Anti-Human CD24 monoclonal antibody, preparation method and application thereof

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to an anti-human CD24 monoclonal antibody, a preparation method thereof and an application thereof. The development method of the human CD24 monoclonal antibody provided by the present invention avoids the shortcomings of hybridoma technology and phage display technology, shortens the time-consuming development of the target antibody, and the prepared anti-human CD24 monoclonal antibody has strong affinity and good antigen-antibody binding activity at both the molecular and cellular levels.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to an anti-Human CD24 monoclonal antibody, a preparation method thereof and an application thereof. Background Art

[0002] CD24 is a highly glycosylated protein that is connected to the plasma membrane through glycosyl-phosphatidylinositol and is mainly expressed on immune cells, such as B cells, macrophages, and dendritic cells. Under normal circumstances, CD24 can promote the proliferation and differentiation of B cells, and can also regulate the activation and effector function of T cells. In addition, CD24 also plays a role in signal transduction, mainly due to its localization on lipid rafts. It has been reported that the expression of CD24 may affect the function or localization of other lipid raft-associated proteins, such as CXCR4 receptor and β1-integrin, and interact with the Src kinase family. In addition, researchers have detected that some tumor cells overexpress CD24 protein, such as cervical cancer, bile duct cancer, colorectal and gastric cancer. In tumor cells, CD24 is also involved in signal transduction, which is essential for cell adhesion, migration and differentiation. For example, the interaction between CD24 and P-selectin may help breast cancer cells roll on endothelial cells and adhere to platelets, and promote the adhesion of ovarian cancer cells to the mesothelium, thereby enhancing the spread of tumor cells. In addition, CD24 on the surface of ovarian cancer, triple-negative breast cancer and other cells inhibits macrophage phagocytosis by binding to Siglec-10 on macrophages, thereby achieving immune escape. The release of vesicles is very active on tumor cells. Studies have shown that CD24-positive vesicles have been detected in the serum of patients with ovarian cancer and breast cancer.

[0003] Therefore, it is possible to detect tumors through CD24 in medical diagnosis. The core reagent antibodies of existing diagnostic test kits cannot be produced by themselves, the price is high, and the quality and stability cannot be guaranteed; some test kits are also limited to the use of polyclonal antibodies, the supply of polyclonal antibodies is limited and the batch-to-batch differences are large, resulting in unstable quality of the kits and affecting the detection effect. If a high-affinity anti-Human CD24 monoclonal antibody is developed, it can provide support for studying the role of CD24 in tumor cells and even diagnosis.

[0004] Among the methods for developing anti-human CD24 monoclonal antibodies, hybridoma technology was the earliest and most commonly used monoclonal antibody development platform, but it is time-consuming and has low cell fusion efficiency, and the applicable species are limited. 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.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a high-affinity anti-Human CD24 monoclonal antibody, a preparation method and an application thereof.

[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-Human CD24 monoclonal antibody is provided, the antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises the following three complementary determining regions CDR-H1, CDR-H2, CDR-H3;

[0009] Amino acid sequence of CDR-H1: NTYMH (SEQ ID NO: 1);

[0010] Amino acid sequence of CDR-H2: RIDPANGNTKYAPKFQG (SEQ ID NO: 2);

[0011] Amino acid sequence of CDR-H3: EDLKTYYAMDY (SEQ ID NO: 3);

[0012] Preferably, the amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:7 or has at least 90% sequence identity.

[0013] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7.

[0014] EVQLQQSVAELVRPGASVNLSCTASGFNIKNTYMHWVKQRPEQGLEWIGRIDPANGNTKYAPKFQ GKATITADTSSNTAYLQLSSLTSEDTAIYYCAREDLKTYYAMDYWGQGTSVTVSS(SEQ ID NO:7)

[0015] In the second aspect of the present invention, a heavy chain of an anti-Human CD24 monoclonal antibody is provided, wherein the heavy chain has the heavy chain variable region and the heavy chain constant region as described in the first aspect of the present invention.

[0016] In a third aspect of the present invention, a light chain variable region of an anti-Human CD24 monoclonal antibody is provided, wherein the light chain variable region of the antibody comprises the following three complementary determining regions CDR-L1, CDR-L2, and CDR-L3;

[0017] Amino acid sequence of CDR-L1: RASESVSIHGTHLMH (SEQ ID NO: 4);

[0018] Amino acid sequence of CDR-L2: AASNLES (SEQ ID NO: 5);

[0019] Amino acid sequence of CDR-L3: QQSIEDPRT (SEQ ID NO: 6);

[0020] Preferably, the light chain variable region amino acid sequence is identical to the amino acid sequence shown in SEQ ID NO: 8 or has at least 90% sequence identity.

[0021] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0022] DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPAR FSGSGSETDFTLNIHPVEEEDAATYFCQQSIEDPRTFGGGTKLEIK. (SEQ ID NO:8)

[0023] In the fourth aspect of the present invention, a light chain of an antibody is provided, wherein the light chain has the light chain variable region and the light chain constant region as described in the third aspect of the present invention.

[0024] In the fifth aspect of the present invention, an antibody is provided, wherein the antibody has the heavy chain variable region as described in the first aspect of the present invention, and / or the light chain variable region as described in the third aspect of the present invention.

[0025] In another preferred embodiment, the antibody has the heavy chain as described in the second aspect of the present invention; and / or the light chain as described in the fourth aspect of the present invention.

[0026] In another preferred embodiment, the antibody is a specific anti-Human CD24 antibody.

[0027] In the sixth aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention.

[0028] In the seventh aspect of the present invention, a vector is provided, which contains the polynucleotide as described in the sixth aspect of the present invention.

[0029] In the eighth aspect of the present invention, a host cell is provided, which contains the vector as described in the seventh aspect of the present invention or the polynucleotide as described in the sixth aspect of the present invention.

[0030] In the ninth aspect of the present invention, a method for preparing an anti-Human CD24 monoclonal antibody is provided, and the specific preparation process is as follows:

[0031] S1. Based on the protein sequence information of Human CD24 (Genbank: P25063), Human Fc-tag was connected to its C-terminus to express Human CD24 / hFc Protein;

[0032] S2. After rapidly immunizing mice with the Human CD24 / hFc Protein obtained in step S1 combined with a water-soluble adjuvant, spleen and bone marrow cells of the mice were extracted;

[0033] S3. Using the mouse cells obtained in step S2 as raw materials, positive cells were sorted by flow cytometry, and an antibody library was constructed by a single-cell library construction system and next-generation sequencing was performed;

[0034] S4. Screening the antibody sequences obtained in step S3, extracting antibody sequences with natural pairing of light and heavy chains, and cloning them into a vector;

[0035] S5. Induce high-throughput expression of anti-human CD24 antibody through mammalian cell high-throughput expression system to obtain anti-human CD24 monoclonal antibody;

[0036] S6. Perform antigen binding experiments and antigen-antibody affinity verification.

[0037] In another preferred embodiment, the antigen binding detection of the present invention is an ELISA method; and the antigen-antibody affinity detection is an SPR method.

[0038] In the tenth aspect of the present invention, there is provided a use of the anti-Human CD24 monoclonal antibody according to the fifth aspect of the present invention in the preparation of a Human CD24 protein detection reagent.

[0039] In the eleventh aspect of the present invention, there is provided a use of the anti-Human CD24 monoclonal antibody according to the fifth aspect of the present invention in the preparation of a product binding to the Human CD24 protein.

[0040] Compared with the prior art, the technical effects of the present invention are as follows: the monoclonal antibody development method provided by the present invention has the advantages of short time consumption, high throughput, high positive rate, etc., and the obtained monoclonal antibody has the advantages of good antigen binding activity and strong affinity, etc. The present invention uses single B cells as the research subject, uses flow cytometry to sort out positive cells, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The results of the mouse serum titer test at different dilutions of the present invention are shown;

[0042] Figure 2 The results of the flow cytometer sorting test of cells expressing positive antibodies are shown;

[0043] Figure 3 The ELISA test result diagram showing the antigen binding activity of the antibody in the antigen binding test of the present invention is shown;

[0044] Figure 4 The figure shows the SPR experimental results in the antigen-antibody affinity detection of the present invention, wherein a is the affinity detection result of the positive control, b is the affinity detection result of Ab1, c is the affinity detection result of Ab2, d is the affinity detection result of Ab3, e is the affinity detection result of Ab4, f is the affinity detection result of Ab5, g is the affinity detection result of Ab6, h is the affinity detection result of Ab7, and i is the affinity detection result of Ab8. DETAILED DESCRIPTION

[0045] The present invention is further explained below in conjunction with specific embodiments, but it should be noted that the following embodiments are only used to explain the present invention but not to limit the present invention, and all technical solutions that are the same or similar to the present invention are within the protection scope of the present invention.

[0046] If no specific techniques or conditions are specified in the present examples, the operations are carried out according to conventional techniques and methods in the art and the contents of the instrument manuals; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0047] Example 1

[0048] The method for developing anti-Human CD24 monoclonal antibodies of the present invention comprises the following steps:

[0049] S1. Antigen modification

[0050] Based on the protein sequence and gene sequence information of Human CD24 (Genbank: P25063), the antigen was expressed and a human Fc-tag was connected to its C-terminus to obtain a modified Human CD24 / hFc protein for subsequent mouse immunization.

[0051] S2, labeled antigen

[0052] The HumanCD24 / hFc protein was fluorescently labeled with AF488 using the AlexaFluor 488 antibody labeling kit (purchased from Thermo Fisher, catalog number A20181), and the obtained labeled antigen AF488-Human CD24 / hFc protein was used for subsequent mouse cell screening.

[0053] S3. Immunization of mice

[0054] The Human CD24 / hFc protein obtained in step S1 was added with a water-soluble adjuvant to rapidly immunize mice. The specific steps are as follows:

[0055] (1) Primary immunization: Human CD24 / hFc protein was mixed with a water-soluble adjuvant and injected intraperitoneally and subcutaneously into mice;

[0056] (2) Secondary immunization: On the 10th day, the mice were immunized again with human CD24 / hFc protein, and blood was collected 3 days later to detect the titer of anti-human CD24 / hFc serum;

[0057] S4. ELISA tests serum titer. The testing process is as follows:

[0058] (1) The ELISA plate was coated with 5 μg / mL Human CD24 / hFc protein and incubated at 37°C for 2 h;

[0059] (2) Wash with 0.05% PBST and block with 1% BSA at room temperature for 2 h; Wash with 0.05% PBST and add 3-fold serial dilutions of mouse sera before and after immunization, with the first well being 1000-fold diluted, and incubate at room temperature for 1 h;

[0060] (3) Wash with 0.05% PBST, add Goat Anti-mouse IgG (Fc specific) HRP (purchased from Sigma, catalog number A0168), incubate at room temperature for 30 min; wash with 0.05% PBST;

[0061] (4) Add Bio-Tech TMB colorimetric solution and react at room temperature for 10 min. Then add Bio-Tech TMB stop solution to terminate the reaction immediately and read the OD450 immediately.

[0062] The titer of the antiserum after the second immunization was 27,000. The titer of the mouse serum was as follows: Figure 1 It shows that the antigen prepared in step S1 of Example 1 can induce mice to produce high-titer antiserum specific to Human CD24 / hFc protein.

[0063] S5. Collect mouse spleen and bone marrow cells after serum titer is qualified:

[0064] The spleen and bone marrow cells of the immunized mice were collected, centrifuged at 400 g for 5 min, resuspended in 1 mL of red blood cell lysis buffer, lysed on ice for 1 min, terminated red blood cell lysis with a large volume of Macs buffer, centrifuged at 400 g for 5 min, resuspended in 1 mL of Macs buffer, added with the labeled antigen AF488-Human CD24 / hFc protein obtained in step S2 and fluorescent antibodies for B cell-specific molecular markers, incubated at 4°C for 1 h, resuspended in a large volume of Macs buffer, centrifuged at 400 g for 5 min, discarded the supernatant, resuspended in buffer, and antigen-positive cells were sorted using a flow cytometer.

[0065] The flow cytometry results are as follows Figure 2 As shown; the proportion of antigen-positive cells is approximately 1.18%.

[0066] S6. Construction of mouse positive single cell library. The process is as follows:

[0067] (1) Using a single-cell library construction platform to construct a library of the sorted positive mouse cells, using the oil-in-water technique to generate oil droplets containing single mouse positive cells, and completing cell lysis, mRNA capture, and reverse transcription in the oil droplets. The reverse transcription products carrying the cell label barcode are pre-amplified for cDNA and antibody VDJ gene amplification. After interruption, the adapter and library label are connected to obtain an antibody library, and antibodies with natural pairing of light and heavy chains are obtained through second-generation sequencing and data analysis;

[0068] (2) Eight antibody sequences were selected based on their frequencies, and eight antibodies, Ab1 to Ab8, were obtained by expressing and purifying them using a mammalian high-throughput antibody expression platform.

[0069] Example 2

[0070] Antigen binding test: The antigen binding activity of 8 antibodies was tested by ELISA. The specific steps are as follows:

[0071] (1) The ELISA plate was coated with 2 μg / mL Human CD24 / hFc protein at 4°C overnight;

[0072] (2) Wash with 0.05% PBST and block with 1% BSA at room temperature for 2 h;

[0073] (3) Wash with 0.05% PBST, add 8 antibodies diluted 5-fold, Anti-HEL Human IgG1-Kappa Isotype, and positive control, and incubate at room temperature for 1 h;

[0074] (4) Wash with 0.05% PBST, add Anti-human IgG (Fc specific) HRP (purchased from Sigma, catalog number A0170), and incubate at room temperature for 30 min; wash with 0.05% PBST, add TMB colorimetric solution, react at room temperature for 10 min, and then add TMB stop solution to stop the reaction immediately, and read OD450 immediately.

[0075] Antigen binding test results Figure 3 As shown: the positive rate of 8 antibodies was 100% (8 / 8).

[0076] Example 3

[0077] Antigen-antibody affinity test: The affinity of 8 antibodies was tested by SPR. The specific steps are as follows:

[0078] (1) The antibody was diluted to 50 μg / mL with 0.1 M MES pH 5.5 (purchased from Carterra, Catalog No. CAT3625), and coupled to the surface of HC200M chip (purchased from Carterra, Catalog No. CAT4287) via NHS / EDC amino coupling. NHS / EDC activation was performed for 15 min, coupling was performed for 20 min, and blocking was performed with 1 M Ethanolamine for 8 min. The capture height was approximately 2000 RU.

[0079] (2) The instrument running buffer was replaced with 10xHBSTE buffer (purchased from Carterra, Catalog No. CAT3630), and 2000 nM antigen was injected into a single channel, with 3-fold gradient dilution, 11 concentrations as analytes, and each concentration of antigen was bound for 180 s, dissociated for 300 s, and regenerated for 20 s / 3 times with 10 mM Glycine HCL (purchased from Carterra, Catalog No. CAT3640), pH 2.0;

[0080] (3) Set up 7 buffer cycles and 11 antigen concentration cycles;

[0081] (4) After the run is completed, open the Carterra LSA analysis software for data analysis.

[0082] Antigen-antibody affinity test results Figure 4 As shown: the KD (M) value of Ab1 is 8.25E-10, which is close to the positive antibody Anti-Human CD24 (clone number ATG-031) with a KD value of 8.95E-10, and has the advantage of strong affinity.

[0083] The sequence of the anti-human CD24 monoclonal antibody numbered Ab1 is shown in the following table:

[0084]

[0085]

[0086] 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 CD24 monoclonal antibody, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises the following three complementary determining regions CDR-H1, CDR-H2, and CDR-H3; The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1; The amino acid sequence of CDR-H2 is shown in SEQ ID NO:2; The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3; The light chain variable region of the antibody includes the following three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The amino acid sequence of CDR-L1 is shown in SEQ ID NO:4; The amino acid sequence of CDR-L2 is shown in SEQ ID NO:5; The amino acid sequence of CDR-L3 is shown in SEQ ID NO:

6.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:7 or has at least 90% sequence identity; the amino acid sequence of the light chain variable region is identical to the amino acid sequence shown in SEQ ID NO:8 or has at least 90% sequence identity.

3. A polynucleotide, characterized in that The polynucleotide encodes an antibody selected from any one of claims 1-2.

4. A carrier, characterized in that The vector contains the polynucleotide according to claim 3.

5. A host cell, characterized in that The host cell contains the vector according to claim 4 or the polynucleotide according to claim 3.

6. Use of the anti-Human CD24 monoclonal antibody according to any one of claims 1 to 2 in the preparation of a Human CD24 protein detection reagent.

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