WO2025140619A1 - Multispecific antibody containing ccr8 antigen binding domain - Google Patents

Abstract

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

Description

Multispecific antibodies comprising a CCR8 antigen binding domain Technical Field

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

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.

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

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

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.

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 in 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, thereby further producing more VEGF. Therefore, the key role of VEGF in tumor angiogenesis makes it a well-known anti-tumor target. Bevacizumab (trade name Avastin) is a monoclonal antibody developed by Genentech, a subsidiary of Roche, that specifically blocks VEGF to inhibit the formation of tumor blood vessels. 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.

PD-1/PD-L1 is an important target in tumor immunotherapy (IO). Since PD-L1 is highly expressed in most tumors, 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 T cells to kill tumor cells. Since the launch of the first PD-1 antibody Nivolumab in 2014, the development of PD-L1 antibodies has followed closely. Among them, Atezol izumab developed by Roche was approved for marketing in 2016, and Avelumab, developed by Pfizer and Merck, was approved for marketing in 2017.

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

Summary of the invention

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

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

In a first aspect of the present invention, an anti-CCR8 antibody or an antigen-binding fragment thereof is provided, wherein the antibody comprises the following three heavy chain variable regions CDR:

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

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

HCDR3 having 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 having an amino acid sequence as shown in SEQ ID NO:33, 36, 39, 50 or 55;

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

LCDR3 having an amino acid sequence as shown in SEQ ID NO:35, 38, 42, 45, 49, 52, 54 or 57.

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

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.

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:59, and a light chain variable region as shown in SEQ ID NO:73.

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:60, and a light chain variable region as shown in SEQ ID NO:74.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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:71, and a light chain variable region as shown in SEQ ID NO:85.

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:72, and a light chain variable region as shown in SEQ ID NO:86.

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

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

The second aspect of the present invention provides a multispecific antibody, wherein the multispecific antibody comprises the anti-CCR8 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

In another preferred embodiment, the multispecific antibody comprises:

A first targeting domain, wherein the first targeting domain comprises one or more CCR8 antigen binding domains;

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

Optionally, comprising a third targeting domain, said third targeting domain binding to VEGF or PD-L1;

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

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

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

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

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

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

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

And, the following three light chain variable region CDRs:

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

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

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

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

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

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

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

And, the following three light chain variable region CDRs:

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

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

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

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 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.

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

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 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.

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

In another preferred embodiment, the CCR8 antigen binding domain is selected from the following group: scFv, Fab, or a combination thereof.

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

In another preferred embodiment, the CCR8 antigen binding domain is a 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

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

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

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

In another preferred example, 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.

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

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

Y349C/K370E/K409D/K439E,

S354C/D356K/E357K/D399K;

S354C/T366W,

Y349C/T366S/L368A/Y407V.

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

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

Y349C/K370E/R409D/K439E,

S354C/E356K/E357K/D399K; or

S354C/T366W,

Y349C/T366S/L368A/Y407V.

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

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

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

In another preferred embodiment, the bispecific antibody comprises:

A first targeting domain, wherein the first targeting domain comprises one or more CCR8 antigen binding domains; and a second targeting domain, wherein the second targeting domain is a VEGF antigen binding domain.

In another preferred embodiment, the bispecific antibody comprises:

A first targeting domain, wherein the first targeting domain comprises one or more CCR8 antigen binding domains; and a second targeting domain, wherein the second targeting domain is a PD-L1 antigen binding domain.

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

In another preferred embodiment, the trispecific antibody comprises:

A first targeting domain, wherein the first targeting domain comprises one or more CCR8 antigen binding domains;

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

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

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

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

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

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

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

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

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

In another preferred embodiment, 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).

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

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

In another preferred embodiment, the VEGF antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 90-92, 149-150.

In another preferred embodiment, the VEGF antigen binding domain comprises a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:96-102.

In another preferred embodiment, the VEGF antigen binding domain is selected from the following group: scFv, Fab, or a combination thereof.

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.

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.

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

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

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

In another preferred embodiment, the Fab2 is anti-VEGF Fab.

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

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.

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

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.

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

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.

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

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

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

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

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

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

In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula II (eg, b in FIG. 7A ):

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

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

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

Fc1 is the Fc fragment.

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

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

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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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.

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

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

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

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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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.

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

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

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

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

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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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

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

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

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

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

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

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.

In another preferred embodiment, 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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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

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

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

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

In another preferred embodiment, the Fab2 is anti-VEGF Fab.

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

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.

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

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.

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

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.

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

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

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

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

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

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.

In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula VII (eg, g in FIG. 7A ):

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

In another preferred embodiment, the Fab2 is anti-VEGF Fab.

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

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.

In another preferred embodiment, 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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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

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

In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula VIII (eg, h in FIG. 7A ):

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

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

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

Fc1 is the Fc fragment.

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

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

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.

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

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.

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

The anti-VEGF scFv 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.

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

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

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

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

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

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

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

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

Fab2 is the second targeting domain, and the Fab2 is anti-PDL1 Fab or anti-VEGF Fab;

Fc1 is the Fc fragment.

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

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

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.

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

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.

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

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.

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

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

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

In another preferred example, the Fc1 has an amino acid sequence as shown in SEQ ID NO:114.

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

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

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

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

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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

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

In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula XI (b in FIG. 8A ):

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

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

Fab2 is the second targeting domain, and Fab1 is anti-VEGF Fab;

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

Fc1 and Fc2 are each independently an Fc fragment.

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

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.

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

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.

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

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.

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

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

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

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

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

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

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

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

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

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

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

Fc1 and Fc2 are each independently an Fc fragment.

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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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

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

In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula XIII (eg, d in FIG. 8A ):

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

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

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

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

Fc1 is the Fc fragment.

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

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

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.

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

The anti-VEGF scFv 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.

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

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.

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

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

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

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

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

The third aspect of the present invention provides a polynucleotide encoding the anti-CCR8 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the multispecific antibody as described in the second aspect of the present invention.

The fourth aspect of the present invention provides an expression vector, wherein the expression vector comprises the polynucleotide as described in the third aspect of the present invention.

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

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

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

In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, HEK 293T cells, and CHO cells.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In another preferred embodiment, the anti-VEGF antibody mutant comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 90-92, 149-150.

In another preferred embodiment, the anti-VEGF antibody mutant comprises a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:96-102.

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

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

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

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

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

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

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

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

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

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

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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG2 shows the ADCC effect of the CCR8 recombinant monoclonal antibody.

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

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

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

FIG6 shows the sequence peptides with aggregation tendency and high free energy in the Fab sequence of AS-1.

FIG7A shows nine bispecific antibody structures (a-i) composed of a CCR8 antigen binding domain and a VEGF or PD-L1 antigen binding domain;

Among them, a shows the antibody structure of the combination of anti-VEGF Fab (CK-VH+CH1-VL) and anti-CCR8 Fab (CH1-VH+CK-VL), in which IgG1 Fc forms a heterodimer through charge pair or knob and hole mutation; b shows the antibody structure formed by anti-CCR8 Fab and anti-VEGF scFv (Fab-Fc-scFv×Fab-Fc-scFv); c shows the antibody structure formed by anti-CCR8 Fab and anti-VEGF scFv (Fab-Fc-scFv×Fab-Fc) ; d shows the molecular structure formed by anti-VEGF scFv and anti-CCR8 Fab (scFv-Fc×Fab-Fc); e shows the molecular structure formed by anti-CCR8 scFv, VEGF scFv and anti-CCR8 Fab (scFv-scFv-Fc×Fab-Fc); f shows the 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 a shows the antibody structure of anti-VEGF Fab (CH1-VH+CK-VL), anti-CCR8 Fab (CK-VH+CH1-VL) combined with anti-CCR8 scFv (Fab-Fab-Fc×scFv-Fc); h shows the antibody structure of anti-PD-L1scFv, anti-CCR8 Fab combined with anti-PD-L1scFv, anti-CCR8 Fab (scFv-Fab-Fc×scFv-Fab-Fc); i shows the antibody structure of anti-VEGF or PDL1 Fab (CH1-VH+CK-VL), anti-CCR8 Fab (CK-VH+CH1-VL) combined with anti-CCR8 scFv. The antibody structure (Fab-Fab-Fc×Fab-Fab-Fc) is composed of an IgG1 Fc (CH1-VH+CK-VL) and an anti-VEGF or PDL1 Fab (CH1-VH+CK-VL), or an anti-CCR8 Fab (CK-VH+CH1-VL); wherein the IgG1 Fc forms a heterodimer by a charge pair or knob and hole mutation; wherein the IgG1 Fc forms a heterodimer by a charge pair or knob and hole mutation, wherein the IgG1Fc forms a heterodimer by a charge pair or knob and hole mutation.

FIG. 7B shows an exemplary bispecific antibody structure of FIG. 7Aa, designated 8As-1.

FIG. 7C shows an exemplary bispecific antibody structure of FIG. 7A b, designated 8As-2.

[Corrected 06.02.2025 in accordance with Article 91]
FIG. 7D shows an exemplary bispecific antibody structure of FIG. 7A c, designated 8As-3.

FIG. 7E shows an exemplary bispecific antibody structure of FIG. 7A d, designated 8As-4.

FIG. 7F shows an exemplary bispecific antibody structure of FIG. 7A e, designated 8As-5.

FIG. 7G shows an exemplary bispecific antibody structure of FIG. 7Af, designated 8As-6.

FIG. 7H shows an exemplary bispecific antibody structure of FIG. 7A g, designated 8As-7.

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

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

FIG. 7K shows an exemplary bispecific antibody structure of FIG. 7A i, designated 8As-9.

FIG8A shows three triple antibody structures consisting of a CCR8 antigen binding domain, a VEGF antigen binding domain, and a PD-L1 antigen binding domain;

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

FIG8B shows an exemplary trispecific antibody structure of FIG8Aa, designated 8AsPl-1.

FIG8C shows an exemplary trispecific antibody structure of FIG8A b, designated 8AsPl-2.

FIG8D shows an exemplary trispecific antibody structure of FIG8A c, designated 8AsPl-3.

Figure 8E shows an exemplary trispecific antibody structure of Figure 8Ad, designated 8AsPl-4.

FIG. 9 shows the ADCC effect of the monoclonal antibody and the multispecific antibody of the present invention.

FIG. 10 shows the VEGF blocking effect of the multispecific antibodies of the present invention.

FIG. 11 shows the PD-L1 blocking effect of the multispecific antibodies of the present invention.

FIG. 12 shows the in vivo pharmacodynamic effect of the CCR8 monoclonal antibody of the present invention on a mouse tumor model.

FIG. 13 shows the in vivo pharmacodynamic effects of the CCR8 dual antibody of the present invention on a mouse tumor model.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventors unexpectedly developed a class of multispecific antibodies containing CCR8 antigen binding domains for the first time. The multispecific antibody contains a first targeting domain targeting 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 its antigen binding fragment, and also contains a second targeting domain and/or a third targeting domain that binds to VEGF and/or PD-L1. The multispecific 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.

On this basis, the present invention has been completed.

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

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 in 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, thereby further producing more VEGF. The key role of VEGF in tumor angiogenesis makes it a well-known target for anti-tumor drugs.

PD-1/PD-L1 is an important target in tumor immunotherapy (IO). Since PD-L1 is highly expressed in most tumors, 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 T cells to kill tumor cells.

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

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

As used herein, the term "variable" means that certain parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region 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, Volume I, pp. 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.

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

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

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

In naturally occurring antibodies, the six CDRs present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form an "antigen binding domain" as the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain, referred to as the "framework" region, show less inter-molecular variability. The framework region predominantly adopts 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 region acts to form a scaffold that positions the CDRs in the correct orientation through inter-chain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. Having been precisely defined, one of ordinary skill in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively, for any given heavy or light chain variable region.

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

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

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

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 that the original antibody, antibody fragment, or antibody domain specifically binds to. It should be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical", such embodiments include any and all numerical percentages equal to or above the lower limit. In addition, it should be understood that where an amino acid sequence is present in the present application, it should be interpreted as additionally disclosing or comprising an amino acid sequence that has 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.

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

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.

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 when bound to the target molecule. In some embodiments, a bispecific antibody is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or a fragment thereof that specifically binds to a target protein.

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

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

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

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

In another preferred embodiment of the present invention, a trispecific antibody targeting CCR8, VEGF and PD-L1 is constructed, having structures as shown in a-d in FIG8A , and exemplary molecules of structures a-d are shown in FIG8B-E , respectively. The anti-CCR8 Fab therein may 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 may be a Fab/scFv constructed using the VH and VL of any anti-VEGF antibody described in the present invention; and the anti-PD-L1 scFv may be a scFv constructed using the VH and VL of any anti-PD-L1 antibody described in the present invention.

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

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

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

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

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

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

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

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

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

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method, the steps used are well known in the art. Another method is to use MgCl _2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant 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. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

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

The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

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

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

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, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

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

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

When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 8 milligrams/kg body weight, preferably the dosage is about 10 micrograms/kg body weight to about 1 milligram/kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified 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 according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

Sequences used to prepare the multispecific antibodies of the present invention

CCR8 antibody sequences

CCR8 antibody sequences

VEGF antibody sequence

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, 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, respectively, and the mutation sites are marked in bold.

PD-L1 Antibody Sequence

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.

IgG1 CH sequence

The CH1 sequence is as follows:

The Fc region sequence is shown below:

The Fc region may comprise the following mutations:

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

In another preferred embodiment of the present invention, the lysine (K) at the C-terminus of the Fc region is removed to reduce the formation of charge isoforms of the antibody.

IgG4 CH sequence

The Fc region may comprise the following mutations:

CL sequence

Optional linker sequences

Example 1 Preparation and verification of anti-CCR8 monoclonal antibodies

1.1 Screening of anti-CCR8 monoclonal antibodies

The CCR8 antibody of the present invention is obtained by screening humanized mouse immunization hybridoma. The hCCR8 expression plasmid is constructed, DNA is extracted and endotoxin is removed, and the heavy and light chain variable region humanized mice (derived from CN114763558A) are immunized. The spleen of the immunized mouse is selected, separated to obtain spleen cells, and fused with Sp2/0-Ag14 multiple myeloma cells. The fusion is performed by electroporation according to the hybridoma fusion technology method. After fusion, a certain number of cells are seeded in each well of a 96-well plate and cultured in HAT culture medium for 10 days. The hybridoma cell culture supernatant is detected with the HEK293-CCR8 overexpression cell line to obtain positive hybridoma cells. The positive hybridoma cells are cultured, and the supernatant of the cell culture for 5 days is taken, and a small amount of antibodies are purified by Protein A magnetic beads to obtain hybridoma antibodies of human variable region-mouse constant region. The purified antibodies are tested for binding activity function. During the screening process, specific binding molecules were screened against the extracellular region of CCR8 or cell lines overexpressing CCR8 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 one category, C5 and C27 are in one category, and other antibodies are in one category.

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

Further, 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 CCR8 full-length sequence gene fragments of human, mouse and cynomolgus monkey were obtained by full gene synthesis, and then these fragments were inserted into the skeleton of the stable expression vector to obtain the expression plasmid vector. These three plasmids were transfected into HEK293 cells, and monoclonal cell lines (HEK293-hCCR8 cells, HEK293-mCCR8 cells, HEK293-cCCR8 cells) expressing human, mouse and cynomolgus monkey CCR8 were screened under the pressure of puromycin, which were the flow detection cell lines used in this embodiment.

Using the above HEK293-hCCR8 cells, 1x10 ⁵ cells were plated in each well of a 96-well plate, and the antibody was diluted to a starting concentration of 1μg/ml and diluted 2 times. 50μl of the diluted antibody was mixed with the cells and incubated at 4°C for 45 minutes. Wash twice with washing solution. After pouring off the liquid, all cells were resuspended in 50μl 200ng/ml Goat anti-Mouse IgG PE and incubated at 4°C in the dark for 30 minutes. Wash three times with washing solution. After pouring off the liquid, all cells were resuspended in 25μl diluent. Detected by flow cytometry. The FACS binding data of 14 monoclonal antibodies obtained are shown in Figure 1.

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

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

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

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

The antibody was diluted to 5 μg/ml, and the antigen human CCR8 (1-35aa)-mFc or cynomolgus monkey CCR8-mFc was 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, and the well with a concentration of 0 was set as the baseline. Antibody affinity detection was performed using a Gator instrument.

The results are shown in Table 2. The affinity of the candidate antibodies for human CCR8 ranges from 0.0168 to 1.24 nM (Table 1). A number of candidate antibodies can bind to cynomolgus monkey CCR8 (Table 2).

Table 1. Binding affinity of antibodies to human CCR8

Table 2. Binding of Antibodies to Cynomolgus Monkey CCR8

1.4 ADCC activity of anti-CCR8 recombinant human monoclonal antibody

The ADCC activity of 14 anti-CCR8 recombinant human monoclonal antibodies was tested as follows:

CHO-K1 cells overexpressing CCR8 were plated at 2×10 ⁴ cells per well and cultured overnight. The culture medium was removed and 25 μl of complete culture medium was added. Antibody dilution was prepared with complete culture medium to a final concentration 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, and 25 μl of antibody dilution was added to each well. Keep the density of Jurkat-FcγRIIIa-V158 effector cells between 0.2-1.0×10 ⁶ cells/ml, resuspend the effector cells in culture medium to a cell density of 6×10 ⁶ cells/ml, add 25μl of cell suspension to each well of the 96-well white plate, and the final total volume of each sample well is 75μl. Incubate in a cell culture incubator for 6 hours. Add luciferase substrate to the plate, 50μl/well, and mix for 30 seconds. Use a multifunctional microplate reader, Lum-TM channel for detection.

The results are shown in FIG2 . All candidate antibodies have different degrees of ADCC against target cells. Among them, C5 has the smallest EC ₅₀ , and C61 has the highest signal value. Both have good ADCC effects.

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

The blocking effect of 14 anti-CCR8 recombinant human monoclonal antibodies on the binding of CCR8 and its ligand CCL1 was tested as follows:

The blocking experiment used the β-Arrestin eXpress GPCR Assay kit from DiscoverX. Add 11.5 ml of Cell Plating Reagente22 to a 15 ml centrifuge tube. Add 0.5 ml of Cell Plating Reagent to the cryopreservation tube, wait for the PathHunter eXpress β-Arrestin GPCR cells to melt and transfer all to the above 15 ml centrifuge tube, mix well, add 100 μl to each well, put in the cell culture incubator, and incubate for 48 hours. Configure the starting concentration to be 220000 ng/ml (22× final concentration), three-fold dilution, a total of 8 gradients. Add 5 μl of antibody dilution to each well of the white plate, add complete culture medium to the remaining wells, put in the cell culture incubator, and incubate for 30 minutes. Prepare 88 nM CCL1 (22 times the final concentration, the final concentration of CCL1 is 4 nM), add 5 μl to each well, put in the cell culture incubator, and incubate for 90 minutes. Add 55 μl Working Detection Solution to each well, incubate at room temperature in the dark for 60 minutes, and detect using the Lum-TM channel.

The results are shown in Figure 3. The tested antibodies all have a certain blocking effect on the binding of CCL1 and CCR8. The antibodies with a blocking effect of >90% are C5, C20, C23, C57, and C61. The blocking percentages are 96.83%, 90.08%, 91.39%, and 92.46%, respectively. Their _IC50 values are 496.8, 377.4, 216.5, and 183.4 ng/ml, respectively.

Example 2 Preparation and verification of anti-VEGF mutants

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

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

In addition, in order to simultaneously analyze the aggregation tendency and thermal stability of the AS-1 sequence, the inventors used the SolubiS server to analyze the regions in the variable region sequence of the AS-1 structure that are prone to aggregation (high TANGO score) and poor in thermal stability (high free energy ΔG). As shown in Figure 6, 6 peptide segments were identified. Among them, the light chain L46-S57 region has the highest aggregation tendency and the lowest thermal stability. In addition, there are also literatures that have found through site-directed mutagenesis (Dudgeon et al, 2012, PNAS) 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 antibodies.

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

In order to confirm whether these mutations have changed the hydrophobicity of the antibody, the Proteomix HIC Butyl-NP5 column was used to distinguish the retention time of each mutant antibody. After the mutants of the antibody were loaded in a solution of 1.5M NaCl, 25mM Na ₃ PO ₄ pH 7.4, they were eluted with a solution of gradually decreasing salt concentration at pH 7.4. The retention time of the mutants is shown in Table 3. It can be seen that AS-1 has the longest retention time, indicating that it has the strongest hydrophobicity. The retention time of other mutants has decreased. Some combined mutant antibodies, such as AS-6, can reduce the retention time by nearly half, greatly reducing the hydrophobicity of the antibody and its possible aggregation tendency.

Table 3

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

Table 4

Example 3 Preparation of multispecific antibody molecules

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

The molecular sequence fragment gene of the multispecific antibody was obtained by the whole gene synthesis method, and then the target sequence was inserted into the expression vector by conventional gene cloning means (see, for example, Lo.B.K.C methods in Molecular Biology. Volume 248, 2004. Antibody Engineering).

HEK293E cells were transfected with vectors carrying multispecific antibody molecules. The cells were cultured at 37°C and 5% CO ₂ for 7 days to produce the corresponding molecules in F17 medium (1L F17+10mL 10% PF68+30ml 200mM L-glutamine). After the expression, the supernatant was harvested and purified to obtain multispecific antibody molecules, which can be used for various experimental analyses.

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

Using the above methods, the following multispecific molecules were prepared:

(1) a bispecific antibody consisting of a CCR8 antigen binding domain and a VEGF or PD-L1 antigen binding domain having the structure shown in a-i in FIG. 7A ;

A bispecific antibody of structure a in FIG7A is named 8As-1, and its structure is shown in FIG7B ;

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

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

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

e structure of a bispecific antibody named 8As-5, whose structure is shown in Figure 7F;

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

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

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

A bispecific antibody of structure i was named Pl8-9, and its structure is shown in Figure 7J; another bispecific antibody of structure i was named 8As-9, and its structure is shown in Figure 7K; it differs from Pl8-9 in that the anti-PD-L1 VH and VL are replaced with anti-VEGF VH and VL.

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

A bispecific antibody of structure a in FIG8A is named 8AsPl-1, and its structure is shown in FIG8B ;

A bispecific antibody with structure b was named 8AsPl-2, and its structure is shown in FIG8C

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

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

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

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

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

4.1 ADCC experiments of multispecific antibody molecules targeting CCR8 and VEGF/PDL1

The ADCC effect of the multispecific antibody molecules (8As-1, 8As-2, 8As-4, 8As-7, 8As-9, P18-8, P18-9, 8AsP1-1, 8AsP1-3, 8AsP1-4) targeting CCR8 and VEGF/PD-L1 prepared in Example 2 was determined, wherein some antibodies were expressed in Fut8 knockout CHO cells (FKO). The specific method is as follows:

Collect CHOK1-hCCR8 cells in the logarithmic growth phase (human CCR8 overexpressing cells, obtained by transfecting CHOK1 cells with a CCR8 full-length expression plasmid, and the preparation method is the same as the HEK293-hCCR8 cell preparation process described in Example 1.2), centrifuge at 300g for 5 minutes. Take 1ml assay buffer (1640 culture medium + 10% FBS) to resuspend the cells, count, 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 at 25μl/well. Use assay buffer to add the gradient diluted antibodies to the above 96-well white plate in sequence, 25μl/well, and set up double wells. Place in a _CO2 incubator and incubate for 30 minutes. Finally, collect the ADCC Bioassay Effector Cell V Variant (High Affinity)/NFAT Luciferase Reporter Jurkat Cell Line with good growth status into a 50ml centrifuge tube and centrifuge at 300g for 5min. Take 3ml assay buffer (1640medium+10% FBS) to resuspend the cells, count and adjust the cell density to 3E6/ml. After mixing, add to the above 96-well white plate, 50μl/well. Place in a _CO2 incubator and incubate for 5h. Take out the 96-well white plate and add BRITELITE PLUS reagent, 100μl/well. Incubate at room temperature in the dark for 5min, and detect the lumi readings with an enzyme plate reader. Use Prism software to draw a graph and calculate the EC ₅₀ value.

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

4.2 VEGF blocking experiment with multispecific antibody molecules targeting CCR8 and VEGF/PD-L1

The blocking ability of multispecific antibody molecules targeting CCR8 and VEGF/PD-L1 (8As-1, 8As-2, 8As-4, 8As-9, 8AsPl-1, 8AsPl-3, 8AsPl-4) on VEGF signaling was determined, with AS-1 molecule as a control sample. The specific method is as follows:

Collect VEGFR2/NFAT Reporter–HEK293 Recombinant cells in the logarithmic growth phase and centrifuge at 300g for 5min. Resuspend the cells in 1ml assay buffer (MEM/EBSS medium + 10% FBS), count 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 at 50μl/well. Add the gradient diluted antibodies to the above 96-well white plate in sequence with assay buffer at 25μl/well, and set up double wells. Then, dilute the human VEGF165 his Tag protein to 50ng/ml with assay buffer and transfer it to the 96-well white plate at 25μl/well. Incubate in a CO ₂ incubator for 4h. Finally, take out the 96-well white plate and add BRITELITE PLUS reagent at 100μl/well. Incubate at room temperature in the dark for 5 min, and use an ELISA reader to measure the lumi readings. Use Prism software to plot and calculate the IC ₅₀ value.

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

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

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

Collect CHO-hPDL1 cells in the logarithmic growth phase and centrifuge at 300g for 5min. Resuspend the cells in 1ml FACS buffer (1X PBS + 2% FBS), count 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 at 25μl/well. Add 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 at 50μl/well, and set up double wells. Dilute the human PD-1mFc Tag protein to 16μg/ml using FACS buffer and transfer it to a 96-well V-bottom plate at 25μl/well. Place in a 4°C refrigerator and incubate for 30min. Wash twice with FACS buffer, add Goat anti-Mouse IgG Fc Cross-Adsorbed Secondary Antibody PE, place in a 4℃ refrigerator, and continue incubation for 20 minutes. Wash twice with FACS buffer and detect with flow cytometry. Plot the experimental data with Prism software and calculate the IC ₅₀ value.

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

Example 5 In vivo efficacy experiment of antibody molecules targeting CCR8

5.1 In vivo efficacy study in MC38 mouse model

CCR8 humanized C57BL/6 mice were inoculated with MC38 cells (5×10 ⁵ cells/mouse) to construct the MC38 mouse model, and the mice were divided and dosed when the average tumor volume grew to about 100 mm ^3. The intravenous injection was administered at a dose of 10 mg/kg, twice a week, for 5 consecutive times.

The results are shown in FIG. 12 a. On the 14th day after administration, BM-1, C61, and C27 all had anti-tumor effects, and the tumor growth inhibition rates (TGI) were 30%, 34%, and 45%, respectively.

Another group of CCR8 humanized C57BL/6 mice were inoculated with MC38 cells, wherein the mouse inoculation amount, tumor grouping volume and administration dose (equimolar) were the same as those in the experiment shown in Figure 12a above. Intraperitoneal injection was performed twice a week for 6 consecutive times.

The results are shown in FIG. 12 b . On the 20th day after administration, C61, 8AS-2, and 8AsPl-4v all had anti-tumor effects, and the tumor growth inhibition rates (TGI) were 45%, 83.5%, and 89.5%, respectively.

5.2 In vivo efficacy study in CT26 mouse model

CCR8 humanized Balb/c mice were inoculated with CT26 cells (3×10 ⁵ cells/mouse) to construct a CT26 mouse model, and the mice were divided and dosed when the average tumor volume grew to about 60 mm ^3. The intraperitoneal injection was performed, and the dosage of each group was as shown in Figure 13, twice a week for 4 consecutive times.

The results are shown in Figure 13a. On the 14th day after administration, the tumor growth inhibition rate (TGI) of each administration group was 70% (AS-1), 4% (C61), 66% (AS-1+C61), 63% (8As-1), 58% (8As-2), and 74% (8As-4). The weight changes of mice during administration are shown in Figure 13b. It can be seen that the dual-antibody molecule of the present invention also exhibits a good anti-tumor effect in mice.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application.

Claims (15)

An anti-CCR8 antibody or an antigen-binding fragment thereof, wherein the antibody comprises the following three heavy chain variable region CDRs: HCDR1 having an amino acid sequence as shown in SEQ ID NO: 1, 4, 7, 10, 15, 18, 20, 24, 28 or 30; HCDR2 having an amino acid sequence as shown in SEQ ID NO: 2, 5, 8, 11, 13, 16, 21, 23, 25 or 31; and HCDR3 having 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 having an amino acid sequence as shown in SEQ ID NO:33, 36, 39, 50 or 55; LCDR2 having an amino acid sequence as shown in SEQ ID NO:34, 37, 40, 44, 48, 51, 53, 56 or 58; and LCDR3 having an amino acid sequence as shown in SEQ ID NO:35, 38, 42, 45, 49, 52, 54 or 57. The anti-CCR8 antibody or its antigen-binding fragment as described in claim 1 is characterized in that 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. A multispecific antibody, characterized in that the multispecific antibody comprises: A first targeting domain, wherein the first targeting domain comprises one or more CCR8 antigen binding domains, wherein the CCR8 antigen binding domain comprises the anti-CCR8 antibody or antigen binding fragment thereof according to claim 1; a second targeting domain that binds to VEGF or PD-L1; Optionally, comprising a third targeting domain, said third targeting domain binding to VEGF or PD-L1; Furthermore, the second targeting domain and the third targeting domain bind to different antigens respectively. The multispecific antibody according to claim 3, characterized in that the targeting domain is in the form of a single-chain Fv (scFv), a Fab fragment, a single domain antibody (sdAb), a fragment variable (Fv) heterodimer, TriFab or a combination thereof. The multispecific antibody of 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 Comprising 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. The multispecific antibody of 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 Comprising 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. The multispecific antibody according to claim 3, characterized in that the multispecific antibody further comprises an Fc fragment, preferably, the Fc fragment is derived from IgG1 or IgG4. The multispecific antibody according to claim 7, characterized in that the Fc fragment comprises a mutation for forming a knob-in-hole structure and/or a mutation for enhancing ADCC. The multispecific antibody according to claim 3, characterized in that the multispecific antibody is a bi/trispecific antibody. A polynucleotide encoding the anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the multispecific antibody according to claim 3. A use of the anti-CCR8 antibody or antigen-binding fragment thereof as claimed in claim 1, or the multispecific antibody as claimed in claim 3, characterized in that it is used for preparing a drug for treating cancer/tumor. An immunoconjugate, characterized in that the conjugate comprises: (i) the anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the multispecific antibody according to claim 3; and (ii) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: (a) the anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the multispecific antibody according to claim 3; and (b) a pharmaceutically acceptable carrier. Use of the anti-CCR8 antibody or antigen-binding fragment thereof according to claim 1, or the immunoconjugate according to claim 12, for preparing a detection reagent or a kit for detecting CCR8 molecules in a sample. 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 that can reduce the hydrophobicity of the antibody.