breast cancer, uterine corpus cancer, cervical cancer, ovary cancer, prostate cancer, lung cancer, stomach 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, blood cell cancer such as leukemia or lymphoma, bile duct carcinoma, gallbladder carcinoma, thyroid carcinoma, testicular carcinoma, thymic carcinoma, or hepatocarcinoma.

[0021] In some embodiments, the cancer is breast cancer, uterine corpus cancer, ovary cancer, lung cancer, colorectal cancer, kidney cancer and sarcoma. In some embodiments, the solid tumor is colon carcinoma.

[0022] In some embodiments, the administration of the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB reduces the tumor volume in the subject.

[0023] In some embodiments, the administration of the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB reduces more tumor volume than administering a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 alone or administering a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB alone.

[0024] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered one, two, three or more times.

[0025] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered every 1, 2, 3, 4, 5 or more days.

[0026] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered at the same dosage.

[0027] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered intravenously and / or intraperitoneally.

[0028] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered three times, wherein the first administration is performed intravenously and the second and third administrations are performed intraperitoneally.

[0029] In some embodiments, the methods described herein further comprise administering a therapeutically effective amount of an anti-OX40 antibody, an anti-PDl antibody, an anti-PDLl antibody, an anti-PDL2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, and / or an anti-CD40 antibody, to the subject. The present disclosure is also related to pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB, and a pharmaceutically acceptable carrier.

[0030] As used herein, the term “antigen-binding domain” refers to one or more protein domain(s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen(s) (e.g., an effector antigen or tumor antigen). In some examples, an antigenbinding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally- occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. One example of an antigen-binding domain is an antigen-binding domain formed by VH-VL. In some embodiments, an antigen-binding domain can include an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art. In some examples, an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a tumor antigen).

[0031] As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any 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, multi-specific antibodies (e.g., bi-specific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0032] 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, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain). Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F(ab’)2, and Fv fragments.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0034] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0035] DESCRIPTION OF DRAWINGS

[0036] FIG. 1 is a schematic diagram illustrating the anti-tumor study using the CT26 tumor model and dosing regimen.

[0037] FIG. 2 shows the mean tumor volume curve in the anti-tumor study using the CT26 tumor model. The mice were treated with CCR8-1 mAb (1 mg / kg), 4-1BB-1 mAb (1 mg / kg), or the combination of CCR8-1 mAb (1 mg / kg) and 4-1BB-1 mAb (1 mg / kg), with PBS serving as the negative control. Each curve represents the mean tumor volume of each treatment group.

[0038] FIGs. 3A-3C show the individual tumor volume curve in the anti-tumor study using the CT26 tumor model. Each curve represents one individual mouse treated with 1 mg / kg of CCR8-1 mAb (FIG. 3A), 1 mg / kg of 4-1BB-1 mAb (FIG. 3B), or the combination of 1 mg / kg of CCR8-1 mAb and 1 mg / kg of 4- 1BB-1 mAb (FIG. 3C).

[0039] FIG. 4 shows the survival rate of mice in the anti -tumor study using the CT26 tumor model.

[0040] FIG. 5 is a schematic diagram illustrating the anti -tumor study using the MC38 tumor model and dosing regimen.

[0041] FIG. 6 shows the mean tumor volume curve in the anti -tumor study using the MC38 tumor model. The mice were treated with CCR8-1 mAb (10 mg / kg), 4-1BB-1 mAb (10 mg / kg), or the combination of CCR8-1 mAb (10 mg / kg) and 4-1BB-1 mAb (10 mg / kg), with PBS serving as the negative control. Each curve represents the mean tumor volume of each treatment group.

[0042] FIGs. 7A-7C show the individual tumor volume curve in the anti -tumor study using the MC38 tumor model. Each curve represents one individual mouse treated with 10 mg / kg of CCR8-1 mAb (FIG. 7A), 10 mg / kg of 4-1BB-1 mAb (FIG. 7B), or the combination of 10 mg / kg of CCR8-1 mAb and 10 mg / kg of 4-1BB-1 mAb (FIG. 7C).

[0043] FIG. 8 shows the mice survival rate of mice in the anti -tumor study using the MC38 tumor model. FIGs. 9A-9C show the cell killing percentage of the target CHO cells co-expressing human CCR8 and human 4-1BB by effector MO macrophages through antibody dependent cellular phagocytosis (ADCP). The target CHO cells were treated with the combination of CCR8-2 mAb and 4-1BB-2 mAb. The effector and target (E:T) ratio was tested at 1 : 1 (FIG. 9A), 3 : 1 (FIG. 9B), or 5 : 1 (FIG. 9C).

[0044] DETAILED DESCRIPTION

[0045] This disclosure relates to antibodies or antigen -binding fragments thereof that specifically binds to 4-1BB and antibodies or antigen-binding fragments thereof that specifically binds to C-C motif chemokine receptor 8 (CCR8).

[0046] This disclosure also relates to methods of treating a subject having a tumor, the method comprising administering a therapeutically effective amount of: a) a composition comprising an antibody or antigenbinding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB to the subject.

[0047] This disclosure also relates to methods of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of: a) a composition comprising an antibody or antigenbinding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB to the subject.

[0048] This disclosure also relates to methods of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of: a) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigenbinding fragment thereof that specifically binds to 4-1BB to the subject.

[0049] Anti-4-lBB Antibody

[0050] Human 4- IBB (also known as tumor necrosis factor receptor superfamily member 9, TNFRSF59 and CD137) is a tumor necrosis factor (TNF) receptor superfamily member (TNF superfamily member 9) expressed on number of immune cells including activated T cells, B cells, dendritic cells (DC), activated natural killer (NK) cells, as well as nonimmune cells, such as activated endothelial cells. When activated by human 4- IBB ligand, human 4- IBB co-stimulates T cells, thereby providing an effective T cell immune response and generating immune memory by inducing multiple signaling cascades.

[0051] 4-1BB is a member of the TNF receptor superfamily and is an activation-induced T-cell costimulatory molecule. Signaling via 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 underscored in a number of diseases, including cancer.

[0052] Signaling via 4-1BB by agonistic mAb, soluble 4-1BBL, or cell lines expressing 4-1BBL in the presence of anti-T-cell-receptor Ab has been shown to cause T-cell expansion, cytokine induction, and upregulation of antiapoptotic genes, and to prevent activation-induced cell death. Kinetic studies and expression analyses have revealed that although levels of 4-1BB expression are comparable in CD4+ and CD8+ T cells, CD8+ T cells have a considerably higher proliferative potential than CD4+ T cells. A similar effect was observed in vivo. In addition to its effects on T cells, ligation of the 4-1BB receptor on DCs and macrophages induces the production of IL-12 and IL-8, respectively, by these cells.

[0053] To date, the role of 4-1BB signaling has been studied in several autoimmune processes, including rheumatoid arthritis, experimental autoimmune encephalomyelitis, and systemic lupus erythematosus. There is a dual immunoregulatory effects of 4-1BB signaling.

[0054] A detailed review 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.

[0055] The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to 4- 1BB (e.g., human 4- IBB). In one aspect, the disclosure provides an anti-4- IBB antibody or antigenbinding fragment thereof.

[0056] In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).In some embodiments, the heavy chain variable region (VH) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and the light chain variable region (VL) comprises CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs 1, 2, 3 amino acid sequences are one of the following: (1 ) the selected VH CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1 , 2, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

[0057] (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 22, 23, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 24, 25, 26, respectively:

[0058] (3) the selected VH CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 42, 43, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 44, 45, 46, respectively: and

[0059] (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, 66, respectively.

[0060] In some embodiments, the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2 and 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5 and 6, respectively.

[0061] In some embodiments, the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 22, 23, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 24, 25, 26, respectively.

[0062] In some embodiments, the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 42, 43, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 44, 45, 46, respectively.

[0063] In some embodiments, the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, 66, respectively.

[0064] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and the VL 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, 27, 47, or 67, and the selected VL sequence is SEQ ID NO: 8, 28, 48, or 68.

[0065] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1 , VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 7, 27, 47, or 67, and the selected VL sequence is SEQ ID NO: 8, 28, 48, or 68.

[0066] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8.

[0067] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 28.

[0068] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48.

[0069] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 67, and the selected VL sequence is SEQ ID NO: 68.

[0070] In some embodiments, the CDR definition is based on Kabat, Chothia, IMGT, contact or AbM definition.

[0071] In some embodiments, the antigen-binding domain specifically binds to human or mouse 4- IBB.

[0072] In some embodiments, the antigen-binding domain is a human or humanized antigen-binding domain.

[0073] In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv).

[0074] 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 VH of the antigen-binding domain is connected to the Fc region. In some embodiments, the C-terminus of the VH of the antigenbinding domain is connected to the Fc region through a CHI domain.

[0075] The anti-4- IBB antibodies or antigen-binding fragments described herein can be in any suitable structure. In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof is a single- chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) linked by a linker.

[0076] The linkers described herein can be any suitable linkers known in the art. In some embodiments, the linker includes 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: 81.

[0077] In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof includes a heavy chain sequence comprising the VH, wherein the 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: 9, 29, 49, or 69. In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof includes a light chain sequence comprising the VL, wherein the 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, 30, 50, or 70.

[0078] The disclosure provides anti-4-lBB antibodies and the modified antibodies thereof, including, e.g., chimeric antibodies, humanized antibodies, and human antibodies.

[0079] The anti-4-lBB antibodies or antigen-binding fragments thereof can also have various forms. Many different formats of bispecific antibodies or antigen-binding fragments thereof are known in the art, and are described e.g., in 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 the entirety.

[0080] In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof is a BiTE, a (scFv)?, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH- CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. In some embodiments, the anti-4-lBB antibody is a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a crossMab, a DAF (two-in- one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a KX-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a 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, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F(ab’)2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HC Ab, a scDiabody-Fc, a Diabody -Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFvl- PEG-scFv2. In some embodiments, the anti -4- IBB antibody or antigen-binding fragment thereof can be a TrioMab. In a TrioMab, the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.

[0081] In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof has two different heavy chains and two different light chains. Heterodimerization of heavy chains can be based on the knobs-into-holes or some other heavy chain pairing technique.

[0082] The anti-4-lBB antibody or antigen-binding fragment thereof can be a Duobody. The Fab-exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled matter in IgGl antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavylight chains.

[0083] In Dual-variable-domain antibody (DVD-Ig), additional VH and variable light chain (VL) domain are added to each N-terminus for bispecific targeting. This format resembles the IgG-scFv, but the added binding domains are bound individually to their respective N-termini instead of a scFv to each heavy chain N-terminus.

[0084] In scFv-IgG, the two scFv are connected to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites and is consequently also called tetravalent. There are no heavy-chain and light-chain pairing problem in the scFv-IgG.

[0085] In some embodiments, the anti -4- IBB antibody or antigen-binding fragment thereof can have a IgG- IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.

[0086] The anti-4-lBB antibody or antigen-binding fragment thereof can also have a Fab-scFv-Fc format. In Fab-scFv-Fc format, a light chain, heavy chain and a third chain containing the Fc region and the scFv are assembled. It can ensure efficient manufacturing and purification.

[0087] In some embodiments, the anti -4- IBB antibody or antigen-binding fragment thereof can be a TF. Three Fab fragments are linked by disulfide bridges. The TF format does not have an Fc region.

[0088] ADAPTIR has two scFvs bound to each side of an Fc region. It abandons the intact IgG as a basis for its construct, but conserves the Fc region to extend the half-life and facilitate purification.

[0089] Dual affinity retargeting (DART) has two peptide chains connecting the opposite fragments, thus VLA with VHB and VLB with VHA, and a sulfur bond at their C-termini fusing them together. In DART, the sulfur bond can improve stability over BiTEs. In DART-Fc, an Fc region is attached to the DART structure. It can be generated by assembling three chains, two via a disulfide bond, as with the DART. One chain contains half of the Fc region which will dimerize with the third chain, only expressing the Fc region. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.

[0090] In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created with half an Fc region and will dimerize. This format has bivalent binding to both targets, thus it is a tetraval ent molecule.

[0091] Tandem diabody (TandAb) comprises two diabodies. Each diabody consists of an VHA and VLB fragment and a VHA and VLB fragment that are covalently associated. The two diabodies are linked with a peptide chain. It can improve stability over the diabody consisting of two scFvs. It has two bivalent binding sites.

[0092] The scFv-scFv-toxin includes toxin and two scFv with a stabilizing linker. It can be used for specific delivery of payload.

[0093] In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof includes KIH mutations. In some embodiments, the anti -4- IBB antibody or antigen-binding fragment thereof includes a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain includes a heavy chain that including one or more knob mutations (a knob heavy chain), and the second antigen-binding domain includes a heavy chain including one or more hole mutations (a hole heavy chain). In some embodiments, the first antigen-binding domain includes a heavy chain that includes one or more hole mutations (a hole heavy chain), and the second antigen-binding domain that includes a heavy chain including one or more knob mutations (a knob heavy chain).

[0094] In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof includes the antigen-binding domains shown in Table 1.

[0095] The disclosure also provides nucleic acid comprising a polynucleotide encoding an anti -4- IBB antibody. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-4-lBB antibody comprises CDRs as shown in Table 1. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to 4- IBB.

[0096] The anti-4-lBB antibodies or antigen-binding fragments thereof can also be anti-4-lBB antibody variants (including derivatives and conjugates) of anti-4-lBB antibodies or antibody fragments. Additional anti-4-lBB antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The anti-4-lBB antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. In some embodiments, the anti-4-lBB antibody or antigenbinding fragment is an IgG (e.g., IgGl Fc region) antibody or antigen-binding fragment thereof.

[0097] Fragments of anti -4- IBB antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity to 4- IBB. Thus, a fragment of an anti -4- IBB antibody will retain an ability to bind to 4- IBB.

[0098] In some embodiments, the antibodies or antigen-binding fragments thereof have identical light chain variable regions.

[0099] Anti-CCR8 Antibody

[0100] C-C motif chemokine receptor 8 (CCR8) is a protein found mainly on the surface of a highly immunosuppressive subset of regulatory T cells (Tregs) within tumors. It is a regulator of immune response. As a chemokine receptor, CCR8 serves as a mediator of activity in concert with its chemokine CC chemokine ligand 1 (CCL1). CCR8 expression is upregulated on a highly immunosuppressive subset of Tregs in multiple cancer types. CCR8 is found to be particularly upregulated on Tregs in the tumor microenvironment (TME), with lower levels of CCR8+ Tregs found in the blood and normal tissues. Tregs act via many 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 correlated with poor prognosis in multiple cancer types.

[0101] Targeting CCR8 has the potential to be more specific in anti-tumor activity than other current approaches to Treg depletion. Other Treg target molecules are also found on Tregs peripherally (in the blood), in normal tissue and on effector T cells. As such, a highly selective target such as CCR8 is needed that allows for the specific depletion of tumor-infiltrating Tregs without affecting Tregs in the blood and normal tissues or destroying effector T cells.

[0102] More information related to CCR8 can be found e g., in 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; 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;l 14(23):6086-6091, each of which is incorporated herein by reference in the entirety.

[0103] The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CCR8 (e.g., human CCR8). In one aspect, the disclosure provides an anti-CCR8 antibody or antigenbinding fragment thereof.

[0104] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).In some embodiments, the heavy chain variable region (VH) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and the light chain variable region (VL) comprises CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs 1, 2, 3 amino acid sequences are one of the following:

[0105] (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 12. 13, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 14, 15, 16, respectively;

[0106] (2) the selected VH CDRs 1, 2, 3 amino acid sequences arc set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively; (3) the selected VH CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively: and

[0107] (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 71, 72, 73, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 74, 75, 76, respectively.

[0108] In some embodiments, the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 12 and 13, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 14, 15 and 16, respectively.

[0109] In some embodiments, the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively.

[0110] In some embodiments, the selected VH CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56. respectively.

[0111] In some embodiments, the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 71, 72, 73, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 74, 75, 76. respectively.

[0112] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and the VL 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, 37, 57, or 77, and the selected VL sequence is SEQ ID NO: 18, 38, 58, or 78.

[0113] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 17, 37, 57, or 77, and the selected VL sequence is SEQ ID NO: 18, 38, 58, or 78.

[0114] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1 , VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18.

[0115] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38.

[0116] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57, and the selected VL sequence is SEQ ID NO: 58.

[0117] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 77, and the selected VL sequence is SEQ ID NO: 78.

[0118] In some embodiments, the CDR definition is based on Kabat, Chothia, IMGT, contact or AbM definition.

[0119] In some embodiments, the antigen-binding domain specifically binds to human or mouse CCR8.

[0120] In some embodiments, the antigen-binding domain is a human or humanized antigen-binding domain.

[0121] In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv); and / or the second antigen-binding domain is a scFv.

[0122] 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 VH of the antigen-binding domain is connected to the Fc region. In some embodiments, the C-terminus of the VH of the antigenbinding domain is connected to the Fc region through a CHI domain.

[0123] The anti-CCR8 antibodies or antigen-binding fragments described herein can be in any suitable structure. In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof is a singlechain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) linked by a linker. The linkers described herein can be any suitable linkers known in the art. In some embodiments, the linker includes 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: 81.

[0124] In some embodiments, the anti-CCR8 antibody or anti gen -binding fragment thereof includes a heavy chain sequence comprising the VH, wherein the 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, 39, 59, or 79. In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof includes a light chain sequence comprising the VL, wherein the 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, 40, 60, or 80.

[0125] The disclosure provides anti-CCR8 antibodies and the modified antibodies thereof, including, e.g., chimeric antibodies, humanized antibodies, and human antibodies.

[0126] The anti-CCR8 antibodies or antigen-binding fragments thereof can also have various forms. Many different formats of bispecific antibodies or antigen-binding fragments thereof are known in the art, and are described e.g., in 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 the entirety.

[0127] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof is a BiTE, a (SCFV)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH- CL-scFv, a HSAbody, scDiabody -HAS, or a tandem-scFv. In some embodiments, the anti-CCR8 antibody is a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a crossMab, a DAE (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a K -body, an orthogonal Fab, a DVD- IgG, a IgG(H)-scFv, a 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, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F(ab’)2- scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv- Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFvl-PEG- scFv2. In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof can be a TrioMab. In a TrioMab, the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.

[0128] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof has two different heavy chains and two different light chains. Heterodimerization of heavy chains can be based on the knobs-into-holes or some other heavy chain pairing technique.

[0129] The anti-CCR8 antibody or antigen-binding fragment thereof can be a Duobody. The Fab-exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled matter in IgGl antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavylight chains.

[0130] In Dual-variable-domain antibody (DVD-Ig), additional VH and variable light chain (VL) domain are added to each N-terminus for bispecific targeting. This format resembles the IgG-scFv, but the added binding domains are bound individually to their respective N-termini instead of a scFv to each heavy chain N-terminus.

[0131] In scFv-IgG, the two scFv are connected to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites and is consequently also called tetravalent. There are no heavy-chain and light-chain pairing problem in the scFv-IgG.

[0132] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof can have a IgG- IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.

[0133] The anti-CCR8 antibody or antigen-binding fragment thereof can also have a Fab-scFv-Fc format. In Fab-scFv-Fc format, a light chain, heavy chain and a third chain containing the Fc region and the scFv are assembled. It can ensure efficient manufacturing and purification.

[0134] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof can be a TF. Three Fab fragments are linked by disulfide bridges. The TF format does not have an Fc region.

[0135] ADAPTIR has two scFvs bound to each side of an Fc region. It abandons the intact IgG as a basis for its construct, but conserves the Fc region to extend the half-life and facilitate purification.

[0136] Dual affinity retargeting (DART) has two peptide chains connecting the opposite fragments, thus VLA with VHB and VLB with VHA, and a sulfur bond at their C-termini fusing them together. In DART, the sulfur bond can improve stability over BiTEs. In DART-Fc, an Fc region is attached to the DART structure. It can be generated by assembling three chains, two via a disulfide bond, as with the DART. One chain contains half of the Fc region which will dimerize with the third chain, only expressing the Fc region. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.

[0137] In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created with half an Fc region and will dimerize. This format has bivalent binding to both targets, thus it is a tetraval ent molecule.

[0138] Tandem diabody (TandAb) comprises two diabodies. Each diabody consists of an VHA and VLB fragment and a VHA and VLB fragment that are covalently associated. The two diabodies are linked with a peptide chain. It can improve stability over the diabody consisting of two scFvs. It has two bivalent binding sites.

[0139] The scFv-scFv-toxin includes toxin and two scFv with a stabilizing linker. It can be used for specific delivery of payload.

[0140] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof includes KIH mutations. In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof includes a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain includes a heavy chain that including one or more knob mutations (a knob heavy chain), and the second antigen-binding domain includes a heavy chain including one or more hole mutations (a hole heavy chain). In some embodiments, the first antigen-binding domain includes a heavy chain that includes one or more hole mutations (a hole heavy chain), and the second antigen-binding domain that includes a heavy chain including one or more knob mutations (a knob heavy chain).

[0141] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof includes the antigen-binding domains shown in Table 1.

[0142] The disclosure also provides nucleic acid comprising a polynucleotide encoding an anti-CCR8 antibody. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-CCR8 antibody comprises CDRs as shown in Table 1. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to CCR8.

[0143] The anti-CCR8 antibodies or antigen-binding fragments thereof can also be anti-CCR8 antibody variants (including derivatives and conjugates) of anti-CCR8 antibodies or antibody fragments. Additional anti-CCR8 antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bi specific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The anti-CCR8 antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. In some embodiments, the anti-CCR8 antibody or antigen-binding fragment is an IgG (e.g., IgGl Fc region) antibody or antigen-binding fragment thereof.

[0144] Fragments of anti-CCR8 antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity to CCR8. Thus, a fragment of an anti-CCR8 antibody will retain an ability to bind to CCR8.

[0145] In some embodiments, the antibodies or antigen-binding fragments thereof have identical light chain variable regions.

[0146] Antibodies and Antigen Binding Fragments thereof

[0147] In some embodiments, the anti-4-lBB antibody or antigen-binding fragment thereof includes an antigen-binding domain that is derived from an anti-4-lBB antibody. In some embodiments, the anti- CCR8 antibody or antigen-binding fragment thereof includes an antigen-binding domain that is derived from an anti-CCR8 antibody. These anti -4- IBB or anti-CCR8antibodies and antigen-binding fragments thereof can have various forms.

[0148] In general, antibodies (also called immunoglobulins) can be made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting anti-4- IBB or anti-CCR8 antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the anti-4-lBB antibody or anti-CCR8 antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.

[0149] The hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principal antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding domain.

[0150] Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Kabat, E A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(l-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.

[0151] The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen-binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

[0152] In some embodiments, the anti -4- IBB or anti-CCR8 antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., 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.

[0153] The anti -4- IBB or anti-CCR8 antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, or camelid). The antigen-binding domain or antigen binding fragment is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab’, F(ab’)2, and variants of these fragments. Thus, in some embodiments, an anti -4- IBB or anti-CCR8 antibody or antigen binding fragment thereof can comprise e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen-binding domains include, e.g., the heavy chain and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

[0154] In some embodiments, the scFv in an anti -4- IBB or anti-CCR8 antibody has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the anti-4-lBB or anti-CCR8 scFv has two antigen binding regions (Antigen binding regions: A and B), and the two antigen binding regions can bind to the respective target antigens with different affinities.

[0155] In some embodiments, the anti-4- IBB or anti-CCR8 antibodies or antigen-binding fragments thereof can comprises one, two, or three heavy chain variable region CDRs selected from Table 1.

[0156] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof is selected from BMS-986340, LM-108, S-531011, SRF-114, ABBV-514, BAY3375968, CM369, HBM-1022, JTX-1811, PSB-114, ZL-1218, FPA 157, GB2101 and HFB-101110.

[0157] In some embodiments, the anti -4- IBB antibody or antigen-binding fragment thereof is selected from Utomilumab, Urelumab, EU-101, YH-004, ADG-106 and LVGN-6051.

[0158] In some embodiments, the anti -4- IBB or anti-CCR8 antibodies described herein can be conjugated to a therapeutic agent. The anti -4- IBB or anti-CCR8 antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, di one, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, e.g., a VC linker. Details of the linkers used for ADCs can be found, e.g., 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.

[0159] In some embodiments, the anti-4-lBB or anti-CCR8 antibody is a bispecific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96 / 27011, which is incorporated by reference in its entirety.

[0160] Any of the anti-4-lBB or anti-CCR8 antibodies or antigen-binding fragments thereof described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an anti-4- IBB or anti-CCR8 antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).

[0161] Antibody Characteristics

[0162] The anti-4-lBB antibodies can include an anti-4-lBB antigen-binding domain as described herein. The anti-CCR8 antibodies can include an anti-CCR8 antigen-binding domain as described herein. In some embodiments, the anti-4-lBB antibodies or antigen-binding fragments thereof, and / or anti-CCR8 antibodies or antigen-binding fragments thereof can initiate target cell (e.g., tumor cell, such as, CT26 cell or MC38 cell) killing, CDC, ADCC, or ADCP.

[0163] The disclosure provides anti-4-lBB and anti-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 4-1BB interaction with its ligands, the anti-4-lBB antibody can activate 4-lBB-associated signaling pathways, thus treating cancer. In some embodiments, the antibody can reduce tumor volume in a subject. Thus, in some embodiments, the anti-4-lBB antibody described herein is a 4-1BB agonist. In some embodiments, the anti-4-lBB antibody described herein is a 4-1BB antagonist.

[0164] General techniques can be used to measure the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR). Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff / kon). In some implementations, the anti-4-lBB antibodies or antigen- binding fragments thereof 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 0.1 s’1, less than 0.01 s’1, less than 0.001 s’1, less than 0.0001 s’1, or less than 0.00001 s’1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s’1, greater than 0.001 s’1, greater than 0.0001 s’1, greater than 0.00001 s’1, or greater than 0.000001 s’1.

[0165] In some embodiments, kinetic association rates (kon) is greater than 1 x 102 / Ms, greater than 1 x 103 / MS, greater than 1 x 104 / Ms, greater than 1 x 105 / Ms, or greater than 1 x 106 / Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 105 / Ms, less than 1 x 106 / Ms, or less than 1 x 107 / Ms.

[0166] In some embodiments, the anti-4-lBB antibodies or antigen-binding fragments 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 x 10’6M, less than 1 x 10’7M, less than 1 x 10’8M, less than 1 x 10’9M, or less than 1 x 10’10M. 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, KD is greater than 1 x 10’7M, greater than 1 x 10’8M, greater than 1 x 10’9M, or greater than 1 x 10’10M.

[0167] The anti-CCR8 antibodies or antigen-binding fragments thereof can also include an antigen-binding domain that can specifically bind to CCR8. In some embodiments, the antibodies or antigen-binding fragments thereof described herein recruit CCR8-expressing cells (e.g., CCR8-expressing Tregs) and mediate the depletion of such cells. In some embodiments, the antibody can reduce tumor volume in an animal. In some embodiments, the antibody can reduce or slow down the progression of an autoimmune disease. Thus, in some embodiments, the anti-CCR8 antibody described herein is a CCR8 agonist. In some embodiments, the anti-CCR8 antibody described herein is a CCR8 antagonist.

[0168] In some implementations, the anti-CCR8 antibodies or antigen-binding fragments thereof can bind to CCR8 (e.g., human CCR8, monkey CCR8, mouse CCR8, and / or chimeric CCR8) with a dissociation rate (koff) of less than 0.1 s’1, less than 0.01 s’1, less than 0.001 s’1, less than 0.0001 s’1, or less than 0.00001 s’1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s’1, greater than 0.001 s’1, greater than 0.0001 s’1, greater than 0.00001 s’1, or greater than 0.000001 s’1.

[0169] In some embodiments, kinetic association rates (kon) is greater than 1 x 102 / Ms, greater than 1 x 103 / MS, greater than 1 x 104 / Ms, greater than 1 x 105 / Ms, or greater than 1 x 106 / Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 105 / Ms, less than 1 x 106 / Ms, or less than 1 X 107 / MS. Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff / kon). In some embodiments, KD is less than 1 x 10'6M, less than 1 * 10'7M, less than 1 x 10'8M, less than 1 x 10'9M, or less than 1 x io10M. 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, KD is greater than 1 x 10'7M, greater than 1 x 10'8M, greater than 1 x 10'9M, or greater than 1 x 10'10M.

[0170] In some embodiments, the anti-4-lBB or anti-CCR8 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage (TGI%) that is 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, the anti-4-lBB or anti-CCR8 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. The TGI (%) can be determined, e.g., at 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 treatment starts. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:

[0171] TGI (%) = [l-(Ti-T0) / (Vi-V0)]xl00%

[0172] Ti is the average tumor volume in the treatment group on day i. TO is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

[0173] In some embodiments, the anti -4- IBB or anti-CCR8 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 the tumor size with the administration of PBS or without the administration of the anti -4- IBB or anti-CCR8 antibody, antigen-binding fragment, or ADC. Tumor volume is expressed in mm3and can be calculated using the formula: “V = (L x w x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L).

[0174] In some embodiments, the anti-4-lBB or anti-CCR8 antibody, antigen-binding fragment, or ADC has a functional Fc region. In some embodiments, effector function of a functional Fc region is antibodydependent cell-mediated cytotoxicity (ADCC). In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgGl, human IgG2, human IgG3, or human IgG4. In some embodiments, the anti-4-lBB or anti-CCR8 antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the anti-4-lBB or anti-CCR8 antibodies or antigenbinding fragments thereof are Fab, Fab’, F(ab’)2, and Fv fragments. In some embodiments, the anti-4-lBB or anti-CCR8 antibodies or antigen-binding fragments thereof as described herein have an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering).

[0175] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region). In some embodiments, the anti-4-lBB or anti-CCR8 antibody described herein can be designed to have an IgGl subtype structure with the GAALIE mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the anti-4-lBB or anti-CCR8 antibody described herein can be designed to have an IgGl 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.

[0176] Some other modifications to the Fc region can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may have any increased half-life in vitro and / or in vivo.

[0177] In some embodiments, the IgG4 has S228P mutation (EU numbering). The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange.

[0178] In some embodiments, anti-4-lBB or anti-CCR8 antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the anti-4-lBB or anti-CCR8 antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A).

[0179] In some embodiments, the methods described herein are designed to make a bispecific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96 / 27011, which is incorporated by reference in its entirety.

[0180] In some embodiments, the anti -4- IBB or anti-CCR8 antibody, antigen-binding fragment, or ADC promotes antibody-dependent cellular phagocytosis (ADCP), wherein the anti -4- IBB or anti- CCR8 antibody, antigen-binding fragment, or ADC bind to target cells that co-express 4-1BB and / or CCR8 (e.g., CT26 cells, MC38 cells, or CHO cells), marking them for destruction by phagocytic cells (e.g., macrophages, dendritic cells, or neutrophils). In ADCP, the Fab region of the antibody binds to antigens on the surface of the target cell co-expressing 4-1BB and / or CCR8 (e.g., CT26 cells, MC38 cells, or CHO cells), while the Fc region interacts with Fc receptors (like CD64 or CD32) on the surface of the phagocytes. Once engaged, this interaction triggers the engulfment and digestion of the target cell co-expressing 4-1BB and / or CCR8 (e.g., CT26 cells, MC38 cells, or CHO cells) by the phagocyte (e.g., macrophages, dendritic cells, or neutrophils), leading to the clearance of the marked cells (or cell killing). ADCP is a critical mechanism in the immune response and is actively leveraged in therapeutic antibody treatments, particularly in cancer therapy, where antibodies are designed to target and eliminate tumor cells through phagocytosis. ADCP is a key mode of action for many monoclonal antibodies known in arts used in oncology.

[0181] In some embodiments, the anti -4- IBB or anti-CCR8 antibody, antigen-binding fragment, or ADC promotes ADCP of the target cells (e.g., CHO cells) co-expressing hCCR8 and h4-lBB are by effector MO macrophages. In some embodiments, effector cells (e.g., macrophages) to target cells (e.g., CHO cells co-expressing 4-1BB and / or CCR8) ratio or E:T ratio can be any ratio between 1: 10 to 50: 1 (e.g., between 1 :5 to 25: 1, between 1 :2 to 10: 1, or between 1 : 1 to 5: 1). In some embodiments, E:T ratio can be at least 1 : 10, 1 :9, 1 :8, 1 :7, 1 : 6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1 , 3: 1, 4:1 , 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, or 20: 1). In some embodiments, E:T ratio can be less than 50:1, 40: 1, 30: 1, 20: 1, 10: 1, 9:1, 8: 1, 7:1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1:3, 1 :4, or 1 :5).

[0182] In some embodiments, knobs-into-holes (KIH) technology can be used, which involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization. The KIH technique is described e.g., in 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, which is incorporated by reference in its entirety. In some embodiments, one heavy chain has a T366W, and / or S354C (knob) substitution (EU numbering), and the other heavy chain has an Y349C, T366S, L368A, and / or Y407V (hole) substitution (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 can have one or more the following substitutions E356C, T366S, L368A, and Y407V (EU numbering). Furthermore, a substitution (-ppcpScp— >-ppcpPcp-) can also be introduced at the hinge regions of both substituted IgG.

[0183] Recombinant Vectors

[0184] The present disclosure also provides recombinant vectors (e.g., expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and / or a vector comprising the polynucleotide), and the production of anti-4-lBB or anti-CCR8 antibody polypeptides or fragments thereof by recombinant techniques.

[0185] The full length sequence of human CCR8 is known in the art (e.g., NCBI Gene ID: 1237, NCBI Reference Sequence: NM 005201.4). In some embodiments, a His-tagged human CCR8 protein is used as the immunogen.

[0186] The full length sequence of human 4-1BB is known in the art (e.g., GenBank Accession No.: U03397.1). In some embodiments, a His-tagged human 4-1BB protein is used as the immunogen.

[0187] As used herein, a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

[0188] A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0189] In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, 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 will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. 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., 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 may also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

[0190] For expression, the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. colt and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurhim cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

[0191] Non-limiting vectors for use in 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. Nonlimiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

[0192] Non-limiting bacterial promoters suitable for use include the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0193] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997).

[0194] Introduction of the construct into the host cell can be effected 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 etal., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0195] Transcription of DNA encoding an anti-4-lBB or anti-CCR8 antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cisacting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0196] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into 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.

[0197] The polypeptide (e.g., an anti-4-lBB or anti-CCR8 antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

[0198] The disclosure also provides a nucleic 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 nucleotide sequence as described herein, and 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 amino acid sequence as described herein.

[0199] The disclosure also provides a nucleic acid sequence that has a homology of 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% to any nucleotide sequence as described herein, and an amino acid sequence that has a homology of 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% to any amino acid sequence as described herein.

[0200] In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as 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, 1 0, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0201] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

[0202] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

[0203] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then 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 percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0204] The disclosure provides one or more nucleic acid encoding any of the polypeptides as described herein. In some embodiments, the nucleic acid (e.g., cDNA) includes a polynucleotide encoding a polypeptide of a heavy chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a polypeptide of a light chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a scFv polypeptide as described herein.

[0205] In some embodiments, the vector can have two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to 4-1BB. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to 4- 1BB. In some embodiments, the vector includes two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to CCR8. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to CCR8.

[0206] Methods of Treatment

[0207] The methods described herein include methods for the treatment of disorders associated with cancer (e.g., treating a subject having tumor, decreasing the rate of tumor growth or killing a tumor cell). Generally, the methods include administering a therapeutically effective amount of a composition a) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

[0208] As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with cancer. Often, cancer results in death; thus, a treatment can result in an increased life expectancy or survival (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by 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 the treatment of a condition associated with cancer will result in decreased number of cancer cells, reduced tumor volume, alleviated symptoms, and / or improved life expectancy or survival.

[0209] As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers (e g., colon carcinoma), renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0210] In some embodiments, the cancer is a chemotherapy resistant cancer.

[0211] In one aspect, the disclosure also provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and / or duration of one or more symptoms of the cancer in a subject.

[0212] In one aspect, the disclosure features methods that include administering a therapeutically effective amount of anti-4-lBB and anti-CCR8 antibodies disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., 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.

[0213] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea- pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals. In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

[0214] As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the anti-4- IBB and anti-CCR8 antibodies, anti-4-lBB and anti-CCR8 antigen binding fragments, anti-4-lBB and anti-CCR8 antibody-encoding polynucleotides, vector comprising the polynucleotide, and / or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

[0215] An effective amount can be administered in one or more administrations. By way of example, an effective amount of an anti-4-lBB or anti-CCR8 antibody or antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and / or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and / or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount of an anti-4-lBB or anti-CCR8 antibody, antigen binding fragment, or antibody-drug conjugate may vary, depending on, inter alia, patient history as well as other factors such as the type (and / or dosage) of the agent used.

[0216] Effective amounts and schedules for administering the anti-4-lBB and anti-CCR8 antibodies, antigen-binding fragments thereof, antibody-encoding polynucleotides, and / or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the anti-4- IBB and anti-CCR8 antibodies, antigen-binding fragments thereof, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of the agent or compositions disclosed herein used and other drugs being administered to the mammal.

[0217] A typical daily dosage of an effective amount of each of an anti-4- IBB and anti-CCR8 antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage can 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 dosage can 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.

[0218] In some embodiments, the anti-4- IBB antibody and the anti-CCR8 antibody described herein kill tumor cells and / or reduces tumor size (e.g., through the activation of the 4-lBB-related signaling pathway or the recruiting and depletion of CCR8-expressing immune cells (e.g., CCR8-expressing tumor infiltrating Tregs)) in a subject receiving the antibody. 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 the tumor cells are killed by the administration of the anti -4- IBB antibody and anti-CCR8 antibody described herein.

[0219] In some embodiments, the antitumor effect of the anti-4- IBB antibody and the anti-CCR8 antibody described herein is through the modulation of Treg / Teff cells (e.g., in mice or in humans) to enhance tumor immunity and increase the survival rate.

[0220] In some embodiments, the administration of the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB reduces the tumor volume (tumor size) in the subject. In some embodiments, the tumor volume in a subject receiving the anti-4- IBB antibody and 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 the tumor size without the administration of the anti-4-lBB antibody and anti-CCR8 antibody. In some embodiments, the tumor size in a subject receiving the anti-4-lBB antibody and 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 than the tumor size reduced by the administration of an anti -4- IBB antibody alone or an anti-CCR8 antibody alone.

[0221] In some embodiments, the administration of the anti-4-lBB antibody and anti-CCR8 antibody described herein increases the survival rate of the subject receiving the antibody. In some embodiments, the survival rate is increased by about or at least 5%, 10%o, 15%, 20%, 25%, 30%, 35%o, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, compared the survival rate of a subject without the administration of the anti -4- IBB antibody and anti-CCR8 antibody or by the administration of an anti-4-lBB antibody alone or an anti-CCR8 antibody alone.

[0222] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein and the composition comprising the antibody or antigen- binding fragment thereof that specifically binds to 4-1BB described herein are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times.

[0223] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein and the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to 4-1BB described herein are administered every 1, 2, 3, 4, 5 or more days. In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4- IBB described herein are administered once a week, twice a week, three times a week, four times a week or more. In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein and the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to 4-1BB described herein are administered weekly, every two week, every three weeks, every month, every two months, every three months or more.

[0224] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein and the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to 4- IBB described herein are administered at the same dosage, or at a ratio of dosages of about 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1 or any other suitable dosage ratio.

[0225] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein and the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to 4-1BB described herein are administered intravenously and / or intraperitoneally.

[0226] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein and the composition comprising the antibody or antigenbinding fragment thereof that specifically binds to 4-1BB described herein are administered three times, wherein the first administration is performed intravenously and the second and third administrations are performed intraperitoneally.

[0227] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, a 4- IBB inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K- Ras, an inhibitor of c-Met, an inhibitor of CCR8, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of a phosphatidylinositol 3-kinase (PI3K), an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K / mT0R inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK), and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and / or Isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3 -dioxygenase- 1) (IDO1) (e.g., epacadostat).

[0228] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of 4-1BB, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

[0229] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0230] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, 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 treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

[0231] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

[0232] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-Ll antibody, 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.

[0233] Pharmaceutical Compositions and Routes of Administration

[0234] Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the anti-4-lBB and / or anti-CCR8 antibodies, antigen-binding fragments, or antibody-drug conjugates described herein. The pharmaceutical compositions may be formulated in any manner known in the art.

[0235] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, 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), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e g., U.S. Patent No. 4,522,811). Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant.

[0236] Compositions containing one or more of any of the anti -4- IBB and anti-CCR8 antibodies or antigenbinding 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 quantity of active compound for ease of administration and uniformity of dosage).

[0237] Toxicity and therapeutic efficacy of compositions 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 in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0238] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of the antibodies or antigen-binding fragments, or antibody-drug conjugates (e.g., anti-4-lBB and anti-CCR8 antibodies, antigen-binding fragments, or antibody-drug conjugates) will be an amount that treats the disease (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer), or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured), decreases the severity, frequency, and / or duration of one or more symptoms of a disease in a subject (e.g., a human). The effectiveness and dosing of any of the antibodies or antigenbinding fragments, or antibody-drug conjugates (e.g., anti-4-lBB and anti-CCR8 antibodies, antigenbinding fragments, or antibody-drug conjugates) described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0239] Exemplary doses include milligram or microgram amounts of any of the antibodies or antigenbinding fragments, or antibody-drug conjugates (e.g., anti-4-lBB and anti-CCR8 antibodies, antigenbinding fragments, or antibody-drug conjugates) described herein per kilogram of the subject’s weight (e.g., about 1 pg / kg to about 500 mg / kg; about 100 pg / kg to about 500 mg / kg; about 100 pg / kg to about 50 mg / kg; about 10 pg / kg to about 5 mg / kg; about Img / kg to about 5 mg / kg, about 10 pg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the therapeutic agent in vivo.

[0240] The antibodies or antigen-binding fragments, or antibody-drug conjugates (e.g., anti-4-lBB and anti- CCR8 antibodies, antigen-binding fragments, or antibody-drug conjugates) described herein can be administered one or more times during the course of treatment. For example, the antibodies or antigenbinding fragments, or antibody-drug conjugates (e.g., anti-4-lBB and anti-CCR8 antibodies, antigenbinding fragments, or antibody-drug conjugates) described herein can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In some embodiments, the antibodies or antigen-binding fragments, or antibody - drug conjugates (e.g., anti-4-lBB and anti-CCR8 antibodies, antigen-binding fragments, or antibody-drug conjugates) is administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

[0241] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen-binding fragments, or antibody-drug conjugates (e.g., anti-4-lBB and anti-CCR8 antibodies, antigen-binding fragments, or antibody-drug conjugates) for various uses as described herein.

[0242] EXAMPLES

[0243] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0244] Example 1: Molecule Constructions

[0245] To generate the anti-CCR8 monoclonal antibody (e.g., CCR8-1 mAb), the VH region was linked to a wild-type m!gG2a Fc, while the VL region was linked to a mouse lambda light constant region. To generate the anti-4-lBB monoclonal antibody (e.g., 4-1BB-1 mAb), the VH region was linked to a wildtype m!gG2a Fc, while the VL region was linked to a mouse kappa light constant region.

[0246] For the generation of additional anti-CCR8 monoclonal antibodies (e.g., CCR8-2 mAb, CCR8-3 mAb, and CCR8-4 mAb), the VH region was linked to a wild-type hlgGl Fc, while the VL region was linked to human kappa light constant region. For the generation of other anti-4-lBB monoclonal antibodies (e g., 4-1BB-2 mAb, 4-1BB-3 mAb, and 4-1BB-4 mAb) the VH was linked to a wild-type hlgGl Fc while the VL region was linked to human kappa light constant region.

[0247] Fragments were synthesized (IDT) and assembled (NEB NEBuilder® HiFi DNA Assembly) into pcDNA3.4 or pcDNA3.4 carrying desired fragments as described above. The assembled plasmid was transformed to Competent E. coli (NEB® 5-alpha). Clones with the correct sequence, confirmed by sequencing results (Elim Biopharm), were further cultured with lysogeny broth (LB) containing Carbenicillin (100 pg / l) for plasmid amplification, followed by plasmid purification (QIAGEN Plasmid Plus Kits). Plasmids were eluted in Nuclease-free H2O (Sigma) and stored at -80 °C. Example 2: Transfection and Purification of Antibodies

[0248] All antibody light chain and heavy chain coding sequences were generated by direct DNA synthesis and cloned into a mammalian expression vector. The cloned sequences were verified by Sanger Sequencing.

[0249] Chinese hamster ovary (CHO) cells were grown in CHOgro medium (Mirus) complemented with 4mM L-Glutamine and 0.33% Poloxamer-188 and diluted to 4xl06cells / ml with fresh cell culture media at the time of transfection. Cells were transfected by lipofection with Ipg ofplasmid DNAper 4xl06cells. Transfected cells were kept in incubator with shaking at 125 rpm in 5% CO2 and 70% humidity environment at 32°C.

[0250] Protein expression titers was measured every 5 days. Cell supernatant was collected by centrifugation after 10-14 days post-transfection and filtered through a 0.22pm PES membrane filter. Expressed antibodies were purified by Protein A chromatography (TOYOPEARL® AF-rProtein A HC-650M resin, Tosoh) and aggregates were removed by anion exchange resin (TOYOPEARL NH2-750F, Tosoh).

[0251] Purified antibodies were analyzed by HPLC (Dionex UltiMate® 3000-RS UHPLC, ThermoFisher) controlled by Chromeleon® software. A size exclusion column TSKgel UP-SW3000, 2 pm, 4.6 mm ID* 15 cm (Tosoh) was used to resolve antibodies purity and aggregation.

[0252] All purified antibodies were 97% or greater monomer purity, sterilized through 0.22pm PES membrane filter, and used for binding and functional assays.

[0253] Example 3. CCR8 mAb and 4-1BB mAb synergistically reduce tumor growth in CT26 colon carcinoma model

[0254] The anti-tumor effects of the combination of anti-CCR8 mAb and anti -4- IBB mAb were examined against CT26 colon carcinoma model using BalB / c mice. Female BalB / c mice, 6-9 weeks old, were obtained from Vital River Laboratories Research Models and Services (Guangdong, China) and used in the experiments. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC). CT26 colon carcinoma cells in logarithmic growth phase were injected subcutaneously (5 x 105cells) into the right rear flank of the mice on day 0 in 0.1 ml of PBS for tumor development. Mice were randomized and assigned to treatment groups when mean tumor size reached approximately 80-120 mm3on day 7. Mice were treated with CCR8-1 mAb, 4-1BB-1 mAb, or the combination of CCR8-1 mAb and 4-1BB-1 mAb on days 7, 10, and 14 at a dose of 1 mg / kg dose. Control group mice were treated with phosphate buffer saline (PBS). First dose was administered intravenously, while the second and third were administered via intraperitoneal injections (FIG. 1). The tumors were measured three times per week in two dimensions using a caliper, and the volume were expressed in mm3using the formula: V = (L x WxW) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Graphing and statistical analysis was performed using GraphPad Prism program. Two-way ANOVA with Dunnett multiple comparisons test was used for analyzing tumor growth, while Kaplan-Meyer survival analysis was used for the percent survival data.

[0255] In the comparison of a mean tumor volume between the single-agent administration groups and CCR8-1 mAb + 4-1BB-1 mAb combination administration group. As shown in FIG. 2, single-agent CCR8-1 mAb or single-agent 4-1BB-1 mAb treatments induced partial anti -tumor activity in the mouse CT26 tumor model, however, administration of CCR8-1 mAb and 4-1BB-1 mAb combination exhibited a synergistic effect in strongly inhibiting tumor growth.

[0256] A complete remission rate of the tumors at days was also compared. By the end of the study on day 49, CCR8-1 mAb and 4-1BB-1 mAb in combination administration led to higher number of tumor free mice (4 tumor free mice out of 6) compared to single-agent treatment with either CCR8-1 mAb (no tumor free mice out of 6) or single-agent 4-1BB-1 mAb (no tumor free mice out of 6) alone (FIGs. 3A-3C). Moreover, CCR8-1 mAb and 4-1BB-1 mAb combination administration group requires shorter time for tumor regression compared with either monoclonal CCR8-1 mAb or 4-1BB-1 mAb alone.

[0257] Percent survival rates during the treatment were also calculated. As shown in FIG. 4, the CCR8-1 mAb and 4-1BB-1 mAb combination treatment resulted in highest percent survival (100%) among mice by the end of the study on day 49, compared to CCR8-1 mAb (16.66 %) or 4-1BB-1 mAb (66.66 %) alone..

[0258] This data strongly demonstrates that the CCR8 mAb and 4- IBB mAb combination treatment can synergistically enhance the anti-tumor activity in mouse CT26 tumor model. Combination treatment with both antibodies together induced a stronger anti-tumor effect, resulting in a higher number of tumor-free mice and 100% overall survival, compared to either CCR8 mAb or 4- IBB mAb alone.

[0259] The major concern of 4- IBB antibody therapy is dose-limiting liver toxicity, as observed in urelumab’s clinical development. Most common adverse events were elevated levels of alanine transaminase (ALT) and aspartate aminotransferase (AST). Blood samples were collected from CT26 cell- implanted mice for ALT and AST measurements after the CCR8-1 mAb and 4-1BB-1 mAb treatment combination. No significant elevation of ALT and AST was observed, suggesting little risk for liver toxicity commonly induced by CCR8 mAb (1 mg / kg) and 4-1BB mAb (1 mg / kg) combination treatment. Example 4. CCR8 mAh and 4-1BB mAb synergistically reduce tumor growth in MC38 colon carcinoma model

[0260] The anti-tumor effects of the anti-CCR8 mAb and anti-4-lBB mAb combination were examined against MC38 colon carcinoma model using BalB / c mice. Female BalB / c mice, 6-9 weeks old, were obtained from Vital River Laboratories Research Models and Services (Guangdong, China), and used in the experiments. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC). MC38 colon carcinoma cells in logarithmic growth phase were injected subcutaneously (5 x 105cells) into the right rear flank of the mice on day 0 in 0.1 ml of PBS for tumor development. Mice were randomized and assigned to the treatment groups when mean tumor size reached approximately 80- 120 mm3on day 7. Mice were treated with CCR8-1 mAb, 4-1BB-1 mAb, or the combination of CCR8-1 mAb and 4-1BB-1 mAb on days 7, 10, 14, 18, 22 and 26 at a dose of 10 mg / kg dose. Control group mice were treated with PBS. First dose was given intravenously, while the second and third doses were administered via intraperitoneal injections (FIG. 5). The tumors were measured three times per week in two dimensions using a caliper, and the volume were expressed in mm3using the formula: V = (L * WxW) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Graphing and statistical analysis was performed using GraphPad Prism program. Two-way ANOVA with Dunnett multiple comparisons test was used for analyzing tumor growth, while Kaplan-Meyer survival analysis was used for the percent survival data.

[0261] In the comparison of a mean tumor volume between the single-agent administration groups and CCR8-1 mAb + 4-1BB-1 mAb combination administration group. As shown in FIG. 6, single-agent CCR8-1 mAb or single-agent 4-1BB-1 mAb treatments induced partial anti-tumor activity in the mouse MC38 tumor model, however, administration of CCR8-1 mAb and 4-1BB-1 mAb combination exhibited a synergistic effect in significantly inhibiting tumor growth.

[0262] A complete remission rate of the tumors at days was also compared. By the end of the study on day 34, CCR8-1 mAb and 4-1BB-1 mAb combination administration group requires shorter time for tumor regression as compared with either monoclonal CCR8-1 mAb or4-lBB-l mAb alone, as shown in FIGs. 7A-7C.

[0263] Percent survival rates during the treatment were also calculated. As shown in FIG. 8, the CCR8-1 mAb and 4-1BB-1 mAb combination treatment resulted in highest percent survival (100%) among mice by the end of the study on day 24. compared to CCR8-1 mAh (33.33 %) and 4-1BB-1 mAh (66.66 %) alone.

[0264] This data strongly demonstrates that the CCR8 mAb and 4- IBB mAb combination treatment can synergistically enhance the anti-tumor activity in mouse MC38 tumor model. Combination treatment with both antibodies together induced a stronger anti-tumor effect, resulting in significant tumor inhibition and 100% overall survival, compared to either CCR8 mAb or 4- IBB mAb alone.

[0265] These results suggest that, as demonstrated by the antitumor effects of the CCR8 mAb and 4-1BB mAb combination treatment administered to mice, Treg / Teff cells in humans are modulated to enhance anti-tumor immunity and increase the survival probability.

[0266] The major concern of 4- IBB antibody therapy is dose-limiting liver toxicity, as observed in urelumab’s clinical development. Most common adverse events were elevated alanine transaminase (ALT) and aspartate aminotransferase (AST). Blood samples were collected for ALT and AST measurements from MC38 mice after the treatment of CCR8-1 mAb (10 mg / kg) and 4-1BB-1 mAb (10 mg / kg) combination. No significant elevation of ALT and AST was observed, suggesting little risk for liver toxicity commonly induced by CCR8 mAb and 4-1BB mAb treatment combination.

[0267] Example 5. The combination of CCR8 mAb and 4-1BB mAb mediated the cell killing of CHO cells co-expressing human CCR8 and 4-1BB by MO macrophages

[0268] The antibody dependent cellular phagocytosis (ADCP) efficacy of target CHO cells co-expressing hCCR8 and h4-lBB by effector MO macrophages via using the combination of anti-CCR8 and anti-4-lBB monoclonal antibodies was assessed. CD 14+ monocytes were purified from human PBMC without CD 16 depletion using EasySep™ Human Monocyte Enrichment Kit (StemCell Technologies, Cat #: 19058)), and then differentiated into M0 macrophages with 50 ng / mL of macrophage colony-stimulating factor (M- CSF, StemCell Technologies, Cat #: 78057) for 7 days. CHO cells were labeled with CFSE (ThermoFisher, Cat #: C34554). About 100,000 macrophages were incubated with either 100,000, 33,333.3 or 20,000 CFSE+ CHO cells to maintain effector to target (E:T) ratios of 1 :1, 3: 1 or 5:1 respectively, in the presence of serially diluted antibodies (the combination of 4-1BB-2 mAb and CCR8-2 mAb) in complete RPMI media (supplemented with 10% heat inactivated FBS and 5% penicillin / streptomycin) for 24 hours at 37°C. After incubation, the cells were washed with FACS buffer (buffer used in fluorescence-activated cell sorting) twice and stained with 50pl of Live dead Near-IR (Thermo Fisher, Cat #: L10119) at 1 :2000 dilution in PBS at room temperature for 10 min, and followed with two washes. Cells were resuspended in l OOpl of FACS buffer and analyzed with a Cytek® cytometer. CHO cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume for all treatment conditions. Percentage of target cell killing was calculated as: (absolute number of CFSE+ CHO cell number in non-treatment group - absolute number of CFSE+ Live / Dead Near-IR- CHO cell number in treatment group) / absolute number of CFSE+ CHO cell number in non-treatment group x 100. As shown in FIGs. 9A-9C, the combination of CCR8-2 and 4-1BB-2 mAb efficiently enhanced the cell killing of CCR8 and 4-1BB co-expressing CHO cells at all three tested E:T ratios.

[0269] Example 6. Additional exemplary CCR8 mAb and 4-1BB mAb Additional 4-1BB mAb and CCR8 mAb pairs including 4-1BB-3 + CCR8-3 mAb pair and 4-1BB-4 + CCR8-4 mAb pair are also tested. Each pair of antibodies shows higher synergistical anti-tumor effects, lower liver toxicity and lower E:T ratio, compared to CCR8 mAb or 4-1BB mAb alone.

[0270] These results suggest that, as demonstrated by the antitumor effects of the CCR8 mAb and 4- IBB mAb combination treatment administered to mice, Treg / Teff cells can be modulated to enhance anti-tumor immunity and increase the survival rate.

[0271] Table 1. Amino acid sequences described in the present disclosure

[0272] OTHER EMBODIMENTS

[0273] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having a tumor, the method comprising administering a therapeutically effective amount of: a) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB to the subject.

2. A method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB to the subject.

3. A method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of: a) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB to the subject.

4. The method of any one of claims 1-3, wherein the 4-1BB is human 4-1BB, and wherein the CCR8 is human CCR8.

5. The method of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof that specifically binds to CCR8 is BMS-986340, LM-108, S-531011, SRF-114, ABBV- 514, BAY3375968, CM369, HBM-1022, JTX-1811, PSB-114, ZL-1218, FPA 157, GB2101 or HFB-101110, and wherein the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB is Utomilumab, Urelumab, EU-101, YH-004, ADG-106 or LVGN-6051.

6. The method of any one of claims 1-5, wherein the tumor is a solid tumor or a blood tumor.

7. The method of claim 6, wherein the cancer is a solid tumor.

8. The method of claim 7, wherein the cancer is breast cancer, uterine corpus cancer, cervical cancer, ovary cancer, prostate cancer, lung cancer, stomach 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, blood cell cancer such as leukemia or lymphoma, bile duct carcinoma, gallbladder carcinoma, thyroid carcinoma, testicular carcinoma, thymic carcinoma, or hepatocarcinoma.

9. The method of claim 8, wherein the cancer is breast cancer, uterine corpus cancer, ovary cancer, lung cancer, colorectal cancer, kidney cancer and sarcoma.

10. The method of claim 6, wherein the solid tumor is colon carcinoma.

11. The method of any one of claims 1-10, wherein the administration of the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4- IBB reduces the tumor volume in the subject.

12. The method of claim 11, wherein the administration of the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4- IBB reduces more tumor volume than administering a composition comprising an antibody or antigenbinding fragment thereof that specifically binds to CCR8 alone or administering a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB alone.

13. The method of any one of claims 1-12, wherein the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprisingthe antibody or antigen-binding fragment thereof that specifically binds to 4-1 BB are administered one, two, three or more times.

14. The method of claim 13, wherein the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered every 1, 2, 3, 4, 5 or more days.

15. The method of any one of claims 1-14, wherein the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered at the same dosage.

16. The method of any one of claims 1-15, wherein the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4- IBB are administered intravenously and / or intraperitoneally.

17. The method of claim 16, wherein the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising the antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered three times, wherein the first administration is performed intravenously and the second and third administrations are performed intraperitoneally.

18. The method of any one of claims 1-17, further comprising administering a therapeutically effective amount of an anti-OX40 antibody, an anti-PDl antibody, an anti-PDLl antibody, an anti- PDL2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, and / or an anti-CD40 antibody, to the subject.

19. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8, an antibody or antigen-binding fragment thereof that specifically binds to 4- IBB, and a pharmaceutically acceptable carrier.

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