CN118598998A

CN118598998A - Anti-CCR 8 antibody and application thereof

Info

Publication number: CN118598998A
Application number: CN202310802616.XA
Authority: CN
Inventors: 宋其峰; 朱永强; 李理想
Original assignee: Beijing Xingji Beiqin Biotechnology Co ltd
Current assignee: Beijing Xingji Beiqin Biotechnology Co ltd
Priority date: 2023-07-03
Filing date: 2023-07-03
Publication date: 2024-09-06

Abstract

The invention discloses an anti-CCR 8 antibody and application thereof. The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody provided by the invention are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 in sequence. The antibody has high specificity and high binding activity to human CCR8 (hCR 8), and can effectively mediate the cytotoxic killing of effector cells to cells expressing hCR 8, thereby having drug formation. Furthermore, the antibodies can also bind efficiently to monkey CCR8 molecules, which is of particular importance for preclinical studies of antibody drugs.

Description

Anti-CCR 8 antibody and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to an anti-CCR 8 antibody and application thereof.

Background

The physiological process of the human body is constantly under the monitoring of the immune system, in which cellular immunity plays a driving role. Often, the occurrence and progression of tumors is such that cancerous cells escape this immune monitoring and in further proliferation and progression the killing effect in this monitoring is exhausted, rested, disabled, or even tumor tissue is provided with a chemo-immune system for use in support of their own growth and proliferation. The immune system of a cancer patient is regulated in a targeted way, and the intervention treatment is carried out, so that a good treatment effect is often brought. In recent years, antibody drugs developed for negative regulatory factors on the surfaces of killer effector T cells such as PD-1 and LAG3 are used for blocking T cell depletion induced by tumor tissues. The target T cell surface (mainly Regulatory T cells, regulatory T cell, treg for short) CTLA-4 molecules are prepared by eliminating Treg, so that negative regulation on effector cells is inhibited, the effector cells are allowed to perform the function of killing tumors, and the aim of treating cancers is fulfilled (Sakaguchi S., et al, regulatory T CELLS AND human disease. Annu. Rev. Immunol.38,541-566 (2020)). However, in clinical application, it was found that although targeting CTLA-4 molecules, clearing tregs showed significant therapeutic effects, its immune-related toxicity (irAE) caused by peripheral immunity (PERIPHERAL IMMUNE SYSTEM) was not negligible, sometimes resulting in drug withdrawal or more serious consequences (Kim J.M.,Rasmussen J.P.,Rudensky A.Y.,Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice.Nat.Immunol.8,191–197(2007).Tanaka A.,Sakaguchi S.,Targeting Treg cells in cancer immunotherapy.Eur.J.Immunol.49,1140–1146(2019).Hodi F.S.,et al.,Improved survival with ipilimumab in patients with metastatic melanoma.N.Engl.J.Med.363,711–723(2010).)., thus finding suitable targets, specifically clearing tumor-related foxp3+ tregs, and being a more ideal drug development approach.

During development of the immune system, a foxp3+ Treg develops automatically from the thymus, and these populations migrate and are stimulated by different antigens or inflammatory processes, thus developing effector tregs, the common phenotype being CD25high, CTLA-4high, and CD45RAlow, acting in different tissues and organs. The effector tregs of healthy humans recognize self-antigens and commensal microorganisms, thus avoiding attacks (Miyara M.,et al.,Functional delineation and differentiation dynamics of human CD4+T cells expressing the FoxP3 transcription factor.Immunity 30,899–911(2009).Fisson S.,et al.,Continuous activation of autoreactive CD4+CD25+regulatory T cells in the steady state.J.Exp.Med.198,737–746(2003).). on them in Treg populations of tumor tissues, the effector tregs are predominant, and worse than actively proliferating tregs associated with tumor prognosis adverse depth (Saito T.,et al.,Two FOXP3(+)CD4(+)T cell subpopulations distinctly control the prognosis of colorectal cancers.Nat.Med.22,679–684(2016).Kumagai S.,et al.,The PD-1expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1blockade therapies.Nat.Immunol.21,1346–1358(2020).)., studies have shown that the tregs of tumor microenvironment (tumor microenvironment, TME) and peripheral immune system are also able to recognize mutated or overexpressed tumor-associated antigens, thereby possibly helping them to circumvent attacks of the immune system (Leonard J.D.,et al.,Identification of natural regulatory T cell epitopes reveals convergence on a dominant autoantigen.Immunity 47,107–117.e8(2017).Legoux F.P.,et al.;CD4+T cell tolerance to tissue-restricted self antigens is mediated by antigen-specific regulatory T cells rather than deletion.Immunity 43,896–908(2015).).

In conclusion, the existing antibody drug-based immunotherapy is difficult to overcome immunosuppression caused by tumor tissues in mechanism, and has low applicable population and response rate in clinical application. Whereas the development of drugs against tumor-specific effector tregs is likely to address a significant portion of unmet clinical needs.

The study of barsheet et al identified chemokine receptor 8 (Chemokine receptor) CCR8 as a driver for immunosuppression. In human peripheral blood cells, more than 30% of regulatory T cells have up-regulated CCR8 expression in the presence of CCL 1. Their studies have also shown that CCL1 is the only/unique ligand for CCR8 molecules that activate Treg proliferation. This function is achieved by STAT3 dependent up-regulation of FOXp expression. In the mouse EAE model, the effect of Treg-mediated activity, i.e., immunosuppressive activity, was seen with CCL1 addition, which confirmed that the CCR8-CCL1 axis is the driver of Treg-mediated immunosuppression (Proc NATL ACAD SCI U S A.2017Jun 6;114 (23): 6086-6091.Doi: 10.1073/pnas.1621280114.), a mechanism involving expression of immunosuppressive molecules such as IL10, TGF-. Beta.and CD39, and downstream upregulation of IL2 and CD25, CTLA-4, PD-1, LAG3, etc. (Wang L,Simons DL,Lu X,et al..Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer.Nat Immunol 2019;20:1220–30.10.1038/s41590-019-0429-7).

Treg cells highly express CCR-8 in a variety of tumor microenvironments including breast, colon, lung cancer tumors (Plitas et al, 2016;De Simone et al, wang et al, 2019). The Treg cells in the tumor microenvironment with high expression of CCR-8 have strong immunosuppressive activity and are predictive of poor patient prognosis (Plitas et al, 2016;De Simone et al, wang et al, 2019). Thus, the use of antibodies to direct specific killing of CCR-8 expressing Treg cells in the tumor microenvironment may be an effective anti-tumor strategy and avoid killing due to low CCR-8 expression of Treg cells in the peripheral blood circulation, thereby avoiding immune related toxicity (irAE).

Disclosure of Invention

The invention aims to provide an anti-CCR 8 antibody and application thereof.

In a first aspect, the invention claims an antibody against CCR 8.

The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the anti-CCR 8 antibody are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 in sequence.

Wherein HCDR1, HCDR2 and HCDR3 are three complementarity determining regions in the heavy chain variable region, and LCDR1, LCDR2 and LHCDR are three complementarity determining regions in the light chain variable region. The sequence of the complementarity determining regions is defined according to Kabat.

Within the scope of the present invention, the antibodies may be in the form of full length antibodies, fab fragments, F (ab') ₂ fragments, or single chain Fv fragments, among others. Preferably, the antibody is a humanized antibody.

Further, the amino acid sequence of the heavy chain variable region may be selected from any one of the following:

(A1) SEQ ID No.7, or at least more than 90% identity to SEQ ID No.7 (inconsistencies may be in the Framework Region (FR));

(A2) SEQ ID No.9, or at least more than 90% identity to SEQ ID No.9 (inconsistencies may be in the Framework Region (FR));

(A3) SEQ ID No.10, or at least more than 90% identity to SEQ ID No.10 (inconsistencies may be in the Framework Region (FR));

(A4) SEQ ID No.11, or at least more than 90% identity to SEQ ID No.11 (inconsistencies may be in the Framework Region (FR));

(A5) SEQ ID No.12, or at least more than 90% identity to SEQ ID No.12 (inconsistencies may be in the Framework Region (FR)).

Further, the amino acid sequence of the light chain variable region may be selected from any one of the following:

(B1) SEQ ID No.8 or the sequence obtained by replacing the 107 th amino acid N of SEQ ID No.8 with K, or has at least more than 90% of the identity with SEQ ID No. 8;

(B2) SEQ ID No.13, or at least more than 90% identity to SEQ ID No. 13;

(B3) SEQ ID No.14, or at least more than 90% identity to SEQ ID No. 14;

(B4) SEQ ID No.15, or at least more than 90% identical to SEQ ID No. 15.

In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID No.9 and the amino acid sequence of the light chain variable region is SEQ ID No.15 (corresponding to humanized antibody Hu9F5-13 in the examples).

In another embodiment of the invention, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID No.7 and the amino acid sequence of the light chain variable region is SEQ ID No.8 (corresponding to murine antibody 9F5 in the examples). In another embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID No.7, and the amino acid sequence of the light chain variable region is a sequence obtained by replacing amino acid N at position 107 of SEQ ID No.8 with amino acid K (corresponding to chimeric antibody 9F5 in examples).

Within the scope of the present invention, the antibody may further comprise a heavy chain constant region selected from the group consisting of IgG (e.g., igG 1) and a light chain constant region comprising a light chain selected from the group consisting of Kappa.

Namely: the antibody may be an IgG antibody. In a specific embodiment of the invention, the IgG antibody is an IgG1 antibody. In a specific embodiment of the invention, the light chain type of the antibody is Kappa type.

In addition, antigen binding fragments derived from the antibodies are also within the scope of the invention.

The antigen binding fragment contains the HCDR1, the HCDR2 and the HCDR3. Further, the antigen binding fragment comprises the LCDR1, the LCDR2, and the LCDR3.

In a second aspect, the invention claims a nucleic acid molecule.

The nucleic acid molecules claimed in the present invention encode an antibody or said antigen binding fragment as described in the first aspect hereinbefore.

Further, in the nucleic acid molecule, the nucleotide sequences encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody may be represented by SEQ ID No.16 (or SEQ ID No.18 or 19 or 20 or 21) at positions 76-105, 148-198 and 295-315 from the 5' end in sequence.

Further, in the nucleic acid molecule, the nucleotide sequences encoding LCDR1, LCDR2 and LHCDR in the light chain variable region of the antibody may be represented by SEQ ID No.17 (or SEQ ID No.22 or 23 or 24) at positions 70-117, 163-183, 280-306 in sequence from the 5' end.

Still further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the antibody may be selected from any one of the following:

(C1) SEQ ID No.16, or at least more than 90% identity to SEQ ID No.16 (inconsistencies may be in the Framework Region (FR));

(C2) SEQ ID No.18, or at least more than 90% identity to SEQ ID No.18 (inconsistencies may be in the Framework Region (FR));

(C3) SEQ ID No.19, or at least more than 90% identity to SEQ ID No.19 (inconsistencies may be in the Framework Region (FR));

(C4) SEQ ID No.20, or at least more than 90% identity to SEQ ID No.20 (inconsistencies may be in the Framework Region (FR));

(C5) SEQ ID No.21, or at least more than 90% identical to SEQ ID No.21 (inconsistencies may be in the Framework Region (FR)).

Still further, in the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region of the antibody may be selected from any one of the following:

(D1) SEQ ID No.17, or at least more than 90% identity to SEQ ID No.17 (inconsistencies may be in the Framework Region (FR));

(D2) SEQ ID No.22, or at least more than 90% identity to SEQ ID No.22 (inconsistencies may be in the Framework Region (FR));

(D3) SEQ ID No.23, or at least more than 90% identity to SEQ ID No.23 (inconsistencies may be in the Framework Region (FR));

(D4) SEQ ID No.24, or at least more than 90% identity to SEQ ID No.24 (inconsistencies may be in the Framework Region (FR)).

In a specific embodiment of the present invention, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the antibody is SEQ ID No.18 and the nucleotide sequence encoding the light chain variable region of the antibody is SEQ ID No.24 (corresponding to humanized antibody Hu9F5-13 in the examples).

In another embodiment of the invention, in the nucleic acid molecule the nucleotide sequence encoding the heavy chain variable region of the antibody is SEQ ID No.16 and the nucleotide sequence encoding the light chain variable region of the antibody is SEQ ID No.17 (corresponding to chimeric antibody 9F5 in the examples).

In a third aspect, the invention claims an expression cassette, recombinant vector, recombinant cell or recombinant bacterium comprising a nucleic acid molecule as described in the second aspect above.

In a specific embodiment of the invention, the nucleotide sequence encoding the heavy chain variable region of the antibody is substituted for the fragment between the BsmBI cleavage recognition sites of vector pHr-IgG1 to yield a recombinant expression vector that expresses the heavy chain of the antibody. The nucleotide sequence encoding the light chain variable region of the antibody is substituted for the fragment between the BsmBI cleavage recognition sites of vector pHr-Kappa to obtain a recombinant expression vector expressing the light chain of the antibody.

Wherein, the vector pHr-IgG1 is a recombinant vector obtained by replacing a fragment between KpnI and Not I cleavage recognition sites of the vector pHR 'CMV GFP with a target DNA molecule with a nucleic acid sequence shown as SEQ ID No.25, and keeping other nucleotide sequences of the vector pHR' CMV GFP unchanged. SEQ ID No.25 shows the signal peptide gene at positions 19-75, the buffer sequence at positions 76-123 (BsmBI cleavage recognition sites are located at both ends of the buffer sequence), and the human IgG1 constant region (CH 1+junction +CH2+CH 3) at positions 124-1116. The vector pHr-Kappa is a recombinant vector obtained by replacing a fragment between KpnI and Not I cleavage recognition sites of vector pHR 'CMV GFP with a target DNA molecule with a nucleic acid sequence shown as SEQ ID No.26, and keeping other nucleotide sequences of the vector pHR' CMV GFP unchanged. SEQ ID No.26 shows the signal peptide gene at 19-84, the buffer sequence at 85-132 (BsmBI cleavage recognition sites at both ends of the buffer sequence) and the human Kappa Constant region (Constant Kappa) at 133-456.

The recombinant cells are obtained by co-transfecting 293F cells with the two recombinant expression vectors respectively expressing the heavy chain and the light chain of the antibody.

In a fourth aspect, the invention claims a pharmaceutical composition.

The pharmaceutical composition claimed in the present invention comprises an antibody as described in the first aspect hereinbefore and a pharmaceutically acceptable excipient, diluent or carrier.

In a fifth aspect, the invention also claims an application as shown in any one of the following:

(A1) Use of a nucleic acid molecule according to the second aspect of the foregoing or an expression cassette, recombinant vector, recombinant cell or recombinant bacterium according to the third aspect of the foregoing for the preparation of an antibody or antigen-binding fragment according to the first aspect of the foregoing or a pharmaceutical composition according to the fourth aspect of the foregoing;

(A2) The use of an antibody as described in the first aspect hereinbefore for the preparation of a pharmaceutical composition as described in the fourth aspect hereinbefore;

(A3) Use of an antibody as described in the first aspect hereinbefore or of an antigen binding fragment as described or of a nucleic acid molecule as described in the second aspect hereinbefore or of an expression cassette as described in the third aspect hereinbefore, of a recombinant vector, of a recombinant cell or of a recombinant bacterium or of a pharmaceutical composition as described in the fourth aspect hereinbefore, for the preparation of a product for the prophylaxis and/or treatment of a disease associated with CCR8 overexpression;

in a specific embodiment of the invention, the CCR8 high expression associated disease is colorectal cancer. It should be noted that the scope of the invention is not limited to colorectal cancer.

(A4) Use of an antibody as described in the first aspect hereinbefore or of an antigen binding fragment as described or of a nucleic acid molecule as described in the second aspect hereinbefore or of an expression cassette, recombinant vector, recombinant cell or recombinant bacterium as described in the third aspect hereinbefore or of a pharmaceutical composition as described in the fourth aspect hereinbefore for the manufacture of a product for the detection of CCR 8;

(A5) Use of an antibody as described in the first aspect hereinbefore or of an antigen binding fragment as described or of a nucleic acid molecule as described in the second aspect hereinbefore or of an expression cassette, recombinant vector, recombinant cell or recombinant bacterium as described in the third aspect hereinbefore or of a pharmaceutical composition as described in the fourth aspect hereinbefore for the preparation of a product for binding CCR 8;

(A6) Use of an antibody or said antigen binding fragment of the first aspect of the foregoing or a nucleic acid molecule of the second aspect of the foregoing or an expression cassette, recombinant vector, recombinant cell or recombinant bacterium of the third aspect of the foregoing or a pharmaceutical composition of the fourth aspect of the foregoing for the preparation of a product for blocking the stimulation of CCR8 by CCL 1;

(A7) Use of an antibody or an antigen binding fragment as described in the first aspect of the foregoing or a nucleic acid molecule as described in the second aspect of the foregoing or an expression cassette, recombinant vector, recombinant cell or recombinant bacterium as described in the third aspect of the foregoing or a pharmaceutical composition as described in the fourth aspect of the foregoing for the preparation of a product for activating an effector cell (ADCC effector cell);

(A8) Use of an antibody as described in the first aspect hereinbefore or of an antigen binding fragment as described or of a nucleic acid molecule as described in the second aspect hereinbefore or of an expression cassette, recombinant vector, recombinant cell or recombinant bacterium as described in the third aspect hereinbefore or of a pharmaceutical composition as described in the fourth aspect hereinbefore, for the preparation of a product for targeting regulatory T cells bearing CCR 8;

(A9) Use of an antibody as described in the first aspect hereinbefore or of an antigen binding fragment as described or of a nucleic acid molecule as described in the second aspect hereinbefore or of an expression cassette, recombinant vector, recombinant cell or recombinant bacterium as described in the third aspect hereinbefore or of a pharmaceutical composition as described in the fourth aspect hereinbefore for the preparation of a fusion protein, bispecific antibody or therapeutic chimeric antigen receptor (CHIMERIC ANTIGEN receptor, CAR).

The antibody has high specificity and high binding activity to human CCR8 (hCR 8), and can effectively mediate the cytotoxic killing of effector cells to cells expressing hCR 8, thereby having drug formation. Furthermore, the antibodies can also bind efficiently to monkey CCR8 molecules, which is of particular importance for preclinical studies of antibody drugs.

Drawings

FIG. 1 shows the results of flow cytometry assays for stably transfected cell lines hCR 8-HEK293 and hCR 8-CHOK 1. a is the hCR 8-HEK293 result; b is the hCR 8-CHOK1 result.

FIG. 2 is a SDS-PAGE electrophoresis of hCR 8-Fc fusion proteins. Where M represents the molecular weight standard of the protein, lane 1 is in the non-reduced state (dimer, molecular weight about 60.4 kD) and lane 2 is in the reduced state (monomer, molecular weight about 30.2 kD).

FIG. 3 is a SDS-PAGE electrophoresis of hCR 8-mFc fusion protein. Where M represents the molecular weight standard of the protein, lane 1 is in the non-reduced state (dimer, molecular weight about 60.4 kD) and lane 2 is in the reduced state (monomer, molecular weight about 30.2 kD).

FIG. 4 is a plot of the fit of the chimeric antibody cell ELISA assay OD450 values to the antibody concentration (abscissa).

FIG. 5 is a graph of OD450 values measured in different 9F5 humanized molecular cell ELISA fitted to different antibody concentrations (abscissa). FIG. REFERENCE AB shows the REFERENCE AB-B antibody.

FIG. 6 shows the binding activity of the selected optimal 9F5 humanized molecule Hu9F5-13 to hCR 8-HEK293 at various concentrations as measured by flow cytometry. Log10 for concentration (ng/ml) values is a curve fitted on the abscissa with fluorescence signal intensity on the ordinate. Reference antibody B in the figure indicates REFERENCE AB-B antibody.

FIG. 7 shows the binding activity of the optimal 9F5 humanized molecule Hu9F5-13 to mCCR-HEK 293 (stable monkey CCR8 cells) at various concentrations as measured by flow cytometry. Log10 for concentration (ng/ml) values is a curve fitted on the abscissa with fluorescence signal intensity on the ordinate. Reference antibody B in the figure indicates REFERENCE AB-B antibody.

FIG. 8 is a reporter assay for the binding activity of Hu9F5-13 molecules blocking CCR8 to its ligand. The fluorescence values read by chemiluminescence are on the ordinate and Log10 of antibody concentration (ng/ml) values are on the abscissa as shown.

FIG. 9 shows the activation of luciferase expression in human CD16A-Jurkat cells mediated by Hu9F5-13 molecule binding target cells (hCR 8-HEK 293) as measured by reporter gene method, the luciferase activity values (ordinate) as measured by chemiluminescent method, and Log10 of antibody concentration (ng/ml) values as abscissa, and fitting to obtain a curve. Meanwhile, the 9F5 humanized molecule and the chimeric antibody molecule can effectively activate the expression of the effector cell luciferase gene and characterize the activity of activating antibody-dependent cellular cytotoxicity (ADCC).

FIG. 10 is a graph showing the binding and dissociation curves of the affinity assay of the Hu9F5-13 molecule for hCR 8-Fc (see example 2) using the SPR principle.

FIG. 11 shows the results of the tumor-inhibiting activity of the Hu9F5-13 molecule in the mouse transplanted tumor model MC 38.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 construction of CCR8 stable Trans cell lines

The human CCR 8-encoding gene cDNA clone (NCBI access No. NM-005201.4) was purchased from Yinqiao Shenzhou. Primers were designed containing primers specific for the 5 'and 3' -ends of the genes (lowercase) and primers homologous to the vector (uppercase) and the sequences were as follows:

Upstream primer F:5'-CTCGAGACGCGTGGCGCCACCatg gat tat aca cttgacctcagt-3' (underlined is the recognition sequence for the cleavage site MluI);

The downstream primer R:5'-GATCCGGCGGCCGCtcacaaaatgtagtc tac gctgga-3' (underlined is the recognition sequence for the cleavage site NotI).

The primer was used for Polymerase Chain Reaction (PCR) amplification of genes, and the reaction system is shown in Table 1.

TABLE 1 PCR reaction System for amplifying CCR8 Gene

Reagent(s)	Volume of
		Upstream primer F (10. Mu.M)	2.5μl
Downstream primer R (10. Mu.M)	2.5μl
		Stencil (20 ng/. Mu.l)	1μl
Deionized water (ddH ₂ O)	19μl
		Phusion 2xMM	25μl

Wherein Phusion 2xMM is a 2-fold premixed reaction solution of Phusion high-fidelity DNA polymerase (NEB, cat. No. M0536).

The reaction procedure was: 98 ℃ for 30s; 8s at 98 ℃, 20s at 56 ℃ and 40s at 72 ℃, and the steps 2-4 are repeatedly circulated for 25 times; further, the sample was stored at 4℃after being extended at 72℃for 8 minutes.

The amplified DNA fragment was recovered and purified using a gel recovery kit (OMEGA), and the concentration of the purified DNA fragment was 98.1 ng/. Mu.l. pTargeT plasmid (Promega) was digested with MluI and NotI restriction enzymes and reacted overnight at 37℃to give linearized vector. The cleavage system is shown in Table 2.

TABLE 2 enzyme digestion System

Reagent(s)	Volume of
		MluI	3μl
NotI	3μl
		Buffer	4μl
PTargeT plasmid	10μg
		Deionized water (ddH ₂ O)	Up to 100. Mu.l

And (3) recovering and purifying the linearized vector by using an OMEGA gel recovery kit. Purified linearized support was obtained at a concentration of 46.70 ng/. Mu.l. After the gene fragment and the linearized vector were ligated with T4 ligase (purchased from NEB), 2.5. Mu.l of transformed E.coli competent cells (DH 5. Alpha., beijing Soy Biotechnology Co., ltd.) were taken, the whole transformation system was plated on a2 XYT solid culture plate, and cultured overnight at 37℃to obtain transformant colonies. The monoclonal colonies were picked, plasmids were extracted (OMEGA plasmid extraction kit), and were sent to sequencing and verification by Shanghai, inc. The plasmid which was confirmed to be correct by sequencing was designated pTargeT-hCR 8. pTargeT-hCR 8 is described as: a recombinant vector was obtained by inserting the CDS sequence (136-1203 of NCBI access No. NM-005201.4) of the CCR 8-encoding gene between the cleavage sites MluI and NotI of the pTargeT plasmid.

CHO-K1 and HEK293 cells were cultured in cell culture plates or six well plates with the addition of 10% Fetal Bovine Serum (FBS) and green streptomycin (P/S, i.e. penicillin penicillin and streptomycin streptomycin) in DMEM or RPMI1640 medium. Cell transfection was performed according to instructions with lipofectamin3000 (Inlet's strapdown) as a lipofection reagent, and pTargeT-hCCR8 expression plasmid was transferred into the cells. After 24 hours of transfection, 10. Mu.g/ml Neomycin (Neomycin) was added and the culture continued. After 5 days, subcloning was performed by limiting dilution. Limiting dilution by cell counting, sucking the culture volume containing 50 cells (if 10. Mu.l containing 50 cells, then sucking 10. Mu.l of culture) to 15ml of fresh culture medium, reversing and mixing uniformly, sub-packaging the diluted culture medium into 96-well cell culture plates with 150. Mu.l of each well, placing into a 37 ℃ incubator with 5% CO ₂, and culturing for 10 days to obtain monoclonalized hCR 8-CHO-K1 and hCR 8-HEK293 stably transformed cells. Expression of target molecules on the surfaces of hCCR8 stably transformed CHO-K1 and HEK293 cells was detected by flow cytometry with commercially available anti-CCR 8 antibodies (Abcam, ab 140796). Specifically, anti-CCR 8 antibodies (Abcam, ab 140796) were diluted to 1% fetal bovine serum (fetal bovine serum FBS purchased from Gibco, cat No. 10099-141C, diluted to PBS (cell culture, cat No. P1020) at a volume/volume ratio) at final concentrations of 20 microgram/ml, 4 microgram/ml, 0.8 microgram/ml, 0.16 microgram/ml, 0.032 microgram/ml, 0.0064 microgram/ml, 0.00128 microgram/ml and 0, respectively. 100. Mu.l of each concentration of antibody solution was incubated with 500000 cells (4 ℃ C., 30 minutes), the cells were washed with 1% FBS, resuspended in 200. Mu.l of washing solution during washing, centrifuged at 300 Xg, and the supernatant was discarded, and the washing was performed twice. Binding of the antibody to the cells was then detected with a fluorescent secondary antibody. Fluorescent secondary antibodies were purchased from Jackson Immunoresearch (Cat: 115-603-003), diluted 200-fold with 1% FBS, added to the cells, 100 μl of each sample was added and incubated at 4deg.C for 30 minutes. cells were then washed with 1% FBS, resuspended in 200. Mu.L of wash solution, and centrifuged at 300 Xg, and the supernatant was discarded, and the cells were washed twice. Finally, the cells are resuspended by PBS, and the average fluorescence signal value of each sample is read by an on-line (Welgrow Easycell) analyzer to obtain curves of the antibody binding stable cell lines with different concentrations as shown in figure 1a and figure 1 b. As can be seen from the figure: stably transformed HEK293 and CHO-K1 both expressed hCR 8 higher, whereas wild type cells did not detect any expression of CCR8 molecules.

To facilitate the study of species cross-reactions, we also constructed stable transgenic cell lines of monkey CCR8 (mCCR, a homologous molecule to cynomolgus monkey (mucaca mulatta)). The method is the same as the construction of human CCR8 (hCR 8) stable transgenic cell strain, and mCCR-HEK 293 cell strain is finally obtained. mCCR8 can be found in Uniprot database O97665 CCR8-MACMU.

Example 2 preparation of hCR 8 (1-35 aa) -fusion protein

The N-terminal sequence 1-35 amino acids and the first loop domain (loop 1) of the human CCR8 (hCR 8) receptor are the regions that bind in contact with the ligand, especially the N-terminal 1-35aa, and thus schemes were devised to clone these 35aa, express them in Fc and mFc-fusion and obtain purified fusion proteins for immunological and anti-serum, antibody binding assays. The coding genes of 1-35aa are designed by using an online design tool, and the following overlapped primers are designed and synthesized, and the coding genes are synthesized by using the primers as overlapped PCR (overlapping PCR).

HCCR81-35aa encoding gene synthesis overlap primers:

C8-hF-P1：5’-CTTGTCGCGATTCTTAAGGGTGTCCAGTGCATGGACTACACCC-3’；

C8-hF-P2：5’-GGATAGTAATAGTCGGTGACTGTAGTGACACTCAGGTCCAGGGTGTAGTCCATGCA CTG-3’；

C8-hF-P3：5’-TACAGTCACCGACTATTACTATCCCGACATTTTCTCCAGCCCTTGTGACGCCGAA CTGA-3’；

C8-hF-P4：5’-GTTTTGTCACTAGATTTGGGCTCCTTGCCATTTGTTTGAATCAGTTCGGCGTCAC AAG-3’。

The gene synthesis was carried out in two steps, and the first reaction system is shown in Table 3.

TABLE 3 first-step reaction System

The procedure is: 98 ℃ for 30s; repeating the steps 2-4 for 4 cycles at 98 ℃ for 10s,55 ℃ for 25s and 72 ℃ for 10 s; an additional 8 minutes at 72 ℃. Preserving at 4 ℃.

The second-stage reaction system is shown in Table 4.

TABLE 4 second step reaction System

Reagent(s)	Volume of
		Reaction product of the first step	2μl
C8-hF-P1(10μM)	2.5μl
		C8-hF-P4(10μM)	2.5μl
2×PCR mastermix(Phusion，NEB)	25μl
		Distilled water	18μl

The procedure is: steps 2-4 were repeated for 16 cycles at 98℃30s,98℃10s,55℃25s,72℃10s, and an additional extension at 72℃for 8 minutes. Preserving at 4 ℃.

The PCR products were separated by agarose gel electrophoresis to obtain fragments of the correct size and recovered and purified using a gel recovery kit (OMEGA).

Vector pHr-hG1Fc (also known as pHr-Fc vector) and pHr-mG2aFc (also known as pHr-mFc vector) were engineered on the basis of vector pHR' CMV GFP (derived from Addgene, plasmid # 14858), vector pHr-Fc, in particular: the DNA molecule with the nucleic acid sequence shown in SEQ ID No.27 is used for replacing a fragment between KpnI and Not I cleavage recognition sites of vector pHR 'CMV GFP (a small fragment between KpnI and Not I cleavage recognition sites), and other nucleotide sequences of vector pHR' CMV GFP are kept unchanged to obtain the recombinant vector pHr-Fc. Wherein, the 19 th to 75 th positions of SEQ ID No.27 are signal peptide genes, the 76 th to 123 th positions are buffer sequences (BsmBI restriction enzyme cleavage recognition sites are arranged at two ends of the buffer sequences), and the 124 th to 819 th positions are human IgG1 constant regions (CH 2+CH 3). The modification of PHr-mFc is similar, and the sequence between KpnI and Not I cleavage recognition sites is shown in SEQ ID No. 28. The 19 th to 75 th positions of SEQ ID No.28 are signal peptide genes, the 76 th to 123 th positions are buffer sequences (BsmBI restriction enzyme recognition sites are arranged at two ends of the buffer sequences), and the 124 th to 822 th positions are mouse IgG2a constant regions (CH 2+CH 3).

In order to clone the fragment of hCR 81-35 coding gene into pHr-Fc and pHr-mFc, respectively, a linear vector was obtained by digestion with BsmBI restriction enzymes at the multiple cloning sites of pHr-Fc and pHr-mFc, the target DNA fragment (hCR 81-35aa coding gene) was provided with a stretch (about 30 nt) of nucleotide sequence homologous to the vector at both ends, the extended gene was transformed into Transblue (full gold) DH 5. Alpha. Cells (from Solabao organism) together with the linear vector, the transformant colonies were picked up, cultured and plasmid extracted and sequenced to obtain the expression plasmids correctly ligated into the vector, designated pHr-hCR 8-1-35aa-Fc and pHr-hCR 8-1-35aa-mFc.

And preparing enough expression plasmids pHr-hCR 8-1-35aa-Fc and pHr-hCR 8-1-35aa-mFc, transfecting 293F cells, expressing hCR 8-Fc and hCR 8-mFc fusion proteins, secreting the fusion proteins into culture supernatant, and performing affinity purification to obtain the target protein. Specifically, 100. Mu.g of expression plasmid pHr-hCR 8-1-35aa (i.e., pHr-hCR 8-1-35aa-Fc or pHr-hCR 8-1-35 aa-mFc) was mixed with the transfection reagent PEI, diluted with the medium, allowed to stand, added to 293F cell culture broth, cultured with shaking at 37℃for 24 hours with 5% carbon dioxide, and supplemented with feed A (available from kai R.pearls, cat No. K40001) and shake-cultured for 3 days. The culture was centrifuged at an extreme speed (12000 rpm) for 30 minutes and the supernatant was collected into a new shake flask. For hCR 8-Fc or hCR 8-mFc fusion proteins, 1ml of ProteinA gel filler (Boglaung AA 402305) was added, and after incubation for 1-3 hours with shaking (145 rpm) at room temperature, it was passed through a gravity chromatography column and the effluent was collected for cryopreservation for further testing. The column was rinsed with 5ml of PBS and the wash solution was allowed to flow slowly through the gel column. 3ml glycine eluent (pH 3.2) was added and the captured proteins were eluted. All eluates were centrifuged in ultrafiltration concentrate centrifuge tubes (Millipore), and replaced in sterile pyrogen-free PBS. The ultraviolet light absorption at 280nm wavelength is measured, and divided by the extinction coefficient to obtain the concentration value, PBS is used for adjusting the concentration value to 2mg/ml, and the obtained product is split-packaged and frozen in a refrigerator at the temperature of minus 80 ℃.

2. Mu.g of the fusion protein was taken, a proper amount of loading buffer (next holy) was added according to instructions, treated at 95℃for 8 minutes, and stored at 4℃for 2 minutes. SDS-PAGE was performed on this sample. The gel was stained with coomassie blue fast dye (next holy) to check sample purity and molecular size. hCR 8-Fc is shown in FIG. 2. hCR 8-mFc is shown in FIG. 3. It can be seen that both fusion proteins are normally expressed and purified to give a product with a purity of over 95%, the size of the electrophoretic migration being slightly larger than expected, possibly through normal glycosylation and other post-translational modifications, which are usually necessary for their activity. It is believed that these fusion proteins can be used in immunological and antisera, antibody detection assays.

Example 3 animal immunization

Monoclonal hCR 8-CHO-K1 cells (constructed in example 1) were expanded, passaged 2X 10 ⁵, grown for about 3-4 days at 37℃in 5% CO ₂ to reach 2X 10 ⁷ plates, adherent hCR 8-CHO-K1 cells were trypsinized, all cells collected by centrifugation, counted, centrifuged at 300 Xg, the supernatant removed, and resuspended in 2ml of non-heat phosphate buffer PBS. 10 mice (Balb/c mice were purchased from Venlhua, total of 10) were injected with a suspension of hCCR8-CHO-K1 cells, subcutaneously injected in multiple spots in their forelimbs, under the neck and back, and a total of 1X 10 ⁷ cells, in a volume of about 100. Mu.l. The injection is performed every two weeks. About 20. Mu.l of blood was taken from the vein of the animal on day 7 after the second injection immunization, and 20. Mu.l of blood was taken on the seventh day after each immunization, and serum titer detection was performed. The serum titer detection method is as follows:

1. Serum was prepared by centrifuging whole blood, placing the whole blood in a small tube, placing the whole blood in a centrifuge, and centrifuging at 13000 Xg for 2 minutes. Carefully aspirate the supernatant and transfer to a new tube to obtain serum.

2. HCR 8-HEK293 cells 50000/well were dispensed to 96-well cell culture plates, incubated for 48 hours, the supernatant was discarded, washed with PBS, and 4% tissue fixative (solarbio), RT,30min was added.

3. The supernatant was discarded, rinsed twice with 200. Mu.l PBS, and the mixture was dried by pipetting. Mu.l of 5% skim milk was added and incubated at 4℃overnight or 37℃for 2 h.

4. The supernatant was discarded and washed twice with 300. Mu.l/well of PBS-T. To this end, the cell ELISA plate can be frozen or the ELISA assay can be continued.

5. Serum was diluted with 1% BSA (w/v in PBS-T (0.05% Tween 20, hereinafter the same) at a maximum concentration of 1:500, diluted 5-fold. 7 different dilutions of serum solutions were obtained.

6. The diluted serum solution was added to the wells of the cell ELISA plate at 50. Mu.l/well. Incubate at 37℃for 1h.

7. The supernatant was discarded, wells were washed, 300. Mu.l/well PBST, 5 times.

8. The ELISA plates were dried and secondary antibodies were added. The secondary antibody was HRP-goat anti-mouse IgG (Jackson immunoresearch, cat# 115-035-062). 20000-fold dilution with 1% bsa. Incubate at 37℃for 1h.

9. The supernatant was discarded, wells were washed, 300. Mu.l/well PBST, 5 times.

10. The ELISA plate was dried on absorbent paper, 50. Mu.l of the color development solution was added, and developed at 37℃or RT for 2min.

11. Add 50. Mu.l/Kong Zhongzhi (ready-to-use, solarbio) and read OD450.

The signal value per well divided by the signal background at the 0 concentration point gives a factor F, F greater than 2.1, which is considered significantly positive. The maximum dilution of the positive wells was recorded as the serum titer of the animal.

Table 5 is the OD450 readings measured for each mouse. Table 6 shows the calculated F values. It can be seen that all mouse sera had a binding response to hCCR8 stable cells, meaning that IgG antibodies were produced in vivo. Wherein, the titers of 5# and 8# and 10# are highest, the titers of 8 and 10# reach 156 ten thousand, and 5# reaches 31 ten thousand.

TABLE 5 OD450 readings from each mouse

TABLE 6F values calculated from the results of TABLE 5

Example 4 preparation of hybridomas and selection by spleen cell fusion

1. Preparation of SP2/0 cells

5 Bottles of SP/20 cells (SKU: SNL-120, available from Wohang Shang Biotechnology Co., ltd.) with a growth of 80-90% were taken, the supernatant was aspirated, the cells were scraped off by beating on a table, the cells were collected with PBS to a 50ml centrifuge tube, the cells were collected by centrifugation at 1500rpm for 3 minutes, and the cells were resuspended in 40ml PBS, and the SP/20 cells were counted as: 3.9X10 ⁶/ml, activity 94.57%.

2. Treatment of spleen cells

(1) The number 8 mouse obtained in example 3 was selected, sacrificed by cervical removal, immersed in 75% alcohol for 5min, and the spleens of the mice were removed with sterile ophthalmic scissors and forceps, respectively, to remove connective tissue around the spleens.

(2) The spleen was ground with a 5ml syringe plunger on a 200 mesh stainless steel cell screen (PBS immersed screen), and the spleen cells remaining on the screen and syringe plunger were rinsed off with PBS, passed through a 40um cell screen into a 50ml graduated centrifuge tube, 300 Xg, and centrifuged for 7min.

(3) The supernatant was aspirated, 2ml RBC lysis buffer (Sigma) was added and the cells resuspended and after 1min lysis was stopped by the addition of 20ml PBS. 300 Xg, centrifuged for 7min, the supernatant was aspirated, and the cells were resuspended in 2ml PBS and fixed to 20ml. The spleen cells of the mice were counted as: 3.16X10 ⁶/ml, activity 60.54%; SP2/0 is: 3.41 multiplied by 10 ⁶/ml, the activity rate is more than 66.16%.

3. Fusion of

Spleen cells from CCR8 immunized mice (8#) were mixed with SP2/0 cells in a 2:1 ratio, 300 Xg, centrifuged for 7min, and resuspended in 15ml Medium C (BTX), 300 Xg, centrifuged for 7min. The procedure was repeated twice. Finally resuspended in 12ml Medium C. 6ml of the cell suspension was added to 9ml of the fusion electrode (BTX ECM 2001) and fused according to the parameters shown in Table 1 to obtain hybridoma cells. The fused hybridoma cells were resuspended in 600ml of complete medium (PRIM 1640+15% FBS+1. Times. HAT; sigma), and the cells were inoculated in 200. Mu.l/well into 30 96-well plates and incubated in a 5% CO ₂ incubator at 37℃for 7 days. Wherein, the cell fusion parameters are set forth in Table 7.

TABLE 7 cell fusion parameter settings

AC	AC duration	DC	Pulse length	Post-fusion AC	Number of pulse
						45V	30S	850V	30μs	9s	1

4. Screening

HCR 8-HEK293 cells (constructed in example 1) were dispensed at 5X 10 ⁴/well into 96-well cell culture plates, incubated for 48 hours, the supernatant was discarded, washed with 200. Mu.l PBS, and 4% tissue fixative (solarbio), RT,30min was added. The supernatant was discarded, rinsed twice with 200. Mu.l PBS, and the mixture was dried by pipetting. Mu.l of 5% skim milk (skim milk powder An Jia skim milk powder (New Zealand original entry) was dissolved in PBS-T) was added and incubated at 4℃overnight or 37℃for 2 h. The supernatant was discarded and washed twice with 300. Mu.l/well of PBS-T. To this end, the cell ELISA plate can be frozen or the ELISA assay can be continued.

To test whether hybridomas secrete antibodies that bind hCR 8, we aspirate 50. Mu.l/Kong Zajiao tumor cell culture supernatant and add to an ELISA plate coated with hCR 8-HEK293 cells for 1 hour at 37 ℃. After washing the plate 5 times with PBS-T, adding horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Jackson immunoResearch, cat# 115-035-062), reacting for 1 hour, washing the plate 5 times with PBS-T, adding TMB (50. Mu.l/well, beijing Soy Biotechnology Co., ltd., cat# PR 1200) for color development for 10 minutes, and stopping the reaction at 50. Mu.l/well 2M H ₂SO₄, and reading the OD450 light absorption value by an ELISA reader. Binding activity assay of hybridoma cell culture supernatants to HEK293 cells reference the method described above, thus excluding wells of plate that non-specifically bind to blank HEK293 cells.

The list of ELISA response signals of positive hybridoma clones and hCR 8-HEK293 and blank HEK293 are shown in Table 8. It can be seen that: the culture supernatants of these positive hybridomas contained significantly antibodies that bound hCCR8-HEK 293.

TABLE 8 ELISA reaction signals of each hybridoma clone with hCR 8-HEK293 and blank HEK293

Example 5 subcloning, antibody sequencing and chimeric antibody construction

The hybridomas positive for the primary screening of example 4 were subcloned by limiting dilution to obtain positive monoclonal cell lines. Specifically, cells in positive wells (OD 450 value greater than 0.3) were aspirated and diluted to 50 cells/20 ml in complete medium, and the cell suspension was dispensed into 96-well plates with 200. Mu.l per well, with an average of 0.5 cells per well, i.e., about half of the wells had one cell, and a population of monoclonal cells was desired. After 7 days of culture in an incubator, cell ELISA screening was performed to obtain plate hole data with positive binding activity, positive holes were selected for expansion culture, 1 million cells were harvested after expansion to T75 bottle, lysis and RNA extraction were performed (performed as described in kit, tiangen organism, cat No. DP 430). cDNA synthesis was performed using RNA as a template according to the kit instructions (Semerle, cat. No. 4387406) with a synthesis system of 20. Mu.l. Then, the DNA encoding the variable region of the antibody was amplified by PCR using the cDNA as a template. The primers used to amplify the antibody encoding DNA are shown in table 9.

TABLE 9 primers for amplifying antibody-encoding DNA

Note that: degenerate bases referred to in the primer sequences in the tables are all well known in the art.

Taq DNA polymerase was purchased from Beijing Soy Biotechnology Co., ltd., cat: the PC1100 and PCR reaction system are shown in Table 10.

TABLE 10PCR reaction System

Reagent(s)	Volume of
		10 Xbuffer	5μl
Taq DNA polymerase	1μl
		dNTPs(2.5mM each)	4μl
cDNA	3μl
		Primer mIgG-R122 (heavy chain) or mIg κ -117 (light chain)	2.5μl
Mixed primer (primer combination P1-P7)	2.5μl
		Deionized water	32μl
Total volume of	50μl

The amplification procedure was: (1) 94℃for 1min, (2) 94℃for 15sec, (3) 56℃for 20sec, (4) 72 DEG C

30Sec, 8min at 72℃and 4 ℃. Wherein (2) - (4) are cycled 30 times.

The amplified DNA fragment (PCR product) was separated by electrophoresis in a 1.5% agarose gel (constant voltage 140V, electrophoresis for 20 minutes), a distinct band was visible under an ultraviolet lamp, excised with a blade, and DNA was recovered with a gel recovery kit (OMEGA, cat No.). The samples were sent to the biological engineering (Shanghai) Co.Ltd for sequencing, the heavy chain gene sequencing primer was mIgG-R122 and the light chain gene sequencing primer was mIg kappa-R117.

The variable region DNA sequences were obtained by sequencing and the variable region amino acid sequences of the multiple antibody clones obtained by on-line tool https:// imgt. Org/download/V-QUEST/analysis are shown in Table 11.

TABLE 11 variable region amino acid sequences of multiple antibody clones

Example 7 chimeric antibody construction, expression and binding assays

The heavy chain variable region encoding gene of each of the murine monoclonal antibodies in example 6 (see Table 12, since amino acid N at position 107 of the light chain variable region of the 9F5 antibody is relatively rare at this position, and thus corrected to amino acid K at the time of constructing the chimeric antibody) was constructed into vector pHr-IgG1, and, taking the recombinant expression of the 9F5 heavy chain as an example, the fragment between the BsmBI cleavage recognition sites (small fragment including the BsmBI cleavage recognition site) of vector pHr-IgG1 was replaced with the DNA molecule having the nucleotide sequence of SEQ ID No.16 to obtain recombinant expression vector pHr-IgG1-9F5, and recombinant expression vector pHr-IgG1-9F5 was able to express the heavy chain of chimeric antibody 9F 5.

The light chain variable region gene (see Table 12) of each of the murine monoclonal antibodies of example 6 was constructed into vector pHr-Kappa, and, as an example of recombinant expression of the 9F5 light chain, the fragment between the BsmBI cleavage recognition sites of vector pHr-Kappa (small fragment containing the BsmBI cleavage recognition site) was replaced with a DNA molecule having the nucleotide sequence of SEQ ID No.17 to obtain recombinant expression vector pHr-Kappa-9F5, and recombinant expression vector pHr-Kappa-9F5 was able to express the light chain of chimeric antibody 9F 5.

Vectors pHr-IgG1 and pHr-Kappa were engineered on the basis of vector pHR' CMV GFP (from Addgene, plasmid # 14858), exemplified by vector pHr-IgG1, specifically: the target DNA molecule with the nucleic acid sequence shown in SEQ ID No.25 is used for replacing a fragment between KpnI and Not I cleavage recognition sites of vector pHR 'CMV GFP (a small fragment between KpnI and Not I cleavage recognition sites), and the recombinant vector pHr-IgG1 is obtained after other nucleotide sequences of vector pHR' CMV GFP are kept unchanged. Wherein, the 19 th to 75th positions of SEQ ID No.25 are signal peptide genes, the 76 th to 123 th positions are buffer sequences (BsmBI restriction enzyme recognition sites are arranged at two ends of the buffer sequences), and the 124 th to 1116 th positions are human IgG1 constant regions (CH 1+junction +CH2+CH 3).

Construction of vector pHr-Kappa reference pHr-IgG1, differing only in the substitution of the DNA of interest with the DNA fragment shown in SEQ ID No. 26. Wherein, the 19 th to 84 th positions of SEQ ID No.26 are signal peptide genes, the 85 th to 132 th positions are buffer sequences (BsmBI digestion recognition sites are arranged at two ends of the buffer sequences), and the 133 th to 456 th positions are human Kappa Constant regions (Constant Kappa).

TABLE 12 variable region nucleotide sequences of individual antibody clones

Taking 9F5 as an example, 293F cells were co-transfected with heavy chain expression plasmid pHr-IgG1-9F5 and light chain expression plasmid pHr-Kappa-9F5 at a ratio of 1.5:1, and the cell culture broth was affinity purified to obtain the objective antibody (chimeric antibody). Specifically, the heavy chain expression plasmid and the light chain expression plasmid (wherein the recombinant expression vector pHr-IgG1-9F5 is 150. Mu.g, the recombinant expression vector pHr-Kappa-9F5 is 100. Mu.g) of the chimeric antibody are mixed with the transfection reagent PEI, diluted with a culture medium (opti-mem 5 ml), left to stand, added to 293F cell culture broth, subjected to shake culture at 37 ℃ for 24 hours with 5% carbon dioxide, added with a feed A (purchased from Pinctada kai Rayleigh, cat No. K40001), and subjected to shake culture for 3 days to obtain cell culture broth. The cell culture broth was centrifuged at 12000rpm for 30 minutes and the supernatant was collected into a new shake flask. To the shake flask with the supernatant collected, 1ml of Protein A gel filler (Boguron, AA 0131) was added, and after incubation for 1-3 hours with shaking (145 rpm) at room temperature, it was allowed to flow through the gravity chromatography column and the effluent was collected for sampling and frozen for retesting.

The column was rinsed with 5ml of PBS and the wash solution was allowed to flow slowly through the gel column. 3ml glycine eluent (pH 3.2) was added and the captured proteins were eluted. And (3) carrying out affinity purification by using Protein A gel to obtain a recombinant chimeric antibody solution with the purity of more than 95%, and finally changing the solution into phosphate buffer PBS.

The remaining chimeric antibodies were prepared as described with reference to 9F5, which differs only in: the heavy and light chain expression plasmids pHr-IgG1-9F5 and pHr-Kappa-9F5 of the transfected 293F cells were replaced with the corresponding heavy and light chain encoding gene plasmids.

The binding strength of the antibody to CCR8 molecules on the cell surface is detected by adopting a cell ELISA method. The preparation method of the cell ELISA detection plate comprises the following steps: hCCR8-HEK293 cells (constructed in example 1) were aliquoted into 96-well plates (taan technology, cat# 02047364), 5×10 ⁴ cells per well, and after incubation with 200 μl of complete medium (RPMI 1640, P/S1:100,Puromycin 1mg/ml, FBS 10%) for 24 hours at 37 ℃ with 5% co ₂, 300×g was centrifuged for 4 min and the supernatant was discarded. 100. Mu.l of tissue fixative (Beijing Soy Bao Biotechnology, P1110) was used for each well, incubated at 37℃for 30 minutes, the fixative was discarded, 200. Mu.l of PBS was used for each well for washing, 200. Mu.l of 5% skim milk was added to each well, and the wells were placed in a 37℃incubator for 2 hours. After 200 μl PBST wash per well, the cell ELISA was frozen in-80 ℃ freezer or antibody binding assays continued.

After careful quantification of the chimeric antibody by A280 light absorption, serial concentration dilutions were performed with a maximum concentration of 20. Mu.g/ml, followed by 5X-fold dilution with 1% BSA solution, each antibody gave 8 solutions of different concentrations (maximum concentration of 20. Mu.g/ml, 5-fold dilution, 4. Mu.g/ml, 0.8. Mu.g/ml, 0.16. Mu.g/ml, 0.032. Mu.g/ml, 0.0064. Mu.g/ml, 0.00128. Mu.g/ml and 0. Mu.g/ml of antibody solution (i.e.diluent)). 50 microliters of antibody solutions of different concentrations were added to the wells of the cell ELISA plate and reacted at 37℃for 1 hour. Wash 5 times with PBST, 200 microliters per well. After the last supernatant removal, ELISA plates were dried as much as possible, and 50. Mu.l/well of working solution of horseradish peroxide-labeled anti-human IgG secondary antibody (Jackson immunoresearch, cat# 109-035-190) (i.e.diluting the antibody 20000 times with 1% BSA solution) was added and reacted at 37℃for 1 hour. Wash 5 times with PBST, 200 microliters per well. After the last supernatant removal, ELISA plates were dried as much as possible, 50. Mu.l/well TMB chromogenic solution (Soy Bao, PR 1200) was added and developed for 5-10 min at room temperature or 37 ℃. The ultraviolet light absorption with the wavelength of 450 nanometers is read by an enzyme-labeled instrument. The resulting data was analyzed using GRAPHPAD PRISM software and the fitted curve is shown in figure 4. Table 13 shows the EC50 of each chimeric antibody. From the results, it can be seen that: the chimeric antibody such as 9F5 has a good binding activity.

TABLE 13 EC50 of chimeric antibodies

Note that: representing the fitted EC50 approximation.

Wherein REFERENCE AB-B was prepared with reference to the sequence of 4A19 published in patent WO2021/194942A 1. 4a19, the heavy and light chain variable region amino acid sequences are as follows:

>4A19VH

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDSEMHWVRQATGQGLEWMGAIQPETGGTAYNQKFKARVT MTRDTSISTAYMELSSLRSEDTAVYYCARRRRNFDYWGQGTLVTVSS

>4A19VL

DIVMTQTPLSLSVTPGQPASISCRSSQSLFHSSGNTYLHWYLQKPGQPPQLLIYKVSNRFSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCSQSTHVPFTFGQGTKLEIK

The coding genes of the genes are synthesized as follows:

>4A19vh

CAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAGCCCGGTGCCAGCGTGAAAGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACAGCGAGATGCACTGGGTGAGGCAGGCCACCGGTCAGGGCCTGGAGTGGATGGGCGCCATCCAGCCCGAGACTGGCGGCACAGCCTATAACCAGAAGTTCAAGGCAAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCTTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCGGAGGAACTTCGACTACTGGGGCCAGGGCACCCTCGTGACCGTGAGCTCT

>4A19vl

GACATCGTGATGACCCAGACCCCCCTGAGCTTGAGCGTGACCCCAGGCCAACCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGTTCCACAGCAGTGGCAACACCTACCTTCACTGGTACCTGCAGAAACCCGGTCAACCCCCGCAACTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTTAGCGGCAGCGGGAGCGGCACCGACTTCACCCTGAAGATTAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCAGTCAGTCTACCCACGTGCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG

Following the procedure for the preparation of the chimeric antibody described in example 7, we obtained the 4A19-hG 1-kappa chimeric antibody designated REFERENCE AB-B.

Example 8 humanized engineering and screening

The murine monoclonal antibody 9F5 was humanized. The antibodies were identified for HCDR1 (SEQ ID No. 1), HCDR2 (SEQ ID No. 2), HCDR3 (SEQ ID No. 3) and LCDR1 (SEQ ID No. 4), LCDR2 (SEQ ID No. 5), LCDR3 (SEQ ID No. 6) using IMGT delineation criteria, suitable human IgG germline was searched, and CDR regions were grafted to the human germline IgG variable regions (framework region FR was retained). Key amino acids requiring back mutation (back mutation) were identified by Discovery Studio software, changed to corresponding maternal monoclonal antibody amino acids, paired-combined these light and heavy chain molecules with different back mutated amino acids, co-transfected 293F cells, expressed and purified the corresponding antibody molecules, see above for specific procedures (human IgG1, kappa for the constant region).

Specific information on humanization transformation is as follows:

9F5 murine anti variable region sequence:

>9F5 heavy chain variable region: the amino acid sequence is shown in SEQ ID No.7, see Table 11; the nucleotide sequence is shown in SEQ ID No.16, see Table 12.

>9F5 light chain variable region: the amino acid sequence is shown in SEQ ID No.8, see Table 11; the nucleotide sequence is shown in SEQ ID No.17, see Table 12.

Corresponding human germline antibody variable region genes:

IGHV1-46*01

IGKV2-30*02

Humanized candidate sequences:

Heavy chain variable region VH (amino acid sequence):

>VH1-1

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGAIDPNTGRTAYNQKFKGRAT MTADTSTSTVYMELSSLRSEDTAVYYCARIRRAMDYWGQGTLVTVSS(SEQ ID No.9).

>VH1-2

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWIGAIDPNTGRTAYNQKFKGRAT MTADTSTSTVYMELSSLRSEDTAVYYCTRIRRAMDYWGQGTLVTVSS(SEQ ID No.10).

>VH1-3

QVQLVQSGAEVVKPGASVKVSCKASGYTFTDYEMHWVKQAPGQGLEWIGAIDPNTGRTAYNQKFKGRAT MTADTSTSTVYMELSSLRSEDTAVYYCTRIRRAMDYWGQGTLVTVSS(SEQ ID No.11).

>VH1-4

QVQLVQSGAEVVKPGASVKVSCKASGYTFTDYEMHWVKQAPGQGLEWIGAIDPNTGRTAYNQKFKGRAT LTADTSTSTVYMELSSLRSEDTAVYYCTRIRRAMDYWGQGTLVTVSS(SEQ ID No.12).

Light chain variable region VL (amino acid sequence):

>VL1-1

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSSGTTYLHWFQQRPGQSPRRLIYRVSNRFSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGGGTKVEIK(SEQ ID No.13).

>VL1-2

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSSGTTYLHWHQQRPGQSPRLLIYRVSNRFSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIK(SEQ ID No.14).

>VL1-3

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSSGTTYLHWHLQRPGQSPRLLIYRVSNRFSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYFCSQTTHVPWTFGGGTKVEIK(SEQ ID No.15).

heavy chain variable region VH (nucleotide sequence):

>VH1-1

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGTGCCAGCGTCAAGGTAAGCTGCAAGGCCAGCGGCTACACTTTCACAGACTACGAGATGCACTGGGTGAGGCAGGCTCCCGGCCAAGGCCTCGAGTGGATGGGCGCCATCGACCCCAACACCGGCAGGACCGCCTATAACCAGAAGTTCAAGGGCAGGGCCACCATGACCGCCGACACCAGTACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTCTACTACTGCGCCAGGATCAGGAGGGCCATGGACTACTGGGGCCAGGGAACCCTGGTGACCGTGAGCTCT(SEQ IDNo.18).

>VH1-2

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGTGCCAGCGTCAAGGTAAGCTGCAAGGCCAGCGGCTACACTTTCACAGACTACGAGATGCACTGGGTGAGGCAGGCTCCCGGCCAAGGCCTCGAGTGGATcGGCGCCATCGACCCCAACACCGGCAGGACCGCCTATAACCAGAAGTTCAAGGGCAGGGCCACCATGACCGCCGACACCAGTACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTCTACTACTGCACCAGGATCAGGAGGGCCATGGACTACTGGGGCCAGGGAACCCTGGTGACCGTGAGCTCT(SEQ IDNo.19).

>VH1-3

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGgtGAAGCCCGGTGCCAGCGTCAAGGTAAGCTGCAAGGCCAGCGGCTACACTTTCACAGACTACGAGATGCACTGGGTGAaGCAGGCTCCCGGCCAAGGCCTCGAGTGGATcGGCGCCATCGACCCCAACACCGGCAGGACCGCCTATAACCAGAAGTTCAAGGGCAGGGCCACCATGACCGCCGACACCAGTACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTCTACTACTGCACCAGGATCAGGAGGGCCATGGACTACTGGGGCCAGGGAACCCTGGTGACCGTGAGCTCT(SEQ IDNo.20).

>VH1-4

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGgtGAAGCCCGGTGCCAGCGTCAAGGTAAGCTGCAAGGCCAGCGGCTACACTTTCACAGACTACGAGATGCACTGGGTGAaGCAGGCTCCCGGCCAAGGCCTCGAGTGGATcGGCGCCATCGACCCCAACACCGGCAGGACCGCCTATAACCAGAAGTTCAAGGGCAGGGCCACCcTGACCGCCGACACCAGTACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTCTACTACTGCACCAGGATCAGGAGGGCCATGGACTACTGGGGCCAGGGAACCCTGGTGACCGTGAGCTCT(SEQ IDNo.21).

light chain variable region VL (nucleotide sequence):

>VL1-1

GACGTGGTGATGACCCAAAGCCCCCTGAGCCTGCCCGTGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGGAGCAGTCAAAGCCTGGTGCACTCAAGCGGCACCACCTACCTGCACTGGTTCCAGCAGAGGCCCGGTCAGAGCCCAAGGAGGCTGATCTACAGGGTGAGCAACAGGTTTAGCGGCGTGCCCGACAGGTTCAGCGGTAGCGGCAGCGGGACCGACTTCACCCTTAAAATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCAGCCAGACCACCCACGTGCCCTGGACCTTCGGTGGCGGAACCAAAGTGGAGATCAAG(SEQ ID No.22).

>VL1-2

GACGTGGTGATGACCCAAAGCCCCCTGAGCCTGCCCGTGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGGAGCAGTCAAAGCCTGGTGCACTCAAGCGGCACCACCTACCTGCACTGGcaCCAGCAGAGGCCCGGTCAGAGCCCAAGGctGCTGATCTACAGGGTGAGCAACAGGTTTAGCGGCGTGCCCGACAGGTTCAGCGGTAGCGGCAGCGGGACCGACTTCACCCTTAAAATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTtCTGCAGCCAGACCACCCACGTGCCCTGGACCTTCGGTGGCGGAACCAAAGTGGAGATCAAG(SEQ ID No.23).

>VL1-3

GACGTGGTGATGACCCAAAGCCCCCTGAGCCTGCCCGTGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGGAGCAGTCAAAGCCTGGTGCACTCAAGCGGCACCACCTACCTGCACTGGcaCCTGCAGAGGCCCGGTCAGAGCCCAAGGctGCTGATCTACAGGGTGAGCAACAGGTTTAGCGGCGTGCCCGACAGGTTCAGCGGTAGCGGCAGCGGGACCGACTTCACCCTTAAAATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTtCTGCAGCCAGACCACCCACGTGCCCTGGACCTTCGGTGGCGGAACCAAAGTGGAGATCAAG(SEQ ID No.24).

the resulting humanized antibody molecules are shown in Table 14.

TABLE 14 humanized antibody molecules

	VH1-1	VH1-2	VH1-3	VH1-4
					VL1-1	Hu9F5-11	Hu9F5-21	Hu9F5-31	Hu9F5-41
VL1-2	Hu9F5-12	Hu9F5-22	Hu9F5-32	Hu9F5-42
					VL1-3	Hu9F5-13	Hu9F5-23	Hu9F5-33	Hu9F5-43

The humanized antibody was assayed in parallel with reference antibody REFERENCE AB-B (see above for specific preparation) by ELISA assay at the same series of concentrations as described above for the chimeric antibody. The resulting fitted curve is shown in fig. 5. The EC50 values of each humanized antibody are shown in table 15. From the EC50 values, hu9F5-13 performed best.

TABLE 15 EC50 values for each humanized antibody

reference Ab-B	Hu9F5-43	Hu9F5-13	Hu9F5-11	Hu9F5-12	Hu9F5-21	Hu9F5-22
							EC50	11.55	108	9.033	9.687	10.46	21.17	37.94
Hu9F5-23	Hu9F5-32	Hu9F5-31	Hu9F5-41	Hu9F5-33	Hu9F5-42
							EC50	12.19	19.59	17.48	37.94	12.19	19.59

Flow cytometric assays were also performed using hCR 8-HEK293 cells (constructed in example 1) to determine that Hu9F5-13 was able to bind to CCR8 molecules on the surface of intact cells. The method is as described in example 1 for the detection of stable cell surface CCR8 molecules. Hu9F5-13 antibody was diluted in a multiple ratio at a concentration of 20 micrograms/ml, 4 micrograms/ml, 0.8 micrograms/ml, 0.16 micrograms/ml, 0.032 micrograms/ml, 0.0064 micrograms/ml, 0.00128 micrograms/ml and 0, for a total of 8 species. The average fluorescence signal intensity (MFI) values for flow cytometric assays were curve-fitted as shown in fig. 6. The results show that Hu9F5-13 binds well to CCR8 molecules on the surface of intact cells.

To examine whether Hu9F5-13 could bind to monkey CCR8, we performed flow cytometric assays using mCCR-HEK 293 cells (constructed in example 1). Specific operations are described in the foregoing. The results are shown in FIG. 7. The results indicate that Hu9F5-13 can bind to monkey CCR8 better, whereas reference antibody B binds only weakly.

Example 9, beta-Arrestin assay for ligand binding blocking Activity

Under the stimulation of ligand CCL1, CCR8 recruits beta-Arrestin and binds to the CCL 8, a stable cell strain expressing CCR8 and beta-Arrestin fused with a luciferase reporter gene is constructed according to the principle, beta-Arrestin is not bound with CCR8 when ligand stimulation is lacked, the luciferase fused with beta-Arrestin is in an inactivated conformation, when CCR8 is stimulated by ligand CCL1, beta-Arrestin fused with the luciferase reporter gene is recruited, the luciferase reporter gene is in an activated state, a luminescent signal is enhanced after a substrate is added, and when CCL1 is blocked from stimulating CCR8 by a sample to be tested, a luminescent signal of the reporter gene is inhibited.

The specific experimental process is as follows:

(1) Logarithmic growth of CCR8/β -Arrestin/CHO cells (kobai, product number CBP 71363), pancreatin digestion and termination of pancreatin reaction with f12k+10% fbs medium, centrifugation of the supernatant, resuspension of washed cells with DPBS once, further centrifugation of the supernatant, followed by resuspension of cells in Opti-MEM ^TM reduced serum medium containing 2% fbs, the cell density of the resuspension was adjusted to 3.125×10 ⁵/mL.

(2) Resuspended cells were inoculated into 96-well cell culture plates with clear bottoms on white walls, 80. Mu.L/well cell suspension, and incubated overnight in an incubator at 37 ℃.

(3) The next day, based on the concentration of the sample (all antibodies to be tested), a10 Xconcentration gradient dilution solution was prepared with Opti-MEM ^TM minus serum medium containing 2% FBS, starting from a maximum concentration of 200. Mu.g/mL (10 Xconcentration), 5-fold gradient dilution was performed, 12 concentration gradients were set up (containing 0 concentration medium control) and the gradient diluted samples were added to the 96-well plates of step (2) (10. Mu.L/well) and incubated in an incubator at 37℃for 60 minutes.

(4) CCL1 was prepared with Opti-MEM ^TM minus serum medium containing 2% FBS at 150ng/mL and added to the 96-well plate of step (3) (10. Mu.L/well), and only 1 to 11 columns and 12 columns of E, B, C, D wells, 12 columns of E, F, G, H were added to an equal volume of medium as negative control wells without ligand stimulation, and then the 96-well plate was placed in a cell incubator for further culture for 90 minutes.

(5) Will beLIVE CELL ASSAY SYSTEM in LIVE CELL ASSAY SYSTEMLive Cell Substrate for use inLCS Dilution Buffer was diluted 20 times to prepare a 5X test solution.

(6) The 96-well plate in the step (4) is taken out from the incubator, 25 mu L/well of the 5 Xdetection solution prepared in the step (5) is added, and then the mixture is vigorously shaken for 15 to 30 seconds and put into an enzyme-labeling instrument for reading.

(7) The inhibition rate of the sample corresponding to each well was calculated from the corresponding read value for each gradient concentration well, and then, based on the calculated inhibition rate and the corresponding sample concentration, a gradient curve of the sample for cell inhibition and IC50 values (IC 50 is defined as the corresponding compound concentration when the compound inhibition rate is 50%) were fitted using PRISM GRAPHPAD software.

% Inhibition = [1- (test compound well value-negative control well average)/(DMSO control well average-negative control well average) ]100

Curve fitting was performed with the resulting fluorescent signal values versus antibody concentration using Graphpad software and IC50 was calculated as shown in FIG. 8. As can be seen from the figure: the humanized antibodies Hu9F5-13 and chimeric antibody 9F5 (example 7) showed no significant difference in IC50 values from the reference antibody REFERENCE AB-B and were excellent in activity.

Example 10 ADCC reporter detection

The mechanism of action of CCR8 antibody drugs is to target regulatory T cells (tregs) bearing CCR8 and mediate the killing and clearance of tregs by effector cells, i.e., antibody-mediated cytotoxic killing. The use of a fluorescent reporter gene system (ADCC effector cells, rhino BIO accession number: RA-CK 01) within effector cells can be used to determine whether an antibody is effective in activating effector cells.

1. HCR 8-HEK293 cells (constructed as described in example 1) were collected, the cell concentration was adjusted to 5X 10 ⁵/ml, added to a 96-well plate at 100. Mu.l/well, and incubated overnight at 37℃in a 5% CO ₂ incubator.

2. The next day appropriate amounts of Hu9F5-13, REFERENCE AB-B antibody (see above) were each aspirated and diluted to 10. Mu.g/ml with complete medium (RPMI 1640, 10% FBS), 40. Mu.l was aspirated and diluted to 160. Mu.l of complete medium, and after air-blow mixing, 5-fold gradient dilutions were performed sequentially 8 times. The experiment was also set up with a non-humanized 9F5 chimeric antibody as a control.

3. The hCR 8-HEK293 cell 96-well culture plate was removed, 50. Mu.l of diluted antibody was sequentially added after the supernatant was pipetted off, and a complete medium well was set as a negative control, a cell-free well as a background well, and incubated in a 5% CO ₂ incubator at 37℃for 45 minutes.

4. ADCC effector cells (carrying a fluorescent reporter gene, rhino BIO accession number: RA-CK 01) cultured in advance as described were adjusted to a concentration of 1X 10 ⁶/ml, added to antibody-added hCR 8-HEK293 cell wells at 25. Mu.l/well, and incubated in a 5% CO ₂ incubator at 37℃for 6 hours.

5. The 96-well plate was removed from the incubator, left at room temperature for 15min, and added at 75. Mu.l/wellAfter the One-Step firefly luciferase assay reagent is incubated for 3 minutes at room temperature, the supernatant is sucked and transferred into a detection plate, and then the detection plate is put into a luminescence detector for detection. An activity-concentration dependent fitted curve was made with chemiluminescent values. The results are shown in FIG. 9. As can be seen from the figure: both humanized antibodies Hu9F5-13 and chimeric antibodies are effective in mediating activation of ADCC effector cells.

Example 11, antibody affinity assay

The instrument is adopted: reichert 4SPR, chip is SAM chip (cat# 13206061).

The ambient temperature was set at 25℃and the running buffer was PBS-T. 40mg of EDC ((1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)) and 10mg of NHS (N-hydroxysuccinimide) were dissolved in 1mL of deionized water at a flow rate of 10. Mu.L/min and EDC/NHS was injected for 7min; the antibody was diluted to 10. Mu.g/mL with 10mM sodium acetate solution (sodium acetate as solute, deionized water as solvent, pH 5.0) and injected at a flow rate of 10. Mu.l/min for 7min to immobilize the antibody on the chip surface. Then blocked with pH 8.5.1M ethanolamine for 7min.

The antigen was diluted with PBS-T and 2-fold gradient diluted to give antigen dilutions at concentrations of 6.25nM,12.5nM,25nM,50nM,100nM, at a flow rate of 25. Mu.L/min, combined for 3min, and dissociated for 5min. The affinity of the antibodies was determined. The results are shown in table 16, where ka (1/(m×s)) represents the antigen-antibody binding rate, KD (1/s) represents the antigen-antibody dissociation rate, and KD (M) represents the affinity constant. Wherein the antibody is Hu9F5-13 and the antigen is fusion protein hCR 8-mFc or hCR 8-hFc (see example 2 for preparation). Chip regeneration conditions: the surface was rinsed with pH 2.0.10 mM glycine for 15 seconds.

Table 16, results of affinity measurement of antibodies Hu9F5-13 with antigen

Ka(1/(M*s))	Kd(1/s)	KD(M)
			8.57E+4	3.15E-4	3.68E-9

FIG. 10 is a graph showing the binding and dissociation curves of the affinity assay for the Hu9F5-13 molecule to hCR 8-Fc (see example 2) using Reichert. Wherein the ordinate is the binding response signal value and the abscissa is time(s), the curves represent the antigen hCCR8-Fc dilution gradient from low to high, respectively: 6.25nM,12.5nM,25nM,50nM,100nM. From the figure, the Hu9F5-13 molecule can be obviously combined with the antigen, the response signal value is high, and the KD value calculation is suitable.

The results show that: hu9F5-13 has a strong affinity for the antigen.

Example 12 homologous model drug efficacy

In order to test the efficacy of Hu9F5-13 in a murine homolog model, the present invention constructs a chimeric antibody expressing Hu9F5-13-mG2a to facilitate its mediation of the ADCC effect in mice. The light and heavy chain variable regions of Hu9F5-13 were linked to murine kappa and mG2a constant regions, respectively, to construct chimeric antibodies and expressed as described in example 7. Wherein the sequence of the murine kappa constant region gene is as follows:

AGGGCCGACGCTGCGCCAACCGTCAGCATCTTCCCCCCCAGCAGCGAACAGTTGACCTCCGGCGGTGCAAGCGTTGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGCGTGCTGAACAGCTGGACCGACCAGGACAGCAAAGATAGCACTTACAGCATGTCCAGCACCCTTACCCTCACCAAAGACGAATACGAGCGACACAACAGCTACACCTGCGAGGCCACGCACAAGACAAGTACCAGCCCTATAGTGAAGAGCTTTAACCGCAACGAGTGCTGA

the sequence of the murine mG2a constant region is as follows:

GCAAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATAA

the complete expressed gene was constructed in pHr vectors as described in example 7.

A model MC38 of murine colorectal carcinoma transplantable tumor was constructed using a C57 transgenic mouse (with human CCR8 gene, shanghai Prinsepia Biotechnology Co., ltd.). MC38 cells were cultured in RPMI1640 complete medium (Soxhibao), the MC38 cells were collected and inoculated subcutaneously on the back side of mice, each mouse was inoculated with 400,000 cells, and after about 4-5 days, the volume of the transplanted tumor mass was measured at 100mm ³. At this time, animals were randomly divided into two groups of 6 animals, and the animals were dosed at 10mG/kg (i.e., 10mG of chimeric Hu9F5-13-mG2a antibody was dosed per kg of mouse body weight) starting on the day of the grouping, and the control group was injected with an equal volume of PBS phosphate buffer, twice weekly (i.e., BIW), and tumor volumes were measured. The experiment was ended 26 days after grouping. The tumor growth curves of the two groups are plotted as shown in fig. 11. The result shows that Hu9F5-13 can effectively inhibit the growth of MC38 tumor, and the tumor growth inhibition rate (TGI) can reach 55.8%.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. An anti-CCR 8 antibody, characterized in that: the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 in sequence.

2. The antibody of claim 1, wherein: the amino acid sequence of the heavy chain variable region is selected from any one of the following:

(A1) SEQ ID No.7, or at least more than 90% identity to SEQ ID No. 7;

(A2) SEQ ID No.9, or at least more than 90% identity to SEQ ID No. 9;

(A3) SEQ ID No.10, or at least more than 90% identity to SEQ ID No. 10;

(A4) SEQ ID No.11, or at least more than 90% identity to SEQ ID No. 11;

(A5) SEQ ID No.12, or at least more than 90% identity to SEQ ID No. 12;

the amino acid sequence of the light chain variable region is selected from any one of the following:

(B2) SEQ ID No.13, or at least more than 90% identity to SEQ ID No. 13;

(B3) SEQ ID No.14, or at least more than 90% identity to SEQ ID No. 14;

(B4) SEQ ID No.15, or at least more than 90% identical to SEQ ID No. 15.

3. The antibody of claim 1 or 2, wherein: the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID No.9, and the amino acid sequence of the light chain variable region is SEQ ID No.15;

Or (b)

The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID No.7, and the amino acid sequence of the light chain variable region is SEQ ID No.8 or a sequence obtained by replacing the 107 th amino acid N of SEQ ID No.8 with K.

4. An antibody according to any one of claims 1-3, characterized in that: the antibody is an IgG antibody;

Further, the IgG antibody is an IgG1 antibody;

And/or

The light chain type of the antibody is Kappa type.

5. An antigen binding fragment, characterized in that: the antigen binding fragment is from the antibody of any one of claims 1-4;

the antigen binding fragment contains the HCDR1, the HCDR2 and the HCDR3; and/or

The antigen binding fragment contains the LCDR1, the LCDR2, and the LCDR3.

6. A nucleic acid molecule characterized in that: the nucleic acid molecule encodes the antibody of any one of claims 1-4 or the antigen-binding fragment of claim 5.

7. The nucleic acid molecule of claim 6, wherein: in the nucleic acid molecule, the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are shown in SEQ ID No.16 at positions 76-105, 148-198 and 295-315 from the 5' end;

And/or

In the nucleic acid molecule, the nucleotide sequences of LCDR1, LCDR2 and LHCDR in the light chain variable region of the antibody are shown as 70-117, 163-183 and 280-306 from the 5' end of SEQ ID No. 17;

And/or

Further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the antibody is selected from any one of the following:

(C1) SEQ ID No.16, or at least more than 90% identity to SEQ ID No. 16;

(C2) SEQ ID No.18, or at least more than 90% identity to SEQ ID No. 18;

(C3) SEQ ID No.19, or at least more than 90% identity to SEQ ID No. 19;

(C4) SEQ ID No.20, or at least more than 90% identity to SEQ ID No. 20;

(C5) SEQ ID No.21, or at least more than 90% identity to SEQ ID No. 21;

And/or

Further, in the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region of the antibody is selected from any one of the following:

(D1) SEQ ID No.17, or at least more than 90% identity to SEQ ID No. 17;

(D2) SEQ ID No.22, or at least more than 90% identity to SEQ ID No. 22;

(D3) SEQ ID No.23, or at least more than 90% identity to SEQ ID No. 23;

(D4) SEQ ID No.24, or at least more than 90% identity to SEQ ID No. 24;

And/or

Still further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the antibody is SEQ ID No.18 and the nucleotide sequence encoding the light chain variable region of the antibody is SEQ ID No.24;

And/or

Still further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the antibody is SEQ ID No.16 and the nucleotide sequence encoding the light chain variable region of the antibody is SEQ ID No.17.

8. An expression cassette, recombinant vector, recombinant cell or recombinant bacterium comprising the nucleic acid molecule of claim 6 or 7.

9. A pharmaceutical composition characterized in that: the pharmaceutical composition comprising the antibody of any one of claims 1-4 and a pharmaceutically acceptable excipient, diluent or carrier.

10. The application is as follows:

(A1) Use of a nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacterium according to any one of claims 6-8 for the preparation of an antibody according to any one of claims 1-4 or an antigen binding fragment according to claim 5 or a pharmaceutical composition according to claim 9;

(A2) Use of an antibody according to any one of claims 1-4 for the preparation of a pharmaceutical composition according to claim 9;

(A3) Use of an antibody or said antigen-binding fragment or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacterium or pharmaceutical composition according to any one of claims 1-9 for the preparation of a product for the prevention and/or treatment of a disease associated with CCR8 overexpression;

(A4) Use of the antibody or the antigen-binding fragment or the nucleic acid molecule or the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium or the pharmaceutical composition of any one of claims 1-9 for the preparation of a product for detecting CCR 8;

(A5) Use of the antibody or the antigen-binding fragment or the nucleic acid molecule or the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium or the pharmaceutical composition of any one of claims 1-9 for the preparation of a product for binding CCR 8;

(A6) Use of the antibody or the antigen-binding fragment or the nucleic acid molecule or the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium or the pharmaceutical composition of any one of claims 1-9 for the preparation of a product for blocking the stimulation of CCR8 by CCL 1;

(A7) Use of an antibody or said antigen-binding fragment or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacterium or pharmaceutical composition according to any one of claims 1-9 for the preparation of a product for activating effector cells;

(A8) Use of the antibody or the antigen-binding fragment or the nucleic acid molecule or the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium or the pharmaceutical composition of any one of claims 1-9 for the preparation of a product for targeting regulatory T cells bearing CCR 8;

(A9) Use of an antibody or said antigen binding fragment or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacterium or pharmaceutical composition according to any one of claims 1-9 for the preparation of a fusion protein, bispecific antibody or therapeutic chimeric antigen receptor.