Anti-human CCR8 monoclonal antibody and preparation method thereof

Abstract

The invention 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 disclosed by the invention comprises VHCDR1, VHCDR2 and VHCDR3, wherein the amino acid sequences of the VHCDR1, the VHCDR2 and the VHCDR3 are shown as SEQ ID NO: 1-3; the amino acid sequences of the VLCDR1, the VLCDR2 and the VLCDR3 are as shown in SEQ ID NO: 4-6. The antibody development process comprises the following steps: synthesizing Human CCR8mRNA, immunizing a mouse, collecting spleen, bone marrow and lymph node cells of the mouse, wrapping a single plasma cell suspension, a fluorescence labeled secondary antibody and a Human CCR8HEK293 overexpression cell line in water-in-oil liquid drops together by adopting a microfluidic liquid drop sorting system, carrying out dielectrophoresis sorting, and enriching plasma cells secreting the target cell binding antibody; carrying out single-cell library construction on plasma cells, and carrying out NGS sequencing to obtain antibody sequences with naturally paired light and heavy chains; and selecting an antibody sequence for high-throughput expression of mammalian cells, and carrying out flow detection verification. The monoclonal antibody provided by the invention has good antigen-antibody binding activity and specificity on the cellular level.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an anti-human CCR8 monoclonal antibody and a preparation method thereof. Background Art

[0002] CCR8 (CC chemokine receptor type 8) is a chemokine receptor that belongs to the G protein-coupled receptor (GPCR) family. It is mainly expressed on the surface of regulatory T cells (Tregs), certain 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 a variety of tumor microenvironments, especially on tumor-infiltrating regulatory T cells (Tregs). These Tregs help tumor cells escape immune surveillance by inhibiting the function of effector T cells, thereby promoting tumor growth and metastasis. Therefore, therapeutic strategies targeting CCR8 are expected to achieve therapeutic goals by reducing Tregs in the tumor microenvironment and enhancing anti-tumor immune responses.

[0004] Many studies are devoted to developing monoclonal antibodies that can specifically bind to CCR8 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 order to achieve similar effects. Although the above research has made some progress, there is still a lack of efficient, specific and safe anti-CCR8 treatments. Existing antibodies and small molecule inhibitors have varying degrees of limitations in terms of affinity, specificity, stability and production costs.

[0005] The existing anti-CCR8 antibodies still need to be improved in terms of affinity and specificity, and it is difficult to ensure that they can accurately identify and bind 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 are of great significance to promote the development of tumor immunotherapy. Summary of the invention

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

[0007] The present invention also provides a method for preparing the antibody, including the steps of immunizing mice, screening plasma cells, antibody sequencing analysis and recombinant expression. The monoclonal antibody development method provided by the present invention has the advantages of short time consumption and high throughput, and has good antigen-antibody binding activity at the cellular level, providing support for ELISA, FACS and other detection of human CCR8 protein.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect of the present invention, an anti-human CCR8 monoclonal antibody is provided, which comprises VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1-3; and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4-6.

[0010] Furthermore, the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7.

[0011] Furthermore, the antibody comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8.

[0012] In the second aspect of the present invention, a method for preparing an anti-human CCR8 monoclonal antibody as described in the first aspect is also provided, and the process is as follows: synthesizing Human CCR8 mRNA, immunizing mice by intramuscular injection, collecting mouse spleen, bone marrow, and lymph node cells, and using a microfluidic droplet sorting system to encapsulate a single plasma cell suspension with a fluorescently labeled secondary antibody and a Human CCR8 HEK293 overexpressing cell line in an oil-in-water droplet, incubating for 2-4 hours, and performing dielectrophoresis sorting based on the fluorescence signal intensity to achieve efficient enrichment of plasma cells that secrete target cell binding antibodies; single-cell library construction of plasma cells, and obtaining antibody sequences with natural pairing of light and heavy chains by NGS sequencing; selecting antibody sequences based on evolutionary trees and antibody sequence frequencies 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 based on the amino acid sequence information of Human CCR8;

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

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

[0017] S4, analyzing the antibody sequences obtained in step S3, selecting some antibody sequences with natural pairing of light and heavy chains according to the evolutionary tree and the frequency of occurrence, and cloning them into a vector;

[0018] S5. The antibody was expressed in high throughput through a mammalian cell high throughput expression system, and cell binding was verified by FACS method to finally obtain a monoclonal antibody against Human CCR8.

[0019] In the third aspect of the present invention, a nucleic acid encoding the anti-human CCR8 monoclonal antibody as described in the first aspect is also provided.

[0020] In the fourth aspect of the present invention, an expression vector containing the nucleic acid as described in the third aspect is also provided, and preferably the expression vector is pcDNA3.4.

[0021] In the fifth aspect of the present invention, a host cell containing the expression vector as described in the fourth aspect is also provided, and preferably the host cell is a CHO cell.

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

[0023] In the seventh aspect of the present invention, there is also provided the use of the anti-human CCR8 monoclonal antibody as described in the first aspect in the preparation of a product binding to human CCR8 protein.

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

[0025] Figure 1 This is the test result of the final immune serum titer of mice;

[0026] Figure 2 The results of FACS detection of antibody binding to cells; DETAILED DESCRIPTION

[0027] The present invention is further explained below in conjunction with specific examples, but it should be noted that the following examples are only used to explain the present invention, and cannot be used to limit the present invention, and all technical solutions that are the same or similar to the present invention are within the scope of protection of the present invention. If no specific technology or conditions are specified in this embodiment, the operation is carried out according to the conventional technical methods and instrument instructions in the art; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0028] Example 1

[0029] The method for developing anti-Human CCR8 monoclonal antibodies of the present invention comprises the following steps:

[0030] S1. Based on the protein sequence and gene sequence information of Human CCR8 (UniProt: P51685), mRNA synthesis was performed for subsequent mouse immunization.

[0031] S2. Based on the protein sequence and gene sequence information of Human CCR8 (UniProt: P56747), a HEK293 overexpression cell line was constructed for subsequent mouse cell screening and antibody specific binding verification.

[0032] S3. Construction of overexpression cell line: Synthesize pLVX-IRES-puro-hCCR8 plasmid by gene recombination. The operation process is as follows:

[0033] Take psPAX2 plasmid, PMD2G plasmid, and pLVX-IRES-puro-hCCR8 plasmid and dilute them into culture medium, add PEI, and incubate at room temperature for 15 minutes; add the above mixture to 293T cells, collect the supernatant of lentiviral particles after 72 hours, and filter with a 0.45μM filter; inoculate the lentiviral particles into HEK293 cells, and add 1 / 100 volume of F108 infection enhancement solution, discard and replace with fresh culture medium after 6-8 hours, expand culture after 48 hours, and add 10μg / mL puromycin for resistance screening. The cells that can continue to grow and passage are HEK293-hCCR8 overexpressing cells.

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

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

[0036] S5. Grind spleen, bone marrow and lymph node cells from mice immunized with #452-#455 into single cells, pass through a 70 μm cell strainer, collect the filtrate, centrifuge at 400 g for 5 min, resuspend in 1 mL MACS Buffer, enrich CD138 positive cells using CD138 kit (stemcell), and take 1×10 6 Plasma cells, 5×10 6 Each target cell was resuspended in 1 mL of coating medium; the cell suspension was filtered through a 40 μm mesh and added to a microfluidic chip, and 2312 positive droplets were sorted on the chip, and then the emulsion was broken to recover the antigen-enriched plasma cells;

[0037] S6. Use the single-cell library construction platform to construct a library of the sorted positive mouse cells, and perform second-generation sequencing on the constructed library. Obtain antibodies with natural pairing of light and heavy chains through data analysis. Select antibody sequences based on the evolutionary tree and frequency, recombinant them into the pcDNA3.4 expression vector, and transfect the HEK293 cell line. Finally, 11 antibodies were expressed, namely BY-CCR8-1 to BY-CCR8-11.

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

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

[0040]

[0041] according to Figure 2 As can be seen from the results in the table above, BY-CCR8-10 has a stronger signal value and a lower EC50 of 0.1313 nM than the positive reference Anti-Human CCR8 antibody (TPP-21360). Therefore, the anti-CCR8 monoclonal antibody numbered BY-CCR8-10 has both antigen and cell binding activity and exhibits higher binding activity than the positive reference.

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

[0043]

[0044]

[0045] Finally, it should be noted that the above embodiments are only illustrative of the principles, performance and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by 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 VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1-3; and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4-6.

2. The anti-human CCR8 monoclonal antibody according to claim 1, characterized in that The antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:

7.

3. The anti-human CCR8 monoclonal antibody according to claim 1, characterized in that The antibody comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

8.

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

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

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

7. The method for preparing an anti-human CCR8 monoclonal antibody according to any one of claims 1 to 3, characterized in that: The process is as follows: Human CCR8 mRNA is synthesized, mice are immunized by intramuscular injection, spleen, bone marrow, and lymph node cells of mice are collected, and a microfluidic droplet sorting system is used to encapsulate a single plasma cell suspension with a fluorescently labeled secondary antibody and a Human CCR8 HEK293 overexpressing cell line in an oil-in-water droplet. After 2-4 hours of incubation, dielectrophoresis sorting is performed based on the intensity of the fluorescent signal to achieve efficient enrichment of plasma cells that secrete target cell-binding antibodies; Single-cell libraries were constructed for plasma cells, and antibody sequences with natural pairing of light and heavy chains were obtained through NGS sequencing. Antibody sequences were selected based on the evolutionary tree and antibody sequence frequency for high-throughput expression in mammalian cells, and finally verified through flow cytometry.

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

9. Use of the anti-human CCR8 monoclonal antibody according to any one of claims 1 to 3 in the preparation of a product that binds to human CCR8 protein.

10. Use of the anti-human CCR8 monoclonal antibody according to any one of claims 1 to 3 in tumor immunotherapy.

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