An anti-cd25 monoclonal antibody, its preparation method and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to an anti-CD25 monoclonal antibody, a preparation method thereof and application thereof. The anti-CD25 monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region VH comprises three complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO: 1-3. The light chain variable region VL also comprises three complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO: 4-6. The application further provides a preparation method of the anti-CD25 monoclonal antibody. The monoclonal antibody development method has the advantages of short time consumption, high throughput and high positive rate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-CD25 monoclonal antibody, its preparation method, and its application. Background Technology

[0002] CD25, also known as the interleukin-2 receptor α chain (IL-2Rα), is a key factor in the immune system. It is primarily found on regulatory T cells (Tregs), which play a crucial regulatory role in the immune system. Tregs inhibit the activity of other immune cells by producing inhibitory cytokines, such as transforming growth factor-beta (TGF-β) and interleukin-10 (IL-10), and through mechanisms such as cell-cell contact, thus preventing excessive immune responses and autoimmune diseases. Furthermore, CD25 expression serves as an important indicator for assessing immune status. When the immune system is subjected to infection, inflammation, or other stimuli, T cell activation leads to an upregulation of CD25 expression levels. Therefore, detecting CD25 expression levels can reveal the degree of T cell activation, as well as the number and functional status of regulatory T cells, helping to assess the function and disease status of the immune system, such as infections and autoimmune diseases.

[0003] In cancer patients, an increase in regulatory T cells can suppress the immune system's ability to kill tumor cells. By blocking CD25, the activity of regulatory T cells can be inhibited, thereby reducing the effects of immunosuppression, enhancing the immune system's ability to kill tumor cells, and improving the efficacy of immunotherapy. The combined use of CD25 antibodies and PD-1 antibodies has shown synergistic anti-tumor effects, indicating that combination therapy may become a new trend in cancer immunotherapy in the future. In particular, the development of the ADC-drug conjugate Cami (Camidanlumab Tesirine), which targets CD25 to treat relapsed or refractory Hodgkin's lymphoma, demonstrates the great potential of antibody drugs in cancer treatment.

[0004] Hybridoma technology is currently the most mature and widely used platform for monoclonal antibody development; however, it is time-consuming, has low cell fusion efficiency, and is limited in the species it can be applied to. While phage display technology shortens antibody development time, it affects antibody specificity and affinity because it cannot guarantee the natural pairing of light and heavy chains. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an anti-CD25 monoclonal antibody, its preparation method, and its applications. The monoclonal antibody development method provided by this invention has advantages such as short processing time, high throughput, and high positive rate.

[0006] The inventive concept of this invention is as follows: This invention uses a single B cell as the research subject, uses flow cytometry to sort out B cells expressing positive antibodies, and adopts a single-cell antibody library construction method to maintain the natural pairing of antibody light and heavy chains, and replaces the inefficient, time-consuming, and easily contaminated hybridoma cell fusion and culture.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect of the invention, an anti-CD25 monoclonal antibody is provided, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region.

[0009] The heavy chain variable region VH contains three complementarity-determining regions (CDRs), namely:

[0010] VHCDR1, whose amino acid sequence is shown in SEQ ID NO: 1;

[0011] VHCDR2, whose amino acid sequence is shown in SEQ ID NO: 2;

[0012] VHCDR3, whose amino acid sequence is shown in SEQ ID NO: 3;

[0013] The light chain variable region (VL) also contains three complementary determinant regions (CDRs), namely:

[0014] VLCDR1, whose amino acid sequence is shown in SEQ ID NO: 4;

[0015] VLCDR2, whose amino acid sequence is shown in SEQ ID NO: 5;

[0016] VLCDR3, whose amino acid sequence is shown in SEQ ID NO: 6.

[0017] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 7.

[0018] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 8.

[0019] A second aspect of the present invention also provides a method for preparing an anti-CD25 monoclonal antibody as described in the first aspect.

[0020] The specific steps of the method are as follows: expressing human CD25-Fc and immunizing mice with Freund's adjuvant, collecting mouse spleen and bone marrow cells; screening mouse B cells that bind to CD25 antigen using flow cytometry; constructing a single-cell library from positive B cells, obtaining naturally paired antibody sequences of light and heavy chains by NGS sequencing; further screening the sequences, expressing the antibodies using a mammalian cell high-throughput expression system; and finally verifying the results by ELISA.

[0021] Specifically, the preparation method includes the following steps:

[0022] S1. Based on the CD25 protein sequence information, express the human CD25-Fc protein;

[0023] S2. After immunizing mice with the human CD25-Fc obtained in step S1 combined with Freund's adjuvant, the spleen and bone marrow cells of the mice were extracted.

[0024] S3. Using the mouse cells obtained in step S2 as raw materials, positive antibody-secreting cells were screened by flow cytometry, and antibody libraries were constructed and next-generation sequencing was performed using a single-cell library construction system.

[0025] S4. Screen the antibody sequences obtained in step S3, extract the naturally paired antibody sequences of the light and heavy chains, and clone them into the vector;

[0026] S5. High-throughput expression of anti-CD25 antibody is induced through a mammalian cell high-throughput expression system, and antigen binding is verified by antibody detection technology such as ELISA, ultimately obtaining a monoclonal antibody against CD25.

[0027] In a third aspect of the invention, a nucleic acid encoding an anti-CD25 monoclonal antibody as described in the first aspect is also provided.

[0028] In a fourth aspect of the invention, an expression vector containing nucleic acids as described in the third aspect is also provided.

[0029] In a fifth aspect of the invention, a host cell containing the expression vector as described in the fourth aspect is also provided.

[0030] In a sixth aspect of the invention, the use of monoclonal antibodies as described in the first aspect is also provided:

[0031] 1) Application in the preparation of CD25 protein detection reagents.

[0032] 2) Application in the preparation of products that bind to human CD25 protein.

[0033] 3) Application in the preparation of tumor immunotherapy drugs.

[0034] Compared with the prior art, the technical effects of the present invention are as follows:

[0035] 1) This invention screened and obtained a new anti-CD25 monoclonal antibody, which has better antigen-binding activity than existing anti-CD25 monoclonal antibodies;

[0036] 2) The method for developing anti-CD25 monoclonal antibodies of the present invention shortens the development time of the target antibody, and is time-saving, high-throughput, and has a high positive rate. Attached Figure Description

[0037] Figure 1 The results show the serum titer of mice at different dilutions; where NC is the negative control and 1 is the serum.

[0038] Figure 2 This is the result of flow sorting.

[0039] Figure 3 This refers to the results of an antigen binding test. Detailed Implementation

[0040] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated in accordance with conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0041] Example 1

[0042] The method for developing the anti-CD25 monoclonal antibody of the present invention includes the following steps:

[0043] S1. Based on the protein and gene sequence information of CD25 (Genbank: P01589), the antigen was expressed and an Fc-tag was attached to its C-terminus to obtain the modified human CD25-Fc protein, which was used for subsequent mouse immunization.

[0044] S2. 100 μL of human CD25-Fc protein at a concentration of 1 mg / mL was labeled with AF488 fluorescent labeling for subsequent mouse cell screening.

[0045] S3. Immunize mice with the human CD25-Fc protein obtained in step S1: Dilute 60 μg of human CD25-Fc protein with Freund's adjuvant and inject mice intraperitoneally and subcutaneously. After the serum titer is qualified, collect the spleen and bone marrow cells of the mice for screening.

[0046] Serum titer of anti-human CD25-Fc protein was detected by ELISA. The detection procedure was as follows: 2 μg / mL human CD25-Fc protein was coated onto an ELISA plate and incubated at 37°C for 2 h; the plate was washed with 0.05% PBST and blocked with 1% BSA at room temperature for 2 h; after washing with 0.05% PBST, mouse serum before and after immunization was added in serially diluted 3-fold (100-fold and 1000-fold dilutions for the first two wells) and incubated at room temperature for 1 h; after washing with 0.05% PBST, anti-mouse IgG (Fc specific) HRP was added and incubated at room temperature for 30 min; after washing with 0.05% PBST, Beyotime TMB chromogenic solution was added and reacted at room temperature for 10 min, then Beyotime TMB stop solution was added to immediately stop the reaction, and the OD450 was read immediately.

[0047] The titer of the antiserum after immunization was 512,000, as shown in the results. Figure 1 As shown in the figure, this antigen can induce mice to produce high-titer antiserum specifically targeting human CD25-Fc.

[0048] S4. Collect spleen and bone marrow cells from immunized mice, centrifuge at 400g for 5 min, resuspend in 1 mL of erythrocyte lysis buffer, stop erythrocyte lysis with a large volume of MACS buffer, centrifuge at 400g for 5 min, take 5E7 cells and resuspend in 1 mL of MACS buffer, add fluorescent antibody of AF488-human CD25-Fc protein and B cell-specific molecular marker, incubate at 4℃ for 1 h, resuspend in a large volume of MACS buffer, centrifuge at 400g for 5 min, discard the supernatant, resuspend the cells in the buffer, and sort the cells using a flow cytometer.

[0049] Flow sorting results are as follows Figure 2 As shown, the final positive rate of sorted cells was approximately 0.68%.

[0050] S5. Using a single-cell library construction platform, the positive mouse cells obtained from the sorting were used to construct a library, and the constructed library was subjected to next-generation sequencing. Through data analysis, antibodies with natural pairing of light and heavy chains were obtained. Nine antibody sequences were selected for high-throughput expression in mammalian cells.

[0051] S6. ELISA was used to detect the antigen-binding activity of nine antibodies. The specific steps are as follows: The ELISA plate was coated with 2 μg / mL human CD25-Fc protein and incubated overnight at 4°C; washed with 0.05% PBST and blocked with 1% BSA at room temperature for 2 hours; washed with 0.05% PBST and added 3-fold serially diluted versions of the nine antibodies, positive control (Daclizumab, 7G7B6-hIgG1), and isotype control, respectively, and incubated at room temperature for 1 hour; washed with 0.05% PBST and added Anti-human IgG (Fc specific) HRP, and incubated at room temperature for 30 minutes; washed with 0.05% PBST and added Beyotime TMB chromogenic solution, reacted at room temperature for 10 minutes, and then the reaction was immediately stopped by adding Beyotime TMB stop solution, and the OD450 was read immediately.

[0052] Antigen binding test results as follows Figure 3 As shown in the figure, the positive rate of the nine antibodies was 88.9% (8 / 9). Among them, the EC50 values ​​of CD25-3, CD25-4, CD25-6, and CD25-8 were lower than those of the positive control, at 0.09066, 0.1051, 0.1322, and 0.1239, respectively. Therefore, the anti-human CD25-Fc monoclonal antibody designated CD25-3 exhibited higher binding activity than the positive control.

[0053] The six CDR sequences, heavy chain variable region sequence, and light chain variable region sequence of the anti-human CD25-Fc monoclonal antibody designated CD25-3 are shown in the table below:

[0054] type Serial Number VHCDR1 SEQ ID NO:1 VHCDR2 SEQ ID NO:2 VHCDR3 SEQ ID NO:3 VLCDR1 SEQ ID NO:4 VLCDR2 SEQ ID NO:5 VLCDR3 SEQ ID NO:6 VH SEQ ID NO:7 VL SEQ ID NO:8

[0055] 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-CD25 monoclonal antibody, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The heavy chain variable region VH contains three complementarity-determining regions (CDRs), namely: VHCDR1, whose amino acid sequence is shown in SEQ ID NO: 1; VHCDR2, whose amino acid sequence is shown in SEQ ID NO: 2; VHCDR3, whose amino acid sequence is shown in SEQ ID NO: 3; The light chain variable region (VL) also contains three complementary determinant regions (CDRs), namely: VLCDR1, whose amino acid sequence is shown in SEQ ID NO: 4; VLCDR2, whose amino acid sequence is shown in SEQ ID NO: 5; VLCDR3, whose amino acid sequence is shown in SEQ ID NO:

6.

2. The anti-CD25 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:

7.

3. The anti-CD25 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

8.

4. The nucleic acid encoding the anti-CD25 monoclonal antibody as described in any one of claims 1-3.

5. An expression vector containing the nucleic acid as described in claim 4.

6. A host cell containing the expression vector as described in claim 5.

7. The use of the anti-CD25 monoclonal antibody as described in any one of claims 1-3 in the preparation of CD25 protein detection reagents.

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