Anti-CD25 monoclonal antibody as well as preparation method and application thereof

Abstract

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

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-CD25 monoclonal antibody, a preparation method thereof and an application thereof. Background Art

[0002] The CD25 molecule, also known as the interleukin-2 receptor alpha chain (IL-2Rα), is a key factor in the immune system. It is mainly present on regulatory T cells (Tregs), which play an important 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 contact, to prevent excessive immune responses and the occurrence of autoimmune diseases. Secondly, the expression of CD25 molecules can also be used as an important indicator for evaluating immune status. When the immune system is infected, inflamed, or otherwise stimulated, the activation of T cells will lead to an upregulation of the expression level of CD25. Therefore, by detecting the expression level of CD25, we can understand the degree of activation of T cells and the number and functional status of regulatory T cells, which is helpful to evaluate the function and disease state of the immune system, such as infection, autoimmune diseases, etc.

[0003] In tumor patients, the increase of regulatory T cells will inhibit the immune system from killing tumors. By blocking CD25, the activity of regulatory T cells can be inhibited, thereby reducing the impact of immunosuppression, enhancing the immune system's killing of tumor cells, and improving the effect of immunotherapy. The combined use of CD25 antibodies and PD-1 antibodies showed a synergistic anti-tumor effect, indicating that combination therapy may become a new trend in tumor immunotherapy in the future. In particular, the development of the ADC-conjugated drug Cami (Camidanlumab Tesirine), which treats relapsed or refractory Hodgkin's lymphoma by targeting CD25, shows the great potential of antibody drugs in tumor treatment.

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

[0005] In view of the common defects of the prior art, the present invention provides an anti-CD25 monoclonal antibody, a preparation method thereof and an application thereof. The monoclonal antibody development method provided by the present invention has the advantages of short time consumption, high throughput and high positive rate.

[0006] The inventive concept of the present invention is as follows: The present invention uses single B cells 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 hybridoma cell fusion and culture that is inefficient, time-consuming, and susceptible to contamination.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

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

[0009] The heavy chain variable region VH comprises three complementary determining regions CDRs, namely:

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

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

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

[0013] The light chain variable region VL also comprises three complementary determining regions CDRs, namely:

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

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

[0016] VLCDR3, the amino acid sequence of which 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] The second aspect of the present invention further provides a method for preparing the anti-CD25 monoclonal antibody as described in the first aspect.

[0020] The method comprises the following specific steps: expressing human CD25-Fc, immunizing mice with Freund's adjuvant, and collecting mouse spleen and bone marrow cells; screening mouse B cells binding to CD25 antigen using a flow cytometer; constructing a single-cell library for positive B cells, and obtaining antibody sequences of natural pairing of light and heavy chains by NGS sequencing; further screening the sequences, and expressing the antibodies by a mammalian cell high-throughput expression system; and finally verifying the results by ELISA detection.

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

[0022] S1. Express human CD25-Fc protein based on the protein sequence information of CD25;

[0023] S2, immunizing mice with the human CD25-Fc obtained in step S1 combined with Freund's adjuvant, and extracting spleen and bone marrow cells from the mice;

[0024] S3, using the mouse cells obtained in step S2 as raw materials, screening positive antibody-secreting cells by flow cytometry, constructing an antibody library through a single-cell library construction system, and performing next-generation sequencing;

[0025] S4, screening the antibody sequences obtained in step S3, extracting antibody sequences of natural pairing of light and heavy chains, and cloning them into a vector;

[0026] S5. Induce high-throughput expression of anti-CD25 antibodies through a mammalian cell high-throughput expression system, verify antigen binding through antibody detection technology such as ELISA, and finally obtain anti-CD25 monoclonal antibodies.

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

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

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

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

[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) The present invention screens and obtains a new anti-CD25 monoclonal antibody, which has better antigen binding activity than the existing anti-CD25 monoclonal antibody;

[0036] 2) The method for developing anti-CD25 monoclonal antibodies of the present invention shortens the development time of the target antibody and has short development time, high throughput and high positive rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figures are the results of mouse serum titer detection at different dilutions; NC is the negative control and 1 is the serum.

[0038] Figure 2 The results of flow cytometry sorting.

[0039] Figure 3 Antigen binding test results. DETAILED DESCRIPTION

[0040] 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.

[0041] Example 1

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

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

[0044] S2. 100 μL of human CD25-Fc protein at a concentration of 1 mg / mL was fluorescently labeled with AF488 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, inject intraperitoneally and subcutaneously into mice, and collect spleen and bone marrow cells of mice for screening after the serum titer is qualified.

[0046] The titer of Anti-human CD25-Fc Protein serum was detected by ELISA, and the detection process was as follows: the ELISA plate was coated with 2μg / mL human CD25-Fc protein and incubated at 37℃ for 2h; washed with 0.05% PBST and blocked with 1% BSA at room temperature for 2h; washed with 0.05% PBST, and 3-fold gradient dilutions of mouse sera before and after immunization were added, with the first 2 wells being 100 and 1000-fold dilutions, and incubated at room temperature for 1h; washed with 0.05% PBST, and Anti-mouse IgG (Fc specific) HRP was added, and incubated at room temperature for 30min; washed with 0.05% PBST, and Beyotime TMB colorimetric solution was added, and reacted at room temperature for 10min, and then Beyotime TMB stop solution was added to immediately stop the reaction, and OD450 was read immediately.

[0047] The titer of the antiserum after immunization was 512000. Figure 1 As shown. It can be seen that the antigen can induce mice to produce high-titer antiserum specific to 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 red blood cell lysis buffer, stop red blood cell 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 AF488-human CD25-Fc protein and fluorescent antibodies of B cell-specific molecular markers, incubate at 4°C for 1 h, resuspend in a large volume of MACS buffer, centrifuge at 400g for 5 min, discard the supernatant, resuspend the cells in buffer, and sort the cells using a flow cytometer.

[0049] The flow cytometry results are as follows Figure 2 As shown, the final positive proportion of sorted cells was about 0.68%.

[0050] S5. Use the single-cell library construction platform to construct a library for 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 9 antibody sequences for high-throughput expression in mammalian cells.

[0051] S6. ELISA was used to detect the antigen binding activity of 9 antibodies. The specific steps were as follows: the ELISA plate was coated with 2 μg / mL human CD25-Fc protein at 4°C overnight; washed with 0.05% PBST and blocked with 1% BSA at room temperature for 2 h; washed with 0.05% PBST, and 3-fold gradient dilutions of 9 antibodies, positive control (Daclizumab, 7G7B6-hIgG1), and isotype control were added, and incubated at room temperature for 1 h; washed with 0.05% PBST, Anti-human IgG (Fc specific) HRP was added, and incubated at room temperature for 30 min; washed with 0.05% PBST, TMB colorimetric solution was added, and the reaction was allowed to react at room temperature for 10 min, and then TMB stop solution was added to stop the reaction immediately, and the OD450 was read immediately.

[0052] Antigen binding test results Figure 3 As shown in the figure, the positive rate of 9 antibodies was 88.9% (8 / 9), among which the EC50 of CD25-3, CD25-4, CD25-6, and CD25-8 were lower than that of the positive control, which were 0.09066, 0.1051, 0.1322, and 0.1239, respectively. Therefore, the anti-human CD25-Fc monoclonal antibody numbered CD25-3 showed higher binding activity than the positive control.

[0053] The 6 CDR sequences, heavy chain variable region sequence and light chain variable region sequence of the anti-human CD25-Fc monoclonal antibody numbered CD25-3 are shown in the following table:

[0054] type Sequence 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 V L SEQ ID NO:8

[0055] 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-CD25 monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region VH comprises three complementary determining regions CDRs, namely: VHCDR1, the amino acid sequence of which is shown in SEQ ID NO: 1; VHCDR2, the amino acid sequence of which is shown in SEQ ID NO: 2; VHCDR3, the amino acid sequence of which is shown in SEQ ID NO: 3; The light chain variable region VL also comprises three complementary determining regions CDRs, namely: VLCDR1, the amino acid sequence of which is shown in SEQ ID NO: 4; VLCDR2, the amino acid sequence of which is shown in SEQ ID NO: 5; VLCDR3, the amino acid sequence of which 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 method for preparing an anti-CD25 monoclonal antibody according to any one of claims 1 to 3, characterized in that: The steps are as follows: express human CD25-Fc, immunize mice with Freund's adjuvant, collect mouse spleen and bone marrow cells; use flow cytometry to screen mouse B cells that bind to the CD25 antigen; construct a single-cell library for positive B cells, and obtain the antibody sequence of natural pairing of light and heavy chains through NGS sequencing; after further screening the sequence, express the antibody through a mammalian cell high-throughput expression system; and finally verify the result through ELISA detection.

5. A nucleic acid encoding the anti-CD25 monoclonal antibody according to any one of claims 1 to 3.

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

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

8. Use of the anti-CD25 monoclonal antibody according to any one of claims 1 to 3 in the preparation of a CD25 protein detection reagent.

9. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a product that binds to human CD25 protein.

10. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of tumor immunotherapy drugs.

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