Multispecific antibody containing CCR8 antigen binding domain

Abstract

Provided are a multispecific antibody and the use thereof. Specifically, provided is a multispecific antibody, which contains a first targeting domain that targets a chemokine (C-C motif) receptor 8 (CCR8) molecule highly expressed on the surface of a tumor-infiltrating regulatory T cell (Treg), and a second targeting domain that binds to VEGF and / or a third targeting domain that binds to PD-L1. Various functional domains of the CCR8-related multispecific antibody block a PD-1 / PD-L1 immunosuppressive pathway, etc. by means of killing the tumor-infiltrating Treg, so that a T cell is activated, and the generation of a tumor vessel is inhibited. Therefore, the CCR8-related multispecific antibody can be used as an effective therapeutic agent for tumor treatment.

Description

Multispecific antibodies comprising a CCR8 antigen-binding domain Technical Field

[0001] The present invention relates to the field of cancer treatment, and more particularly, to a multispecific antibody comprising an anti-CCR8 antibody or an immunoreactive fragment thereof for treating cancer. Background Art

[0002] Cancer is generally defined as a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Conventional cancer treatments aim to remove cancerous tissue and prevent its spread. These treatment options include surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapies, and palliative care. Treatment is typically tailored to the type, location, and grade of the cancer, as well as the patient's health and preferences. However, these therapies have limitations, as they may be ineffective, particularly when the cancer has metastasized. Furthermore, chemotherapy and radiation therapy have a range of side effects related to cytotoxicity.

[0003] Current promising areas of cancer treatment include antibody-mediated targeted therapy and therapies that harness the immune system to attack and kill tumor cells.

[0004] Chemokine (CC motif) receptor 8 (CCR8) belongs to the G protein-coupled receptor (GPCR) family and is a G protein-coupled 7-transmembrane protein. High expression of CCR8 is negatively correlated with the survival rate of various tumors, including breast cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, etc. In cancer patients, compared with normal tissues and peripheral blood, CCR8 is highly expressed on regulatory T cells (Treg) residing in the tumor site, and tumor-infiltrating Treg is one of the main immunosuppressive cell populations in the tumor microenvironment. Anti-CCR8 antibodies kill tumor-infiltrating Treg through antibody-mediated cytotoxicity (ADCC), which can effectively relieve their inhibition on T cells, thereby restoring the ability of T cells to kill tumor cells. At present, no CCR8 monoclonal antibody or multispecific antibody has been approved for marketing, but several have entered the clinical research stage.

[0005] IgG1 subtype antibodies have a strong ADCC effect, which is generated by the binding of Fc to Fc-γ receptors, with the binding force influenced by N-glycans in the CH2 domain. Studies have shown that reducing or removing fucose from the Fc core sugar structure can enhance ADCC. Fucose is catalyzed by fucosyltransferase (Fut8). Therefore, by knocking out the Fut8 gene in expressing cells such as CHO cells, ADCC-enhancing therapeutic antibodies can be expressed, thereby enhancing their ability to kill Tregs and relieve or alleviate T cell suppression.

[0006] Vascular endothelial growth factor (VEGF) is a member of the platelet-derived growth factor (PDGF) family. VEGF is a key mediator of angiogenesis in tumors, mediating the continuous formation of new vascular systems in and around tumors. Abnormalities in the structure and function of tumor blood vessels formed under the action of VEGF can lead to poor tumor bleeding and hypoxia, thereby further producing more VEGF. Therefore, the key role of VEGF in tumor angiogenesis makes it a well-known anti-tumor target. Bevacizumab (trade name Avastin) is a monoclonal antibody developed by Genentech, a subsidiary of Roche, that specifically blocks VEGF to inhibit the formation of tumor blood vessels. To date, the approved indications for bevacizumab include colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, etc.

[0007] PD-1 / PD-L1 are important targets in immuno-oncology (IO) therapy. PD-L1 is highly expressed in most tumors, and the binding of PD-L1 to PD-1 on the surface of T cells transmits inhibitory signals to T cells. Therefore, blocking PD-1 / PD-L1 can effectively activate T cells to kill tumor cells. Since the launch of the first PD-1 antibody, nivolumab, in 2014, the development of PD-L1 antibodies has followed closely. Atezolizumab, developed by Roche, was approved for marketing in 2016, and avelumab, developed jointly by Pfizer and Merck, was approved for marketing in 2017.

[0008] Due to the complexity of the tumor microenvironment, current monoclonal antibody therapies have become increasingly difficult to meet the growing clinical needs. In order to achieve better therapeutic effects, the field urgently needs to develop multispecific antibodies that simultaneously target multiple tumor therapeutic targets. Summary of the Invention

[0009] The present invention constructs bi- / tri-specific antibodies to inhibit or kill tumor cells from different directions at the same time, thereby achieving better therapeutic effects.

[0010] The present invention provides bi- / tri-specific antibodies comprising a CCR8 antigen-binding domain. These bi- / tri-specific antibodies can bind to CCR8 and simultaneously block the VEGF and / or PDL1 pathways. Also provided are methods for treating diseases, such as cancer, using the antibodies and antibody conjugates of the present invention, as well as pharmaceutical compositions and products thereof.

[0011] In a first aspect, the present invention provides an anti-CCR8 antibody or an antigen-binding fragment thereof, wherein the antibody comprises the following three heavy chain variable region CDRs:

[0012] HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, 4, 7, 10, 15, 18, 20, 24, 28 or 30;

[0013] HCDR2 having the amino acid sequence shown in SEQ ID NO: 2, 5, 8, 11, 13, 16, 21, 23, 25 or 31; and

[0014] HCDR3 having the amino acid sequence shown in SEQ ID NO: 3, 6, 9, 12, 14, 17, 19, 22, 26, 27, 29 or 32;

[0015] and, the following three light chain variable region CDRs:

[0016] LCDR1 having the amino acid sequence shown in SEQ ID NO: 33, 36, 39, 50 or 55;

[0017] LCDR2 having the amino acid sequence shown in SEQ ID NO: 34, 37, 40, 44, 48, 51, 53, 56 or 58; and

[0018] LCDR3 having the amino acid sequence shown in SEQ ID NO: 35, 38, 42, 45, 49, 52, 54 or 57.

[0019] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises three heavy chain variable region CDRs (HCDRs) and three light chain variable region CDRs (LCDRs) selected from the following group:

[0020] In another preferred example, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 59-72, and / or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 73-86.

[0021] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73.

[0022] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 60, and a light chain variable region as shown in SEQ ID NO: 74.

[0023] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 61, and a light chain variable region as shown in SEQ ID NO: 75.

[0024] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 62, and a light chain variable region as shown in SEQ ID NO: 76.

[0025] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 63, and a light chain variable region as shown in SEQ ID NO: 77.

[0026] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 64, and a light chain variable region as shown in SEQ ID NO: 78.

[0027] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0028] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 66, and a light chain variable region as shown in SEQ ID NO: 80.

[0029] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 67, and a light chain variable region as shown in SEQ ID NO: 81.

[0030] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 68, and a light chain variable region as shown in SEQ ID NO: 82.

[0031] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 69, and a light chain variable region as shown in SEQ ID NO: 83.

[0032] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 70, and a light chain variable region as shown in SEQ ID NO: 84.

[0033] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 71, and a light chain variable region as shown in SEQ ID NO: 85.

[0034] In another preferred embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 72, and a light chain variable region as shown in SEQ ID NO: 86.

[0035] In another preferred embodiment, the antibody or antigen-binding fragment thereof is a human, murine, humanized or chimeric antibody.

[0036] In another preferred embodiment, the antibody or antigen-binding fragment thereof is a human antibody.

[0037] The second aspect of the present invention provides a multispecific antibody, wherein the multispecific antibody comprises the anti-CCR8 antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0038] In another preferred embodiment, the multispecific antibody comprises:

[0039] a first targeting domain comprising one or more CCR8 antigen binding domains;

[0040] a second targeting domain that binds to VEGF or PD-L1;

[0041] Optionally, comprising a third targeting domain that binds to VEGF or PD-L1;

[0042] Furthermore, the second targeting domain and the third targeting domain bind to different proteins respectively.

[0043] In another preferred embodiment, the targeting domain is in the form of a single domain antibody (sdAb), a fragment variable (Fv) heterodimer, a single chain Fv (scFv), a Fab fragment, a TriFab or a combination thereof.

[0044] In another preferred embodiment, the CCR8 antigen-binding domain comprises the anti-CCR8 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0045] In another preferred embodiment, the CCR8 antigen binding domain comprises the following three heavy chain variable region CDRs:

[0046] HCDR1 having the amino acid sequence shown in SEQ ID NO: 1;

[0047] HCDR2 having the amino acid sequence shown in SEQ ID NO: 2; and

[0048] HCDR3 having the amino acid sequence shown in SEQ ID NO: 3;

[0049] and, the following three light chain variable region CDRs:

[0050] LCDR1, which has the amino acid sequence shown in SEQ ID NO: 33;

[0051] LCDR2 having the amino acid sequence shown in SEQ ID NO: 34; and

[0052] LCDR3 having the amino acid sequence shown in SEQ ID NO:35.

[0053] In another preferred embodiment, the CCR8 antigen binding domain comprises the following three heavy chain variable region CDRs:

[0054] HCDR1 having the amino acid sequence shown in SEQ ID NO: 18;

[0055] HCDR2 having the amino acid sequence shown in SEQ ID NO: 5; and

[0056] HCDR3 having the amino acid sequence shown in SEQ ID NO: 19;

[0057] and, the following three light chain variable region CDRs:

[0058] LCDR1, which has the amino acid sequence shown in SEQ ID NO:46;

[0059] LCDR2 having the amino acid sequence shown in SEQ ID NO: 34; and

[0060] LCDR3 having the amino acid sequence shown in SEQ ID NO:38.

[0061] In another preferred example, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 59, and / or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 73.

[0062] In another preferred example, the CCR8 antigen binding domain comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73.

[0063] In another preferred example, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 65, and / or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 79.

[0064] In another preferred example, the CCR8 antigen binding domain comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0065] In another preferred embodiment, the CCR8 antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0066] In another preferred embodiment, the CCR8 antigen binding domain is scFv.

[0067] In another preferred embodiment, the CCR8 antigen-binding domain is a Fab comprising a heavy chain variable region as shown in SEQ ID NO: 59 and a light chain variable region as shown in SEQ ID NO: 73, or a heavy chain variable region as shown in SEQ ID NO: 65 and a light chain variable region as shown in SEQ ID NO: 76; and

[0068] The heavy chain constant region CH1 shown in SEQ ID NO: 111 or 119, and the light chain constant region CL shown in SEQ ID NO: 121 or 122; or the heavy chain constant region CH1 shown in SEQ ID NO: 112, and the light chain constant region CL shown in SEQ ID NO: 123.

[0069] In another preferred embodiment, the multispecific antibody further comprises an Fc fragment.

[0070] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0071] In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG1, which has an amino acid sequence as shown in SEQ ID NO: 113 or 114.

[0072] In another preferred embodiment, the Fc fragment comprises a mutation for forming a knob-in-hole structure and / or a mutation for enhancing ADCC.

[0073] In another preferred embodiment, the Fc fragment derived from IgG1 has a mutation selected from the following group:

[0074] Y349C / K370E / K409D / K439E,

[0075] S354C / D356K / E357K / D399K;

[0076] S354C / T366W,

[0077] Y349C / T366S / L368A / Y407V.

[0078] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 115-118.

[0079] In another preferred embodiment, the IgG4 Fc fragment has a mutation selected from the following group:

[0080] Y349C / K370E / R409D / K439E,

[0081] S354C / E356K / E357K / D399K; or

[0082] S354C / T366W,

[0083] Y349C / T366S / L368A / Y407V.

[0084] In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG4, which has the amino acid sequence shown in SEQ ID NO: 120.

[0085] In another preferred embodiment, the multispecific antibody is a bi / trispecific antibody.

[0086] In another preferred embodiment, the multispecific antibody is a bispecific antibody.

[0087] In another preferred embodiment, the bispecific antibody comprises:

[0088] a first targeting domain comprising one or more CCR8 antigen binding domains; and a second targeting domain comprising a VEGF antigen binding domain.

[0089] In another preferred embodiment, the bispecific antibody comprises:

[0090] and a first targeting domain comprising one or more CCR8 antigen binding domains; and a second targeting domain comprising a PD-L1 antigen binding domain.

[0091] In another preferred embodiment, the multispecific antibody is a trispecific antibody.

[0092] In another preferred embodiment, the trispecific antibody comprises:

[0093] a first targeting domain comprising one or more CCR8 antigen binding domains;

[0094] a second targeting domain, wherein the second targeting domain is a VEGF antigen binding domain;

[0095] The third targeting domain is a PD-L1 antigen binding domain.

[0096] In another preferred example, the VEGF antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 87 or 88, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 93 or 94.

[0097] In another preferred example, the VEGF antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 89, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 95.

[0098] In another preferred embodiment, the VEGF antigen binding domain comprises a mutation that can reduce the hydrophobicity of the antibody.

[0099] In another preferred example, the mutation capable of reducing the hydrophobicity of the antibody occurs in the non-CDR3 region of the anti-VEGR antigen binding domain having a heavy chain variable region as shown in SEQ ID NO: 89 and a light chain variable region as shown in SEQ ID NO: 95.

[0100] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position selected from the following group: position 28, 30, 31, 32, 33, 35, or a combination thereof in the heavy chain variable region as shown in SEQ ID NO: 89.

[0101] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position selected from the following group: position 24, 49, 50, 51, 52, 53, 56, or a combination thereof in the light chain variable region as shown in SEQ ID NO: 95.

[0102] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in the region of positions 46-57 of the light chain variable region as shown in SEQ ID NO:95.

[0103] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody is to mutate the above amino acid sites into hydrophilic amino acids, such as aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).

[0104] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 30 in the heavy chain variable region as shown in SEQ ID NO: 89. Preferably, serine (S) at position 30 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).

[0105] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 50 and / or serine (S) at position 52 in the light chain variable region as shown in SEQ ID NO: 95; preferably, serine (S) at position 50 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R), and / or serine (S) at position 52 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).

[0106] In another preferred embodiment, the VEGF antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 90-92 and 149-150.

[0107] In another preferred embodiment, the VEGF antigen binding domain comprises a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 96-102.

[0108] In another preferred embodiment, the VEGF antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0109] In another preferred example, the PD-L1 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 103 or 104, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 107 or 108.

[0110] In another preferred example, the PD-L1 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 105 or 106, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 109 or 110.

[0111] In another preferred embodiment, the PD-L1 antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0112] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula I (e.g., a in FIG. 7A ):

[0113] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0114] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0115] Fab2 is a second targeting domain, and the Fab2 is an anti-VEGF Fab or an anti-PD-L1 Fab;

[0116] Fc1 and Fc2 are each independently an Fc fragment.

[0117] In another preferred embodiment, the Fab2 is an anti-VEGF Fab.

[0118] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0119] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0120] In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0121] The anti-VEGF Fab comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0122] In another preferred embodiment, the anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0123] The anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0124] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0125] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0126] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0127] In another preferred example, the Fc1 has the amino acid sequence shown in SEQ ID NO: 115, and the Fc2 has the amino acid sequence shown in SEQ ID NO: 117.

[0128] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1" in the "Fab1-Fc1" is shown in SEQ ID NO: 125, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0129] In another preferred example, the amino acid sequence of "HC2 (heavy chain)-Fc2" in the "Fab2-Fc2" is shown in SEQ ID NO: 126, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO: 127.

[0130] In another preferred embodiment, the multispecific antibody has a structure as shown in the following Formula II (e.g., b in FIG. 7A ):

[0131] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0132] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0133] scFv2 is a second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0134] Fc1 is the Fc fragment.

[0135] In another preferred embodiment, the “║” is a disulfide bond.

[0136] In another preferred embodiment, the scFv2 is an anti-VEGF scFv.

[0137] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0138] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0139] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0140] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0141] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0142] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0143] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0144] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0145] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0146] In another preferred embodiment, the Fc1 has the amino acid sequence shown in SEQ ID NO: 113.

[0147] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv2" in the "Fab1-Fc1-scFv2" is shown in SEQ ID NO: 128, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0148] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula III (eg, c in FIG. 7A ):

[0149] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0150] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0151] scFv2 is a second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0152] Fc1 and Fc2 are each independently an Fc fragment.

[0153] In another preferred embodiment, the scFv2 is an anti-VEGF scFv.

[0154] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0155] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0156] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0157] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0158] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0159] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0160] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0161] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0162] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0163] In another preferred example, the Fc1 has the amino acid sequence shown in SEQ ID NO: 115, and the Fc2 has the amino acid sequence shown in SEQ ID NO: 118.

[0164] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv2" in the "Fab1-Fc1-scFv2" is shown in SEQ ID NO: 128, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0165] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is shown in SEQ ID NO: 125, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0166] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula IV (eg, d in FIG. 7A ):

[0167] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0168] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0169] scFv2 is a second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0170] Fc1 and Fc2 are each independently an Fc fragment.

[0171] In another preferred embodiment, the scFv2 is an anti-VEGF scFv.

[0172] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0173] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0174] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0175] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0176] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0177] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0178] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0179] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0180] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0181] In another preferred example, the Fc1 has the amino acid sequence shown in SEQ ID NO: 115, and the Fc2 has the amino acid sequence shown in SEQ ID NO: 118.

[0182] In another preferred embodiment, the amino acid sequence of the "scFv2-Fc1" is shown in SEQ ID NO:129.

[0183] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is shown in SEQ ID NO: 125, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0184] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula V (e in FIG. 7A ):

[0185] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0186] scFv1 and Fab1 are the first targeting domains, the scFv1 is an anti-CCR8 scFv, and the Fab1 is an anti-CCR8 Fab;

[0187] scFv2 is a second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0188] Fc1 and Fc2 are each independently an Fc fragment.

[0189] In another preferred embodiment, the scFv2 is an anti-VEGF scFv.

[0190] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0191] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0192] In another preferred embodiment, the anti-CCR8 scFv comprises the heavy chain variable region shown in SEQ ID NO: 59, and the light chain variable region shown in SEQ ID NO: 73; or

[0193] The anti-CCR8 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0194] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0195] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0196] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0197] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0198] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0199] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0200] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0201] In another preferred example, the Fc1 has the amino acid sequence shown in SEQ ID NO: 115, and the Fc2 has the amino acid sequence shown in SEQ ID NO: 118.

[0202] In another preferred example, the amino acid sequence of the "scFv1-scFv2-Fc1" is shown in SEQ ID NO:130.

[0203] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is shown in SEQ ID NO: 125, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0204] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula VI (e.g., f in FIG. 7A ):

[0205] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0206] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0207] Fab2 is a second targeting domain, and the Fab2 is an anti-VEGF Fab or an anti-PD-L1 Fab;

[0208] Fc1 and Fc2 are each independently an Fc fragment.

[0209] In another preferred embodiment, the Fab2 is an anti-VEGF Fab.

[0210] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0211] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0212] In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0213] The anti-VEGF Fab comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0214] In another preferred embodiment, the anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0215] The anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0216] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0217] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0218] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0219] In another preferred example, the Fc1 has the amino acid sequence shown in SEQ ID NO: 116, and the Fc2 has the amino acid sequence shown in SEQ ID NO: 117.

[0220] In another preferred example, the amino acid sequence of "HC2 (heavy chain)-HC1 (heavy chain)-Fc1" in the "Fab2-Fab1-Fc1" is shown as SEQ ID NO: 132, the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO: 131, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO: 134.

[0221] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is as shown in SEQ ID NO: 133, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 131.

[0222] In another preferred embodiment, the multispecific antibody has a structure as shown in the following Formula VII (e.g., g in FIG7A ):

[0223] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0224] Fab1 and scFv1 are the first targeting domains, the Fab1 is an anti-CCR8 Fab, and the scFv1 is an anti-CCR8 scFv;

[0225] Fab2 is a second targeting domain, and the Fab2 is an anti-VEGF Fab or an anti-PD-L1 Fab;

[0226] Fc1 and Fc2 are each independently an Fc fragment.

[0227] In another preferred embodiment, the Fab2 is an anti-VEGF Fab.

[0228] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0229] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0230] In another preferred embodiment, the anti-CCR8 scFv comprises the heavy chain variable region shown in SEQ ID NO: 59, and the light chain variable region shown in SEQ ID NO: 73; or

[0231] The anti-CCR8 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0232] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0233] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0234] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0235] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0236] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0237] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0238] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0239] In another preferred example, the Fc1 has the amino acid sequence shown in SEQ ID NO: 116, and the Fc2 has the amino acid sequence shown in SEQ ID NO: 118.

[0240] In another preferred example, the amino acid sequence of "HC2 (heavy chain)-HC1 (heavy chain)-Fc1" in the "Fab2-Fab1-Fc1" is shown as SEQ ID NO: 132, the amino acid sequence of "LC1 (light chain)" is shown as SEQ ID NO: 131, and the amino acid sequence of "LC2 (light chain)" is shown as SEQ ID NO: 134.

[0241] In another preferred embodiment, the amino acid sequence of the "scFv1-Fc2" is shown in SEQ ID NO:135.

[0242] In another preferred embodiment, the multispecific antibody has a structure as shown in the following Formula VIII (e.g., h in FIG. 7A ):

[0243] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0244] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0245] scFv2 is the third targeting domain, and the scFv2 is an anti-PD-L1 scFv or an anti-VEGF scFv;

[0246] Fc1 is the Fc fragment.

[0247] In another preferred embodiment, the “║” is a disulfide bond.

[0248] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0249] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0250] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0251] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0252] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0253] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0254] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0255] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0256] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0257] In another preferred embodiment, the Fc1 has the amino acid sequence shown in SEQ ID NO: 113.

[0258] In another preferred example, the amino acid sequence of "scFv2-HC1 (heavy chain)-Fc1" in the "scFv2-Fab1-Fc1" is shown in SEQ ID NO: 136, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0259] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula IX (e.g., i in FIG. 7A ):

[0260] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0261] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0262] Fab2 is a second targeting domain, and the Fab2 is an anti-PDL1 Fab or an anti-VEGF Fab;

[0263] Fc1 is the Fc fragment.

[0264] In another preferred embodiment, the “║” is a disulfide bond.

[0265] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0266] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0267] In another preferred embodiment, the anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0268] The anti-PD-L1 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0269] In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0270] The anti-VEGF Fab comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0271] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0272] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0273] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0274] In another preferred embodiment, the Fc1 has the amino acid sequence shown in SEQ ID NO: 114.

[0275] In another preferred example, the amino acid sequence of "HC2 (heavy chain)-HC1 (heavy chain)-Fc1" in the "Fab2-Fab1-Fc1" is shown in SEQ ID NO: 138, the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 137, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO: 139.

[0276] In another preferred example, the amino acid sequence of "HC2 (heavy chain)-HC1 (heavy chain)-Fc1" in the "Fab2-Fab1-Fc1" is shown in SEQ ID NO: 140, the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 137, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO: 134.

[0277] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula X (eg, a in FIG8A ):

[0278] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0279] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0280] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv;

[0281] scFv3 is the third targeting domain, and the scFv3 is an anti-PD-L1 scFv;

[0282] Fc1 and Fc2 are each independently an Fc fragment.

[0283] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0284] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0285] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0286] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0287] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0288] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0289] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0290] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0291] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0292] In another preferred example, the Fc1 fragment has the amino acid sequence shown in SEQ ID NO: 115, and the Fc2 fragment has the amino acid sequence shown in SEQ ID NO: 118.

[0293] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv2" in the "Fab1-Fc1-scFv2" is shown in SEQ ID NO: 141, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0294] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc2-scFv3" in the "Fab1-Fc2-scFv3" is shown in SEQ ID NO: 142, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0295] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula XI (b in FIG8A ):

[0296] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0297] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0298] Fab2 is the second targeting domain, and the Fab1 is an anti-VEGF Fab;

[0299] scFv3 is the third targeting domain, and the scFv3 is an anti-PD-L1 scFv;

[0300] Fc1 and Fc2 are each independently an Fc fragment.

[0301] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0302] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0303] In another preferred embodiment, the anti-VEGF Fab comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0304] The anti-VEGF Fab comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0305] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0306] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0307] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0308] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0309] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0310] In another preferred example, the Fc1 fragment has the amino acid sequence shown in SEQ ID NO: 116, and the Fc2 fragment has the amino acid sequence shown in SEQ ID NO: 118.

[0311] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv3" in the "Fab1-Fc1-scFv3" is shown in SEQ ID NO: 144, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 143.

[0312] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc2-scFv3" in the "Fab2-Fc2-scFv3" is shown in SEQ ID NO: 145, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO: 134.

[0313] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula XII (eg, c in FIG. 8A ):

[0314] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0315] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0316] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv;

[0317] scFv3 is the third targeting domain, and the scFv3 is an anti-PD-L1 scFv;

[0318] Fc1 and Fc2 are each independently an Fc fragment.

[0319] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0320] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0321] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0322] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0323] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0324] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0325] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0326] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0327] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0328] In another preferred example, the Fc1 fragment has the amino acid sequence shown in SEQ ID NO: 115, and the Fc2 fragment has the amino acid sequence shown in SEQ ID NO: 118.

[0329] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv3" in the "Fab1-Fc1-scFv3" is shown in SEQ ID NO: 142, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0330] In another preferred example, the amino acid sequence of the "scFv2-Fc2-scFv3" is shown in SEQ ID NO:146.

[0331] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula XIII (eg, d in FIG8A ):

[0332] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0333] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0334] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv;

[0335] scFv3 is the second targeting domain, and the scFv3 is an anti-PD-L1 scFv;

[0336] Fc1 is the Fc fragment.

[0337] In another preferred embodiment, the “║” is a disulfide bond.

[0338] In another preferred embodiment, the anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 59, and a light chain variable region as shown in SEQ ID NO: 73; or

[0339] The anti-CCR8 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 65, and a light chain variable region as shown in SEQ ID NO: 79.

[0340] In another preferred embodiment, the anti-VEGF scFv comprises a heavy chain variable region as shown in SEQ ID NO: 87 or 88, and a light chain variable region as shown in SEQ ID NO: 93 or 94; or

[0341] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92 and 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0342] In another preferred embodiment, the anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 103 or 104, and a light chain variable region as shown in SEQ ID NO: 107 or 108; or

[0343] The anti-PD-L1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 105 or 106, and a light chain variable region as shown in SEQ ID NO: 109 or 110.

[0344] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0345] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0346] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0347] In another preferred embodiment, the Fc1 fragment has the amino acid sequence shown in SEQ ID NO: 113.

[0348] In another preferred example, the amino acid sequence of "scFv2-HC1 (heavy chain)-Fc1-scFv3" in the "scFv2-Fab1-Fc1-scFv3" is shown in SEQ ID NO: 147, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 124.

[0349] The third aspect of the present invention provides a polynucleotide encoding the anti-CCR8 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the multispecific antibody according to the second aspect of the present invention.

[0350] The fourth aspect of the present invention provides an expression vector, which comprises the polynucleotide as described in the third aspect of the present invention.

[0351] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0352] The fifth aspect of the present invention provides a host cell, which comprises the expression vector as described in the fourth aspect of the present invention, or the polynucleotide as described in the third aspect of the present invention is integrated into its genome.

[0353] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0354] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, HEK 293T cells, and CHO cells.

[0355] In a sixth aspect, the present invention provides a use of the anti-CCR8 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the multispecific antibody according to the second aspect of the present invention, for preparing a medicament for treating cancer / tumor.

[0356] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of CCR8.

[0357] In another preferred embodiment, the cancer / tumor includes solid tumors and blood tumors.

[0358] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0359] In another preferred embodiment, the cancer / tumor is selected from the group consisting of colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or a combination thereof.

[0360] The seventh aspect of the present invention provides an immunoconjugate, wherein the conjugate comprises:

[0361] (i) the anti-CCR8 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the multispecific antibody according to the second aspect of the present invention; and

[0362] (ii) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0363] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0364] In an eighth aspect, the present invention provides a pharmaceutical composition comprising: (a) the anti-CCR8 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the multispecific antibody as described in the second aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0365] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0366] The ninth aspect of the present invention provides a method for treating cancer / tumor, comprising administering the multispecific antibody according to the first aspect of the present invention to a subject in need thereof.

[0367] In another preferred embodiment, the subject in need thereof is a human or non-human mammal.

[0368] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of CCR8.

[0369] In another preferred embodiment, the cancer / tumor includes solid tumors and blood tumors.

[0370] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0371] In another preferred embodiment, the cancer / tumor is selected from the group consisting of colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or a combination thereof.

[0372] The tenth aspect of the present invention provides the use of the anti-CCR8 antibody or antigen-binding fragment thereof as described in the first aspect, or the immunoconjugate as described in the seventh aspect of the present invention, for preparing a detection reagent or kit for detecting CCR8 molecules in a sample.

[0373] In another preferred embodiment, the sample includes an in vitro sample, such as an in vitro tissue or cell sample.

[0374] In another preferred embodiment, the detection reagent or kit is used as a diagnostic reagent for diagnosing cancers / tumors with high CCR8 expression.

[0375] In the eleventh aspect of the present invention, an anti-VEGF antibody mutant is provided, which comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 89, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, and comprises a mutation that can reduce the hydrophobicity of the antibody.

[0376] In another preferred example, the mutation capable of reducing the hydrophobicity of the antibody occurs in the non-CDR3 region of the anti-VEGR antigen binding domain having a heavy chain variable region as shown in SEQ ID NO: 89 and a light chain variable region as shown in SEQ ID NO: 95.

[0377] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position selected from the following group: position 28, 30, 31, 32, 33, 35, or a combination thereof in the heavy chain variable region as shown in SEQ ID NO: 89.

[0378] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid position selected from the following group: position 24, 49, 50, 51, 52, 53, 56, or a combination thereof in the light chain variable region as shown in SEQ ID NO: 95.

[0379] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in the region of positions 46-57 of the light chain variable region as shown in SEQ ID NO:95.

[0380] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody is to mutate one or more (e.g., two, three, four) of the above-mentioned amino acid sites into hydrophilic amino acids, such as aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).

[0381] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 30 in the heavy chain variable region as shown in SEQ ID NO: 89. Preferably, serine (S) at position 30 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).

[0382] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at serine (S) at position 50 and / or serine (S) at position 52 in the light chain variable region as shown in SEQ ID NO: 95; preferably, serine (S) at position 50 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R), and / or serine (S) at position 52 is mutated to aspartic acid (D), glutamic acid (E), lysine (K) or arginine (R).

[0383] In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 90-92 and 149-150.

[0384] In another preferred embodiment, the anti-VEGF antibody mutant comprises a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 96-102.

[0385] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 91, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 95.

[0386] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 89, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 97.

[0387] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 89, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 99.

[0388] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 89, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 101.

[0389] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 91, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 97.

[0390] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 91, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 99.

[0391] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 91, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 101.

[0392] In another preferred embodiment, the anti-VEGF antibody mutant has significantly reduced hydrophobicity and significantly weakened aggregation tendency of antibody molecules compared to the original antibody (i.e., the anti-VEGF antibody with the heavy chain variable region amino acid sequence of SEQ ID NO: 89 and the light chain variable region sequence of SEQ ID NO: 95).

[0393] In another preferred embodiment, the "significantly reduced hydrophobicity of the antibody" refers to the hydrophobicity F1 of the anti-VEGF antibody mutant, compared with the hydrophobicity F0 of the original antibody, F1 / F0 < 1, preferably, F1 / F0 ≤ 0.7, more preferably, F1 / F0 ≤ 0.5.

[0394] In another preferred embodiment, the anti-VEGF antibody mutant is used to construct a multispecific antibody targeting VEGF, for example, the multispecific antibody described in the second aspect of the present invention.

[0395] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0396] Figure 1 shows FACS binding of CCR8 hybridoma monoclonal antibodies to the human CCR8 HEK293 cell line.

[0397] FIG2 shows the ADCC effect of the CCR8 recombinant monoclonal antibody.

[0398] Figure 3 shows the blocking effect of CCR8 recombinant monoclonal antibody on the binding of human CCR8 to human CCL1

[0399] FIG4 shows a schematic diagram of the surface hydrophobicity of the Fab structure of AS-1.

[0400] FIG5 shows the aggregation propensity scores of all amino acids of the scFv of AS-1.

[0401] FIG6 shows the sequential peptide segments with aggregation tendency and high free energy in the Fab sequence of AS-1.

[0402] FIG7A shows nine bispecific antibody structures (ai) consisting of a CCR8 antigen-binding domain and a VEGF or PD-L1 antigen-binding domain;

[0403] Among them, a shows the antibody structure of the combination of anti-VEGF Fab (CK-VH+CH1-VL) and anti-CCR8 Fab (CH1-VH+CK-VL), in which IgG1 Fc forms a heterodimer through charge pair or knob and hole mutation; b shows the antibody structure formed by anti-CCR8 Fab and anti-VEGF scFv (Fab-Fc-scFv×Fab-Fc-scFv); c shows the antibody structure formed by anti-CCR8 Fab and anti-VEGF scFv (Fab-Fc-scFv×Fab-Fc); d shows the molecular structure formed by anti-VEGF scFv and anti-CCR8 Fab (scFv-Fc×Fab-Fc); e shows the molecular structure formed by anti-CCR8 scFv, VEGF scFv and anti-CCR8 Fab (scFv-scFv-Fc×Fab-Fc); f shows the anti-VEGF Fab (CH1-VH + CK-VL), the antibody structure of the combination of anti-CCR8 Fab (CK-VH + CH1-VL) and anti-CCR8 Fab (CK-VH + CH1-VL) (Fab-Fab-Fc × Fab-Fc); g shows the antibody structure of the combination of anti-VEGF Fab (CH1-VH + CK-VL), anti-CCR8 Fab (CK-VH + CH1-VL) and anti-CCR8 scFv (Fab-Fab-Fc × scFv-Fc); h shows the antibody structure of the combination of anti-PD-L1 scFv, anti-CCR8 Fab and anti-PD-L1 scFv, anti-CCR8 Fab (scFv-Fab-Fc × scFv-Fab-Fc); i shows the antibody structure of anti-VEGF or PDL1 Fab (CH1-VH + CK-VL), anti-CCR8 Fab (CK-VH + CH1-VL) and anti-VEGF or PDL1 Antibody structure (Fab-Fab-Fc×Fab-Fab-Fc) composed of Fab (CH1-VH+CK-VL) and anti-CCR8 Fab (CK-VH+CH1-VL); wherein IgG1 Fc forms heterodimers through charge pair or knob and hole mutations; wherein IgG1 Fc forms heterodimers through charge pair or knob and hole mutations, wherein IgG1Fc forms heterodimers through charge pair or knob and hole mutations.

[0404] FIG7B shows an exemplary bispecific antibody structure of FIG7Aa, designated 8As-1.

[0405] FIG7C shows an exemplary bispecific antibody structure of FIG7A b, designated 8As-2.

[0406] [Corrected 06.02.2025 according to Rule 91] Figure 7D shows an exemplary bispecific antibody structure of Figure 7A c, designated 8As-3.

[0407] FIG7E shows an exemplary bispecific antibody structure of FIG7A d, designated 8As-4.

[0408] FIG7F shows an exemplary bispecific antibody structure of FIG7Ae, designated 8As-5.

[0409] FIG7G shows an exemplary bispecific antibody structure of FIG7Af, designated 8As-6.

[0410] FIG7H shows an exemplary bispecific antibody structure of FIG7Ag, designated 8As-7.

[0411] Figure 7I shows an exemplary bispecific antibody structure of Figure 7Ah, designated P18-8.

[0412] Figure 7J shows an exemplary bispecific antibody structure of Figure 7Ai, designated P18-9.

[0413] FIG7K shows an exemplary bispecific antibody structure of FIG7Ai, designated 8As-9.

[0414] Figure 8A shows three triple antibody structures consisting of a CCR8 antigen-binding domain, a VEGF antigen-binding domain, and a PD-L1 antigen-binding domain;

[0415] Among them, a shows the molecular structure formed by anti-CCR8 antibody, VEGF antibody and PDL1 antibody (IgG-scFv×IgG-scFv); b shows the molecular structure formed by anti-CCR8 antibody, VEGF antibody and PDL1 antibody (Fab-Fc-scFv×Fab-Fc-scFv); c shows the molecular structure formed by anti-CCR8 antibody, VEGF antibody and PDL1 antibody (IgG-scFv×scFv-Fc-scFv); d shows the molecular structure formed by anti-CCR8 antibody, VEGF antibody and PDL1 antibody (scFv-IgG-scFv×scFv-IgG-scFv).

[0416] FIG8B shows an exemplary trispecific antibody structure of FIG8Aa, designated 8AsP1-1.

[0417] FIG8C shows an exemplary trispecific antibody structure of FIG8A b, designated 8AsP1-2.

[0418] FIG8D shows an exemplary trispecific antibody structure of FIG8Ac, designated 8AsP1-3.

[0419] Figure 8E shows an exemplary trispecific antibody structure of Figure 8Ad, designated 8AsP1-4.

[0420] FIG9 shows the ADCC effect of the monoclonal antibody and the multispecific antibody of the present invention.

[0421] FIG10 shows the VEGF blocking effect of the multispecific antibodies of the present invention.

[0422] FIG11 shows the PD-L1 blocking effect of the multispecific antibodies of the present invention.

[0423] FIG12 shows the in vivo pharmacodynamics of the CCR8 monoclonal antibody of the present invention in a mouse tumor model.

[0424] FIG13 shows the in vivo pharmacodynamics of the CCR8 dual antibody of the present invention in a mouse tumor model. DETAILED DESCRIPTION

[0425] After extensive and in-depth research, the inventors unexpectedly developed a class of multispecific antibodies comprising CCR8 antigen-binding domains for the first time. These multispecific antibodies contain a first targeting domain that targets the chemokine (CC motif) receptor 8 (CCR8) molecule, which is highly expressed on the surface of tumor-infiltrating regulatory T cells. The first targeting domain is a CCR8 antibody or antigen-binding fragment thereof, and further contain a second targeting domain and / or a third targeting domain that binds to VEGF and / or PD-L1. The multispecific antibodies of the present invention can simultaneously bind to Treg cells, tumor cells, and free VEGF molecules, and can be used as effective therapeutic agents for tumor treatment.

[0426] On this basis, the present invention was completed.

[0427] As used herein, the term "chemokine (CC motif) receptor 8" or "CCR8" refers to a protein encoded by the CCR8 gene in humans. CCR8 is highly expressed on many tumor-infiltrating Treg cells, but exhibits low or no expression on Treg cells in the thymus, spleen, and peripheral blood.

[0428] VEGF (vascular endothelial growth factor) is a member of the platelet-derived growth factor (PDGF) family. VEGF is a key mediator of angiogenesis in tumors, mediating the continuous formation of new vascular systems within and around tumors. However, structural and functional abnormalities in tumor vessels formed under VEGF's influence can lead to poor tumor bleeding and hypoxia, which in turn leads to the production of more VEGF. VEGF's crucial role in tumor angiogenesis makes it a well-known anti-tumor target.

[0429] PD-1 / PD-L1 are important targets in immuno-oncology (IO) therapy. PD-L1 is highly expressed in most tumors, and its binding to PD-1 on the surface of T cells transmits inhibitory signals to T cells. Therefore, blocking PD-1 / PD-L1 can effectively activate T cells to kill tumor cells.

[0430] The term "Fc fragment" or "Fc" refers to a portion of an antibody that does not have antigen binding activity but was initially observed to crystallize readily, and was therefore named an Fc fragment (for fragment crystallizability). This fragment corresponds to the paired CH2 and CH3 domains and is the portion of the antibody molecule that interacts with effector molecules and cells. The Fc fragments described herein can be derived from IgG1, IgG2, and IgG4 antibodies. For specific uses, specific IgG subclasses may be preferred. For example, IgG1 is more effective than IgG2 and IgG4 in mediating ADCC and CDC. Therefore, when effector function is undesirable, IgG2 Fc may be preferred. However, molecules containing IgG2 Fc are generally more difficult to prepare and may not be as stable as molecules containing IgG1 Fc. In addition, the effector function of an antibody can be increased or decreased by introducing one or more mutations into the Fc (see, e.g., Strohl, Curr. Opin. Biotech., 20: 685-691, 2009).

[0431] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0432] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0433] The antibodies of the present application may include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, bispecific, human, humanized, primatized, chimeric, and single-chain antibodies. The antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

[0434] The term "antibody fragment" or "antigen-binding fragment" is used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DARTs and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein comprising an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex. "Single-chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, the domains are connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH or the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, this protein still retains the specificity of the original immunoglobulin. Regarding IgG, the standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually connected by disulfide bonds in a "Y" configuration, wherein the light chain is connected to the heavy chain from the mouth of the "Y" and extends through the variable region.

[0435] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to the epitope on the antigen. That is, the VL domain and VH domain of the antibody or the complementary determining region (CDR) subset of the antibody combine to form a variable region that limits the three-dimensional antigen binding site. This four-membered antibody structure forms the antigen binding site present at the end of each arm of each Y configuration. More specifically, the antigen binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) on each of the VH and VL chains. In some cases, for example, some immunoglobulin molecules are derived from camelid species or are engineered based on camelid immunoglobulins. Alternatively, the immunoglobulin molecule can be composed of a heavy chain without a light chain or a light chain without a heavy chain.

[0436] In naturally occurring antibodies, the six CDRs present in each antigen-binding domain are short, non-continuous amino acid sequences that are specifically positioned to form an "antigen-binding domain" because the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domain, referred to as the "framework" region, exhibit less inter-molecular variability. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Therefore, the framework region plays the role of forming a scaffold that positions the CDRs in the correct direction through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. Since it has been precisely defined, a person of ordinary skill in the art can easily identify the amino acids comprising the CDRs and framework regions, respectively, for any given heavy or light chain variable region.

[0437] As used herein, the term "light chain constant region (CL)" includes the amino acid sequence CL (SEQ ID NO: 121 or 122) derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0438] As used herein, the term "heavy chain constant region (CH)" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain (SEQ ID NO: 111 or 119), a hinge region (e.g., an upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. It should be understood that the heavy chain constant region can be modified so that its amino acid sequence differs from that of a naturally occurring immunoglobulin molecule.

[0439] In one embodiment of the present invention, the prepared multispecific antibody comprises a crossmab structure, i.e., the heavy chain CH1 and the light chain CL exchange part of the amino acid sequence to prevent mispairing. Such a structure comprises CH1 as shown in SEQ ID NO: 112 and CL as shown in SEQ ID NO: 113.

[0440] As used herein, a "variant" of an antibody, antibody fragment, or antibody domain refers to an antibody, antibody fragment, or antibody domain that: (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target to which the original antibody, antibody fragment, or antibody domain specifically binds. It will be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical," such embodiments include any and all numerical percentages equal to or above the lower limit. Furthermore, it will be understood that where an amino acid sequence is presented in this application, it should be construed as further disclosing or encompassing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that amino acid sequence.

[0441] Included within the scope of the multispecific molecules of the present invention are various compositions and methods, including: asymmetric IgG-like antibodies (e.g., triomabs / quadromas); knobs-into-holes antibodies; cross monoclonal antibodies (Cross MAbs); electrostatically matched antibodies; LUZ-Y; chain exchange engineered domain (SEED) bodies; Fab exchange antibodies, symmetric IgG-like antibodies; two-in-one antibodies; cross-linked monoclonal antibodies, mAb2; Cov X-body; dual variable domain (DVD)-Ig fusion protein; IgG-like bispecific antibody; Ts2Ab; BsAb; scFv / Fc fusion; double (scFv)2-Fabs; F(ab)2 fusion protein; dual-action or Bis-Fab; Dock-and-Lock (DNL); Fab-Fv; scFv-based antibodies and diabody-based antibodies (e.g., bispecific antibodies (BiTEs); tandem diabodies (Tandab); DARTs; single-chain diabodies; TCR-like antibodies; human serum albumin scFv fusion proteins, combodies and IgG / non-IgG fusion proteins.

[0442] As used herein, the phrase "multispecific antibody" refers to a molecule comprising at least two targeting domains with different binding specificities, wherein at least one targeting domain specifically binds to the Treg cell surface antigen CCR8. In some embodiments, a multispecific antibody is a polypeptide comprising a scaffold and two or more immunoglobulin antigen-binding domains that target different antigens or epitopes. In some embodiments, the multispecific antibody is a bispecific antibody. In other embodiments, the multispecific antibody is a trispecific antibody.

[0443] As used herein, the phrase "bispecific" refers to a molecule comprising at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, upon binding to the target molecule, inhibiting the biological function of the target molecule. In some embodiments, a bispecific antibody is a polymer molecule comprising two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or fragment thereof that specifically binds to a target protein.

[0444] The terms "bispecific antibody", "bispecific molecule" and "diabody" are used interchangeably herein and refer to antibodies that can specifically bind to two different antigens (or epitopes). In some embodiments, a bispecific antibody is a full-length antibody that binds to one antigen (or epitope) on one of its two binding arms (a pair of HC / LC) and binds to a different antigen (or epitope) on its second arm (another pair of HC / LC). In these embodiments, the bispecific antibody has two different antigen-binding arms (both in specificity and CDR sequences) and is monovalent for each antigen it binds.

[0445] In other embodiments, bispecific antibodies are full-length antibodies that can bind to two different antigens (or epitopes) in each of their two binding arms (two pairs of HC / LC). In these embodiments, the bispecific antibodies have two identical antigen-binding arms with the same specificity and the same CDR sequences, and are bivalent for each antigen to which they bind.

[0446] The terms "trispecific antibody," "trispecific molecule," and "tri-antibody" are used interchangeably herein and refer to molecules comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, when bound to the target molecule, inhibits the biological function of the target molecule. In some embodiments, a trispecific antagonist is a polymer molecule comprising two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or fragment thereof that specifically binds to a target protein.

[0447] In a preferred embodiment of the present invention, a bispecific antibody targeting CCR8 and VEGF / PD-L1 is constructed, having the structure shown in Figure 7A ai, and exemplary molecules of the ai structure are shown in Figures 7B-K, respectively. The anti-CCR8 Fab / anti-CCR8 scFv can be a Fab / scFv constructed using the VH and VL of any anti-CCR8 antibody described herein; the anti-VEGF Fab / anti-VEGF scFv can be a Fab / scFv constructed using the VH and VL of any anti-VEGF antibody described herein; and the anti-PD-L1 Fab / anti-PD-L1 scFv can be a Fab / scFv constructed using the VH and VL of any anti-PD-L1 antibody described herein.

[0448] In another preferred embodiment of the present invention, a trispecific antibody targeting CCR8, VEGF, and PD-L1 was constructed, having the structures shown in Figures 8A-8A. Exemplary molecules of the structures of Figures 8B-8E are shown, respectively. The anti-CCR8 Fab can be constructed using the VH and VL of any anti-CCR8 antibody described herein; the anti-VEGF Fab / anti-VEGF scFv can be constructed using the VH and VL of any anti-VEGF antibody described herein; and the anti-PD-L1 scFv can be constructed using the VH and VL of any anti-PD-L1 antibody described herein.

[0449] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence of the antibody of the present invention or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to that of the polypeptide of the present invention, but having a different coding region sequence.

[0450] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0451] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0452] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.

[0453] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0454] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0455] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0456] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0457] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0458] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0459] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0460] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0461] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0462] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0463] Therapeutic agents that can be conjugated include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0464] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0465] The pharmaceutical composition of the present invention can be used to treat cancer / tumor, especially solid tumor, especially solid tumor with high expression of LCRR15.

[0466] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 10 mg / kg body weight per day. In addition, the pharmaceutical composition of the present invention can also be used in conjunction with other therapeutic agents.

[0467] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 8 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0468] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0469] Sequences used to prepare the multispecific antibodies of the present invention

[0470] CCR8 antibody sequences

[0471] CCR8 antibody sequences

[0472] VEGF antibody sequence

[0473] AVACH and AVACL are mutants of the heavy chain variable region AVAH and light chain variable region AVAL of the antibody molecule AVA, respectively. The heavy chain variable region ASH and light chain variable region ASL of the antibody molecule AS-1 are derived from U.S. Patent No. 7,758,859. ASCH, AS2H, AS2CH, AS3H, and AS3CH are mutants of their VH regions, while ASCL, AS2L, AS2CL, AS3L, AS3CL, AS4L, and AS4CL are mutants of their VL regions. The underlined portions represent the CDR1, CDR2, and CDR3 of the heavy chain variable region or light chain variable region, respectively, and the mutation sites are indicated in bold.

[0474] PD-L1 antibody sequence

[0475] Among them, PL1CH and PL1CL are mutants of the heavy chain variable region PL1H and light chain variable region PL1L of the antibody molecule PL1, respectively; PL2CH and PL2CL are mutants of the heavy chain variable region PL2H and light chain variable region PL2L of the antibody molecule PL2, respectively.

[0476] IgG1 CH sequence

[0477] The CH1 sequence is as follows:

[0478] The Fc region sequence is shown below:

[0479] The Fc region may comprise the following mutations:

[0480] In a preferred embodiment of the present invention, the Fc region has the following sequence:

[0481] In another preferred embodiment of the present invention, the C-terminal lysine (K) of the Fc region is removed to reduce the formation of charge variants of the antibody.

[0482] IgG4 CH sequence

[0483] The Fc region may comprise the following mutations:

[0484] CL sequence

[0485] Optional linker sequences

[0486] Example 1 Preparation and Validation of Anti-CCR8 Monoclonal Antibodies

[0487] 1.1 Screening of anti-CCR8 monoclonal antibodies

[0488] The CCR8 antibody of the present invention is obtained by screening hybridomas immunized with humanized mice. An hCCR8 expression plasmid is constructed, DNA is extracted and endotoxin-free, and mice with humanized heavy and light chain variable regions (derived from CN114763558A) are immunized. The spleens of the immunized mice are isolated and harvested to obtain splenocytes, which are then fused with Sp2 / 0-Ag14 multiple myeloma cells. The fusion is performed by electroporation according to hybridoma fusion technology. After fusion, a certain number of cells are seeded in each well of a 96-well plate and cultured in HAT medium for 10 days. The hybridoma cell culture supernatant is tested using the HEK293-CCR8 overexpressing cell line to obtain positive hybridoma cells. The positive hybridoma cells are cultured, and the supernatant obtained after 5 days of cell culture is purified in small amounts using Protein A magnetic beads to obtain hybridoma antibodies with human variable regions and mouse constant regions. The purified antibodies are then tested for binding activity. During the screening process, specific binding molecules were screened against the extracellular region of CCR8 or cell lines overexpressing CCR8 using ELISA and FACS methods, ultimately yielding 14 monoclonal antibodies (C5, C6, C20, C23, C24, C27, C39, C40, C46, ​​C53, C54, C57, C61, and C62). Sequence analysis of these 14 antibodies allowed them to be divided into five categories: C24, C39, and C57 were each classified into one category, C5 and C27 into another, and all other antibodies into one category.

[0489] 1.2 Binding ability of anti-CCR8 monoclonal antibodies to CCR8-expressing HEK cells

[0490] Furthermore, the binding ability of the CCR8 antibody of the present invention to HEK293 cells expressing CCR8 was tested. HEK293 cells expressing CCR8 were prepared by the following method: full-length CCR8 gene fragments from humans, mice, and cynomolgus monkeys were obtained by whole gene synthesis, and these fragments were then inserted into the backbone of a stable expression vector to obtain expression plasmid vectors. These three plasmids were transfected into HEK293 cells, and monoclonal cell lines expressing human, mouse, and cynomolgus monkey CCR8 (HEK293-hCCR8 cells, HEK293-mCCR8 cells, and HEK293-cCCR8 cells) were screened under puromycin pressure to obtain the flow cytometry cell lines used in this example.

[0491] The HEK293-hCCR8 cells were plated in each well of a 96-well plate at 1×10 5 Cells were diluted with antibodies to a starting concentration of 1 μg / ml and then diluted 2-fold. 50 μl of the diluted antibodies were mixed with the cells and incubated at 4°C for 45 minutes. The cells were washed twice with washing buffer. After discarding the liquid, all cells were resuspended in 50 μl of 200 ng / ml Goat anti-Mouse IgG PE and incubated at 4°C in the dark for 30 minutes. The cells were washed three times with washing buffer. After discarding the liquid, all cells were resuspended in 25 μl of diluent. Detection was performed using a flow cytometer. The FACS binding data obtained for the 14 monoclonal antibodies are shown in Figure 1.

[0492] The results showed that the Emax and EC of antibodies C20, C24, C46, ​​C53, C54, C61, C62, C40, C6, and C23 50 Both are superior to BM-1 (antibody sequence derived from WO2021194942A1); the EC of C57 and C27 50 Better than BM-1; Emax of C5 is better than BM-1.

[0493] The antibody sequences of these positive hybridoma clones were sequenced, and the antibody sequences were cloned into human IgG1 expression vectors. FUT8- / -CHO cells were used for antibody expression. The supernatant after cell culture was centrifuged to remove the precipitate, and the supernatant was filtered using a 0.22μm filter membrane and purified on a protein purifier to obtain defucosed human IgG1 Fc antibody, i.e., recombinant human monoclonal antibody.

[0494] 1.3 Binding affinity of anti-CCR8 recombinant human monoclonal antibody to CCR8 protein

[0495] Affinity testing was performed on 14 recombinant human monoclonal antibodies using the following method:

[0496] The antibody was diluted to 5 μg / ml, and the antigens human CCR8(1-35aa)-mFc or cynomolgus CCR8-mFc were diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, 1.56 nM, 0.78 nM or 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.2 nM, and 15.6 nM, respectively. A well with a concentration of 0 was set as the baseline. Antibody affinity testing was performed using a Gator instrument.

[0497] The results are shown in Table 2. The affinities of the candidate antibodies for human CCR8 ranged from 0.0168 to 1.24 nM (Table 1). Several candidate antibodies could bind to cynomolgus monkey CCR8 (Table 2).

[0498] Table 1. Binding affinity of antibodies to human CCR8

[0499] Table 2. Binding of Antibodies to Cynomolgus Monkey CCR8

[0500] 1.4 ADCC activity of anti-CCR8 recombinant human monoclonal antibody

[0501] The ADCC activity of 14 anti-CCR8 recombinant human monoclonal antibodies was tested using the following method:

[0502] CHO-K1 cells overexpressing CCR8 were plated at 2×10 4 Plate cells and culture overnight. Remove the culture medium and add 25 μl of complete culture medium. Prepare antibody dilutions in complete culture medium to final concentrations of 4000 ng / ml, 1333.3 ng / ml, 444.4 ng / ml, 148.1 ng / ml, 49.38 ng / ml, 16.5 ng / ml, 5.48 ng / ml, 1.82 ng / ml, 0.61 ng / ml, 0.20 ng / ml, 0.06 ng / ml, and 0.02 ng / ml. Add 25 μl of antibody dilution to each well. Maintain the density of Jurkat-FcγRIIIa-V158 effector cells at 0.2-1.0 × 10 6 cells / ml, and resuspend the effector cells in culture medium to a cell density of 6×10 6 cells / ml, add 25 μl of cell suspension to each well of a 96-well white plate, for a final total volume of 75 μl per well. Incubate cells in a cell incubator for 6 hours. Add luciferase substrate to the plate at 50 μl / well and mix for 30 seconds. Detect using a multi-function microplate reader using the Lum-TM channel.

[0503] The results are shown in Figure 2. All candidate antibodies have different degrees of ADCC against target cells; among them, C5 has the smallest EC 50 , C61 has the highest signal value, and both have good ADCC effects.

[0504] 1.5 Blocking effect of anti-CCR8 recombinant human monoclonal antibody on the binding between CCR8 and its ligand CCL1

[0505] Fourteen anti-CCR8 recombinant human monoclonal antibodies were tested for their blocking effect on the binding between CCR8 and its ligand CCL1 as follows:

[0506] The blocking experiment used the DiscoverX β-Arrestin eXpress GPCR Assay kit. Add 11.5 ml of Cell Plating Reagent e22 to a 15 ml centrifuge tube. Add 0.5 ml of Cell Plating Reagent to the cryovial. Once the PathHunter eXpress β-Arrestin GPCR cells have thawed, transfer them all to the 15 ml centrifuge tube. Mix thoroughly, add 100 μl to each well, place in a cell culture incubator, and incubate for 48 hours. Prepare a starting concentration of 220,000 ng / ml (22× final concentration) and perform three-fold dilutions for a total of 8 steps. Add 5 μl of antibody dilution to each well of a white plate, add complete culture medium to the remaining wells, place in a cell culture incubator, and incubate for 30 minutes. Prepare 88 nM CCL1 (22 times the final concentration, for a final CCL1 concentration of 4 nM), add 5 μl to each well, place in a cell culture incubator, and incubate for 90 minutes. Add 55 μl of Working Detection Solution to each well, incubate at room temperature in the dark for 60 minutes, and detect using the Lum-TM channel.

[0507] The results are shown in Figure 3. The tested antibodies all have a certain blocking effect on the binding of CCL1 and CCR8. The antibodies with a blocking effect of >90% are C5, C20, C23, C57, and C61, with blocking percentages of 96.83%, 90.08%, 91.39%, and 92.46%, respectively. Their IC 50 The values ​​are: 496.8, 377.4, 216.5, and 183.4 ng / ml respectively.

[0508] Example 2 Preparation and Verification of Anti-VEGF Mutants

[0509] The AS-1 antibody (heavy chain variable region: SEQ ID NO: 89, light chain variable region: SEQ ID NO: 95) is inherently hydrophobic and prone to aggregation. To improve the quality of the antibody and facilitate subsequent application of this antibody sequence in bispecific and trispecific antibody constructs, the inventors designed a series of mutations.

[0510] The hydrophobic areas on the surface of the Fab structure of AS-1 (PDB: 2FJG) are marked with the Color_h.py code on the PyMOL Wiki. The higher the hydrophobicity, the darker the color, which is gray-black. The lower the hydrophobicity, the lighter the color, which is gray-white, see Figure 4. Generally speaking, high hydrophobicity of protein sequences easily leads to aggregation, reducing protein stability. In addition, in order to further confirm the hydrophobicity of the relevant amino acids on the antibody surface after conversion to scFv, the inventors used AlphaFold to predict the scFv structure of AS-1 (the scFv sequence is consistent with the scFv sequence of AS-1 used in the bi- and tri-antibody molecules of the present invention) and analyzed it using the Aggrescan server, as shown in Figure 5. All amino acids with a score greater than 0 are hydrophobic, which makes the antibody tend to aggregate. The present invention obtained a large number of hydrophobic amino acid sites from the Fab structure and scFv structure of AS-1, which can be used as potential mutation points.

[0511] Furthermore, to simultaneously analyze the aggregation propensity and thermal stability of the AS-1 sequence, the inventors used the SolubiS server to analyze regions of the AS-1 variable region sequence that are prone to aggregation (high TANGO scores) and have poor thermal stability (high free energy ΔG). As shown in Figure 6, six peptide segments were identified. Among them, the light chain L46-S57 region had the highest aggregation propensity and the lowest thermal stability. Furthermore, a literature review (Dudgeon et al., 2012, PNAS) has found that amino acid mutations at positions 24, 49, 50, 51, 52, 53, and 56 of the light chain and at positions 28, 30, 31, 32, 33, and 35 of the heavy chain can effectively improve antibody aggregation through site-directed mutagenesis.

[0512] Based on the above structural and sequence analysis, the present invention mutated the L46-S57 region, as well as some amino acids in other regions (such as hydrophobic sites in the structure or reported aggregation sites), to aspartic acid (D) to reduce hydrophobicity. Furthermore, the structure diagram published by 2FJG on the PDB indicates that the VH-CDR3 region plays a crucial role in VEGF binding and that many amino acids are buried within it. Therefore, the present invention primarily targeted non-VH-CDR3 regions and other regions unrelated to VEGF binding to maintain the antibody's VEGF binding activity. In this example, seven AS-1 mutants were obtained through site-directed mutagenesis.

[0513] To confirm whether these mutations altered the hydrophobicity of the antibody, the retention times of the various mutant antibodies were analyzed using a Proteomix HIC Butyl-NP5 column. Each mutant was loaded onto the column in a solution of 1.5M NaCl, 25mM Na₃PO₄, pH 7.4, and eluted with solutions of gradually decreasing salt concentration at pH 7.4. The retention times of the mutants are shown in Table 3. AS-1 has the longest retention time, indicating the strongest hydrophobicity. The retention times of the other mutants decreased, and some combined mutants, such as AS-6, showed a reduction of nearly half, significantly reducing the antibody's hydrophobicity and its potential aggregation tendency.

[0514] Table 3

[0515] To further confirm that the mutants' binding to VEGF was not affected, affinity testing was performed on each mutant. His-tagged human VEGF protein was loaded onto an anti-His BLI probe. The mutants were allowed to bind to VEGF for 150 seconds and then dissociate for 300 seconds in a solution containing 1X PBS, 0.1% BSA, 0.02% Tween-20, and 0.05% sodium azide. KD values ​​were calculated and are shown in Table 4. Table 4 shows that only mutants AS-3, AS-6, and AS-8 showed a significant decrease in their VEGF binding affinity, while the other mutants maintained a good affinity compared to AS-1.

[0516] Table 4

[0517] Example 3 Preparation of multispecific antibody molecules

[0518] Based on the 14 different CCR8 antibody sequences obtained in Example 1, a variety of multispecific antibody molecules were designed.

[0519] The molecular sequence fragment gene of the multispecific antibody is obtained by the method of total gene synthesis, and then the target sequence is inserted into the expression vector by conventional gene cloning means (see, for example, Lo. BKC methods in Molecular Biology. Volume 248, 2004. Antibody Engineering).

[0520] HEK293E cells were transfected with a vector carrying a multispecific antibody molecule. Cultured for 7 days at 37°C, 5% CO2, in F17 medium (1 L F17 + 10 mL 10% PF68 + 30 mL 200 mM L-glutamine) to produce the desired molecule. After expression, the supernatant was harvested and purified to yield the multispecific antibody molecule, which was subsequently used for various assays.

[0521] The anti-VEGF sequence comes from the molecule AVA (heavy chain variable region is SEQ ID NO: 87, light chain variable region is SEQ ID NO: 93) and mutants of the VEGF antibody variable region and its CDR region in patent US7758859. The anti-PDL1 sequence comes from the molecule PL1 (heavy chain variable region is SEQ ID NO: 103, light chain variable region is SEQ ID NO: 107) or PL2 (heavy chain variable region is SEQ ID NO: 105, light chain variable region is SEQ ID NO: 109).

[0522] Using the above methods, the following multispecific molecules were prepared:

[0523] (1) a bispecific antibody consisting of a CCR8 antigen-binding domain and a VEGF or PD-L1 antigen-binding domain having the structure shown in ai in FIG7A ;

[0524] A bispecific antibody with structure a in Figure 7A is named 8As-1, and its structure is shown in Figure 7B ;

[0525] A bispecific antibody with structure b was named 8As-2, and its structure is shown in Figure 7C ;

[0526] A bispecific antibody with structure c was named 8As-3, and its structure is shown in Figure 7D ;

[0527] A bispecific antibody with structure d was named 8As-4, and its structure is shown in Figure 7E ;

[0528] A bispecific antibody with structure e was named 8As-5, and its structure is shown in Figure 7F;

[0529] A bispecific antibody with structure f was named 8As-6, and its structure is shown in Figure 7G ;

[0530] A bispecific antibody with g structure was named 8As-7, and its structure is shown in Figure 7H ;

[0531] A bispecific antibody with h structure was named P18-8, and its structure is shown in FIG7I ;

[0532] A bispecific antibody with structure i was named P18-9, and its structure is shown in FIG7J ; another bispecific antibody with structure i was named 8As-9, and its structure is shown in FIG7K ; it differs from P18-9 in that the anti-PD-L1 VH and VL are replaced with anti-VEGF VH and VL.

[0533] (2) A trispecific antibody consisting of a CCR8 antigen-binding domain, a VEGF antigen-binding domain, and a PD-L1 antigen-binding domain as shown in ad in FIG8A .

[0534] A bispecific antibody with structure a in FIG8A is named 8AsPl-1, and its structure is shown in FIG8B ;

[0535] A bispecific antibody with structure b was named 8AsPl-2, and its structure is shown in Figure 8C

[0536] A bispecific antibody with structure c was named 8AsPl-3, and its structure is shown in Figure 8D;

[0537] A bispecific antibody with the d structure is named 8AsPl-4 or 8AsPl-4v, and its structure is shown in Figure 8E.

[0538] The exemplary molecules prepared in this example are as follows, wherein the CCR8 antigen-binding domain uses the VH and VL of C61 and / or C27:

[0539] The structure of the 8AsPl-4v molecule is identical to that of 8AsPl-4, except that the anti-VEGF antigen-binding domain comprises VH as shown in SEQ ID NO: 91 and VL as shown in SEQ ID NO: 99, and the K at the C-terminus of the Fc region is removed.

[0540] Example 4 Performance testing of multispecific antibody molecules targeting CCR8 and VEGF / PD-L1

[0541] 4.1 ADCC experiments with multispecific antibodies targeting CCR8 and VEGF / PDL1

[0542] The ADCC effects of the multispecific antibodies targeting CCR8 and VEGF / PD-L1 (8As-1, 8As-2, 8As-4, 8As-7, 8As-9, P18-8, P18-9, 8AsP1-1, 8AsP1-3, and 8AsP1-4) prepared in Example 2 were determined, with some of the antibodies being expressed in Fut8 knockout CHO cells (FKO). The specific methods are as follows:

[0543] Collect CHOK1-hCCR8 cells (human CCR8 overexpressing cells, obtained by transfecting CHOK1 cells with a full-length CCR8 expression plasmid, prepared using the same method as the HEK293-hCCR8 cell preparation described in Example 1.2) in the logarithmic growth phase and centrifuge at 300g for 5 minutes. Resuspend the cells in 1ml of assay buffer (1640 medium + 10% FBS), count the cells, and adjust the cell density to 1E6 / ml using assay buffer. After mixing thoroughly, plate the CHOK1-hCCR8 cell suspension into a 96-well plate at 25μl / well. Add serially diluted antibodies to the 96-well plate using assay buffer at 25μl / well in duplicate. Incubate in a CO2 incubator for 30 minutes. Finally, collect the ADCC Bioassay Effector Cell V Variant (High Affinity) / NFAT Luciferase Reporter Jurkat Cell Line in good growth condition into a 50ml centrifuge tube and centrifuge at 300g for 5min. Take 3ml assay buffer (1640medium + 10% FBS) to resuspend the cells, count and adjust the cell density to 3E6 / ml. After mixing, add to the above-mentioned 96-well white plate, 50μl / well. Place in a CO2 incubator and incubate for 5h. Take out the 96-well white plate and add BRITELITE PLUS reagent, 100μl / well. Incubate at room temperature in the dark for 5min, and detect lumi readings with an enzyme reader. Use prism software to draw a graph and calculate the EC 50 value.

[0544] The results in Figure 9 show that the ADCC effect of the antibody expressed in Fut8 knockout CHO cells (with FKO after the name) is significantly higher than that of the same antibody expressed in wild-type CHO cells (Figure 9e). The bispecific antibodies of various structures all showed strong ADCC effects (Figure 9ad). Among them, 8As-1, 8As-4, Pl8-8, and Pl8-9 were similar to monoclonal antibodies in ADCC effects on CCR8 targets, and their bispecific antibody structures did not affect the ADCC effect. For the triple antibody molecule 8AsPl-4, while showing good ADCC against CCR8, its ADCC effect on PD-L1 was very weak (Figure 9f and g).

[0545] 4.2 VEGF blocking experiments with multispecific antibodies targeting CCR8 and VEGF / PD-L1

[0546] The ability of multispecific antibodies targeting CCR8 and VEGF / PD-L1 (8As-1, 8As-2, 8As-4, 8As-9, 8AsPl-1, 8AsPl-3, and 8AsPl-4) to block VEGF signaling was determined, with AS-1 serving as a control. The specific method is as follows:

[0547] Harvest VEGFR2 / NFAT Reporter–HEK293 Recombinant cells in the logarithmic growth phase and centrifuge at 300g for 5 minutes. Resuspend the cells in 1ml of assay buffer (MEM / EBSS medium + 10% FBS), count the cells, and adjust the cell density to 6E5 / ml using assay buffer. After mixing thoroughly, seed the VEGFR2 / NFAT Reporter–HEK293 Recombinant Cell Line cell suspension into a 96-well plate at 50μl / well. Add serially diluted antibodies using assay buffer to the same 96-well plate at 25μl / well in duplicate. Next, dilute human VEGF165 his Tag protein to 50ng / ml using assay buffer and transfer 25μl / well to the 96-well plate. Incubate in a CO2 incubator for 4 hours. Finally, remove the 96-well plate and add BRITELITE PLUS reagent at 100μl / well. Incubate at room temperature in the dark for 5 minutes, and use a microplate reader to detect the lumi readings. Use Prism software to plot and calculate the IC 50 value.

[0548] The results are shown in Figure 10. Figure 10 a and b show that the blocking effects of each bispecific antibody on VEGF were very similar, while for the triple antibody molecule ( Figure 10 c), 8AsPl-4 showed a better blocking effect, similar to that of the monospecific antibody molecule.

[0549] 4.3 PD-L1 blocking experiment with multispecific antibody molecules targeting CCR8 and VEGF / PD-L1

[0550] The ability of multispecific antibodies targeting CCR8 and VEGF / PD-L1 to block PD-1 / PD-L1 binding was determined, using the aforementioned PL1 molecule as a control sample. The specific method is as follows:

[0551] Collect CHO-hPDL1 cells in the logarithmic growth phase and centrifuge at 300g for 5 minutes. Resuspend the cells in 1ml of FACS buffer (1X PBS + 2% FBS), count, and adjust the cell density to 2E6 / ml using FACS buffer. After mixing, seed the CHO-hPDL1 cell suspension into a 96-well V-bottom plate at 25μl / well. Add serially diluted antibodies (8AsPl-1, 8AsPl-3, 8AsPl-4, or Pl8-8-FKO, Pl8-9-FKO) to the 96-well V-bottom plate using FACS buffer at 50μl / well in duplicate. Dilute human PD-1mFc Tag protein to 16μg / ml using FACS buffer and transfer 25μl / well to a 96-well V-bottom plate. Incubate at 4°C for 30 minutes. Wash twice with FACS buffer, add Goat anti-Mouse IgG Fc Cross-Adsorbed Secondary Antibody PE, place in a 4°C refrigerator, and continue incubation for 20 minutes. After washing twice with FACS buffer, detect using flow cytometry. The experimental data were plotted using Prism software to calculate IC 50 value.

[0552] The results are shown in Figure 11. The results show that the blocking effect of the two bispecific antibodies on PD-L1 is basically the same as that of the monoclonal antibody (Figure 11a). Among the three-antibody molecules, 8AsPl-3 and 8AsPl-4 have better blocking effects than 8AsPl-1 but lower than the monoclonal antibody control sample (Figure 11b).

[0553] Example 5 In vivo efficacy experiments of antibody molecules targeting CCR8

[0554] 5.1 In vivo efficacy experiments in MC38 mouse models

[0555] CCR8 humanized C57BL / 6 mice were inoculated with MC38 cells (5×10 5 cells / mouse) to establish MC38 mouse model, and wait until the average tumor volume grows to about 100mm 3 The patients were divided into groups and given intravenous injections at a dose of 10 mg / kg twice a week for 5 consecutive times.

[0556] The results are shown in FIG12 a. On the 14th day after administration, BM-1, C61, and C27 all had anti-tumor effects, with tumor growth inhibition rates (TGI) of 30%, 34%, and 45%, respectively.

[0557] Another group of CCR8 humanized C57BL / 6 mice were inoculated with MC38 cells. The mouse inoculation number, tumor grouping volume, and administration dose (equimolar) were the same as those in the experiment shown in Figure 12a. MC38 cells were injected intraperitoneally twice a week for 6 consecutive times.

[0558] The results are shown in FIG12 b. On the 20th day after administration, C61, 8AS-2, and 8AsPl-4v all had anti-tumor effects, with tumor growth inhibition rates (TGI) of 45%, 83.5%, and 89.5%, respectively.

[0559] 5.2 In vivo efficacy experiments in CT26 mouse models

[0560] CCR8 humanized Balb / c mice were inoculated with CT26 cells (3×10 5 cells / mouse) to establish a CT26 mouse model, and wait until the average tumor volume grows to about 60 mm 3 The drugs were administered intraperitoneally, with the dosages for each group shown in Figure 13, twice a week for 4 consecutive times.

[0561] The results are shown in Figure 13(a). On day 14 after administration, the tumor growth inhibition rates (TGI) in each treatment group were 70% (AS-1), 4% (C61), 66% (AS-1+C61), 63% (8As-1), 58% (8As-2), and 74% (8As-4), respectively. The changes in mouse body weight during administration are shown in Figure 13(b). This indicates that the bispecific antibody molecules of the present invention also exhibited significant anti-tumor activity in mice.

[0562] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-CCR8 antibody or an antigen-binding fragment thereof, wherein the antibody comprises the following three heavy-chain variable region CDRs: HCDR1, which has an amino acid sequence as shown in SEQ ID NO: 1, 4, 7, 10, 15, 18, 20, 24, 28 or 30; HCDR2, which has an amino acid sequence as shown in SEQ ID NO: 2, 5, 8, 11, 13, 16, 21, 23, 25 or 31; and HCDR3, which has an amino acid sequence as shown in SEQ ID NO: 3, 6, 9, 12, 14, 17, 19, 22, 26, 27, 29 or 32; And the following three light-chain variable region CDRs: LCDR1, which has an amino acid sequence as shown in SEQ ID NO: 33, 36, 39, 50 or 55; LCDR2, which has an amino acid sequence as shown in SEQ ID NO: 34, 37, 40, 44, 48, 51, 53, 56 or 58; and LCDR3, which has an amino acid sequence as shown in SEQ ID NO: 35, 38, 42, 45, 49, 52, 54 or 57.

2. The anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-CCR8 antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 59-72, and / or a light-chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 73-86.

3. A multispecific antibody, characterized in that, The multispecific antibody comprises: A first targeting domain, the first targeting domain comprising one or more CCR8 antigen-binding domains, the CCR8 antigen-binding domain comprising the anti-CCR8 antibody or an antigen-binding fragment thereof as claimed in claim 1; A second targeting domain, the second targeting domain binding to VEGF or PD-L1; Optionally, comprising a third targeting domain, the third targeting domain binding to VEGF or PD-L1; And the second targeting domain and the third targeting domain bind to different antigens respectively.

4. The multispecific antibody according to claim 3, wherein, The targeting domain is in the form of a single-chain Fv (scFv), Fab fragment, single-domain antibody (sdAb), fragment variable (Fv) heterodimer, TriFab or a combination thereof.

5. The multispecific antibody according to claim 3, characterized in that, The VEGF antigen-binding domain comprises a heavy-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 87 or 88, and a light-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 93 or 94; or Comprises a heavy-chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 89-92, 149-150, and a light-chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 95-102.

6. The multispecific antibody according to claim 3, wherein, The PD-L1 antigen-binding domain comprises a heavy-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 103 or 104, and a light-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 107 or 108; or comprises a heavy-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 105 or 106, and a light-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 109 or 110.

7. The multispecific antibody according to claim 3, characterized in that, The multispecific antibody further comprises an Fc fragment. Preferably, the Fc fragment is derived from IgG1 or IgG4.

8. The multispecific antibody according to claim 7, wherein, The Fc fragment comprises a mutation for forming a knob-in-hole structure and / or a mutation for enhancing ADCC.

9. The multispecific antibody according to claim 3, wherein, The multispecific antibody is a bispecific / trispecific antibody.

10. A polynucleotide encoding the anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the multispecific antibody according to claim 3.

11. Use of an anti-CCR8 antibody or antigen-binding fragment thereof as claimed in claim 1, or of a multispecific antibody as claimed in claim 3, characterized in that, For preparing a drug for treating cancer / tumor.

12. An immunoconjugate, characterized in that, The conjugate comprises: (i) an anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the multispecific antibody according to claim 3; and (ii) a conjugate moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) an anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the multispecific antibody according to claim 3; and (b) a pharmaceutically acceptable carrier.

14. Use of the anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the immunoconjugate according to claim 12, for preparing a detection reagent or kit for detecting CCR8 molecules in a sample.

15. An anti-VEGF antibody mutant, characterized in that, The anti-VEGF antibody mutant comprises a heavy-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 89, and a light-chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, and comprises a mutation capable of reducing the hydrophobicity of the antibody.

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