An anti-human ccr8 monoclonal antibody and a preparation method thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to an anti-human CCR8 monoclonal antibody and a preparation method thereof. The anti-human CCR8 monoclonal antibody of the application comprises VHCDR1, VHCDR2 and VHCDR3 with the amino acid sequences shown in SEQ ID NO: 1-3; and VLCDR1, VLCDR2 and VLCDR3 with the amino acid sequences shown in SEQ ID NO: 4-6. The antibody development process is as follows: Human CCR8 mRNA is synthesized, mice are immunized, and mouse spleen, bone marrow and lymph node cells are collected; a microfluidic droplet sorting system is used to wrap single plasma cell suspension, fluorescently labeled secondary antibody and Human CCR8 HEK293 overexpression cell line in water-in-oil droplets, and dielectrophoresis sorting is performed to enrich plasma cells secreting target cell binding antibodies; single cell library construction is performed on the plasma cells, and NGS sequencing is performed to obtain light and heavy chain naturally paired antibody sequences; the antibody sequences are selected for mammalian cell high-throughput expression, and flow cytometry detection verification is performed. The monoclonal antibody of the application has good antigen-antibody binding activity and specificity at the cell level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to an anti-human CCR8 monoclonal antibody and a preparation method thereof. BACKGROUND

[0002] CCR8 (C-C chemokine receptor type 8) is a kind of chemokine receptor, belonging to the G protein-coupled receptor (GPCR) family. It is mainly expressed on the surface of regulatory T cells (Tregs), some types of helper T cells (Th2 cells) and other immune cells. CCR8 binds to its ligand CCL1 (CC chemokine ligand 1), mediates cell migration and activation, and plays an important role in immune regulation and inflammatory response.

[0003] In recent years, studies have shown that CCR8 is highly expressed in the microenvironment of various tumors, especially on tumor-infiltrating regulatory T cells (Tregs). These Tregs help tumor cells evade immune surveillance by suppressing the function of effector T cells, thereby promoting tumor growth and metastasis. Therefore, therapeutic strategies targeting CCR8 are expected to enhance anti-tumor immune response by reducing Tregs in the tumor microenvironment, achieving the purpose of treatment.

[0004] Many studies have been devoted to the development of monoclonal antibodies that can specifically bind to CCR8, in order to block the binding of CCR8 to its ligand, or selectively kill cells expressing CCR8 through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent (CDC) effects. In addition, some research teams are exploring small molecule compounds that can inhibit CCR8 signaling, in the hope of achieving similar results. Although the above research has made some progress, there is still a lack of efficient, specific and safe anti-CCR8 therapeutic means. The existing antibodies and small molecule inhibitors have different degrees of limitations in affinity, specificity, stability and production cost.

[0005] The existing anti-CCR8 antibodies still need to be improved in terms of affinity and specificity, and it is difficult to ensure their accurate recognition and binding to target cells in complex biological environments. Therefore, the development of an anti-human CCR8 monoclonal antibody with high affinity, high specificity, high stability and low production cost and its preparation method is of great significance for promoting the development of tumor immunotherapy. SUMMARY

[0006] The purpose of the present application is to provide an anti-human CCR8 monoclonal antibody and a preparation method thereof, which has high affinity and high specificity, can effectively target CCR8 molecules on the surface of cell membranes, and enhance anti-tumor immune response, providing a new means for tumor immunotherapy.

[0007] The application also provides a preparation method of the antibody, which comprises the steps of immunizing a mouse, screening a plasma cell, sequencing analysis of the antibody, and recombinant expression.

[0008] To achieve the above-mentioned purposes, the technical scheme adopted by the application is as follows:

[0009] In a first aspect of the application, an anti-human CCR8 monoclonal antibody is provided, which comprises a VHCDR1, a VHCDR2 and a VHCDR3 with the amino acid sequences shown in SEQ ID NO: 1-3; and a VLCDR1, a VLCDR2 and a VLCDR3 with the amino acid sequences shown in SEQ ID NO: 4-6.

[0010] Further, the antibody comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 7.

[0011] Further, the antibody comprises a light chain variable region with the amino acid sequence shown in SEQ ID NO: 8.

[0012] In a second aspect of the application, a preparation method of the anti-human CCR8 monoclonal antibody of the first aspect is also provided, which comprises the following steps: synthesizing Human CCR8 mRNA, immunizing a mouse by intramuscular injection, collecting spleen, bone marrow and lymph node cells of the mouse, using a microfluidic droplet sorting system, and realizing efficient enrichment of plasma cells secreting target cell binding antibodies by encapsulating a single plasma cell suspension, a fluorescently labeled secondary antibody and a Human CCR8 HEK293 overexpression cell line in water-in-oil droplets, incubating for 2-4 hours, and performing dielectrophoresis sorting based on the fluorescence signal intensity; performing single cell library construction on the plasma cells, obtaining light and heavy chain naturally paired antibody sequences by NGS sequencing; selecting antibody sequences according to the phylogenetic tree and antibody sequence frequency for high-throughput expression in mammalian cells, and finally verifying by flow cytometry.

[0013] Specifically, the preparation method comprises the following steps:

[0014] S1, expressing Human CCR8 mRNA according to the amino acid sequence information of Human CCR8;

[0015] S2, immunizing a mouse with the Human CCR8 mRNA obtained in step S1, and dissecting to obtain spleen, bone marrow and lymph node cells of the mouse;

[0016] S3, using the mouse mixed cells obtained in step S2 as raw materials, enriching mouse plasma cells through CD138 magnetic beads, screening the antigen-specific plasma cells using the AbDrop platform based on microfluidic technology, and screening the plasma cells through a single-cell library system to construct an antibody library and perform second-generation sequencing;

[0017] S4, analyzing the antibody sequences obtained in step S3, selecting part of the light and heavy chain naturally paired antibody sequences according to the phylogenetic tree and frequency, and cloning them into a vector;

[0018] S5, performing high-throughput expression of the antibodies through a mammalian cell high-throughput expression system, and performing cell binding verification through a FACS method, and finally obtaining a monoclonal antibody against Human CCR8.

[0019] In a third aspect of the present application, a nucleic acid encoding the anti-human CCR8 monoclonal antibody according to the first aspect is also provided.

[0020] In a fourth aspect of the present application, an expression vector containing the nucleic acid according to the third aspect is also provided, and preferably the expression vector is pcDNA3.4.

[0021] In a fifth aspect of the present application, a host cell containing the expression vector according to the fourth aspect is also provided, and preferably the host cell is a CHO cell.

[0022] In a sixth aspect of the present application, the use of the anti-human CCR8 monoclonal antibody according to the first aspect in the preparation of a human CCR8 protein detection reagent is also provided.

[0023] In a seventh aspect of the present application, the use of the anti-human CCR8 monoclonal antibody according to the first aspect in the preparation of a product combined with human CCR8 protein is also provided.

[0024] Compared with the prior art, the technical effects of the present application are as follows: the development method of the anti-Human CCR8 monoclonal antibody provided by the present application shortens the development time of the target antibody, has high throughput, and has good antigen-antibody binding activity at the cell level. The antibody can be used for diagnosis and treatment of diseases related to CCR8, especially in the application of tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The results of the mouse terminal serum titer detection;

[0026] Figure 2 The results of the antibody and cell binding FACS detection; DETAILED DESCRIPTION

[0027] The application will be further explained in connection with specific embodiments, but it should be noted that the following embodiments are only used to explain the application and cannot be used to limit the application, all technical solutions identical or similar to the application are within the protection scope of the application. The specific techniques or conditions not mentioned in the embodiments are operated according to the conventional technical methods and instrument instruction book contents; the reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by market purchase.

[0028] Example 1

[0029] The development method of the anti-Human CCR8 monoclonal antibody of the application comprises the following processes:

[0030] S1, according to the protein sequence and gene sequence information (UniProt: P51685) of Human CCR8, mRNA synthesis is carried out for subsequent mouse immunization.

[0031] S2, according to the protein sequence and gene sequence information (UniProt: P56747) of Human CCR8, a HEK293 overexpression cell line is constructed for subsequent cell screening of mice and verification of specific binding of antibodies.

[0032] S3, construct overexpression cell line: synthesize pLVX-IRES-puro-hCCR8 plasmid by gene recombination, the operation process is as follows:

[0033] Dilute psPAX2 plasmid, PMD2G plasmid and pLVX-IRES-puro-hCCR8 plasmid into culture medium, and add PEI, incubate at room temperature for 15 min; add the above mixture to 293T cells, collect the slow virus particle supernatant after 72 h, and filter with a 0.45 μM filter head; inoculate the slow virus particles into HEK293 cells, and add 1 / 100 volume of F108 infection enhancer, discard and replace fresh culture medium after 6-8 h, and perform expansion culture after 48 h, and add 10 μg / mL puromycin for resistance screening, and the HEK293-hCCR8 overexpression cells that can continuously grow and pass are obtained.

[0034] S4, use the Human CCR8 mRNA obtained in step S1 to immunize mice by intramuscular injection, immunize once every 2 weeks, take blood, and use FACS to detect the titer of the overexpression cell line. After the serum titer is qualified, collect the mouse spleen and bone marrow cells for enrichment and screening.

[0035] Anti-Human CCR8 serum titer was detected by FACS, and the detection process was as follows: CCR8 high expression cells and negative control cells were taken, the cells were collected by trypsin digestion without EDTA, centrifuged (400g, 5min), and the culture medium was discarded; the cells were resuspended with PBS (containing 2% FBS) and the cell concentration was counted to 1×10 6 cells / mL; 100μL cell suspension (about 10 5 cells) was taken to a 96-well V-bottom plate, gradient-diluted immune mouse serum was added, mixed gently, incubated at 4℃ for 60min; the cells were washed twice with PBS (containing 2% FBS) (300g, 5min), Anti-Mouse IgG FITC fluorescent secondary antibody was added, and incubated at 4℃ for 30min in the dark; washed twice again, and finally resuspended the cells with 200μL PBS, and detected by flow cytometry. The detection results are shown in Figure 1 The detection results show that except for #451 mouse which did not reach the sorting titer, the other mice reached the sorting titer.

[0036] S5, the spleen, bone marrow and lymph node cells of #452-#455 immune mice were ground into single cells, filtered through a 70μm cell filter, centrifuged at 400g for 5min, resuspended with 1mL MACS Buffer, and CD138 positive cells were enriched by CD138 kit (stemcell). 1×10 6 cells of plasma cells and 5×10 6 cells of target cells were resuspended with 1mL coating medium; the cell suspension was filtered through a 40μm sieve and added to a microfluidic chip, and 2312 positive droplets were obtained by sorting, and then the antigen-enriched plasma cells were recovered by demulsification;

[0037] S6, the positive mouse cells obtained by sorting were banked by using a single cell banking platform, and the constructed library was sequenced by next-generation sequencing, and the light and heavy chain naturally paired antibodies were obtained by data analysis; the antibody sequences were selected according to the phylogenetic tree and frequency and recombined into pcDNA3.4 expression vectors, and HEK293 cell strains were transfected, and finally 11 antibodies were expressed, which were BY-CCR8-1~BY-CCR8-11;

[0038] S7, the cell binding activity of the antibody was detected by flow cytometry, and the specific steps were as follows: 1E5 HEK293-hCCR8 overexpression cells were inoculated in each well of a 96-well plate, centrifuged at 400g for 5min, 50μL of 5-fold gradient-diluted antibody was added to each well, incubated at 4℃ for 1h, washed 3 times, 100μL of diluted Goat anti-Human IgG Alexa Fluor 647 was added to each well, incubated at 4℃ for 30min, washed 5 times, resuspended with 200μL 1×PBS, and detected by flow cytometry. The detection results are shown inFigure 2 The positivity rate of the 11 antibodies was 63.63% (7 / 11).

[0039] The specific signal values ​​of the 11 antibodies are shown in the table below:

[0040]

[0041] according to Figure 2 As shown in the table above, BY-CCR8-10 exhibits a stronger signal value and a lower EC50 (0.1313 nM) than the positive control Anti-Human CCR8 antibody (TPP-21360). Therefore, the anti-CCR8 monoclonal antibody designated BY-CCR8-10 possesses both antigen- and cell-binding activities, and demonstrates higher binding activity than the positive control.

[0042] The sequence of the anti-CCR8 monoclonal antibody designated BY-CCR8-10 is shown in the table below:

[0043]

[0044]

[0045] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An anti-human CCR8 monoclonal antibody, characterized in that, The monoclonal antibody comprises a VH CDR1 of an amino acid sequence as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 2; a VH CDR3 as set forth in SEQ ID NO: 3; and a VL CDR1 of an amino acid sequence as set forth in SEQ ID NO: 4; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO:

6. The antibody comprises a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO:

7.

2. The anti-human CCR8 monoclonal antibody according to claim 1, characterized by, The antibody comprises a light chain variable region of an amino acid sequence as set forth in SEQ ID NO:

8.

3. The anti-human CCR8 monoclonal antibody according to claim 1, characterized by, 4. A nucleic acid encoding the anti-human CCR8 monoclonal antibody according to any one of claims 1-3.

5. An expression vector comprising the nucleic acid according to claim 4.

6. A host cell comprising the expression vector according to claim 5.

7. Use of the anti-human CCR8 monoclonal antibody according to any one of claims 1-3 in the preparation of a detection reagent for human CCR8 protein. ​

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