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

Abstract

The present disclosure relates to antibodies, or antigen-binding fragments thereof, that specifically bind to 4-1BB and antibodies, or antigen-binding fragments thereof, that specifically bind to CCR8, and methods of use thereof.

Description

[0001] Priority claim

[0002] This application claims priority to CN202311268301.8 filed on September 26, 2023. The entire contents of the foregoing are incorporated herein by reference. Technical Field

[0003] The present invention relates to anti-4-1BB antibodies or antigen-binding fragments thereof, anti-CCR8 antibodies or antigen-binding fragments thereof, and methods of using the antibodies described herein. background

[0005] The recent clinical and commercial success of anti-cancer antibodies has generated considerable interest in antibody-based therapeutics.

[0006] There is a need to develop antibodies for use in various antibody-based therapies to treat cancer.

[0007] summary

[0008] The present invention relates to an antibody or an antigen binding fragment thereof, wherein the antibody or its antigen binding fragment specifically binds to 4-1BB. The present invention also relates to an antibody or its antigen binding fragment, wherein the antibody or its antigen binding fragment specifically binds to CC motif chemokine receptor 8 (CCR8).

[0009] The present invention also relates to methods of treating a subject in need thereof using an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB and / or an antibody or an antigen-binding fragment thereof that specifically binds to CCR8.

[0010] Therefore, provided herein is a method for treating a subject having a tumor, the method comprising administering a therapeutically effective amount of: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; b) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB of the subject.

[0011] The present invention also relates to a method for reducing tumor growth rate, the method comprising treating tumor cells with an effective amount of: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; b) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB of a subject.

[0012] The present invention also relates to a method for killing tumor cells, comprising contacting the tumor cells with an effective amount of the following substances: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; b) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB of a subject.

[0013] In some embodiments, 4-1BB is human 4-1BB, and wherein CCR8 is human CCR8.

[0014] 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, FPA157, 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.

[0015] In some embodiments, the tumor is a solid tumor or a hematological tumor.

[0016] In some embodiments, the cancer is a solid tumor.

[0017] In some embodiments, the cancer is breast cancer, uterine corpus cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, gastric cancer, non-small cell lung cancer, spleen cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer, sarcoma, a blood cell cancer such as a leukemia or lymphoma, bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, or liver cancer.

[0018] In some embodiments, the cancer is breast cancer, uterine corpus cancer, ovarian cancer, lung cancer, colorectal cancer, renal cancer, and sarcoma.

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

[0020] In some embodiments, administration of a composition comprising an antibody or antigen-binding fragment thereof that specifically binds CCR8 and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds 4-1BB reduces tumor volume in the subject.

[0021] In some embodiments, administration of a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB reduces tumor volume more than administration of a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 alone, or administration of a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB alone.

[0022] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered once, twice, three times, or more.

[0023] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof that specifically binds CCR8 and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds 4-1BB are administered every 1, 2, 3, 4, 5, or more days.

[0024] In some embodiments, the composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered at the same dose.

[0025] In some embodiments, the composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered intravenously and / or intraperitoneally.

[0026] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB are administered three times, wherein the first administration is intravenous and the second and third administrations are intraperitoneal.

[0027] In some embodiments, the methods described herein further comprise administering to the subject a therapeutically effective amount of an anti-OX40 antibody, an anti-PD1 antibody, an anti-PDL1 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.

[0028] The present invention also relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8 and an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB, and a pharmaceutically acceptable carrier.

[0029] As used herein, the term "antigen binding domain" refers to one or more protein domains (e.g., formed by amino acids from a single polypeptide or formed by amino acids from two or more polypeptides (e.g., the same or different polypeptides). It is capable of specifically binding to one or more different antigens (e.g., effector antigens or tumor antigens). In some examples, the antigen binding domain can bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen binding domain can be an antibody or a fragment thereof. An example of an antigen binding domain is an antigen binding domain formed by VH-VL. In some embodiments, the antigen binding domain may include an alternative scaffold. In some embodiments, the antigen binding domain is a VHH. Non-limiting examples of antigen binding domains are described herein. Antigen binding domains are known in the art. In some embodiments, the antigen binding domain can bind to a single antigen (e.g., one of an effector antigen and a tumor antigen).

[0030] As used herein, the term "antibody" refers to an antibody that contains at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In certain embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, double antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0031] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, an antigen binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0032] Unless otherwise defined, all technical and scientific terms used herein are generally understood to have the same meaning as those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0033] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of anti-tumor studies using the CT26 tumor model and dosing regimen.

[0035] Figure 2 The average tumor volume curves in the anti-tumor study of the CT26 tumor model are shown. Mice were treated with CCR8-1 mAb (1 mg / kg), 4-1BB-1 mAb (1 mg / kg), or a combination of CCR8-1 mAb (1 mg / kg) and 4-1BB-1 mAb (1 mg / kg), and PBS was used as a negative control. Each curve represents the average tumor volume of each treatment group.

[0036] Figures 3A-3C Individual tumor volume curves from an antitumor study using the CT26 tumor model are shown. Each curve represents an antitumor effect of 1 mg / kg CCR8-1 mAb ( Figure 3A )、1mg / kg 4-1BB-1mAb( Figure 3B ) or a combination of 1 mg / kg CCR8-1 mAb and 1 mg / kg 4-1BB-1 mAb ( Figure 3C ) treated mice.

[0037] Figure 4 Shown are the percentages of mouse survival in antitumor studies using the CT26 tumor model.

[0038] Figure 5 It is a schematic diagram of anti-tumor research using the MC38 tumor model and drug administration regimen.

[0039] Figure 6 The average tumor volume curves in the anti-tumor study of the MC38 tumor model are shown. Mice were treated with CCR8-1 mAb (10 mg / kg), 4-1BB-1 mAb (10 mg / kg), or a combination of CCR8-1 mAb (10 mg / kg) and 4-1BB-1 mAb (10 mg / kg), and PBS was used as a negative control. Each curve represents the average tumor volume of each treatment group.

[0040] Figures 7A-7C Individual tumor volume curves from an antitumor study using the MC38 tumor model are shown. Each curve represents the volume of tumors in the presence of 10 mg / kg CCR8-1 mAb ( Fig. 7A )、10mg / kg 4-1BB-1mAb( Figure 7B) or a combination of 10 mg / kg CCR8-1 mAb and 10 mg / kg 4-1BB-1 mAb ( Figure 7C ) treated mice.

[0041] Figure 8 Shown are the survival rates of mice in the anti-tumor study using the MC38 tumor model.

[0042] Figures 9A-9C Figure 2 shows the cell killing rate of target CHO cells co-expressing human CCR8 and human 4-1BB by effector M0 macrophages via antibody-dependent cellular phagocytosis (ADCP). Target CHO cells were treated with a combination of CCR8-2 mAb and 4-1BB-2 mAb. The effector cell to target cell (E:T) ratio was tested at 1:1 ( Fig.9A )、3:1( Fig. 9B ) or 5:1( Fig. 9C ). Detailed description

[0044] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to 4-1BB and antibodies or antigen-binding fragments thereof that specifically bind to CC motif chemokine receptor 8 (CCR8).

[0045] The present invention also relates to a method for treating a subject having a tumor, the method comprising administering to the subject a therapeutically effective amount of: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB.

[0046] The present invention also relates to a method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB.

[0047] The present invention also relates to a method for killing tumor cells, comprising contacting the tumor cells with an effective amount of: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; and b) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB.

[0048] Anti-4-1BB Antibody

[0049] Human 4-1BB (also known as tumor necrosis factor receptor superfamily member 9, TNFRSF59 and CD137) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNF superfamily member 9) and is expressed in many immune cells, including activated T cells, B cells, dendritic cells (DCs), activated natural killer (NK) cells, and non-immune cells such as activated endothelial cells. When activated by human 4-1BB ligands, human 4-1BB co-stimulates T cells, thereby providing an effective T cell immune response and generating immune memory by inducing multiple signaling cascades.

[0050] 4-1BB is a member of the TNF receptor superfamily and is an activation-induced T cell co-stimulatory molecule. Signaling through 4-1BB can upregulate survival genes, enhance cell division, induce cytokine production, and prevent activation-induced cell death in T cells. The importance of the 4-1BB pathway in a variety of diseases, including cancer, has been highlighted.

[0051] Studies have shown that signaling through 4-1BB in the presence of anti-T cell receptor antibodies, agonist mAbs, soluble 4-1BBL, or cell lines expressing 4-1BBL, can lead to T cell expansion, cytokine induction, and upregulation of anti-apoptotic genes and protection against activation-induced cell death. Kinetic studies and expression analysis showed that although CD4 + and CD8 + The expression levels of 4-1BB in T cells were comparable, but CD8 + The proliferation potential of T cells is significantly higher than that of CD4 + T cells. Similar effects were observed in vivo. In addition to the effects on T cells, ligation of 4-1BB receptors on DCs and macrophages induced the production of IL-12 and IL-8 by these cells, respectively. In addition to the effects on T cells, ligation of 4-1BB receptors on DCs and macrophages induced the production of IL-12 and IL-8 by these cells, respectively.

[0052] To date, the role of 4-1BB signaling in a variety of autoimmune processes has been studied, including rheumatoid arthritis, experimental autoimmune encephalomyelitis, and systemic lupus erythematosus. 4-1BB signaling has dual immune regulatory effects.

[0053] A detailed description of 4-1BB and its functions can be found in, for example, Vinay et al., Mol Cancer Ther. 2012 May; 11(5): 1062-70; and Bartkowiak and Curran 2015, Frontiers in Oncology, 5: 117.

[0054] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to 4-1BB (e.g., human 4-1BB). In one aspect, the present invention provides anti-4-1BB antibodies or antigen-binding fragments thereof.

[0055] In some embodiments, the anti-4-1BB 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 at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VHCDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3 amino acid sequence; the light chain variable region (VL) comprises CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence,

[0056] Wherein, the selected VH CDR 1, 2, 3 amino acid sequence and the selected VL CDR 1, 2, 3 amino acid sequence are one of the following:

[0057] (1) The selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 5, 6, respectively;

[0058] (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 21, 22, 23, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 25, 26, respectively;

[0059] (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 41, 42, 43, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 45, 46, respectively;

[0060] (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62, 63, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 64, 65, 66, respectively;

[0061] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 5, and 6, respectively.

[0062] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 21, 22, 23, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 25, 26, respectively.

[0063] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 41, 42, 43, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 45, 46, respectively.

[0064] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 61, 62, 63, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 64, 65, 66, respectively.

[0065] 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.

[0066] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 7, 27, 47, or 67, and the selected VL sequence is SEQ ID NO: 8, 28, 48, or 68.

[0067] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8.

[0068] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0069] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0070] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0071] In some embodiments, CDR definitions are based on Kabat, Chothia, IMGT, contact, or AbM definitions.

[0072] In some embodiments, the antigen binding domain specifically binds human or mouse 4-1BB.

[0073] In some embodiments, the antigen binding domain is a human or humanized antigen binding domain.

[0074] In some embodiments, the antigen binding domain is a single chain variable fragment (scFv).

[0075] 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 antigen-binding domain is connected to the Fc region via a CH1 domain.

[0076] Anti-4-1BB antibodies or their antigen-binding fragments described herein can be any suitable structure. In certain embodiments, anti-4-1BB antibodies or their antigen-binding fragments are single-chain fragment variable (scFv) domains, which include light chain variable domains (VL) and heavy chain variable domains (VH) connected by joints.

[0077] The linkers described herein can be any suitable linkers known in the art. In some embodiments, the linkers include 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.

[0078] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain sequence of 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-4-1BB antibody or antigen-binding fragment thereof comprises a light chain sequence of 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.

[0079] The present invention provides anti-4-1BB antibodies and modified antibodies thereof, including, for example, chimeric antibodies, humanized antibodies and human antibodies.

[0080] Anti-4-1BB antibodies or their antigen-binding fragments may also have a variety of structures. A variety of bispecific antibodies or their antigen-binding fragments of different structures are known in the art, for example, described 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 its entirety.

[0081] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof is a BiTE, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HSA, or tandem-scFv. In some embodiments, the anti-4-1BB antibody is VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-hole common light chain, knob-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Trio, Minibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0082] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof can be a TrioMab. In TrioMab, the two heavy chains are from different species, wherein different sequences restrict heavy chain-light chain pairing.

[0083] In some embodiments, the anti-4-1BB antibody or its antigen-binding fragment has two different heavy chains and two different light chains. The heterodimerization of the heavy chain can be based on the knob-in-hole technique or some other heavy chain pairing techniques.

[0084] The anti-4-1BB antibody or antigen-binding fragment thereof can be a Duobody. The naturally occurring Fab exchange mechanism in IgG4 antibodies is mimicked in IgG1 antibodies with a controlled substance, and this mechanism is called controlled Fab exchange. This structure can ensure a specific pairing between heavy chains and light chains.

[0085] In dual variable domain antibodies (DVD-Ig), additional VH and variable light (VL) domains are added to each N-terminus to achieve bispecific targeting. This structure is similar to IgG-scFv, but the added binding domains are bound to each N-terminus, instead of scFv binding to each heavy chain N-terminus.

[0086] In scFv-IgG, two scFvs are attached to the C-terminus (CH3) of the heavy chain. The scFv-IgG structure has two different bivalent binding sites, so it is also called tetravalent. There is no problem of pairing heavy and light chains in scFv-IgG.

[0087] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof can have an IgG-IgG structure. Two complete IgG antibodies are combined by chemically linking the C-termini of the heavy chains.

[0088] The anti-4-1BB antibody or its antigen-binding fragment may also have a Fab-scFv-Fc structure. In the Fab-scFv-Fc structure, a light chain, a heavy chain, and a third chain containing an Fc region and scFv are assembled. This can ensure efficient manufacturing and purification.

[0089] In some embodiments, the anti-4-1BB antibody or its antigen-binding fragment can be TF. The three Fab fragments are connected by disulfide bonds. The TF structure does not have an Fc region.

[0090] ADAPTIR has two scFvs on either side of the Fc region. It abandons the complete IgG as its construction basis, but retains the Fc region to extend the half-life and facilitate purification.

[0091] Dual affinity retargeting (DART) has two peptide chains connecting opposite segments, so VLA is linked to VHB, VLB is linked to VHA, and there is a sulfide bond at the C-terminus fusing them together. In DART, the sulfide bond can improve the stability of the BiTE.

[0092] In DART-Fc, the Fc region is attached to the DART structure. It can be made by assembling three chains, two of which are linked by disulfide bonds, just like DART. One chain contains half of the Fc region, which will dimerize with the third chain to express only the Fc region. The addition of the Fc region can extend the half-life, thereby extending the effective concentration and avoiding continuous intravenous injection.

[0093] Four peptide chains are assembled in a tetravalent DART. Basically, two DART molecules are composed of half of the Fc region and dimerize. This structure has bivalent binding to both targets, making it a tetravalent molecule.

[0094] Tandem diabodies (TandAb) are composed of two diabodies, each of which is composed of covalently bound VHA and VLB fragments and VHA and VLB fragments. The two diabodies are connected by a peptide chain. Compared with diabodies composed of two scFvs, they are more stable and have two bivalent binding sites.

[0095] ScFv-scFv-toxin consists of a toxin and two scFvs with a stabilizing linker. It can be used for specific delivery of payloads.

[0096] In some embodiments, the anti-4-1BB antibody or its antigen-binding fragment comprises a KIH mutation. In some embodiments, the anti-4-1BB antibody or its antigen-binding fragment comprises a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain comprises one or more heavy chains with knob mutations (knob heavy chain), and the second antigen-binding domain comprises one or more heavy chains with hole mutations (hole heavy chain). In some embodiments, the first antigen-binding domain comprises one or more heavy chains with hole mutations (hole heavy chain), and the second antigen-binding domain comprises one or more heavy chains with knob mutations (knob heavy chain).

[0097] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof comprises an antigen-binding domain shown in Table 1.

[0098] The present invention also provides a nucleic acid comprising a polynucleotide encoding an anti-4-1BB antibody, wherein the immunoglobulin heavy chain or immunoglobulin light chain in the anti-4-1BB antibody comprises a CDR as shown in Table 1, and when the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to 4-1BB.

[0099] Anti-4-1BB antibody or its antigen binding fragment can also be anti-4-1BB antibody variant (including derivatives and conjugates), which is anti-4-1BB antibody or antibody fragment.Other anti-4-1BB antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific antibodies (polymers, such as bispecific antibodies), human antibodies, chimeric antibodies (such as human-mouse chimeras), single-chain antibodies, intracellular manufacturing antibodies (i.e., intracellular antibodies) and their antigen binding fragments.Anti-4-1BB antibodies can be any type (such as IgG, IgE, IgM, IgD, IgA and IgY), categories (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses.In certain embodiments, anti-4-1BB antibody or antigen binding fragment is IgG (for example, IgG1 Fc region) antibody or its antigen binding fragment.

[0100] Fragments of anti-4-1BB antibodies are suitable for use in the provided methods as long as they retain the desired affinity and specificity for 4-1BB. Thus, fragments of anti-4-1BB antibodies will retain the ability to bind to 4-1BB.

[0101] In some embodiments, the antibodies or antigen-binding fragments thereof have the same light chain variable regions.

[0102] Anti-CCR8 Antibody

[0103] CC motif chemokine receptor 8 (CCR8) is a protein found primarily on the surface of a highly immunosuppressive subset of regulatory T cells (Tregs) within tumors. It is a regulator of immune responses. As a chemokine receptor, CCR8 acts in concert with its chemokine CC chemokine ligand 1 (CCL1) to act as an active mediator. CCR8 expression is upregulated on a highly immunosuppressive subset of Tregs in multiple cancer types. Studies have found that CCR8 is specifically upregulated on Tregs in the tumor microenvironment (TME), with lower levels of CCR8+ Tregs found in blood and normal tissues. Tregs act through multiple mechanisms within the TME, including inhibition of antigen presenting cell (APC) function, consumption of interleukin 2 (IL-2), and production of immunosuppressive cytokines and metabolites. High Treg infiltration in the TME is associated with poor prognosis in multiple cancer types.

[0104] Compared with other current Treg depletion approaches, the anti-tumor activity of targeting CCR8 may be more specific. Other Treg target molecules are also present in peripheral Treg (in the blood), normal tissues, and effector T cells. Therefore, a highly selective target (such as CCR8) is needed to specifically deplete tumor-infiltrating Tregs without affecting Tregs in the blood and normal tissues or damaging effector T cells.

[0105] More information related to CCR8 can be found in the following literature, such as Li HL 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,SpringerMS.The chemokine / chemokine-receptor family:potential and progress fortherapeutic intervention.Curr Opin Chem Biol.2000;4(4):420-427;Campbell JR etal.Fc-optimized anti-CCR8antibody depletes regulatory T cells in human tumormodels.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-infiltratingregulatory T cells elicits antitumor immunity and synergizes with anti-PD-1therapy.J Immunother Cancer.2021;9(2):e001749;and Barsheshet Y et al. CCR8+FOXp3+Treg cells as master drivers of immune regulation. Proc Natl Acad Sci. 2017; 114(23): 6086-6091, each of which is incorporated herein by reference in its entirety.

[0106] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to CCR8 (eg, human CCR8). In one aspect, the present invention provides anti-CCR8 antibodies or antigen-binding fragments thereof.

[0107] 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 at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence 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 at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDR 1, 2, 3 amino acid sequence and the selected VL CDR 1, 2, 3 amino acid sequence are one of the following:

[0108] (1) The selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12, 13, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15, 16, respectively;

[0109] (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 35, 36, respectively;

[0110] (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 54, 55, 56, respectively; and

[0111] (4) The selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72, 73, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 74, 75, 76, respectively.

[0112] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 11, 12, and 13, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 15, and 16, respectively.

[0113] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 31, 32, and 33, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 35, and 36, respectively.

[0114] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 51, 52, and 53, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 54, 55, and 56, respectively.

[0115] In some embodiments, the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 71, 72, and 73, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 74, 75, and 76, respectively.

[0116] 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.

[0117] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0118] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0119] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0120] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0121] In some embodiments, the VH comprises VHCDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprises VL CDR1, VL CDR2, and VL CDR3 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.

[0122] In some embodiments, CDR definitions are based on Kabat, Chothia, IMGT, contact, or AbM definitions.

[0123] In some embodiments, the antigen binding domain specifically binds human or mouse CCR8.

[0124] In some embodiments, the antigen binding domain is a human or humanized antigen binding domain.

[0125] In some embodiments, the antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0126] 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 antigen-binding domain is connected to the Fc region via a CH1 domain.

[0127] The anti-CCR8 antibody or antigen-binding fragment thereof described herein can be any suitable structure. In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof is a single-chain fragment variable (scFv) domain, which includes a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a linker.

[0128] The linkers described herein can be any suitable linkers known in the art. In some embodiments, the linker 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: 81.

[0129] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain sequence of 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 comprises a light chain sequence of 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.

[0130] The present invention provides anti-CCR8 antibodies and modified antibodies thereof, including, for example, chimeric antibodies, humanized antibodies and human antibodies.

[0131] Anti-CCR8 antibodies or antigen-binding fragments thereof may also have a variety of structures. Various bispecific antibodies or antigen-binding fragments thereof of different structures are known in the art, for example, described 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 its entirety.

[0132] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof is a BiTE, (scFv)2, Nanobody, Nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HSA, or tandem-scFv. In some embodiments, the anti-CCR8 antibody is VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-hole common light chain, knob-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Trio, Minibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0133] 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 heavy chain-light chain pairing.

[0134] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof has two different heavy chains and two different light chains. Heterodimerization of the heavy chains can be based on the knob-in-hole technique or some other heavy chain pairing technique.

[0135] The anti-CCR8 antibody or antigen-binding fragment thereof can be a Duobody. The naturally occurring Fab exchange mechanism in IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies, and this mechanism is called controlled Fab exchange. This structure can ensure specific pairing between heavy chain and light chain.

[0136] In dual variable domain antibodies (DVD-Ig), additional VH and variable light (VL) domains are added to each N-terminus to achieve bispecific targeting. This structure is similar to IgG-scFv, but the added binding domains are bound to each N-terminus, instead of scFv binding to each heavy chain N-terminus.

[0137] In scFv-IgG, two scFvs are attached to the C-terminus (CH3) of the heavy chain. The scFv-IgG structure has two different bivalent binding sites, so it is also called tetravalent. There is no problem of pairing heavy and light chains in scFv-IgG.

[0138] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof may have an IgG-IgG structure. Two intact IgG antibodies are combined by chemically linking the C-termini of the heavy chains.

[0139] The anti-CCR8 antibody or antigen-binding fragment thereof may also have a Fab-scFv-Fc structure, in which a light chain, a heavy chain, and a third chain comprising an Fc region and scFv are assembled, which can ensure efficient production and purification.

[0140] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof may be TF. The three Fab fragments are linked by disulfide bonds. The TF structure does not have an Fc region.

[0141] ADAPTIR has two scFvs on either side of the Fc region. It abandons the complete IgG as its construction basis, but retains the Fc region to extend the half-life and facilitate purification.

[0142] Dual affinity retargeting (DART) has two peptide chains connecting opposite segments, so VLA is linked to VHB, VLB is linked to VHA, and there is a sulfide bond at the C-terminus fusing them together. In DART, the sulfide bond can improve the stability of the BiTE.

[0143] In DART-Fc, the Fc region is attached to the DART structure. It can be made by assembling three chains, two of which are linked by disulfide bonds, just like DART. One chain contains half of the Fc region, which will dimerize with the third chain to express only the Fc region. The addition of the Fc region can extend the half-life, thereby extending the effective concentration and avoiding continuous intravenous injection.

[0144] Four peptide chains are assembled in the tetravalent DART. Basically, two DART molecules are composed of half of the Fc region and dimerize. This form has two valences to bind to both targets, so it is a tetravalent molecule.

[0145] Tandem diabodies (TandAb) are composed of two diabodies, each of which is composed of covalently bound VHA and VLB fragments and VHA and VLB fragments. The two diabodies are connected by a peptide chain. Compared with diabodies composed of two scFvs, they are more stable and have two bivalent binding sites.

[0146] scFv-scFv-toxin consists of a toxin and two scFvs with a stabilizing linker. It can be used for specific delivery of payloads.

[0147] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a KIH mutation. In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain comprises one or more heavy chains with knob mutations (knob heavy chain), and the second antigen-binding domain comprises one or more heavy chains with hole mutations (hole heavy chain). In some embodiments, the first antigen-binding domain comprises one or more heavy chains with hole mutations (hole heavy chain), and the second antigen-binding domain comprises one or more heavy chains with knob mutations (knob heavy chain).

[0148] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof comprises an antigen-binding domain shown in Table 1.

[0149] The present invention also provides a nucleic acid comprising a polynucleotide encoding an anti-CCR8 antibody, wherein the immunoglobulin heavy chain or immunoglobulin light chain in the anti-CCR8 antibody comprises a CDR as shown in Table 1, and when the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CCR8.

[0150] Anti-CCR8 antibodies or antigen-binding fragments thereof may also be anti-CCR8 antibodies, anti-CCR8 antibody variants (including derivatives and conjugates) or antibody fragments. Other anti-CCR8 antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific antibodies (polymers, such as bispecific antibodies), human antibodies, chimeric antibodies (such as human-mouse chimeras), single-chain antibodies, intracellular antibodies (i.e., intracellular antibodies) and antigen-binding fragments thereof. Anti-CCR8 antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In some embodiments, anti-CCR8 antibodies or antigen-binding fragments are IgG (e.g., IgG1 Fc region) antibodies or antigen-binding fragments thereof.

[0151] Fragments of anti-CCR8 antibodies are suitable for use in the provided methods as long as they retain the desired affinity and specificity for CCR8. Thus, fragments of anti-CCR8 antibodies will retain the ability to bind to CCR8.

[0152] In some embodiments, the antibodies or antigen-binding fragments thereof have the same light chain variable regions.

[0153] Antibodies and their antigen-binding fragments

[0154] In some embodiments, anti-4-1BB antibodies or their antigen binding fragments include antigen binding domains from anti-4-1BB antibodies. In some embodiments, anti-CCR8 antibodies or their antigen binding fragments include antigen binding domains from anti-CCR8 antibodies. These anti-4-1BB or anti-CCR8 antibodies and their antigen binding fragments can have various forms.

[0155] In general, antibodies (also referred to as immunoglobulins) can be composed of two types of polypeptide chains, light chains and heavy chains. The non-limiting anti-4-1BB or anti-CCR8 antibodies of the present invention can be complete tetra-immunoglobulin chain antibodies, including two heavy chains and two light chains. The heavy chain of anti-4-1BB antibodies or anti-CCR8 antibodies can be any isotype, including IgM, IgG, IgE, IgA or IgD or subisotypes, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.

[0156] The hypervariable regions are called complementarity determining regions (CDRs), which form loops and constitute the main antigen binding surface of the antibody. The four framework regions mainly adopt a beta-sheet conformation, and the CDRs form loops that connect the beta-sheet structure and in some cases form part of the beta-sheet structure. The CDRs in each chain are closely connected to the framework region and, together with the CDRs in the other chain, form the antigen binding domain.

[0157] Methods for identifying the CDR regions of antibodies by analyzing the amino acid sequence of the antibody are well known, and a number of CDR definitions are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, for example, Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol.275(2):269-94(Jan.1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety.

[0158] CDR is very important for identifying antigen epitopes. "Epitope" as used herein is the smallest part of a target molecule that can specifically bind to the antigen binding domain of an antibody. The smallest epitope may be about three, four, five, six or seven amino acids, but these amino acids do not have to be in a continuous linear sequence of the primary structure of the antigen, because the epitope may depend on the three-dimensional structure of the antigen, which in turn is based on the secondary and tertiary structures of the antigen.

[0159] In some embodiments, anti-4-1BB or anti-CCR8 antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3 and IgG4) are highly conserved, with different constant regions, particularly in the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described, for example, 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.

[0160] Anti-4-1BB or anti-CCR8 antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, or camelids).Antigen binding domains or antigen binding fragments are a part of an antibody that retains the specific binding activity of a complete antibody, i.e., any part of an antibody that can specifically bind to an epitope on a target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Therefore, in some embodiments, anti-4-1BB or anti-CCR8 antibodies or their antigen binding fragments may include, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide including a binding domain, wherein the binding domain is an antibody binding domain or is homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., heavy and / or light chain CDRs of an intact antibody, heavy and / or light chain variable regions of an intact antibody, the full length heavy or light chain of an intact antibody, or a single CDR of a heavy or light chain of an intact antibody.

[0161] In some embodiments, the scFv in the anti-4-1BB or anti-CCR8 antibody has two heavy chain variable regions and two light chain variable regions. In some embodiments, the anti-4-1BB or anti-CCR8 scFv has two antigen binding regions (antigen binding regions: A and B), and the two antigen binding regions can bind to their respective target antigens with different affinities.

[0162] In some embodiments, the anti-4-1BB or anti-CCR8 antibody or antigen-binding fragment thereof may comprise one, two or three heavy chain variable region CDRs selected from Table 1.

[0163] 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.

[0164] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof is selected from Utomilumab, Urelumab, EU-101, YH-004, ADG-106 and LVGN-6051.

[0165] In some embodiments, the anti-4-1BB or anti-CCR8 antibodies described herein may be coupled to a therapeutic agent.Anti-4-1BB or anti-CCR8 antibody-drug conjugates comprising antibodies or their antigen-binding fragments may be covalently or non-covalently bound to a therapeutic agent.In some embodiments, the therapeutic agent is a cytotoxic agent or a cell inhibitor (e.g., monomethyl chlortetracycline E, monomethyl chlortetracycline F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, adriamycin, daunorubicin, dihydroxy anthrax, maytansinoids (e.g., DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like).In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker (e.g., a VC linker). Detailed information on the linker used in ADCs can be found in Su, Z. et al. "Antibody–drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference.

[0166] In some embodiments, anti-4-1BB or anti-CCR8 antibodies are bispecific antibodies. Bispecific antibodies can be manufactured by designing the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface may include at least a portion of the CH3 domains of an antibody constant domain. In the method, one or more small amino acid side chains on the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers, exceeding other unwanted end products, such as homodimers. The method is described in, for example, WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0167] Any anti-4-1BB or anti-CCR8 antibody or its antigen-binding fragment described herein may be combined with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). The binding of stabilizing molecules can increase the half-life of anti-4-1BB or anti-CCR8 antibodies or antigen-binding fragments in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body) or extend their biological activity.

[0168] Antibody characteristics

[0169] Anti-4-1BB antibodies may include anti-4-1BB antigen binding domains as described herein. Anti-CCR8 antibodies may include anti-CCR8 antigen binding domains as described herein. In certain embodiments, anti-4-1BB antibodies or their antigen binding fragments and / or anti-CCR8 antibodies or their antigen binding fragments can initiate target cell (e.g., tumor cells, such as CT26 cells or MC38 cells) killing, CDC, ADCC or ADCP.

[0170] The present invention provides anti-4-1BB and anti-CCR8 antibodies and antigen-binding fragments thereof that can specifically bind to 4-1BB. In some embodiments, anti-4-1BB antibodies can activate 4-1BB-related signaling pathways by binding to 4-1BB or activating the interaction of 4-1BB with its ligand, thereby treating cancer. In some embodiments, the antibody can reduce the tumor volume of the subject. Therefore, in some embodiments, the anti-4-1BB antibody described herein is a 4-1BB agonist. In some embodiments, the anti-4-1BB antibody described herein is a 4-1BB antagonist.

[0171] The affinity of the antibody for the antigen can be measured using common techniques, such as ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some embodiments, the anti-4-1BB antibody or its antigen-binding fragment can bind to 4-1BB (e.g., human 4-1BB, monkey 4-1BB, mouse 4-1BB, and / or chimeric 4-1BB) with a dissociation rate (koff) of less than 0.1s -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0172] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0173] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof can be present in an amount of less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 - 8M, greater than 1×10 -9 M or greater than 1×10 -10 M.

[0174] Anti-CCR8 antibodies or their antigen-binding fragments may also include an antigen-binding domain that can specifically bind to CCR8. In some embodiments, the antibodies or their antigen-binding fragments described herein recruit cells expressing CCR8 (e.g., Tregs expressing CCR8) and mediate the consumption of such cells. In some embodiments, the antibodies can reduce tumor volume in animals. In some embodiments, the antibodies can reduce or slow the progression of autoimmune diseases. Therefore, in some embodiments, the anti-CCR8 antibodies described herein are CCR8 agonists. In some embodiments, the anti-CCR8 antibodies described herein are CCR8 antagonists.

[0175] In some embodiments, the anti-CCR8 antibody or antigen-binding fragment 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 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0176] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0177] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10-8 M, less than 1×10-9M or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M or greater than 1×10 -10 M.

[0178] In some embodiments, anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADCs have a tumor growth inhibition rate or percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADCs have a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150%. TGI (%) can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 days or more after the start of treatment. As used herein, tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:

[0179] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0180] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

[0181] In some embodiments, the anti-4-1BB 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 to administration of PBS or no administration of the anti-4-1BB or anti-CCR8 antibody, antigen binding fragment or ADC. Tumor volume is measured in mm 3 The following formula can be used to calculate V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L).

[0182] In some embodiments, anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADCs have functional Fc regions. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3 or human IgG4.

[0183] In some embodiments, anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADCs do not have a functional Fc region. For example, anti-4-1BB or anti-CCR8 antibodies or their antigen binding fragments are Fab, Fab', F(ab')2 and Fv fragments. In some embodiments, according to anti-4-1BB or anti-CCR8 antibodies or their antigen binding fragments as described herein, there is an Fc region without effector function. In some embodiments, Fc is human IgG4 Fc. In some embodiments, 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).

[0184] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region). In some embodiments, the anti-4-1BB or anti-CCR8 antibodies described herein may be designed to have an IgG1 subtype structure having a GAALIE mutation (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the anti-4-1BB or anti-CCR8 antibodies described herein may be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, glutamic acid (E) at position 332, glutamic acid (E) at position 332, and glutamic acid (E) at position 332.

[0185] Several other modifications may be made to the Fc region, for example, cysteine ​​residues may be introduced into the Fc region to allow interchain disulfide bond formation in said region, so that the resulting homodimeric fusion protein may have an increased half-life in vitro and / or in vivo.

[0186] In some embodiments, the IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0187] In some embodiments, anti-4-1BB or anti-CCR8 antibody variants having a carbohydrate structure and lacking fucose directly or indirectly attached to the Fc region are provided. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297, relative to the sum of all sugar structures attached to Asn 297 (e.g., complex, mixed, and high mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to the small sequence variation of the antibody, Asn297 may also be located at approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. In some embodiments, in order to reduce glycan heterogeneity, the Fc region of anti-4-1BB or anti-CCR8 antibodies can be further modified to replace the asparagine at position 297 with alanine (N297A).

[0188] In certain embodiments, the methods described herein are intended to manufacture bispecific antibodies. Bispecific antibodies can be manufactured by designing the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can comprise at least a portion of the CH3 domains 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 by larger side chains (such as tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine), a compensation "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule. This provides a mechanism for increasing the output of heterodimers, exceeding other unwanted final products, such as homodimers. The method is described in, for example, WO96 / 27011, which is incorporated herein by reference in its entirety.

[0189] In some embodiments, anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADCs promote antibody-dependent cellular phagocytosis (ADCP), wherein anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADCs are combined with target cells (e.g., CT26 cells, MC38 cells or CHO cells) that co-express 4-1BB and / or CCR8, and are marked for destruction by phagocytes (e.g., macrophages, dendritic cells or neutrophils). In ADCP, the Fab region of the antibody binds to the antigen on the surface of the target cell that co-expresses 4-1BB and / or CCR8 (e.g., CT26 cells, MC38 cells or CHO cells), and the Fc region interacts with the Fc receptors (e.g., CD64 or CD32) on the surface of the phagocyte. Once involved in this interaction, phagocytes (e.g., macrophages, dendritic cells or neutrophils) are triggered to engulf and digest target cells (e.g., CT26 cells, MC38 cells or CHO cells) that co-express 4-1BB and / or CCR8, thereby removing labeled cells (or cell killing). ADCP is a key mechanism in the immune response and is actively exploited in therapeutic antibody therapy, particularly in cancer therapy, where antibodies are designed to target and eliminate tumor cells through phagocytosis. ADCP is the primary mode of action for many monoclonal antibodies known in the field of oncology.

[0190] In some embodiments, anti-4-1BB or anti-CCR8 antibodies, antigen binding fragments or ADC promote ADCP of target cells (e.g., CHO cells) co-expressing hCCR8 and h4-1BB by effector M0 macrophages. In some embodiments, the ratio of effector cells (e.g., macrophages) to target cells (e.g., CHO cells co-expressing 4-1BB and / or CCR8) or E:T ratio can be any ratio between 1:10 and 50:1 (e.g., between 1:5 and 25:1, between 1:2 and 10:1, or between 1:1 and 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, the E:T ratio may 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).

[0191] In some embodiments, the knob-into-hole (KIH) technology can be used, which involves engineering the CH3 domain to create a "knob" or "hole" in each heavy chain to promote heterodimerization. The KIH technology is described in, for example, Xu, Yiren, et al. "Production of bispecific antibodies in'knobs-into-holes'using a cell-freeexpression system." MAbs. Vol. 7. No. 1. Taylor & Francis, 2015, which is incorporated herein by reference in its entirety. In some embodiments, one heavy chain has T366W and / or S354C (knob) substitutions (EU numbering), while the other heavy chain has Y349C, T366S, L368A and / or Y407V (hole) substitutions (EU numbering). In some embodiments, one heavy chain has one or more of the following substitutions: Y349C and T366W (EU numbering). The other heavy chain may have one or more of the following substitutions: E356C, T366S, L368A and Y407V (EU numbering). In addition, substitutions may be introduced in the hinge region of both substituted IgGs (-ppcpScp-->-ppcpPcp-).

[0192] Recombinant vector

[0193] The present invention also provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide described herein (e.g., a polynucleotide encoding a polypeptide described herein), a host cell introduced with the recombinant vector (i.e., such that the host cell contains the polynucleotide and / or a vector comprising the polynucleotide), and production of an anti-4-1BB or anti-CCR8 antibody polypeptide or fragment thereof by recombinant technology.

[0194] The full-length sequence of human CCR8 is known in the art (eg, NCBI Gene ID: 1237, NCBI Reference Sequence: NM_005201.4). In some embodiments, a His-tagged human CCR8 protein is used as an immunogen.

[0195] The full-length sequence of human 4-1BB is known in the art (eg, GenBank Accession No.: U03397.1). In some embodiments, a His-tagged human 4-1BB protein is used as an immunogen.

[0196] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Therefore, in an expression vector, the polynucleotide of interest is positioned in the vector for expression by being operably connected to a regulatory element (e.g., a promoter, an enhancer, and / or a poly-A tail), the regulatory element being located at or near or flanking the integration site of the polynucleotide of interest in the vector or in the host cell genome, so that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

[0197] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection and infection and / or transduction (e.g., using recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0198] In some embodiments, a polynucleotide described herein (e.g., a polynucleotide encoding a polypeptide described herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or a replication-defective virus may be used. In the latter case, viral propagation typically occurs only in the complementing virus packaging cells. Suitable systems are described, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; US ​​Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; US Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al. al., 1991, Science, 252: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads for efficient delivery into cells.

[0199] For expression, the DNA insert comprising the polypeptide encoding polynucleotide described herein can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, and the promoters of the retroviral LTR, to name a few. Other suitable promoters are known to those skilled in the art. The expression construct may also include a transcription start site, a termination site, and a translation ribosome binding site in the transcribed region. The coding portion of the mature transcript expressed by the construct may include a translation start codon at the beginning and a termination codon (UAA, UGA or UAG) at an appropriate position at the end of the polypeptide to be translated.

[0200] As noted, the expression vector may include at least one selection marker. These markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells, such as Drosophila S2 and Spodoptera frugiperda Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Culture media and conditions suitable for host cells described herein are known in the art.

[0201] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0202] Non-limiting bacterial promoters suitable for use include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters, and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTR promoters (e.g., promoter of Rous sarcoma virus (RSV)), and metallothionein promoters (e.g., mouse metallothionein-I promoter).

[0203] In yeast, a variety of vectors containing constitutive or inducible promoters (such as α factor, alcohol oxidase, and PGH) can be used. For details, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153: 516-544 (1997).

[0204] The introduction of the construct into the host cell can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0205] By inserting an enhancer sequence in a vector, it is possible to increase the transcription of the DNA of the anti-4-1BB or anti-CCR8 antibodies of the content of the present invention encoded by higher eukaryotes.Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, which act to increase the transcriptional activity of promoters in a given host cell type.The example of enhancer includes SV40 enhancer (located at the downstream side of the replication origin, base pairs 100 to 270), cytomegalovirus early promoter enhancer, polyoma enhancer and adenovirus enhancer at the downstream side of the replication origin.

[0206] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. These signals can be endogenous to the polypeptide or heterologous signals.

[0207] Polypeptide (for example, anti-4-1BB or anti-CCR8 antibody) can be expressed in a modified form, such as fusion protein (for example, GST fusion) or with a histidine tag, and may include not only a secretion signal, but also an additional heterologous functional region. For example, an additional amino acid region (particularly charged amino acids) can be added to the N-terminus of the polypeptide to improve stability and durability during the host cell, during purification or subsequent processing and storage. In addition, the peptide portion can be added to the polypeptide to promote purification. Such regions can be removed before the final preparation of the polypeptide. Peptide portions are added to the polypeptide to cause secretion or excretion, improve stability and promote purification, etc. are conventional techniques familiar in the art.

[0208] The present invention also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein. The present invention relates to the invention to an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences described herein.

[0209] The present invention also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein. The present invention relates to the invention to any of the amino acid sequences described herein, as well as amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences described herein.

[0210] In some embodiments, the present disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0211] 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 described herein.

[0212] 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 described herein.

[0213] In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned to achieve the best comparison purpose (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve the best comparison, and non-homologous sequences can be ignored for comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at the position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, wherein the number of gaps and the length of each gap are taken into account, and these gaps need to be introduced to achieve the best comparison of the two sequences. For example, the Blossum62 scoring matrix can be used to complete the comparison of the sequences and the determination of the percentage identity between the two sequences, wherein the gap penalty is 12, the gap extension penalty is 4, and the frameshift gap penalty is 5.

[0214] The present invention provides one or more nucleic acids encoding any polypeptide described herein. In some embodiments, nucleic acids (e.g., cDNA) include polynucleotides encoding heavy chain polypeptides described herein. In some embodiments, nucleic acids include polynucleotides encoding light chain polypeptides described herein. In some embodiments, nucleic acids include polynucleotides encoding scFv polypeptides described herein.

[0215] In some embodiments, the vector may have two nucleic acids described herein, wherein the vector encodes a VL region and a VH region that jointly bind 4-1BB. In some embodiments, a pair of vectors is provided, wherein each vector comprises a nucleic acid described herein, wherein the vector pair jointly encodes and binds to the VL region and VH region of 4-1BB.

[0216] In some embodiments, the vector comprises two nucleic acids described herein, wherein the vector encodes a VL region and a VH region that jointly bind to CCR8. In some embodiments, a pair of vectors is provided, wherein each vector comprises a nucleic acid described herein, wherein the vector pair jointly encodes and binds to the VL region and VH region of CCR8.

[0217] Treatment

[0218] The methods described herein include methods for treating cancer-related diseases (e.g., treating subjects with tumors, reducing tumor growth rate, or killing tumor cells). Generally, the method includes administering a therapeutically effective amount of a composition to a subject who needs or has been determined to need such treatment, a) comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 described herein; and b) comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB described herein.

[0219] As used herein, "treating" refers to improving at least one symptom of a disease associated with cancer. Cancer usually results in death; therefore, treatment can extend life expectancy or survival time (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 treating a condition associated with cancer will result in a reduction in the number of cancer cells, a reduction in tumor volume, relief of symptoms, and / or an extension of life expectancy or survival time.

[0220] As used herein, the term "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid proliferation of cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of the histopathological type or invasion stage. The term "tumor" used herein refers to cancer cells, such as a large number of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems, such as those affecting lung cancer, breast cancer, thyroid cancer, lymphoma, gastrointestinal cancer and urogenital tract cancer, and adenocarcinomas, including most colon cancers (e.g., colon cancer), renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, small intestine cancer and esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer and melanoma. In some embodiments, cancer is renal cancer or melanoma. Exemplary cancers include cancers formed from cervical, lung, prostate, breast, head and neck, colon and ovarian cancer tissue. The term also includes carcinosarcoma, for example, including malignant tumors composed of carcinoma tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer or tumor cell derived from glandular tissue that forms a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from mesenchyme.

[0221] In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0222] In one aspect, the present disclosure also provides a method for treating a subject's cancer, a method for reducing the rate at which a subject's tumor volume increases over time, a method for reducing the risk of metastasis, or a method for reducing the risk of additional metastasis in a subject. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of a subject's cancer.

[0223] In one aspect, the present disclosure describes a method comprising administering a therapeutically effective amount of an anti-4-1BB and anti-CCR8 antibody as described herein to a subject in need thereof, such as a subject suffering from or identified or diagnosed with cancer, such as 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, cancer is a solid tumor, lung cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer or brain cancer.

[0224] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe animals, humans or non-humans treated by the methods of the present invention. The present invention contemplates veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., people under 18 years of age). In addition to humans, patients 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., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), equines, canines, felines, bovines and other domestic, farm and zoo animals.

[0225] In some embodiments, the compositions and methods described herein can be used to treat patients at risk for cancer. Various methods known in the art can be used to identify patients with cancer.

[0226] "Effective amount" used herein refers to an amount or dosage sufficient to produce a beneficial or desired result, including stopping, slowing down, delaying or inhibiting the progress of a disease (e.g., cancer). The effective amount will vary according to, for example, the age and weight of the subject of anti-4-1BB and anti-CCR8 antibodies, anti-4-1BB and anti-CCR8 antigen-binding fragments, anti-4-1BB and anti-CCR8 antibody encoding polynucleotides, carriers comprising polynucleotides and / or their compositions, the severity of symptoms and route of administration, so it can be determined according to individual circumstances to be administered.

[0227] The effective amount can be applied once or multiple times.For example, the effective amount of anti-4-1BB or anti-CCR8 antibodies or Fab is enough to improve, stop, stabilize, reverse, suppress, slow down and / or delay the amount of patient's autoimmune disease or cancer progression, or enough to improve, stop, stabilize, reverse, slow down and / or delay the amount of in vitro cell (such as biopsy cells, any cancer cell or cell line (such as cancer cell line) described herein) propagation.As understood in the art, the effective amount of anti-4-1BB or anti-CCR8 antibodies, Fab or antibody-drug conjugates may be different, depending on patient medical history and other factors, such as the type (and / or dosage) of the medicament used.

[0228] The effective amount and schedule of administering the anti-4-1BB and anti-CCR8 antibodies, antigen-binding fragments thereof, antibody encoding polynucleotides and / or compositions described herein can be determined empirically, and making such determinations is within the capabilities of those skilled in the art. It will be understood by those skilled in the art that the dose that must be administered will vary, for example, depending on the mammal to which the anti-4-1BB and anti-CCR8 antibodies, antigen-binding fragments thereof, antibody encoding polynucleotides and / or compositions described herein will be administered, the route of administration, the specific type of agent or composition described herein, and other drugs administered to the mammal.

[0229] The typical daily dose of each effective amount of anti-4-1BB antibody and anti-CCR8 antibody is 0.01mg / kg to 100mg / kg. In some embodiments, the dosage may be less than 100mg / kg, 30mg / kg, 20mg / kg, 10mg / kg, 9mg / kg, 8mg / kg, 7mg / kg, 6mg / kg, 5mg / kg, 4mg / kg, 3mg / kg, 2mg / kg, 1mg / kg, 0.5mg / kg or 0.1mg / kg. In some embodiments, the dosage may be greater than 10mg / kg, 9mg / kg, 8mg / kg, 7mg / kg, 6mg / kg, 5mg / kg, 4mg / kg, 3mg / kg, 2mg / kg, 1mg / kg, 0.5mg / kg, 0.1mg / kg, 0.05mg / kg or 0.01mg / kg. In some embodiments, the dose 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.

[0230] In some embodiments, the anti-4-1BB antibodies and anti-CCR8 antibodies described herein kill tumor cells and / or reduce tumor size in subjects receiving the antibodies (e.g., by activating 4-1BB-related signaling pathways or recruiting and depleting immune cells expressing CCR8 (e.g., tumor-infiltrating Tregs expressing CCR8)). In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of tumor cells are killed by administering the anti-4-1BB antibodies and anti-CCR8 antibodies described herein.

[0231] In some embodiments, the anti-tumor effects of the anti-4-1BB antibodies and anti-CCR8 antibodies described herein are to enhance tumor immunity and improve survival rate by regulating Treg / Teff cells (e.g., in mice or humans).

[0232] In some embodiments, administration of a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB will reduce the tumor volume (tumor size) of the subject. In some embodiments, the tumor volume of a subject receiving an anti-4-1BB antibody and an anti-CCR8 antibody as described herein is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the tumor size without administration of anti-4-1BB antibody and anti-CCR8 antibody. In some embodiments, the tumor size of a subject receiving an anti-4-1BB antibody and an anti-CCR8 antibody described herein is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the tumor size reduced by administration of the anti-4-1BB antibody alone or the anti-CCR8 antibody alone.

[0233] In some embodiments, the administration of anti-4-1BB antibodies and anti-CCR8 antibodies described herein can increase the survival rate of subjects receiving the antibodies. In some embodiments, the survival rate is increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the survival rate of subjects not administered anti-4-1BB antibodies and anti-CCR8 antibodies or only administered anti-4-1BB antibodies or only administered anti-CCR8 antibodies.

[0234] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 as described herein and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB as described herein are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more.

[0235] In some embodiments, a composition comprising an antibody or antigen binding fragment thereof specifically binding to CCR8 as described herein and a composition comprising an antibody or antigen binding fragment thereof specifically binding to 4-1BB as described herein are administered every 1, 2, 3, 4, 5 or more days. In some embodiments, a composition comprising an antibody or antigen binding fragment thereof specifically binding to CCR8 as described herein and a composition comprising an antibody or antigen binding fragment thereof specifically binding to 4-1BB as described herein are administered once a week, twice a week, three times a week, four times a week or more. In some embodiments, a composition comprising an antibody or antigen binding fragment thereof specifically binding to 4-1BB as described herein is administered once a week, twice a week, three times a week, four times a week or more.

[0236] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 as described herein and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB as described herein are administered at the same dose, or at a dose ratio of about 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, or any other suitable dose ratio.

[0237] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 as described herein and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB as described herein are administered intravenously and / or intraperitoneally.

[0238] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CCR8 as described herein and a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB as described herein are administered three times, wherein the first administration is performed intravenously and the second and third administrations are performed intraperitoneally.

[0239] In certain embodiments, one or more additional therapeutic agents may be administered to a subject. Additional therapeutic agents may include one or more inhibitors selected from the following groups: B-Raf inhibitors, 4-1BB inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, CCR8 inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In certain embodiments, additional therapeutic agents are inhibitors (e.g., epacadostat) of indoleamine 2,3-dioxygenase-1) (IDO1).

[0240] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of 4-1BB inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, activated Hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0241] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of trabectedin, albumin-bound paclitaxel, trabanib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzatalem.

[0242] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0243] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0244] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-TIM3 antibody, or an anti-GITR antibody.

[0245] Pharmaceutical compositions and routes of administration

[0246] Also provided herein is a pharmaceutical composition comprising at least one (e.g., one, two, three or four) anti-4-1BB and / or anti-CCR8 antibody, antigen binding fragment or antibody-drug conjugate as described herein. The pharmaceutical composition can be formulated in any manner known in the art.

[0247] The preparation of the pharmaceutical composition is compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, an antibacterial or antifungal agent (e.g., benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), an antioxidant (e.g., ascorbic acid or sodium bisulfite), a chelating agent (e.g., ethylenediaminetetraacetic acid), a buffer (e.g., acetate, citrate or phosphate) and an isotonic agent (e.g., sugar (e.g., glucose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride) or any combination thereof). Liposomal suspensions may also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition may be formulated and packaged in an ampoule, a disposable syringe or a multidose vial. Where necessary (for example, in the preparation of injectables), proper fluidity can be maintained by the use of a coating such as lecithin or a surfactant.

[0248] Compositions containing one or more anti-4-1BB and anti-CCR8 antibodies or antigen-binding fragments described herein can be formulated in dosage unit form (i.e., physically discrete units containing predetermined quantities of the active compounds for ease of administration and uniformity of dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration.

[0249] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents with high therapeutic indices are preferred. When an agent exhibits adverse side effects, care should be taken to minimize the potential for harm (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0250] The data obtained from cell culture tests and animal studies can be used to formulate the appropriate dose of any given agent for a subject (e.g., human).Antibody or antigen binding fragment or antibody-drug conjugate (e.g., anti-4-1BB and anti-CCR8 antibodies, antigen binding fragments or antibody-drug conjugates) The therapeutically effective amount will be the amount of the disease (e.g., cancer cell killing) of the subject (e.g., determined to be a human subject with cancer) or the subject (e.g., previously had cancer but now has been cured) determined to be at risk of illness, reducing the severity of one or more symptoms of the subject (e.g., human) disease, frequency and / or duration.Healthcare professionals or veterinary professionals can use methods known in the art and by observing one or more disease symptoms of the subject (e.g., human) to determine the effectiveness and dosage of any antibody or antigen binding fragment or antibody-drug conjugate described herein (e.g., anti-4-1BB and anti-CCR8 antibodies, antigen binding fragments or antibody-drug conjugates). Certain factors may influence the dosage and duration required to effectively treat a subject (e.g., the severity of the disease or condition, previous treatments, the general health and / or age of the subject, and the presence of other medical conditions).

[0251] Exemplary dosages include any antibody or antigen-binding fragment or antibody-drug conjugate (e.g., anti-4-1BB and anti-CCR8 antibodies, antigen-binding fragments or antibody-drug conjugates) in milligrams or micrograms per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 1 mg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg). Although these dosages cover a wide range, those of ordinary skill in the art will understand that the efficacy of therapeutic agents varies, and the effective amount can be determined by methods known in the art. Typically, a relatively low dose is first administered, and then the attending health care professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the development stage) can gradually increase the dose until an appropriate response is obtained. Furthermore, it is understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion and the half-life of the therapeutic agent in vivo.

[0252] Antibodies or antigen binding fragments or antibody-drug conjugates (e.g., anti-4-1BB and anti-CCR8 antibodies, antigen binding fragments or antibody-drug conjugates) described herein may be administered once or multiple times during treatment. For example, antibodies or antigen binding fragments or antibody-drug conjugates (e.g., anti-4-1BB and anti-CCR8 antibodies, antigen binding fragments or antibody-drug conjugates) described herein may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. In some embodiments, antibodies or antigen binding fragments or antibody-drug conjugates (e.g., anti-4-1BB and anti-CCR8 antibodies, antigen binding fragments or antibody-drug conjugates) are administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more.

[0253] The pharmaceutical composition may be included in a container, package or dispenser together with the instructions for administration. The present invention also provides methods for making antibodies or antigen binding fragments or antibody-drug conjugates (e.g., anti-4-1BB and anti-CCR8 antibodies, antigen binding fragments or antibody-drug conjugates) for various uses described herein. Example

[0254] The present invention will be further described by the following examples, but these examples are not intended to limit the scope of the invention described in the claims.

[0255] Example 1: Molecular Construction

[0256] To prepare anti-CCR8 monoclonal antibodies (CCR8-1 mAb), the VH region was linked to wild-type mIgG2a Fc and the VL region was linked to the mouse lambda light chain constant region. To generate anti-4-1BB monoclonal antibodies (e.g., 4-1BB-1 mAb), the VH region was linked to wild-type mIgG2a Fc and the VL region was linked to the mouse kappa light chain constant region.

[0257] To prepare other anti-CCR8 monoclonal antibodies (e.g., CCR8-2 mAb, CCR8-3 mAb, and CCR8-4 mAb), the VH region was linked to wild-type hIgG1 Fc and the VL region was linked to the human kappa light chain constant region. To prepare other anti-4-1BB monoclonal antibodies (e.g., 4-1BB-2mAb, 4-1BB-3mAb, and 4-1BB-4mAb), the VH region was linked to wild-type hIgG1 Fc and the VL region was linked to the human kappa light chain constant region.

[0258] Fragments were synthesized (IDT) and assembled (NEB) as described above. HiFi DNA Assembly) to pcDNA3.4 or pcDNA3.4 carrying the required fragment. Transform the assembled plasmid into competent E. coli ( 5-alpha). The clones with correct sequences confirmed by sequencing results (Elim Biopharm) were further cultured with lysogeny broth (LB medium) containing carbenicillin (100 μg / ml) for plasmid amplification and then plasmid purification (QIAGEN Plasmid Plus Kits). The plasmids were eluted in nuclease-free H2O (Sigma) and stored at -80°C.

[0259] Example 2: Transfection and purification of antibodies

[0260] All antibody light and heavy chain coding sequences were generated by direct DNA synthesis and cloned into mammalian expression vectors. The cloned sequences were verified by Sanger sequencing.

[0261] Chinese hamster ovary (CHO) cells were grown in CHOgro medium (Mirus) supplemented with 4 mM L-glutamine and 0.33% Poloxamer-188 and diluted to 4 × 10 in fresh cell culture medium for transfection. 6 cells / ml. By lipofectamine transfection, 4×10 6 The cells contained 1 μg of plasmid DNA. The transfected cells were kept in a shaking incubator at 32°C, 5% CO2 and 70% humidity at 125 rpm.

[0262] Protein expression titer was measured every 5 days. Cell supernatant was collected by centrifugation 10-14 days after transfection and filtered through a 0.22 μm PES membrane filter. The expressed antibody was purified by Protein A chromatography ( AF-rProtein A HC-650M resin, Tosoh), and anion exchange resin (TOYOPEARL NH2-750F, Tosoh) was used to remove aggregates.

[0263] The purified antibodies were Software-controlled HPLC (Dionex The antibody purity and aggregation were determined using a size exclusion column TSKgel UP-SW3000, 2 μm, 4.6 mm ID×15 cm (Tosoh).

[0264] All purified antibodies had a monomer purity of 97% or greater, were sterilized through 0.22 μm PES membrane filters, and used for binding and functional assays.

[0265] Example 3: CCR8 mAb and 4-1BB mAb synergistically reduce tumor growth in the CT26 colon cancer model

[0266] BalB / c mice were used to verify the anti-tumor effect of the combination of anti-CCR8 mAb and anti-4-1BB mAb on the CT26 colon cancer model. Female BalbC mice aged 6-9 years from the Weitong Lihua Laboratory Research Model and Service Center (Guangdong, China) were used in the experiment. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC). On day 0, CT26 colon cancer cells (5×10 5 The cells were subcutaneously injected into the right posterior abdomen of mice in 0.1 ml PBS for tumor growth. When the average tumor size reached about 80-120 mm on day 7, 3 At 10 days, mice were randomized and assigned to treatment groups. On days 7, 10, and 14, mice were treated with CCR8-1 mAb, 4-1BB-1 mAb, and CCR8-1 mAb and 4-1BB-1 mAb combination at a dose of 1 mg / kg. Control mice were treated with phosphate-buffered saline (PBS). The first dose was administered intravenously, and the second and third doses were administered intraperitoneally ( Figure 1 Tumors were measured in two dimensions three times a week using calipers, and the volume was expressed in cubic millimeters using the following formula: V = (L × W × W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Graphs and statistical analyses were performed using the GraphPad Prism program. Tumor growth was analyzed using two-way analysis of variance and Dunnett's multiple comparison test, and survival percentage data were analyzed using Kaplan-Meyer survival analysis.

[0267] Comparison of the average tumor volume between the single-drug group and the CCR8-1 mAb + 4-1BB-1 mAb combined group. Figure 2 As shown, single-agent CCR8-1 mAb or single-agent 4-1BB-1 mAb treatment induced partial antitumor activity in the mouse CT26 tumor model, however, combined administration of CCR8-1 mAb and 4-1BB-1 mAb had a synergistic effect and strongly inhibited tumor growth.

[0268] The complete remission rate of the tumors was also compared over several days. At the end of the study on day 49, the combination of CCR8-1 mAb and 4-1BB-1 mAb resulted in an increase in the number of tumor-free mice (4 out of 6 mice were tumor-free) compared with treatment with either CCR8-1 mAb alone (none of 6 mice were tumor-free) or 4-1BB-1 mAb alone (none of 6 mice were tumor-free). Figure 3A-3B). In addition, the time required for tumor regression was shorter in the group treated with the combination of CCR8-1 mAb and 4-1BB-1 mAb compared with treatment with either monoclonal CCR8-1 mAb or 4-1BB-1 mAb alone.

[0269] The percentage survival during treatment was also calculated. Figure 4 As shown, at the end of the study on day 49, combined treatment with CCR8-1 mAb and 4-1BB-1 mAb resulted in the highest survival rate of mice (100%) compared to either CCR8-1 mAb (16.66% survival rate) or 4-1BB-1 mAb (66.66% survival rate) alone.

[0270] The data strongly demonstrated that the combination of CCR8 mAb and 4-1BB mAb synergistically enhanced the anti-tumor activity in the mouse CT26 tumor model. Compared with CCR8 mAb or 4-1BB mAb alone, the combination of the two antibodies induced a stronger anti-tumor effect, resulting in a greater number of tumor-free mice and a 100% overall survival rate.

[0271] As observed in the clinical development of Urelumab, the main problem of 4-1BB antibody treatment is dose-limiting hepatotoxicity. The most common adverse events were increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. After combined treatment with CCR8-1mAb and 4-1BB-1mAb, blood samples were collected from CT26 cell-implanted mice to measure ALT and AST. No significant increase in ALT and AST was observed, suggesting that the risk of hepatotoxicity caused by combined treatment with CCR8 mAb (1 mg / kg) and 4-1BB mAb (1 mg / kg) is generally small.

[0272] Example 4: CCR8 mAb and 4-1BB mAb synergistically reduce tumor growth in the MC38 colon cancer model

[0273] BalB / c mice were used to verify the anti-tumor effect of the combination of anti-CCR8 mAb and anti-4-1BB mAb on the MC38 colon cancer model. Female BalbC mice aged 6-9 years from the Weitong Lihua Laboratory Research Model and Service Center (Guangdong, China) were used in the experiment. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC). On day 0, MC38 colon cancer cells (5×10 5 The cells were subcutaneously injected into the right posterior abdomen of mice in 0.1 ml PBS for tumor growth. When the average tumor size reached about 80-120 mm on day 7, 3At 10 days, mice were randomized and assigned to treatment groups. Mice were treated with CCR8-1 mAb, 4-1BB-1 mAb, CCR8-1 mAb, and 4-1BB-1 mAb combination at a dose of 10 mg / kg on days 7, 10, 14, 18, 22, and 26. Control mice were treated with phosphate-buffered saline (PBS). The first dose was administered intravenously, while the second and subsequent doses were administered intraperitoneally ( Figure 5 Tumors were measured three times a week in two dimensions using a caliper and expressed in mm using the formula “V = (L×W×W) / 2”. 3 V represents volume, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Graphs and statistical analyses were performed using the GraphPad Prism program. Tumor growth and Kaplan-Meyer survival were tested using two-way ANOVA with Dunnett's multiple comparison test to obtain survival data.

[0274] Comparison of the average tumor volume between the single-drug group and the CCR8-1 mAb + 4-1BB-1 mAb combined group. Figure 6 As shown in the results, single-agent CCR8-1 mAb or single-agent 4-1BB-1 mAb treatment induced partial antitumor activity in the mouse MC38 tumor model, however, the combined administration of CCR8-1 mAb and 4-1BB-1 mAb had a synergistic effect and strongly inhibited tumor growth. At the end of the study on day 34, the time required for tumor regression in the combined CCR8-1 mAb and 4-1BB-1 mAb group was shorter than that of monoclonal CCR8-1 mAb or 4-1BB-1 mAb alone, as shown in Figure 2. Figures 7A-7C shown.

[0275] The percentage survival during treatment was also calculated. Figure 8 As shown, at the end of the study on day 34, combined treatment with CCR8-1 mAb and 4-1BB-1 mAb resulted in the highest survival rate of mice (100%) compared to CCR8-1 mAb (33.33% survival rate) or 4-1BB-1 mAb (66.66% survival rate) alone.

[0276] The data strongly demonstrated that the combination of CCR8 mAb and 4-1BB mAb synergistically enhanced the anti-tumor activity in the mouse MC38 tumor model. Compared with the use of CCR8 mAb or 4-1BB mAb alone, the combination of the two antibodies produced a stronger anti-tumor effect, resulting in significant tumor inhibition and an overall survival rate of 100%.

[0277] These results suggest that human Treg / Teff cells are regulated to enhance antitumor immunity and improve survival probability, as demonstrated by the antitumor effects of combined CCR8 mAb and 4-1BB mAb therapy in mice.

[0278] As observed in the clinical development of Urelumab, the main problem of 4-1BB antibody treatment is dose-limiting hepatotoxicity. The most common adverse events were increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. After combined treatment with CCR8-1mAb and 4-1BB-1mAb, blood samples were collected from CT26 cell-implanted mice to measure ALT and AST. No significant increase in ALT and AST was observed, suggesting that the risk of hepatotoxicity caused by combined treatment with CCR8 mAb (10 mg / kg) and 4-1BB mAb (10 mg / kg) is generally small.

[0279] Example 5: CCR8 and 4-1BB mAb combination mediates killing of CHO cells co-expressing human CCR8 and 4-1BB by M0 macrophages

[0280] The antibody-dependent cellular phagocytosis (ADCP) efficacy of effector M0 macrophages, using a combination of CCR8 and 4-1BB monoclonal antibodies, against target CHO cells co-expressing hCCR8 and h4-1BB was evaluated. + Monocytes (using EasySep without CD16 depletion) TM Human monocyte enrichment kit (StemCell Technologies, Cat#: 19058) was purified from human PBMC) and differentiated into M0 macrophages after 7 days of action of 50ng / mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). CHO cells were labeled with CFSE (ThermoFisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 100,000, 33,333.3 or 20,000 CFSE+CHO cells and incubated in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin / streptomycin) in the presence of serially diluted antibodies (4-1BB-2 mAb and CCR8-2 mAb) for 24 hours at 37°C. After incubation, cells were washed twice with FACS buffer and stained with 50 μL Live dead Near-IR (Thermo Fisher, Cat#: L10119) at a dilution of 1:2000 in PBS for 10 min at room temperature and then washed twice. Flow cytometry analysis. CHO cells were sorted into CFSE by FACS + CFSE was obtained by collecting a fixed volume under all treatment conditions. + The target cell killing percentage was calculated as: (absolute number of CFSE+CHO cells in the non-treatment group - absolute number of CFSE+CHO cells in the treatment group + Livedear Near-IR-CHO cell absolute number) / non-treatment group CFSE+CHO cell absolute number × 100. Figures 9A-9C As shown, the CCR8 and 4-1BB mAb combination can effectively kill CCR8 and 4-1BB co-expressing CHO cells at all three E:T ratios tested.

[0281] Example 6: Other Exemplary CCR8 mAbs and 4-1BB mAbs

[0282] Several other 4-1BB and CCR8 mAb pairs were also tested (4-1BB-3 and CCR8-3 mAbs, 4-1BB-4 and CCR8-4 mAbs), and each pair showed synergistically higher antitumor effects, lower hepatotoxicity, and lower E:T ratios compared to CCR8 mAb and 4-1BB mAb alone.

[0283] These results suggest that, like the antitumor effects of the combined CCR8 mAb and 4-1BB mAb treatment in mice, human Treg / Teff cells are regulated to enhance tumor immunity and improve survival probability.

[0284] Table 1: Some amino acid sequences described in the present invention

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] Other embodiments

[0296] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than 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 appended claims.

Claims

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 an antigen-binding fragment thereof that specifically binds to CCR8; and b) administering a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB to a subject.

2. A method for reducing tumor growth rate, the method comprising treating tumor cells with an effective amount of: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; and b) administering a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB to a subject.

3. A method for killing tumor cells, comprising treating the tumor cells with an effective amount of: a) a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8; and b) administering a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB to a subject.

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

5. The method according to any one of claims 1 to 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 according to any one of claims 1 to 5, wherein the tumor is a solid tumor or a hematological 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, ovarian cancer, prostate cancer, lung cancer, gastric cancer, non-small cell lung cancer, spleen cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial cancer, sarcoma, a blood cell cancer such as a leukemia or lymphoma, bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, or liver cancer.

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

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

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

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

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

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

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

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

17. The method of claim 16, wherein the composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8 and the composition comprising an antibody or an 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 according to any one of claims 1-17, further comprising administering to the subject a therapeutically effective amount of an anti-OX40 antibody, an anti-PD1 antibody, an anti-PDL1 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.

19. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CCR8, an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB, and a pharmaceutically acceptable carrier.

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