Multispecific antibodies comprising CCR8 antigen binding domains

Abstract

The invention relates to a multispecific antibody and application thereof. Specifically, the invention provides a multispecific antibody which comprises a first targeting structural domain for targeting chemokine (C-C motif) receptor 8 (CCR8) molecules highly expressed on the surface of tumor infiltration regulatory T cells (Treg), and also comprises a second targeting structural domain combined with VEGF and / or a third targeting structural domain combined with PD-L1. According to the CCR8 related multispecific antibody disclosed by the invention, each functional domain is used for killing Treg infiltrated by tumors, blocking a PD-1 / PD-L1 immunosuppression pathway and the like, so that T cells are activated, the generation of tumor blood vessels is inhibited, and the CCR8 related multispecific antibody can be used as an effective therapeutic agent for tumor treatment.

Description

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 that has the potential to invade or spread to other parts of the body. Conventional cancer treatments aim to remove cancerous tissue and prevent it from spreading. Such treatment options include surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and palliative care. Treatment is usually based on the type, location, and grade of the cancer, as well as the patient's health and preferences. But these therapies have limitations as they may not be effective, especially when the cancer has metastasized. In addition, 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 treatments 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 its inhibition on T cells, thereby restoring the ability of T cells to kill tumor cells. At present, no CCR8 monoclonal antibody or multi-specific antibody has been approved for marketing, but many have entered the clinical research stage.

[0005] IgG1 subtype antibodies have a strong ADCC effect, and ADCC is generated by the binding of Fc and Fc-γ receptors, and its binding force is affected by N-glycans in the CH2 domain. Studies have shown that reducing / removing fucose in the Fc core sugar structure can improve the ADCC effect, and fucose can be formed by catalysis of fucosyltransferase (Fut8). Therefore, by knocking out the Fut8 gene in expression cells such as CHO cells, ADCC-enhanced therapeutic antibodies can be expressed to enhance their killing of Tregs and relieve or reduce T cell inhibition.

[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, which can mediate the continuous formation of new vascular systems inside and around tumors. The abnormalities in the structure and function of tumor blood vessels formed under the action of VEGF will lead to poor tumor bleeding and hypoxia, thus further generating 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 that specifically blocks VEGF developed by Genentech under Roche, to inhibit the formation of tumor blood vessels. So far, the approved indications of 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 is an important target in tumor immunotherapy (IO). Since PD-L1 is highly expressed in most tumors, and the binding of PD-L1 to PD-1 on the surface of T cells will transmit inhibitory signals to T cells. Therefore, blocking PD-1 / PD-L1 can effectively activate the killing of tumor cells by T cells. Since the first PD-1 antibody nivolumab was launched in 2014, the development of PD-L1 antibodies has followed closely. Among them, atezolizumab developed by Roche was approved for marketing in 2016, and avelumab developed by the cooperation of Pfizer and Merck was approved for marketing in 2017.

[0008] Due to the complexity of the tumor microenvironment, current monoclonal antibody therapies are increasingly difficult to meet the growing clinical needs. In order to achieve better treatment effects, there is an urgent need in this field to develop multi-specific antibodies that target multiple tumor treatment targets simultaneously. Summary of the Invention

[0009] The present invention constructs bispecific / trispecific antibodies, which simultaneously inhibit or kill tumor cells from different directions, so as to achieve better treatment effects.

[0010] The present invention provides bispecific / trispecific antibodies comprising a CCR8 antigen-binding domain. The bispecific / trispecific antibody molecule can bind to CCR8 and simultaneously block the VEGF or / and PDL1 pathway. Also provided are methods of using the antibodies and antibody conjugates of the present invention, their pharmaceutical compositions and articles for treating diseases such as cancer.

[0011] In a first aspect of the present invention, there is provided an antibody against CCR8 or an antigen-binding fragment thereof, the antibody comprising the following three heavy-chain variable region CDRs:

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

[0013] 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

[0014] 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;

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

[0016] LCDR1, which has an amino acid sequence as shown in SEQ ID NO: 33, 36, 39, 50 or 55;

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

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

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

[0020]

[0021] In another preferred embodiment, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0022] In another preferred embodiment, the anti-CCR8 antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 59, and the light chain variable region shown in SEQ ID NO: 73.

[0023] In another preferred embodiment, the anti-CCR8 antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 60, and the light chain variable region shown in SEQ ID NO: 74.

[0024] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0025] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0026] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0027] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0028] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0029] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0030] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0031] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0032] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0033] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0034] In another preferred embodiment, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0035] In another preferred embodiment, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0036] In another preferred embodiment, the antibody or its antigen-binding fragment is a human, murine, humanized or chimeric antibody.

[0037] In another preferred embodiment, the antibody or its antigen-binding fragment is a human antibody.

[0038] In a second aspect of the present invention, there is provided a multispecific antibody, which comprises the anti-CCR8 antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0039] In another preferred embodiment, the multispecific antibody comprises:

[0040] a first targeting domain, which comprises one or more CCR8 antigen-binding domains;

[0041] a second targeting domain, which binds to VEGF or PD-L1;

[0042] optionally, a third targeting domain, which binds to VEGF or PD-L1;

[0043] and, the second targeting domain and the third targeting domain bind to different proteins respectively.

[0044] In another preferred embodiment, the targeting domain is in the form of a single-domain antibody (sdAb), fragment variable (Fv) heterodimer, single-chain Fv (scFv), Fab fragment, TriFab or a combination thereof.

[0045] In another preferred embodiment, the CCR8 antigen-binding domain comprises the anti-CCR8 antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0046] In another preferred embodiment, the CCR8 antigen-binding domain comprises the following three heavy chain variable region CDRs:

[0047] HCDR1, which has the amino acid sequence as shown in SEQ ID NO:1;

[0048] HCDR2, which has the amino acid sequence shown in SEQ ID NO:2; and

[0049] HCDR3, which has the amino acid sequence shown in SEQ ID NO:3;

[0050] And the following three light chain variable region CDRs:

[0051] LCDR1, which has the amino acid sequence shown in SEQ ID NO:33;

[0052] LCDR2, which has the amino acid sequence shown in SEQ ID NO:34; and

[0053] LCDR3, which has the amino acid sequence shown in SEQ ID NO:35.

[0054] In another preferred example, the CCR8 antigen-binding domain comprises the following three heavy chain variable region CDRs:

[0055] HCDR1, which has the amino acid sequence shown in SEQ ID NO:18;

[0056] HCDR2, which has the amino acid sequence shown in SEQ ID NO:5; and

[0057] HCDR3, which has the amino acid sequence shown in SEQ ID NO:19;

[0058] And the following three light chain variable region CDRs:

[0059] LCDR1, which has the amino acid sequence shown in SEQ ID NO:46;

[0060] LCDR2, which has the amino acid sequence shown in SEQ ID NO:34; and

[0061] LCDR3, which has the amino acid sequence shown in SEQ ID NO:38.

[0062] In another preferred example, the anti-CCR8 antibody or its antigen-binding fragment 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.

[0063] In another preferred example, the CCR8 antigen-binding domain comprises a heavy chain variable region shown in SEQ ID NO:59 and a light chain variable region shown in SEQ ID NO:73.

[0064] In another preferred embodiment, 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.

[0065] In another preferred embodiment, 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.

[0066] In another preferred embodiment, the CCR8 antigen-binding domain is selected from the group consisting of: scFv, Fab, or a combination thereof.

[0067] In another preferred embodiment, the CCR8 antigen-binding domain is scFv.

[0068] In another preferred embodiment, the CCR8 antigen-binding domain is Fab, which 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 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

[0069] a heavy chain constant region CH1 as shown in SEQ ID NO: 111 or 119 and a light chain constant region CL as shown in SEQ ID NO: 121 or 122; or a heavy chain constant region CH1 as shown in SEQ ID NO: 112 and a light chain constant region CL as shown in SEQ ID NO: 123.

[0070] In another preferred embodiment, the multispecific antibody further comprises an Fc fragment.

[0071] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0072] 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.

[0073] In another preferred embodiment, the Fc fragment comprises mutations for forming a knob-in-hole structure and / or mutations for enhancing ADCC.

[0074] In another preferred embodiment, the Fc fragment derived from IgG1 has mutations selected from the group consisting of:

[0075] Y349C / K370E / K409D / K439E,

[0076] S354C / D356K / E357K / D399K;

[0077] S354C / T366W,

[0078] Y349C / T366S / L368A / Y407V.

[0079] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO: 115-118.

[0080] In another preferred embodiment, the IgG4-derived Fc fragment has a mutation selected from the group consisting of:

[0081] Y349C / K370E / R409D / K439E,

[0082] S354C / E356K / E357K / D399K; or

[0083] S354C / T366W,

[0084] Y349C / T366S / L368A / Y407V.

[0085] In another preferred embodiment, the Fc fragment is an IgG4-derived Fc fragment having an amino acid sequence as shown in SEQ ID NO: 120.

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

[0087] In another preferred embodiment, the multispecific antibody is a bispecific antibody.

[0088] In another preferred embodiment, the bispecific antibody comprises:

[0089] A first targeting domain comprising one or more CCR8 antigen-binding domains; and a second targeting domain which is a VEGF antigen-binding domain.

[0090] In another preferred embodiment, the bispecific antibody comprises:

[0091] A first targeting domain comprising one or more CCR8 antigen-binding domains; and a second targeting domain which is a PD-L1 antigen-binding domain.

[0092] In another preferred embodiment, the multispecific antibody is a trispecific antibody.

[0093] In another preferred embodiment, the trispecific antibody comprises:

[0094] a first targeting domain comprising one or more CCR8 antigen-binding domains;

[0095] a second targeting domain, which is a VEGF antigen-binding domain;

[0096] a third targeting domain, which is a PD-L1 antigen-binding domain.

[0097] 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 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.

[0098] 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 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.

[0099] In another preferred embodiment, the VEGF antigen-binding domain comprises a mutation capable of reducing the hydrophobicity of the antibody.

[0100] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in the non-CDR3 region of the anti-VEGR antigen-binding domain having the heavy chain variable region shown in SEQ ID NO: 89 and the light chain variable region shown in SEQ ID NO: 95.

[0101] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid site selected from the group consisting of: positions 28, 30, 31, 32, 33, 35 in the heavy chain variable region shown in SEQ ID NO: 89, or a combination thereof.

[0102] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at an amino acid site selected from the group consisting of: positions 24, 49, 50, 51, 52, 53, 56 in the light chain variable region shown in SEQ ID NO: 95, or a combination thereof.

[0103] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in the region of positions 46-57 in the light chain variable region shown in SEQ ID NO: 95.

[0104] In another preferred example, the mutation capable of reducing the hydrophobicity of the antibody is to mutate the above amino acid site into a hydrophilic amino acid, such as aspartic acid (D), glutamic acid (E), lysine (K), or arginine (R).

[0105] In another preferred example, 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 into aspartic acid (D), glutamic acid (E), lysine (K), or arginine (R).

[0106] In another preferred example, 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 into aspartic acid (D), glutamic acid (E), lysine (K), or arginine (R), and / or serine (S) at position 52 is mutated into aspartic acid (D), glutamic acid (E), lysine (K), or arginine (R).

[0107] 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 any one of SEQ ID NOs: 90-92, 149-150.

[0108] In another preferred example, 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.

[0109] In another preferred example, the VEGF antigen-binding domain is selected from the group consisting of: scFv, Fab, or a combination thereof.

[0110] In another preferred example, 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.

[0111] In another preferred example, 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: 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.

[0112] In another preferred example, the PD-L1 antigen-binding domain is selected from the group consisting of: scFv, Fab, or a combination thereof.

[0113] In another preferred example, the multispecific antibody has the structure shown in Formula I below (for example Figure 7A in a)):

[0114] Fab1-Fc1

[0115] ║

[0116] Fab2-Fc2(I)

[0117] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0118] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0119] Fab2 is the second targeting domain, and the Fab2 is an anti-VEGF Fab or an anti-PD-L1 Fab;

[0120] Fc1 and Fc2 are each independently an Fc fragment.

[0121] In another preferred example, the Fab2 is an anti-VEGF Fab.

[0122] In another preferred example, 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

[0123] 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.

[0124] In another preferred example, 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

[0125] the anti-VEGF Fab comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NOs: 95-102.

[0126] In another preferred example, 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

[0127] 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.

[0128] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0129] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0130] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113 - 118.

[0131] In another preferred embodiment, the Fc1 has an amino acid sequence as shown in SEQ ID NO: 115, and the Fc2 has an amino acid sequence as shown in SEQ ID NO: 117.

[0132] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc1" in the "Fab1-Fc1" is as shown in SEQ ID NO: 125, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0133] In another preferred embodiment, the amino acid sequence of "HC2 (heavy chain)-Fc2" in the "Fab2-Fc2" is as shown in SEQ ID NO: 126, and the amino acid sequence of "LC2 (light chain)" is as shown in SEQ ID NO: 127.

[0134] In another preferred embodiment, the multispecific antibody has a structure as shown in Formula II below (for example Figure 7A in b):

[0135] Fab1-Fc1-scFv2

[0136] ║

[0137] Fab1-Fc1-scFv2 (II)

[0138] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0139] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0140] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0141] Fc1 is an Fc fragment.

[0142] In another preferred embodiment, the "║" is a disulfide bond.

[0143] In another preferred embodiment, the scFv2 is an anti-VEGF scFv.

[0144] 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

[0145] 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.

[0146] 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

[0147] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO:89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NO:95-102.

[0148] 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

[0149] 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.

[0150] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0151] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0152] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO:113-118.

[0153] In another preferred embodiment, the Fc1 has an amino acid sequence as shown in SEQ ID NO:113.

[0154] 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.

[0155] In another preferred example, the multispecific antibody has the structure shown in formula III below (for example Figure 7A in c):

[0156] Fab1-Fc1-scFv2

[0157] ║

[0158] Fab1-Fc2(III)

[0159] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0160] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0161] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0162] Fc1 and Fc2 are each independently an Fc fragment.

[0163] In another preferred example, the scFv2 is an anti-VEGF scFv.

[0164] In another preferred example, 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

[0165] 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.

[0166] In another preferred example, 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

[0167] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO: 89-92, 149-150 and a light chain variable region as shown in any one of SEQ ID NO: 95-102.

[0168] 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

[0169] 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.

[0170] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0171] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0172] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 113-118.

[0173] In another preferred embodiment, the Fc1 has an amino acid sequence as shown in SEQ ID NO: 115, and the Fc2 has an amino acid sequence as shown in SEQ ID NO: 118.

[0174] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv2" in the "Fab1-Fc1-scFv2" is as shown in SEQ ID NO: 128, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0175] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is as shown in SEQ ID NO: 125, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0176] In another preferred embodiment, the multispecific antibody has a structure as shown in formula IV (for example Figure 7A in d):

[0177] scFv2-Fc1

[0178] ║

[0179] Fab1-Fc2 (IV)

[0180] In the formula, "-" is independently a peptide bond or a linker peptide; "║" is a linking bond between peptide chains;

[0181] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0182] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0183] Fc1 and Fc2 are each independently an Fc fragment.

[0184] In another preferred example, the scFv2 is an anti-VEGF scFv.

[0185] In another preferred example, 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

[0186] 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.

[0187] In another preferred example, 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

[0188] The anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO:89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NO:95-102.

[0189] In another preferred example, 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

[0190] 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.

[0191] In another preferred example, the Fc fragment is derived from IgG1 or IgG4

[0192] In another preferred example, the Fc fragment is derived from IgG1.

[0193] In another preferred example, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO:113-118.

[0194] In another preferred example, the Fc1 has an amino acid sequence as shown in SEQ ID NO:115, and the Fc2 has an amino acid sequence as shown in SEQ ID NO:118.

[0195] In another preferred example, the amino acid sequence of the "scFv2-Fc1" is as shown in SEQ ID NO: 129.

[0196] In another preferred example, the amino acid sequence of the "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is as shown in SEQ ID NO: 125, and the amino acid sequence of the "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0197] In another preferred example, the multispecific antibody has the structure shown in Formula V below (for example Figure 7A in e):

[0198] scFv1-scFv2-Fc1

[0199] ║

[0200] Fab1-Fc2(V)

[0201] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a linking bond between peptide chains;

[0202] scFv1 and Fab1 are the first targeting domains, the scFv1 is an anti-CCR8 scFv, and the Fab1 is an anti-CCR8 Fab;

[0203] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv or an anti-PD-L1 scFv;

[0204] Fc1 and Fc2 are each independently an Fc fragment.

[0205] In another preferred example, the scFv2 is an anti-VEGF scFv.

[0206] In another preferred example, 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

[0207] 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.

[0208] In another preferred example, the anti-CCR8 scFv 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

[0209] 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.

[0210] 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

[0211] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO: 89-92, 149-150 and a light chain variable region as shown in any one of SEQ ID NO: 95-102.

[0212] 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

[0213] 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.

[0214] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0215] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0216] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO: 113-118.

[0217] In another preferred embodiment, the Fc1 has an amino acid sequence as shown in SEQ ID NO: 115, and the Fc2 has an amino acid sequence as shown in SEQ ID NO: 118.

[0218] In another preferred embodiment, the amino acid sequence of the "scFv1-scFv2-Fc1" is as shown in SEQ ID NO: 130.

[0219] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is as shown in SEQ ID NO: 125, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0220] In another preferred embodiment, the multispecific antibody has a structure as shown in formula VI (for example Figure 7A in f):

[0221] Fab2 - Fab1 - Fc1

[0222] ║

[0223] Fab1 - Fc2(VI)

[0224] Wherein, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0225] Fab1 is the first targeting domain, and the Fab1 is an anti - CCR8 Fab;

[0226] Fab2 is the second targeting domain, and the Fab2 is an anti - VEGF Fab or an anti - PD - L1 Fab;

[0227] Fc1 and Fc2 are each independently an Fc fragment.

[0228] In another preferred example, the Fab2 is an anti - VEGF Fab.

[0229] In another preferred example, 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

[0230] 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.

[0231] In another preferred example, 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

[0232] The anti - VEGF Fab comprises a heavy - chain variable region as shown in any one of SEQ ID NO:89 - 92, 149 - 150, and a light - chain variable region as shown in any one of SEQ ID NO:95 - 102.

[0233] In another preferred example, 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

[0234] 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.

[0235] In another preferred example, the Fc fragment is derived from IgG1 or IgG4

[0236] In another preferred example, the Fc fragment is derived from IgG1.

[0237] In another preferred example, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO: 113-118.

[0238] In another preferred example, the Fc1 has an amino acid sequence as shown in SEQ ID NO: 116, and the Fc2 has an amino acid sequence as shown in SEQ ID NO: 117.

[0239] In another preferred example, the amino acid sequence of "HC2 (heavy chain)-HC1 (heavy chain)-Fc1" in the "Fab2-Fab1-Fc1" is as shown in SEQ ID NO: 132, the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 131, and the amino acid sequence of "LC2 (light chain)" is as shown in SEQ ID NO: 134.

[0240] 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.

[0241] In another preferred example, the multispecific antibody has a structure as shown in formula VII (for example Figure 7A in g):

[0242] Fab2-Fab1-Fc1

[0243] ║

[0244] scFv1-Fc2(VII)

[0245] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0246] Fab1 and scFv1 are the first targeting domains, the Fab1 is an anti-CCR8 Fab, and the scFv1 is an anti-CCR8 scFv;

[0247] Fab2 is the second targeting domain, and the Fab2 is an anti-VEGF Fab or an anti-PD-L1 Fab;

[0248] Fc1 and Fc2 are each independently an Fc fragment.

[0249] In another preferred example, the Fab2 is an anti-VEGF Fab.

[0250] In another preferred example, 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

[0251] 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.

[0252] In another preferred example, the anti-CCR8 scFv 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

[0253] 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.

[0254] In another preferred example, 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

[0255] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO:89-92, 149-150 and a light chain variable region as shown in any one of SEQ ID NO:95-102.

[0256] In another preferred example, 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

[0257] 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.

[0258] In another preferred example, the Fc fragment is derived from IgG1 or IgG4

[0259] In another preferred example, the Fc fragment is derived from IgG1.

[0260] In another preferred example, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO:113-118.

[0261] 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.

[0262] In another preferred example, in the "Fab2-Fab1-Fc1", the amino acid sequence of "HC2 (heavy chain)-HC1 (heavy chain)-Fc1" is shown in SEQ ID NO: 132, the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 131, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO: 134.

[0263] In another preferred example, the amino acid sequence of the "scFv1-Fc2" is shown in SEQ ID NO: 135.

[0264] In another preferred example, the multispecific antibody has the structure shown in Formula VIII (for example Figure 7A in h):

[0265] scFv2-Fab1-Fc1

[0266] ║

[0267] scFv2-Fab1-Fc1 (VIII)

[0268] In the formula, "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0269] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0270] scFv2 is the third targeting domain, and the scFv2 is an anti-PD-L1 scFv or an anti-VEGF scFv;

[0271] Fc1 is an Fc fragment.

[0272] In another preferred example, the "║" is a disulfide bond.

[0273] In another preferred example, the anti-CCR8 Fab comprises a heavy chain variable region shown in SEQ ID NO: 59 and a light chain variable region shown in SEQ ID NO: 73; or

[0274] the anti-CCR8 Fab comprises a heavy chain variable region shown in SEQ ID NO: 65 and a light chain variable region shown in SEQ ID NO: 79.

[0275] 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

[0276] 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.

[0277] 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

[0278] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO: 89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NO: 95-102.

[0279] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0280] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0281] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO: 113-118.

[0282] In another preferred embodiment, the Fc1 has an amino acid sequence as shown in SEQ ID NO: 113.

[0283] In another preferred embodiment, in the "scFv2-Fab1-Fc1", the amino acid sequence of "scFv2-HC1 (heavy chain)-Fc1" is as shown in SEQ ID NO: 136, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0284] In another preferred embodiment, the multispecific antibody has a structure as shown in Formula IX (for example Figure 7A in i):

[0285] Fab2-Fab1-Fc1

[0286] ║

[0287] Fab2-Fab1-Fc1 (IX)

[0288] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a linking bond between peptide chains;

[0289] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0290] Fab2 is the second targeting domain, and the Fab2 is an anti-PDL1 Fab or an anti-VEGF Fab;

[0291] Fc1 is an Fc fragment.

[0292] In another preferred example, the "║" is a disulfide bond.

[0293] In another preferred example, 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

[0294] 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.

[0295] In another preferred example, 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

[0296] 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.

[0297] In another preferred example, 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

[0298] the anti-VEGF Fab comprises a heavy chain variable region as shown in any one of SEQ ID NO:89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NO:95-102.

[0299] In another preferred example, the Fc fragment is derived from IgG1 or IgG4

[0300] In another preferred example, the Fc fragment is derived from IgG1.

[0301] In another preferred example, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO:113-118.

[0302] In another preferred example, the Fc1 has the amino acid sequence shown in SEQ ID NO: 114.

[0303] In another preferred example, in the "Fab2 - Fab1 - Fc1", the amino acid sequence of "HC2 (heavy chain) - HC1 (heavy chain) - 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.

[0304] In another preferred example, in the "Fab2 - Fab1 - Fc1", the amino acid sequence of "HC2 (heavy chain) - HC1 (heavy chain) - 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.

[0305] In another preferred example, the multispecific antibody has the structure shown in Formula X below (for example Figure 8A in a)):

[0306] Fab1 - Fc1 - scFv2

[0307] ║

[0308] Fab1 - Fc2 - scFv3(X)

[0309] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0310] Fab1 is the first targeting domain, and the Fab1 is an anti - CCR8 Fab;

[0311] scFv2 is the second targeting domain, and the scFv2 is an anti - VEGF scFv;

[0312] scFv3 is the third targeting domain, and the scFv3 is an anti - PD - L1 scFv;

[0313] Fc1 and Fc2 are each independently an Fc fragment.

[0314] In another preferred example, the anti - CCR8 Fab comprises a heavy - chain variable region shown in SEQ ID NO: 59 and a light - chain variable region shown in SEQ ID NO: 73; or

[0315] the anti - CCR8 Fab comprises a heavy - chain variable region shown in SEQ ID NO: 65 and a light - chain variable region shown in SEQ ID NO: 79.

[0316] 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

[0317] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO: 89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NO: 95-102.

[0318] 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

[0319] 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.

[0320] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0321] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0322] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO: 113-118.

[0323] In another preferred embodiment, the Fc1 fragment has an amino acid sequence as shown in SEQ ID NO: 115, and the Fc2 fragment has an amino acid sequence as shown in SEQ ID NO: 118.

[0324] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv2" in the "Fab1-Fc1-scFv2" is as shown in SEQ ID NO: 141, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0325] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc2-scFv3" in the "Fab1-Fc2-scFv3" is as shown in SEQ ID NO: 142, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0326] In another preferred embodiment, the multispecific antibody has a structure as shown in Formula XI (Figure 8A in b):

[0327] Fab1-Fc1-scFv3

[0328] ║

[0329] Fab2-Fc2-scFv3(XI)

[0330] wherein, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0331] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0332] Fab2 is the second targeting domain, and the Fab1 is an anti-VEGF Fab;

[0333] scFv3 is the third targeting domain, and the scFv3 is an anti-PD-L1 scFv;

[0334] Fc1 and Fc2 are each independently an Fc fragment.

[0335] In another preferred example, 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

[0336] 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.

[0337] In another preferred example, 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

[0338] the anti-VEGF Fab comprises a heavy chain variable region as shown in any one of SEQ ID NO: 89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NO: 95-102.

[0339] In another preferred example, 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

[0340] 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.

[0341] In another preferred example, the Fc fragment is derived from IgG1 or IgG4

[0342] In another preferred example, the Fc fragment is derived from IgG1.

[0343] In another preferred example, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO: 113 - 118.

[0344] In another preferred example, the Fc1 fragment has an amino acid sequence as shown in SEQ ID NO: 116, and the Fc2 fragment has an amino acid sequence as shown in SEQ ID NO: 118.

[0345] In another preferred example, the amino acid sequence of "HC1 (heavy chain) - Fc1 - scFv3" in the "Fab1 - Fc1 - scFv3" is as shown in SEQ ID NO: 144, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 143.

[0346] In another preferred example, the amino acid sequence of "HC1 (heavy chain) - Fc2 - scFv3" in the "Fab2 - Fc2 - scFv3" is as shown in SEQ ID NO: 145, and the amino acid sequence of "LC2 (light chain)" is as shown in SEQ ID NO: 134.

[0347] In another preferred example, the multispecific antibody has a structure as shown in Formula XII (for example Figure 8A in c):

[0348] Fab1 - Fc1 - scFv3

[0349] ║

[0350] scFv2 - Fc2 - scFv3 (XII)

[0351] In the formula, each "-" is independently a peptide bond or a linker peptide; "║" is a connecting bond between peptide chains;

[0352] Fab1 is the first targeting domain, and the Fab1 is an anti - CCR8 Fab;

[0353] scFv2 is the second targeting domain, and the scFv2 is an anti - VEGF scFv;

[0354] scFv3 is the third targeting domain, and the scFv3 is an anti - PD - L1 scFv;

[0355] Fc1 and Fc2 are each independently an Fc fragment.

[0356] 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

[0357] 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.

[0358] 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

[0359] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO:89-92, 149-150 and a light chain variable region as shown in any one of SEQ ID NO:95-102.

[0360] 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

[0361] 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.

[0362] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0363] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0364] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO:113-118.

[0365] In another preferred embodiment, the Fc1 fragment has an amino acid sequence as shown in SEQ ID NO:115, and the Fc2 fragment has an amino acid sequence as shown in SEQ ID NO:118.

[0366] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv3" in the "Fab1-Fc1-scFv3" is as shown in SEQ ID NO:142, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO:124.

[0367] In another preferred example, the amino acid sequence of the "scFv2-Fc2-scFv3" is as shown in SEQ ID NO: 146.

[0368] In another preferred example, the multispecific antibody has the structure shown in the following formula XIII (for example Figure 8A in d):

[0369] scFv2-Fab1-Fc1-scFv3

[0370] ║

[0371] scFv2-Fab1-Fc1-scFv3 (XIII)

[0372] In the formula, "-" is independently a peptide bond or a linker peptide; "║" is a linking bond between peptide chains;

[0373] Fab1 is the first targeting domain, and the Fab1 is an anti-CCR8 Fab;

[0374] scFv2 is the second targeting domain, and the scFv2 is an anti-VEGF scFv;

[0375] scFv3 is the second targeting domain, and the scFv3 is an anti-PD-L1 scFv;

[0376] Fc1 is an Fc fragment.

[0377] In another preferred example, the "║" is a disulfide bond.

[0378] In another preferred example, 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

[0379] 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.

[0380] In another preferred example, 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

[0381] the anti-VEGF scFv comprises a heavy chain variable region as shown in any one of SEQ ID NO: 89-92, 149-150, and a light chain variable region as shown in any one of SEQ ID NO: 95-102.

[0382] 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

[0383] 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.

[0384] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0385] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0386] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NO: 113-118.

[0387] In another preferred embodiment, the Fc1 fragment has an amino acid sequence as shown in SEQ ID NO: 113.

[0388] In another preferred embodiment, the amino acid sequence of "scFv2-HC1 (heavy chain)-Fc1-scFv3" in the "scFv2-Fab1-Fc1-scFv3" is as shown in SEQ ID NO: 147, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 124.

[0389] In a third aspect of the present invention, there is provided a polynucleotide encoding the anti-CCR8 antibody or an 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.

[0390] In a fourth aspect of the present invention, there is provided an expression vector comprising the polynucleotide as described in the third aspect of the present invention.

[0391] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0392] In a fifth aspect of the present invention, there is provided a host cell comprising 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 integrated into the genome.

[0393] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0394] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, HEK 293T cells, and CHO cells.

[0395] The sixth aspect of the present invention provides a use of an anti-CCR8 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a multispecific antibody as described in the second aspect of the present invention, for preparing a medicament for treating cancer / tumor.

[0396] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high CCR8 expression.

[0397] In another preferred embodiment, the cancer / tumor includes solid tumors and hematological tumors.

[0398] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0399] In another preferred embodiment, the cancer / tumor is selected from the group consisting of rectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or a combination thereof.

[0400] The seventh aspect of the present invention provides an immunoconjugate, the conjugate comprising:

[0401] (i) an anti-CCR8 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a multispecific antibody as described in the second aspect of the present invention; and

[0402] (ii) a conjugate moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0403] In another preferred embodiment, the conjugate is selected from: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, a prodrug-activating enzyme (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (for example, cisplatin), or any form of nanoparticles, etc.

[0404] The eighth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising: (a) an anti-CCR8 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a multispecific antibody as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0405] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0406] In a ninth aspect of the present invention, there is provided a method for treating cancer / tumor, comprising administering to a subject in need thereof the multispecific antibody described in the first aspect of the present invention.

[0407] In another preferred embodiment, the subject in need thereof is a human or non-human mammal.

[0408] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high CCR8 expression.

[0409] In another preferred embodiment, the cancer / tumor includes solid tumors and hematological tumors.

[0410] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0411] In another preferred embodiment, the cancer / tumor is selected from the group consisting of rectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or a combination thereof.

[0412] In a tenth aspect of the present invention, there is provided the use of the anti-CCR8 antibody or its antigen-binding fragment described in the first aspect, or the immunoconjugate described in the seventh aspect of the present invention, for preparing a detection reagent or kit for detecting CCR8 molecules in a sample.

[0413] In another preferred embodiment, the sample includes an ex vivo sample, such as an ex vivo tissue or cell sample.

[0414] In another preferred embodiment, the detection reagent or kit is used as a diagnostic reagent for diagnosing cancer / tumor with high CCR8 expression.

[0415] In an eleventh aspect of the present invention, there is provided an anti-VEGF antibody mutant, 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 further comprises a mutation capable of reducing the hydrophobicity of the antibody.

[0416] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs in the non-CDR3 region of the anti-VEGR antigen-binding domain having the heavy chain variable region shown in SEQ ID NO: 89 and the light chain variable region shown in SEQ ID NO: 95.

[0417] In another preferred embodiment, the mutation capable of reducing the hydrophobicity of the antibody occurs at the amino acid sites selected from the group consisting of the 28th, 30th, 31st, 32nd, 33rd, 35th sites, or a combination thereof in the heavy chain variable region shown in SEQ ID NO: 89.

[0418] In another preferred example, the mutations capable of reducing the hydrophobicity of the antibody occur at amino acid sites selected from the group consisting of: the 24th, 49th, 50th, 51st, 52nd, 53rd, 56th sites, or combinations thereof, in the light chain variable region as shown in SEQ ID NO: 95.

[0419] In another preferred example, the mutations capable of reducing the hydrophobicity of the antibody occur in the region of positions 46 - 57 in the light chain variable region as shown in SEQ ID NO: 95.

[0420] In another preferred example, the mutations capable of reducing the hydrophobicity of the antibody are mutations that mutate one or more (e.g., two, three, four) of the above amino acid sites to hydrophilic amino acids, such as aspartic acid (D), glutamic acid (E), lysine (K), or arginine (R).

[0421] In another preferred example, the mutations capable of reducing the hydrophobicity of the antibody occur 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).

[0422] In another preferred example, the mutations capable of reducing the hydrophobicity of the antibody occur 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).

[0423] In another preferred example, 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, 149 - 150.

[0424] In another preferred example, 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.

[0425] In another preferred example, the anti - VEGF antibody mutant comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 91, and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 95.

[0426] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:89, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:97.

[0427] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:89, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:99.

[0428] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:89, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:101.

[0429] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:91, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:97.

[0430] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:91, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:99.

[0431] In another preferred example, the anti-VEGF antibody mutant comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:91, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:101.

[0432] In another preferred example, relative to the original antibody (i.e., the anti-VEGF antibody having a heavy chain variable region amino acid sequence of SEQ ID NO:89 and a light chain variable region sequence of SEQ ID NO:95), the anti-VEGF antibody mutant has a significantly reduced antibody hydrophobicity and a significantly weakened antibody molecule aggregation tendency.

[0433] In another preferred example, the "significantly reduced antibody hydrophobicity" means that for 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.

[0434] In another preferred example, the anti-VEGF antibody mutant is used to construct a multi-specific antibody targeting VEGF, for example, the multi-specific antibody as described in the second aspect of the present invention.

[0435] 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 specifically described hereinafter (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 repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0436] Figure 1 Shows the FACS binding of the CCR8 hybridoma monoclonal antibody to the human CCR8 HEK293 cell line.

[0437] Figure 2 Shows the ADCC effect of the CCR8 recombinant monoclonal antibody.

[0438] Figure 3 Shows the blocking effect of the CCR8 recombinant monoclonal antibody on the binding of human CCR8 to human CCL1

[0439] Figure 4 Shows a schematic diagram of the surface hydrophobicity of the Fab structure of AS-1.

[0440] Figure 5 Shows the aggregation propensity scores of all amino acids of the scFv of AS-1.

[0441] Figure 6 Shows the peptide segments with aggregation propensity and high free energy in the Fab sequence of AS-1.

[0442] Figure 7A Shows nine bispecific antibody structural forms (a-i) composed of the CCR8 antigen-binding domain and the VEGF or PD-L1 antigen-binding domain;

[0443] 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), where IgG1 Fc forms a heterodimer through charge pair or knob and hole mutations; 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 antibody structure of the combination of anti-VEGF Fab (CH1-VH+CK-VL), 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 the combination of anti-VEGF or PDL1 Fab (CH1-VH+CK-VL), anti-CCR8 Fab (CK-VH+CH1-VL) and anti-VEGF or PDL1 Fab (CH1-VH+CK-VL), anti-CCR8 Fab (CK-VH+CH1-VL) (Fab-Fab-Fc×Fab-Fab-Fc); where IgG1 Fc forms a heterodimer through charge pair or knob and hole mutations; where IgG1 Fc forms a heterodimer through charge pair or knob and hole mutations, where IgG1 Fc forms a heterodimer through charge pair or knob and hole mutations.

[0444] Figure 7B shows Figure 7A An exemplary bispecific antibody structure of a in the figure is named 8As-1.

[0445] Figure 7C shows Figure 7AAn exemplary bispecific antibody structure of b, named 8As-2.

[0446] Figure 7D Shows Figure 7A An exemplary bispecific antibody structure of c, named 8As-3.

[0447] Figure 7E Shows Figure 7A An exemplary bispecific antibody structure of d, named 8As-4.

[0448] Figure 7F Shows Figure 7A An exemplary bispecific antibody structure of e, named 8As-5.

[0449] Figure 7G Shows Figure 7A An exemplary bispecific antibody structure of f, named 8As-6.

[0450] Figure 7H Shows Figure 7A An exemplary bispecific antibody structure of g, named 8As-7.

[0451] Figure 7I Shows Figure 7A An exemplary bispecific antibody structure of h, named Pl8-8.

[0452] Figure 7J Shows Figure 7A An exemplary bispecific antibody structure of i, named Pl8-9.

[0453] Figure 7K Shows Figure 7A An exemplary bispecific antibody structure of i, named 8As-9.

[0454] Figure 8A Shows three trispecific antibody structural forms composed of the CCR8 antigen-binding domain, the VEGF antigen-binding domain, and the PD-L1 antigen-binding domain;

[0455] Among them, a shows the molecular structure (IgG-scFv×IgG-scFv) formed by the anti-CCR8 antibody, the VEGF antibody, and the PDL1 antibody; b shows the molecular structure (Fab-Fc-scFv×Fab-Fc-scFv) formed by the anti-CCR8 antibody, the VEGF antibody, and the PDL1 antibody; c shows the molecular structure (IgG-scFv×scFv-Fc-scFv) formed by the anti-CCR8 antibody, the VEGF antibody, and the PDL1 antibody; d shows the molecular structure (scFv-IgG-scFv×scFv-IgG-scFv) formed by the anti-CCR8 antibody, the VEGF antibody, and the PDL1 antibody.

[0456] Figure 8B shows Figure 8A An exemplary trispecific antibody structure of a in [reference], named 8AsPl-1.

[0457] Figure 8C shows Figure 8A An exemplary trispecific antibody structure of b in [reference], named 8AsPl-2.

[0458] Figure 8D shows Figure 8A An exemplary trispecific antibody structure of c in [reference], named 8AsPl-3.

[0459] Figure 8E shows Figure 8A An exemplary trispecific antibody structure of d in [reference], named 8AsPl-4.

[0460] Figure 9 shows the ADCC effect of the monoclonal antibody and multispecific antibody of the present invention.

[0461] Figure 10 shows the VEGF blocking effect of the multispecific antibody of the present invention.

[0462] Figure 11 shows the PD-L1 blocking effect of the multispecific antibody of the present invention.

[0463] Figure 12 shows the in vivo efficacy of the anti-CCR8 monoclonal antibody of the present invention in a mouse tumor model.

[0464] Figure 13 shows the in vivo efficacy of the anti-CCR8 bispecific antibody of the present invention in a mouse tumor model. Detailed implementation manners

[0465] After extensive and in-depth research, the present inventors unexpectedly developed for the first time a class of multi-specific antibodies comprising a CCR8 antigen-binding domain. The multi-specific antibody contains a first targeting domain that targets the chemokine (C-C motif) receptor 8 (CCR8) molecule highly expressed on the surface of tumor-infiltrating regulatory T cells. The first targeting domain is a CCR8 antibody or an antigen-binding fragment thereof, and also contains a second targeting domain and / or a third targeting domain that binds to VEGF and / or PD-L1. The multi-specific antibody of the present invention can simultaneously bind to Treg cells, tumor cells, and free VEGF molecules, and can be used as an effective therapeutic agent for tumor treatment.

[0466] Based on this, the present invention was completed.

[0467] As used herein, the term "chemokine (C-C motif) receptor 8" or "CCR8" refers to the protein encoded by the CCR8 gene in humans. CCR8 is highly expressed on many tumor-infiltrating Treg cells, while showing low or no expression in Treg cells in the thymus, spleen, and peripheral blood.

[0468] 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. It can mediate the continuous formation of new vascular systems inside and around tumors. The structural and functional abnormalities of tumor blood vessels formed under the action of VEGF will lead to poor tumor bleeding and hypoxia, thus further generating more VEGF. The key role of VEGF in tumor angiogenesis makes it a well-known target for anti-tumor treatment.

[0469] PD-1 / PD-L1 is an important target in tumor immunotherapy (IO). Since 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 the killing of tumor cells by T cells.

[0470] The term "Fc fragment" or "Fc" refers to the portion of an antibody that does not have antigen - binding activity but was originally observed to be readily crystallizable and was thus named the Fc fragment (for fragment crystallizable). This fragment corresponds to paired CH2 and CH3 domains and is the part 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 a particular use, a particular IgG subclass may be preferred. For example, IgG1 is more effective than IgG2 and IgG4 in mediating ADCC and CDC. Thus, when effector function is not desired, IgG2 Fc may be preferred. However, molecules containing IgG2 Fc are generally more difficult to prepare and may be less stable than 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).

[0471] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric proteins of approximately 150,000 daltons having the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra - chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a number of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; 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.

[0472] As used herein, the term "variable" indicates that certain portions of the variable regions in an antibody differ in sequence and that this forms the binding and specificity of various specific antibodies for their particular antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity - determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable regions are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which generally assume a β - sheet configuration and are connected by three CDRs that form loops and in some cases form part of a β - sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen - binding site of the antibody (see Kabat et al., NIH Publ. No. 91 - 3242, Vol. I, pp. 647 - 669 (1991)). The constant regions do not directly participate in antibody - antigen binding, but they exhibit different effector functions, such as participating in antibody - dependent cytotoxicity.

[0473] The antibodies of the present application can include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, bispecific, human, humanized, primatized, chimeric, and single-chain antibodies. The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, the antibodies are of human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken origin.

[0474] 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 containing VL or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. Regardless of 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 containing immunoglobulin variable regions 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 domain regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility and serine or threonine for solubility, and can link the N-terminus of VH or the C-terminus of VL, and vice versa. Although the constant regions are removed and a linker is introduced, such a protein still retains the specificity of the original immunoglobulin. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000 - 70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chains bracket the heavy chains from the mouth of the "Y" and extend through the variable regions.

[0475] As described above, the variable regions allow antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH domains of an antibody or subsets of the complementarity-determining regions (CDRs) of an antibody bind to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites present at the termini 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, certain immunoglobulin molecules are derived from camelid species or are engineered based on camelid immunoglobulins. Alternatively, an immunoglobulin molecule can consist of only a heavy chain without a light chain or only a light chain without a heavy chain.

[0476] In a naturally occurring antibody, the six CDRs present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form an "antigen-binding domain" as the antibody presents its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domain, called the "framework" region domains, exhibit less intermolecular variability. The framework regions predominantly adopt a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. Thus, the framework regions serve to form a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. Since they have been precisely defined, one of ordinary skill in the art can readily identify the amino acids that comprise the CDRs and framework regions for any given heavy or light chain variable region.

[0477] 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 the constant κ domain or the constant λ domain.

[0478] As used herein, the term "heavy chain constant region (CH)" includes the 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., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or variants or fragments thereof. It should be understood that the heavy chain constant regions can be modified such that their amino acid sequences are different from those of naturally occurring immunoglobulin molecules.

[0479] In one embodiment of the present invention, the prepared multispecific antibody comprises a CrossMab structure, that is, the heavy chain CH1 and the light chain CL exchange partial amino acid sequences to prevent mismatching. Such a structure comprises CH1 as shown in SEQ ID NO: 112 and CL as shown in SEQ ID NO: 113.

[0480] 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 with 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 should be understood that in cases where sequence identity is expressed in the form of "at least x% identical" or "at least x% identity", such embodiments include any and all numerical percentages equal to or higher than the lower limit. In addition, it should be understood that in the case of an amino acid sequence present in the present application, it should be interpreted as additionally disclosing or including an amino acid sequence having 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% identity.

[0481] Within the scope of the multispecific molecules of the present invention, various compositions and methods are included, and these compositions and methods include: asymmetric IgG-like antibodies (e.g., trifunctional monoclonal antibodies / quadromas); knobs-into-holes antibodies; Cross MAb; electrostatic matching antibodies; LUZ-Y; strand exchange engineered domain (SEED) bodies; Fab-exchanged antibodies, symmetric IgG-class antibodies; diabodies; cross-linked monoclonal antibodies, mAb2; Cov X-body; dual variable domain (DVD)-Ig fusion proteins; IgG-like bispecific antibodies; Ts2Ab; BsAb; scFv / Fc fusions; bis(scFv)2-Fabs; F(ab)2 fusion proteins; bis-acting 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.

[0482] 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, the multispecific antibody is a polypeptide comprising a scaffold and two or more immunoglobulin antigen-binding domains targeting different antigens or epitopes. In some embodiments, the multispecific antibody is a bispecific antibody. In some other embodiments, the multispecific antibody is a trispecific antibody.

[0483] 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 inhibiting the biological function of the target molecule upon binding to the target molecule. In some embodiments, the bispecific antibody is a polymeric molecule having two or more peptides. In some embodiments, the targeting domain comprises the antigen-binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or a fragment thereof that specifically binds to a target protein.

[0484] The terms "bispecific antibody", "bispecific molecule" and "bispecific" are used interchangeably herein and refer to an antibody that can specifically bind two different antigens (or epitopes). In some embodiments, the bispecific antibody is a full-length antibody that binds one antigen (or epitope) on one of its two binding arms (a pair of HC / LC) and a different antigen (or epitope) on its second arm (the other pair of HC / LC). In these embodiments, the bispecific antibody has two different antigen-binding arms (both in terms of specificity and CDR sequence) and is monovalent for each antigen it binds.

[0485] In some other embodiments, the bispecific antibody is a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (two pairs of HC / LC). In these embodiments, the bispecific antibody has two identical antigen-binding arms with the same specificity and the same CDR sequence and is bivalent for each antigen it binds.

[0486] The terms "trispecific antibody", "trispecific molecule" and "trispecific" are used interchangeably herein and refer to a molecule comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting the biological function of the target molecule upon binding to the target molecule. In some embodiments, the trispecific antagonist is a polymeric molecule having two or more peptides. In some embodiments, the targeting domain comprises the antigen-binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or a fragment thereof that specifically binds to a target protein.

[0487] In a preferred embodiment of the present invention, a bispecific antibody targeting CCR8 and VEGF / PD-L1 was constructed, having the structures shown in a-i of Figure 7A , and the exemplary molecules of the a-i structures are respectively as shown in Figure 7B -K. The anti-CCR8 Fab / anti-CCR8 scFv therein can be a Fab / scFv constructed using the VH and VL of any anti-CCR8 antibody described in the present invention; the anti-VEGF Fab / anti-VEGF scFv can be a Fab / scFv constructed using the VH and VL of any anti-VEGF antibody described in the present invention; 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 in the present invention.

[0488] In another preferred embodiment of the present invention, a trispecific antibody targeting CCR8, VEGF and PD-L1 was constructed, having the structures shown in a-d of Figure 8A , and the exemplary molecules of the a-d structures are respectively as shown in Figure 8B -E. The anti-CCR8 Fab therein can be a Fab constructed using the VH and VL of any anti-CCR8 antibody described in the present invention; the anti-VEGF Fab / anti-VEGF scFv can be a Fab / scFv constructed using the VH and VL of any anti-VEGF antibody described in the present invention; the anti-PD-L1 scFv can be a scFv constructed using the VH and VL of any anti-PD-L1 antibody described in the present invention.

[0489] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as or a degenerate variant of the coding region sequence of the antibody of the present invention. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but having a different coding region sequence.

[0490] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optionally additional coding sequences) and non-coding sequences.

[0491] The term "polynucleotide encoding a polypeptide" can be a polynucleotide including the polynucleotide encoding this polypeptide, or can also be a polynucleotide further including additional coding and / or non-coding sequences.

[0492] The present invention also relates to polynucleotides that hybridize to the above-described sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher 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, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more.

[0493] The full-length nucleotide sequence or fragment thereof of the antibody of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis methods. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Usually, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0494] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually done by cloning them into a vector, then transferring them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0495] Currently, it is already possible to completely obtain the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) by chemical synthesis. Then, this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0496] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0497] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0498] Transformation of a host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0499] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell. After the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0500] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, 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 refolding treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, 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.

[0501] The antibody of the present invention can be used alone or conjugated or coupled with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination of any of the above substances.

[0502] 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 detectable products.

[0503] Therapeutic agents that can be conjugated include, but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, enteropeptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0504] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances being formulated and the disease 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.

[0505] The pharmaceutical composition of the present invention can be used for the treatment of cancer / tumor, especially solid tumors, particularly solid tumors with high expression of LCRR15.

[0506] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration route. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight - about 10 mg / kg body weight per day. In addition, the pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents.

[0507] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually 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 - about 1 mg / kg body weight. Of course, the specific dosage should also consider factors such as the administration route and the patient's health condition, which are within the scope of the skills of a skilled physician.

[0508] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0509] Sequences used for preparing the multispecific antibodies of the present invention

[0510] CCR8 antibody sequence

[0511]

[0512]

[0513] CCR8 antibody sequence

[0514]

[0515]

[0516]

[0517] VEGF antibody sequence

[0518]

[0519]

[0520] Among them, AVACH and AVACL are mutants of the heavy chain variable region AVAH and the light chain variable region AVAL of the antibody molecule AVA respectively; the heavy chain variable region ASH and the light chain variable region ASL of the antibody molecule AS-1 are derived from US Patent US7758859, and ASCH, AS2H, AS2CH, AS3H, AS3CH are mutants of its VH, and ASCL, AS2L, AS2CL, AS3L, AS3CL, AS4L, AS4CL are mutants of its VL. Among them, the underlined parts are CDR1, CDR2, and CDR3 of the heavy chain variable region or the light chain variable region in turn, and the mutation sites are marked in bold.

[0521] PD-L1 antibody sequence

[0522]

[0523]

[0524] Among them, PL1CH and PL1CL are mutants of the heavy chain variable region PL1H and the light chain variable region PL1L of the antibody molecule PL1 respectively; PL2CH and PL2CL are mutants of the heavy chain variable region PL2H and the light chain variable region PL2L of the antibody molecule PL2 respectively.

[0525] IgG1 CH sequence

[0526] The CH1 sequence is shown as follows:

[0527]

[0528] The Fc region sequence is as follows:

[0529]

[0530] The Fc region may contain the following mutations:

[0531]

[0532] In a preferred embodiment of the present invention, the Fc region has the following sequence:

[0533]

[0534]

[0535] In another preferred embodiment of the present invention, the lysine (K) at the C-terminus of the above Fc region is removed, thereby reducing the formation of antibody charge isomers.

[0536] IgG4 CH sequence

[0537]

[0538] The Fc region may contain the following mutations:

[0539]

[0540] CL sequence

[0541]

[0542]

[0543] Optional linker sequence

[0544] G GS SS GSS GSSSG GGGGS GGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS PGGGGSP PGGGGSPGGGGSPGGGGSP GEPGSGE GEPGSGEGEPGSGE GEPGSGEGEPGSGEEGEPGSGE EGEPGSGEEGEPGSGEEGEPGSGEEGEPGSGE GKPGS GKPGSGKPGS GKPGSGKPGSGKPGS GKPGSGKPGSGKPGSGKPGS GKPGSGKPGSGKPGSGKPGSGKPGS SSSSG SSSSGSSSSG SSSSGSSSSGSSSSG SSSSGSSSSGSSSSGSSSSG GRPGSGPGSGRPGSGRPGS GRPGSGPGSGRPGSGRPGSGRGPS GKPGSGRPGSGKGPSGRPGS

[0545] Example 1 Preparation and Verification of Anti-CCR8 Monoclonal Antibody

[0546] 1.1 Screening of anti-CCR8 monoclonal antibodies

[0547] The CCR8 antibody of the present invention was screened from humanized mouse immunized hybridomas. The hCCR8 expression plasmid was constructed, DNA was extracted and endotoxin was removed, and humanized mice with variable regions of heavy and light chains (derived from CN114763558A) were immunized. The spleens of the immunized mice were selected, and splenocytes were isolated and fused with Sp2 / 0-Ag14 multiple myeloma cells. The fusion was carried out by electroporation according to the hybridoma fusion technology method. After fusion, a certain number of cells were seeded in each well of a 96-well plate and cultured in HAT medium for 10 days. The culture supernatant of hybridoma cells was detected with the HEK293-CCR8 overexpression cell line to obtain positive hybridoma cells. The positive hybridoma cells were cultured, and the supernatant of the cell culture on the 5th day was taken, and the antibody was slightly purified by Protein A magnetic beads to obtain a hybridoma antibody with a human variable region and a murine constant region. The binding activity function of the purified antibody was tested. During the screening process, specific binding molecules were screened against the extracellular region of CCR8 or the CCR8 overexpressing cell line by ELISA and FACS methods, and finally 14 monoclonal antibodies (C5, C6, C20, C23, C24, C27, C39, C40, C46, C53, C54, C57, C61 and C62) were obtained. These 14 antibodies can be divided into 5 categories by sequence analysis. Among them, C24, C39, and C57 are each classified into a separate category, C5 and C27 are in one category, and the other antibodies are in one category.

[0548] 1.2 Binding ability of anti-CCR8 monoclonal antibodies to HEK cells expressing CCR8

[0549] Furthermore, the binding ability of the CCR8 antibody of the present invention and HEK293 cells expressing CCR8 was tested. Among them, the HEK293 cells expressing CCR8 were prepared by the following method: the full-length sequence gene fragments of CCR8 of human, mouse, and cynomolgus monkey were obtained by gene synthesis, and then these fragments were inserted into the backbone of a stable expression vector to obtain an expression plasmid vector. 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, HEK293-cCCR8 cells) were screened under the pressure of puromycin, which were the flow cytometry detection cell lines used in this example.

[0550] Using the above HEK293-hCCR8 cells, 1x10 was plated in each well of a 96-well plate 5Cells, the antibody was diluted to an initial concentration of 1 μg / ml and serially diluted 2-fold. 50 μl of the diluted antibody was mixed with the cells respectively and incubated at 4°C for 45 minutes. The cells were washed twice with the washing buffer. After pouring off the liquid, all the cells were resuspended in 50 μl of Goat anti-Mouse IgG PE at a concentration of 200 ng / ml and incubated at 4°C in the dark for 30 minutes. The cells were washed three times with the washing buffer. After pouring off the liquid, all the cells were resuspended in 25 μl of the dilution buffer. Detection was performed using a flow cytometer. The FACS binding data of the 14 monoclonal antibodies obtained were as Figure 1 .

[0551] The results showed that the Emax and EC 50 of antibodies C20, C24, C46, C53, C54, C61, C62, C40, C6, and C23 were all superior to those of BM-1 (the antibody sequence was derived from WO2021194942A1); the EC 50 of C57 and C27 was superior to that of BM-1; the Emax of C5 was superior to that of BM-1.

[0552] The antibody sequences of these positive hybridoma clones were sequenced, the antibody sequences were cloned into a human IgG1 expression vector, and antibody expression was performed using FUT8- / - CHO cells. After culturing the cells, the supernatant was centrifuged to remove the precipitate, the supernatant was filtered using a 0.22 μm filter membrane, and the supernatant was purified using a protein purification instrument to obtain afucosylated human IgG1 Fc antibody, that is, recombinant human monoclonal antibody.

[0553] 1.3 Binding affinity of anti-CCR8 recombinant humanized monoclonal antibodies to CCR8 protein

[0554] Affinity tests were performed on 14 recombinant human monoclonal antibodies as follows:

[0555] The antibody was diluted to 5 μg / ml, and the antigens human CCR8(1-35aa)-mFc or cynomolgus monkey 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, 15.6 nM respectively. Wells with a concentration of 0 were set as the baseline. Antibody affinity detection was performed using a Gator instrument.

[0556] The results are shown in Table 2. The affinity of the candidate antibodies for human CCR8 was in the range of 0.0168 - 1.24 nM (Table 1). Multiple candidate antibodies could bind to cynomolgus monkey CCR8 (Table 2).

[0557] Table 1. Binding affinity of antibodies for human CCR8

[0558] Antibody name KD(M) C5 1.78E-10 C6 1.24E-09 C20 3.35E-10 C23 7.05E-10 C24 1.68E-11 C27 4.39E-10 C40 8.08E-10 C46 1.02E-09 C53 5.36E-10 C54 3.80E-10 C61 4.44E-10 C62 3.69E-10 C39 NA C57 NA

[0559] Table 2. Binding of Antibodies to Cynomolgus Macaque CCR8

[0560]

[0561]

[0562] 1.4 ADCC activity of anti-CCR8 recombinant humanized monoclonal antibodies

[0563] The ADCC activities of 14 anti-CCR8 recombinant human monoclonal antibodies were detected as follows:

[0564] CHO-K1 cells overexpressing CCR8 were plated at 2×10 4 cells per well and cultured overnight. The medium was removed and 25 μl of complete medium was newly added. Antibody diluents were prepared with complete 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. 25 μl of each antibody diluent was added to each well. The density of Jurkat-FcγRIIIa-V158 effector cells was maintained between 0.2 - 1.0×10 6 cells / ml. The effector cells were resuspended in medium to a cell density of 6×10 6 cells / ml. 25 μl of the cell suspension was added to each well in a 96-well white plate, and the final total volume in each sample well was 75 μl. The cells were cultured in an incubator for 6 hours. Luciferase substrate was added to the plate, 50 μl / well, and mixed for 30 seconds. Detection was performed using a multifunctional microplate reader in the Lum-TM channel.

[0565] The results are as Figure 2 shown. All candidate antibodies had varying degrees of ADCC against the target cells; among them, C5 had a relatively minimum EC 50 , and C61 had a relatively highest signal value, both having good ADCC effects.

[0566] 1.5 Blocking effect of anti-CCR8 recombinant humanized monoclonal antibodies on the binding of CCR8 and its ligand CCL1

[0567] The blocking effects of 14 anti-CCR8 recombinant human monoclonal antibodies on the binding of CCR8 and its ligand CCL1 were tested as follows:

[0568] The blocking experiment used a kit of DiscoverX's β-Arrestin eXpress GPCR Assay. Add 11.5 ml of Cell Plating Reagente22 to a 15-ml centrifuge tube. Add 0.5 ml of Cell Plating Reagent to the cryotube. After the PathHunter eXpressβ-Arrestin GPCR cells thaw, transfer them all to the above 15-ml centrifuge tube, mix well, add 100 μl to each well, place in the cell culture incubator, and incubate for 48 hours. Prepare a starting concentration of 220,000 ng / ml (22× the final concentration), and perform three-fold dilutions for a total of 8 gradients. Add 5 μl of antibody diluent to each well of the white plate, and add complete medium to the remaining wells. Place in the cell culture incubator and incubate for 30 minutes. Prepare 88 nM of CCL1 (22 times the final concentration, the final concentration of CCL1 is 4 nM), add 5 μl to each well, place in the 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.

[0569] The results are as Figure 3 shown. The tested antibodies all have a certain blocking effect on the binding of CCL1 to CCR8. The antibodies with a blocking effect > 90% are C5, C20, C23, C57, and C61, and the blocking percentage values are 96.83%, 90.08%, 91.39%, 92.46% respectively, and their IC 50 values are: 496.8, 377.4, 216.5, 183.4 ng / ml respectively.

[0570] Example 2 Preparation and Verification of Anti-VEGF Mutants

[0571] The AS-1 antibody (heavy chain variable region is SEQ ID NO: 89, light chain variable region is SEQ ID NO: 95) itself has strong hydrophobicity and a tendency to aggregate. In order to improve the quality of the antibody itself and facilitate the subsequent application of this antibody sequence to bispecific or trispecific antibody structures, the inventors designed a series of mutations.

[0572] The hydrophobic regions on the surface of the Fab structure of AS-1 (PDB: 2FJG) were marked with the Color_h.py code on the PyMOL Wiki. The higher the hydrophobicity, the darker the color, showing grayish-black. The lower the hydrophobicity, the lighter the color, showing grayish-white. See Figure 4. Generally speaking, a high hydrophobicity of the protein sequence is likely to cause aggregation and reduce protein stability. In addition, to further confirm the hydrophobicity of the relevant amino acids on the antibody surface after conversion into scFv, the inventors of the present invention predicted the scFv structure of AS-1 using AlphaFold (the scFv sequence is the same as the scFv sequence of AS-1 used in the bispecific and trispecific antibody molecules of the present invention) and analyzed it using the Aggrescan server, as Figure 5 shown. All amino acids with a score greater than 0 are hydrophobic, which makes the antibody tend to aggregate. The present invention has 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 sites.

[0573] In addition, to simultaneously analyze the aggregation tendency and thermal stability of the AS-1 sequence, the inventors of the present invention analyzed the regions in the variable region sequence of the AS-1 structure that are prone to aggregation (high TANGO score) and have poor thermal stability (high free energy ΔG) using the SolubiS server. As Figure 6 shown, 6 peptide segments were identified. Among them, the L46-S57 region of the light chain has the highest aggregation tendency and the lowest thermal stability. In addition, it has also been reported in the literature that amino acid mutations at positions 24, 49, 50, 51, 52, 53, and 56 of the light chain and positions 28, 30, 31, 32, 33, and 35 of the heavy chain can effectively improve the aggregation of the antibody (Dudgeon et al, 2012, PNAS).

[0574] In view of the above structural and sequence analysis, the present invention mutated some of the amino acids in the main L46-S57 region and other regions (such as hydrophobic sites in the structure or reported aggregation-prone sites) to aspartic acid (D) to reduce hydrophobicity. In addition, it can be known from the structure diagram published on PDB of 2FJG that the VH-CDR3 region plays an important role in binding to VEGF and many amino acids are buried inside. Therefore, the present invention mainly mutated the non-VH-CDR3 region and other regions not related to VEGF binding to maintain the binding activity of the antibody to VEGF. In this example, 7 mutants of AS-1 were obtained by site-directed mutagenesis.

[0575] To confirm whether these mutations altered the hydrophobicity of the antibody, the retention times of the mutant antibodies were differentiated using a Proteomix HIC Butyl-NP5 column. After loading each mutant of the antibody onto the column in a solution of 1.5 M NaCl and 25 mM Na3PO4 at pH 7.4, it was eluted with solutions of gradually decreasing salt concentration at pH 7.4. The retention times of the mutants are shown in Table 3. It can be seen that AS-1 had the longest retention time, indicating the strongest hydrophobicity. The retention times of the other mutants all decreased. For some combinatorial mutant antibodies, such as AS-6, etc., the retention time could be reduced by nearly half, greatly reducing the hydrophobicity of the antibody and its possible tendency to aggregate.

[0576] Table 3

[0577]

[0578]

[0579] To further confirm that the binding of the mutants to VEGF was not affected, affinity assays were performed on each mutant. Human VEGF protein with a his tag was loaded onto an anti-his BLI probe. The mutants were incubated with VEGF in a solution containing 1X PBS, 0.1% BSA, 0.02% Tween-20, and 0.05% sodium azide for 150 s and then dissociated for 300 s, and the KD values were calculated as shown in Table 4 below. It can be seen from Table 4 that only the affinities of mutants AS-3, AS-6, and AS-8 for binding to VEGF decreased significantly, while the affinities of the other mutants were maintained well compared to AS-1.

[0580] Table 4

[0581] Mutant KD(M) AS-1 5.15E-11 AS-2 3.29E-11 AS-3 3.23E-10 AS-4 5.14E-11 AS-5 8.53E-11 AS-6 1.56E-10 AS-7 5.37E-11 AS-8 4.61E-10

[0582] Example 3 Preparation of Multispecific Antibody Molecules

[0583] Based on the 14 different CCR8 antibody sequences obtained in Example 1, a variety of multispecific antibody molecules were designed.

[0584] The molecular sequence fragments of the multispecific antibody were obtained by total gene synthesis, and then the target sequence was inserted into the expression vector by conventional gene cloning techniques (see, for example, Lo.B.K.C methods in Molecular Biology. Volume 248, 2004. Antibody Engineering).

[0585] Transfect HEK293E cells with a vector carrying a multi-specific antibody molecule. Culture for 7 days at 37°C with 5% CO2, and the corresponding molecule can be produced in F17 medium (1L F17 + 10mL 10% PF68 + 30ml 200mM L-glutamine). After the expression ends, harvest the supernatant and purify it to obtain the multi-specific antibody molecule, which can be used for various experimental analyses subsequently.

[0586] The anti-VEGF sequence is derived from the molecule AVA (the heavy chain variable region is SEQ ID NO:87, and the light chain variable region is SEQ ID NO:93) and mutants of the VEGF antibody variable region and its CDR regions in Patent US7758859. The anti-PDL1 sequence is derived from the molecule PL1 (the heavy chain variable region is SEQ ID NO:103, and the light chain variable region is SEQ ID NO:107) or PL2 (the heavy chain variable region is SEQ ID NO:105, and the light chain variable region is SEQ ID NO:109).

[0587] Using the above method, the following multi-specific molecules were prepared:

[0588] (1) A bispecific antibody composed of the CCR8 antigen-binding domain with the structure shown in a-i in Figure 7A and the VEGF or PD-L1 antigen-binding domain;

[0589] Figure 7A A bispecific antibody with the structure of a in Figure 7B is named 8As-1, and its structure is as shown in

[0590] A bispecific antibody with the structure of b is named 8As-2, and its structure is as shown in Figure 7C ;

[0591] A bispecific antibody with the structure of c is named 8As-3, and its structure is as shown in Figure 7D ;

[0592] A bispecific antibody with the structure of d is named 8As-4, and its structure is as shown in Figure 7E ;

[0593] A bispecific antibody with the structure of e is named 8As-5, and its structure is as shown in Figure 7F ;

[0594] A bispecific antibody with the structure of f is named 8As-6, and its structure is as shown in Figure 7G ;

[0595] A bispecific antibody with the structure of g is named 8As-7, and its structure is as shown in Figure 7H ;

[0596] A bispecific antibody with an h structure is named Pl8-8, and its structure is as shown in Figure 7I ;

[0597] A bispecific antibody with an i structure is named Pl8-9, and its structure is as shown in Figure 7J ; Another bispecific antibody with an i structure is named 8As-9, and its structure is as shown in Figure 7K ; The difference between it and Pl8-9 is that the VH and VL against PD-L1 are replaced with VH and VL against VEGF.

[0598] (2) A trispecific antibody composed of the CCR8 antigen-binding domain, the VEGF antigen-binding domain, and the PD-L1 antigen-binding domain as shown in a-d of Figure 8A .

[0599] Figure 8A A bispecific antibody with the a structure in Figure 8B is named 8AsPl-1, and its structure is as shown in

[0600] ; A bispecific antibody with the b structure is named 8AsPl-2, and its structure is as shown in Figure 8C ;

[0601] A bispecific antibody with the c structure is named 8AsPl-3, and its structure is as shown in Figure 8D ;

[0602] A bispecific antibody with the d structure is named 8AsPl-4 or 8AsPl-4v, and its structure is as shown in Figure 8E ;

[0603] The exemplary molecules prepared in this example are as follows, where the VH and VL of C61 and / or C27 are used for the CCR8 antigen-binding domain:

[0604] Molecular name Chain 1 (SEQ ID NO:) Chain 2 (SEQ ID NO:) Chain 3 (SEQ ID NO:) Chain 4 (SEQ ID NO:) 8As-1 124 125 126 127 8As-2 124 128 / / 8As-3 124 128 125 / 8As-4 124 129 125 / 8As-5 124 130 125 / 8As-6 131 132 133 134 8As-7 131 132 135 134 Pl8-8 124 136 / / Pl8-9 137 138 139 / 8As-9 137 140 134 / 8AsPl-1 124 141 142 / 8AsPl-2 143 144 145 134 8AsPl-3 124 142 146 / 8AsPl-4 124 147 / / 8AsPl-4v 124 148

[0605] The structure of the 8AsPl-4v molecule is the same as that of 8AsPl-4, except that the VEGF antigen-binding domain therein contains 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.

[0606] Example 4 Performance Detection of CCR8- and VEGF / PD-L1-Targeted Multispecific Antibody Molecules

[0607] 4.1 ADCC experiment of anti-CCR8 and VEGF / PDL1 multispecific antibody molecules

[0608] The ADCC effects of the target CCR8 and VEGF / PD-L1 multi-specific antibody molecules (8As-1, 8As-2, 8As-4, 8As-7, 8As-9, Pl8-8, Pl8-9, 8AsPl-1, 8AsPl-3, 8AsPl-4) prepared in Example 2 were measured, and some of the antibodies were expressed using Fut8-knockout CHO cells (FKO). The specific method is as follows:

[0609] Collect CHOK1-hCCR8 cells (human CCR8 overexpressing cells, obtained by transfecting CHOK1 cells with a full-length CCR8 expression plasmid, and the preparation method is the same as that of HEK293-hCCR8 cells described in Example 1.2) in the logarithmic growth phase, and centrifuge at 300g for 5 min. Resuspend the cells with 1 ml of assay buffer (1640 medium + 10% FBS), count the cells, and adjust the cell density to 1E6 / ml with assay buffer. After mixing evenly, inoculate the CHOK1-hCCR8 cell suspension into a 96-well white plate, 25 μl / well. Add the gradient-diluted antibodies to the above 96-well white plate in sequence with assay buffer, 25 μl / well, and set up duplicate wells. Incubate in a CO2 incubator for 30 min. Finally, collect the well-grown ADCC Bioassay Effector Cell V Variant (High Affinity) / NFAT Luciferase Reporter Jurkat Cell Line into a 50-ml centrifuge tube, and centrifuge at 300g for 5 min. Take 3 ml of assay buffer (1640 medium + 10% FBS) to resuspend the cells, count the cells and adjust the cell density to 3E6 / ml. After mixing evenly, add it to the above 96-well white plate, 50 μl / well. Incubate in a CO2 incubator for 5 h. Take out the 96-well white plate, add BRITELITE PLUS reagent, 100 μl / well. Incubate at room temperature in the dark for 5 min, and detect the lumi reading with a microplate reader. Plot the graph using prism software and calculate the EC 50 value.

[0610] Figure 9 The results showed that the ADCC effect of the antibodies expressed using Fut8-knockout CHO cells (with FKO after the name) was significantly higher than that of the same antibody expressed using wild-type CHO cells ( Figure 9 of e). Bispecific antibodies with various structures all showed strong ADCC effects ( Figure 9of a-d). Among them, 8As-1, 8As-4, Pl8-8, and Pl8-9 are similar to monoclonal antibodies in terms of the ADCC effect on the CCR8 target, and their bispecific antibody structures do not affect the exertion of the ADCC effect. For the trispecific molecule 8AsPl-4, while showing good ADCC against CCR8, the ADCC effect on PD-L1 is very weak ( Figure 9 of f and g).

[0611] 4.2 VEGF Blocking Experiment of the CCR8- and VEGF / PD-L1-Targeting Multispecific Antibody Molecule

[0612] The ability of multi-specific antibody molecules targeting CCR8 and VEGF / PD-L1 (8As-1, 8As-2, 8As-4, 8As-9, 8AsPl-1, 8AsPl-3, 8AsPl-4) to block the VEGF signal was measured, with the AS-1 molecule as a control sample. The specific method is as follows:

[0613] Collect VEGFR2 / NFAT Reporter–HEK293 Recombinant cells in the logarithmic growth phase, centrifuge at 300g for 5 min. Resuspend the cells with 1 ml of assay buffer (MEM / EBSS medium + 10% FBS), count the cells and adjust the cell density to 6E5 / ml with assay buffer. After mixing evenly, inoculate the VEGFR2 / NFAT Reporter–HEK293 Recombinant Cell Line cell suspension into a 96-well white plate, 50 μl / well. Add the antibody diluted in gradient with assay buffer to the above 96-well white plate in turn, 25 μl / well, and set up double replicates. Then, dilute the human VEGF165 his Tag protein to 50 ng / ml with assay buffer and transfer it to the 96-well white plate at 25 μl / well. Incubate in a CO2 incubator for 4 h. Finally, take out the 96-well white plate, add BRITELITE PLUS reagent, 100 μl / well. Incubate at room temperature in the dark for 5 min, and detect the lumi reading with an enzyme-linked immunosorbent assay (ELISA) reader. Plot the graph with prism software and calculate the IC 50 value.

[0614] The results are shown as Figure 10 shown. Figure 10 a and b of show that the difference in the blocking effect of each bispecific antibody molecule on VEGF is very small, while for the trispecific molecule ( Figure 10 of c), 8AsPl-4 shows a good blocking effect, similar to that of the monoclonal antibody molecule.

[0615] 4.3 PD-L1 Blocking Experiment of the CCR8- and VEGF / PD-L1-Targeting Multispecific Antibody Molecule

[0616] The ability of the multi-specific antibody molecules targeting CCR8 and VEGF / PD-L1 to block the binding of PD-1 / PD-L1 was measured, using the above-mentioned PL1 molecule as a control sample. The specific method is as follows:

[0617] Collect CHO-hPDL1 cells in the logarithmic growth phase, centrifuge at 300g for 5 min. Resuspend the cells with 1 ml of FACS buffer (1X PBS + 2% FBS), count the cells and adjust the cell density to 2E6 / ml with FACS buffer. After mixing evenly, inoculate the CHO-hPDL1 cell suspension into a 96-well V-bottom plate, 25 μl / well. Add the gradient-diluted antibodies (8AsPl-1, 8AsPl-3, 8AsPl-4, or Pl8-8-FKO, Pl8-9-FKO) to the above 96-well V-bottom plate in sequence with FACS buffer, 50 μl / well, and set up duplicate wells. Dilute the human PD-1 mFc Tag protein to 16 μg / ml with FACS buffer and transfer it to the 96-well V-bottom plate at 25 μl / well. Place it in a refrigerator at 4°C and incubate for 30 min. Wash twice with FACS buffer, add Goat anti-MouseIgG Fc Cross-Adsorbed Secondary Antibody PE, place it in a refrigerator at 4°C, and continue to incubate for 20 min. After washing twice with FACS buffer, detect with a flow cytometer. Plot the experimental data with prism software and calculate the IC 50 value.

[0618] The results are as Figure 11 shown. The results show that the blockade of PD-L1 by the two bispecific antibody molecules is basically the same as that of the monoclonal antibody ( Figure 11 a). Among the trispecific antibody molecules, the blocking effects of 8AsPl-3 and 8AsPl-4 are better than that of 8AsPl-1 but lower than that of the monoclonal antibody control sample ( Figure 11 b).

[0619] Example 5 In vivo pharmacodynamic experiment of the antibody molecule targeting CCR8

[0620] 5.1 In Vivo Efficacy Experiment in the MC38 Mouse Model

[0621] Inoculate CCR8 humanized C57BL / 6 mice with MC38 cells (5×10 5 cells / mouse) to construct an MC38 mouse model. Wait until the average tumor volume grows to about 100 mm 3 and start grouping for drug administration. Administer the drug by intravenous injection at a dose of 10 mg / kg, twice a week, for 5 consecutive times.

[0622] The results are as Figure 12As shown, on the 14th day after administration, BM-1, C61, and C27 all had antitumor effects, and the tumor growth inhibition rates (TGI) were 30%, 34%, and 45% respectively.

[0623] 5.2 In Vivo Efficacy Experiment in the CT26 Mouse Model

[0624] CT26 cells (3×10 5 cells / mouse) were inoculated into CCR8 humanized Balb / c mice to construct a CT26 mouse model. When the average tumor volume grew to approximately 60 mm 3 , grouping and administration began. Intraperitoneal injection was performed, and the administration doses of each group were as Figure 13 shown, twice a week for 4 consecutive times.

[0625] The results were as Figure 13 shown in a of the figure. On the 14th day after administration, the tumor growth inhibition rates (TGI) of each administration group were 70% (AS-1), 4% (C61), 66% (AS-1 + C61), 63% (8As-1), 58% (8As-2), and 74% (8As-4). The changes in the body weights of the mice during the administration process were as Figure 13 shown in b of the figure. It can be seen that the bispecific molecule of the present invention also exhibited good antitumor effects in mice.

[0626] All documents mentioned in the present invention are cited in this application as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An anti-CCR8 antibody or an antigen-binding fragment thereof, said antibody comprising 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 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.

3. A multispecific antibody, characterized in that, The multispecific antibody comprises: A first targeting domain, said first targeting domain comprising one or more CCR8 antigen-binding domains, said CCR8 antigen-binding domain comprising the anti-CCR8 antibody or an antigen-binding fragment thereof as claimed in claim 1; A second targeting domain, said second targeting domain binding to VEGF or PD-L1; Optionally, comprising a third targeting domain, said 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, wherein, 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 NOs: 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 NOs: 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, wherein The multispecific antibody is a bis / trispecific antibody.

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

9. An immunoconjugate, characterized in that, The conjugate comprises: (i) an anti-CCR8 antibody or an antigen-binding fragment thereof as claimed in claim 1, or a multispecific antibody as claimed in 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.

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

11. 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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