Preparation method and application of anti-human MUC1 antibody

Abstract

The invention relates to the technical field of biology, in particular to a preparation method and application of an anti-human MUC1 antibody, and provides a recombination system which utilizes an in-vitro B cell culture technology, uses ELISA (enzyme-linked immunosorbent assay) to detect the expression condition of the antibody, performs flow detection on the binding capacity level with cells and performs antibody expression and assembly through an expression vector. The monoclonal antibody with high specificity and strong affinity is screened out, and the immunogenicity is reduced on the basis that the high affinity combined with the antigen is reserved through humanized transformation, so that the monoclonal antibody is favorably applied to immunotherapy of a human body; the anti-human MUC1 antibody prepared by the invention plays an important role in immunotherapy of tumors, and solves the technical problems of insufficient specificity and lower affinity of the MUC1 antibody in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of an anti-human MUC1 antibody. Background Art

[0002] Mucin is a high molecular weight glycoprotein distributed in a variety of epithelial cells in the body. As part of the defense barrier of tissues and organs, it plays an important protective role. The human MUC gene family encodes more than 20 proteins. According to their different structures, they can be divided into secretory gel-forming mucins (MUC2, MUC5AC, MUC5B, MUC6, MUC7 and MUC19) and membrane-bound subfamilies (MUC1, MUC3A, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17 and MUC20). Among them, secretory gel-forming mucins serve as a protective barrier for basal mucosal cells, while membrane-bound mucins play a key role in cell signaling pathways and cell-to-cell interactions.

[0003] MUC1, also known as EMA (tumor-associated epithelial membrane antigen) or CD227, is a heterodimeric membrane-bound protein that is usually expressed in glandular or luminal epithelial cells of different tissues. The MUC1 gene encodes a single polypeptide chain that undergoes proteolytic cleavage at the sea urchin sperm protein, enterokinase, agrin (SEA) domain to form two non-covalently bound subunits. Among them, the larger amino-terminal subunit is located outside the cell and contains a signal sequence and tandem repeat domains. The carboxyl-terminal terminal subunit contains a short extracellular domain, a transmembrane domain, and a cytoplasmic tail. It has extensive O-linked glycosylation in its extracellular tandem repeat domain. It is found on the apical surface of epithelial cells in the lungs, stomach, intestines, eyes, and some other organs. Mucins protect the body from infection by binding of pathogens to oligosaccharides in the extracellular domain, thereby preventing pathogens from reaching the cell surface.

[0004] The expression characteristics of MUC1 in cancer progression and metastasis are increased MUC1 levels, altered glycosylation, and abnormal surface distribution patterns. Overexpression of MUC1 is often associated with pancreatic cancer, breast cancer, colon cancer, ovarian cancer, lung cancer, etc. MUC1 has been shown to be present in various adenocarcinomas and is associated with tumor metastasis and recurrence. In addition, MUC1 is also overexpressed in some hematological malignancies. Differences in tumor cell glycosylation also result in reduced cell adhesion ability, providing a basis for tumor metastasis. The anti-adhesion ability of MUC1 in tumor cells is not only important in cancer development, but also may lead to reduced adhesion of immune effector cells to malignant cells, giving malignant cells a survival advantage and playing an important role in tumor invasion and metastasis.

[0005] Therefore, MUC1 is a very promising target with broad application prospects in the field of tumor treatment, but existing MUC1 antibodies have problems of insufficient specificity and low affinity. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method and application of an anti-human MUC1 antibody, aiming to solve the technical problems of insufficient specificity and low affinity of the MUC1 antibody in the prior art.

[0007] To achieve the above object, the present invention adopts a method for preparing an anti-human MUC1 antibody, comprising the following steps:

[0008] Step 1: First, immunize the test object and extract serum during the immunization process;

[0009] Step 2: Perform ELISA titer test on the extracted serum;

[0010] Step 3: After the immunization of the experimental object is completed, B cells are cultured and identified to obtain positive clone cells, which are then collected for later use;

[0011] Step 4: Gene extraction and monoclonal cloning of positive cloned cells to select positive monoclonal antibodies;

[0012] Step 5: Express and purify the positive monoclonal antibody;

[0013] Step 6: Humanize the expressed and purified antibody and detect its binding activity with endogenous expression cells to obtain the anti-human MUC1 antibody.

[0014] Among them, the experimental subjects are animals.

[0015] Among them, immunization of the experimental object includes three immunizations;

[0016] At the first immunization, the immunization dose for each experimental object was: 200 μg protein, the antigen protein was mixed with Freund's complete adjuvant in equal proportions and completely emulsified, and immunized by multiple subcutaneous injections on the back;

[0017] The second immunization should be carried out two weeks after the first immunization. The immunization dose for each experimental object is: 100 μg antigen protein mixed with Freund's incomplete adjuvant in equal proportions and completely emulsified, and injected subcutaneously on the back for multiple times;

[0018] In the third immunization, the test subjects should be boosted three days before the termination of immunization. The immunization dose for each test subject is: 50 μg antigen protein dissolved and injected intraperitoneally.

[0019] The specific method of conducting ELISA titer detection on the extracted test serum is as follows:

[0020] 1 μg / ml MUC1 protein was coated in the ELISA plate, 100 μl was added to each well, and the plate was incubated at 4°C overnight; the supernatant was discarded, and each well was washed 3-5 times with 300 μl PBST; 100 μl of 3% BSA was added to each well, and the plate was blocked for 1 hour at room temperature; the supernatant was discarded, and each well was washed 3-5 times with 300 μl PBST;

[0021] The collected test serum was diluted with an initial ratio of 1:500, and then double-fold gradient dilution was performed. 100 μl of the diluted supernatant was added to each well and incubated at room temperature for 2 hours; the supernatant was discarded, and each well was washed 3-5 times with 300 μl PBST; incubated with anti-rabbit IgG-HRP antibody, 100 μl was added to each well, and incubated at room temperature for 1 hour in the dark; the supernatant was discarded, and each well was washed 5-7 times with 300 μl PBST; 100 μL of TMB colorimetric solution was added to each well for color development, and the color development was carried out at room temperature in the dark for 5-10 minutes; 50 μl of stop solution was added to each well to stop the color development, and the OD450 enzyme label reading was performed and the data was recorded.

[0022] Among them, the specific method of B cell culture and identification after the immunization of the experimental object is as follows:

[0023] After the immunization of the experimental object was completed, the spleen tissue was removed and ground, and the cells were collected and filtered and centrifuged using a 200-mesh sieve. The cells were resuspended and added with red blood cell lysis buffer to remove red blood cells, and the obtained cells were used for subsequent sorting experiments;

[0024] The cells were co-incubated with biotin-labeled MUC1 protein, and the memory B cells of the rabbit were obtained after sorting, and the memory B cells were placed in a 96-cell culture plate and co-incubated with feeder layer cells;

[0025] After two weeks of culture at 37°C and 5% CO2, the binding activity of the clone supernatant was detected by ELISA, and the binding of the clone supernatant to endogenously expressing cells Capan-2 was detected by FACS;

[0026] The clones with positive results from both methods were considered as the final positive clone cells, and the cells were collected for subsequent experiments.

[0027] Among them, the specific method of gene extraction and monocloning of positive clone cells is:

[0028] Positive clones were selected for cell lysis, RNA was extracted using the Trizol method, and reverse transcription was performed;

[0029] Amplify the light chain variable region and heavy chain variable region of the antibody by PCR amplification reaction, construct them into the corresponding light chain and heavy chain expression vectors containing constant regions, perform sequencing and analyze the sequencing results;

[0030] The correctly sequenced light chain and heavy chain plasmids were selected, and the recombinant plasmids were paired for transfection. HEK-293T cells were incubated in a 24-well plate 24 hours before transfection, and the recombinant paired plasmids were transiently transfected with PEI transfection reagent. The supernatant was collected 48 hours later, and the binding activity of the cloned supernatant was detected by ELISA. The binding of the supernatant to the endogenous expression cell Capan-2 was detected by FACS method, and the positive monoclonal antibody was selected.

[0031] The variable region of the monoclonal antibody comprises three CDR light chain hypervariable regions and three CDR heavy chain hypervariable regions, the amino acid sequences of the light chain hypervariable regions CDR1, CDR2, and CDR3 are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the amino acid sequences of the heavy chain hypervariable regions CDR1, CDR2, and CDR3 are SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0032] Among them, the specific method for expressing and purifying the positive monoclonal antibody is:

[0033] The density of 293F cells was adjusted to 5 × 10 5 / mL, incubate in a shaking incubator with 37°C, 120rpm and 5% CO2 for 24 hours, mix 300μg of DNA with PEI and let it stand for 20 minutes, then add it to a 293F culture flask and continue incubating for 72 hours. Collect the cell supernatant by centrifugation, purify the antibody with Protein A medium, determine the antibody concentration and purity by measuring the absorbance with Nanodrop, and evaluate the expression level and purity of the antibody by Coomassie staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0034] Thus, a rabbit anti-human MUC1 monoclonal antibody was obtained, wherein the amino acid sequence of the light chain variable region of the rabbit anti-human MUC1 antibody is SEQ ID NO.7, and its base sequence is SEQ ID NO.8. The amino acid sequence of the heavy chain variable region of the rabbit anti-human MUC1 antibody is SEQ ID NO.9, and its base sequence is SEQ ID NO.10.

[0035] The specific method of obtaining the anti-human MUC1 antibody after humanization of the expressed and purified antibody and detection of binding activity with endogenous expression cells is as follows:

[0036] The rabbit CDR sequence was transplanted into the variable region framework of the human antibody, and the cell binding activity of the humanized antibody was tested by flow cytometry;

[0037] Thus, a humanized anti-human MUC1 monoclonal antibody was obtained, wherein the amino acid sequence of the light chain variable region of the humanized anti-human MUC1 antibody is SEQ ID NO.11, and its base sequence is SEQ ID NO.12. The amino acid sequence of the heavy chain variable region of the humanized anti-human MUC1 antibody is SEQ ID NO.13, and its base sequence is SEQ ID NO.14.

[0038] The antibody expression supernatant was incubated with the Capan-2 cells that highly expressed the antigen at 4°C for 30 minutes, then washed once with PBS and centrifuged at 1200 rpm for 3 minutes. Then, Anti-Human IgG Fc secondary antibody was added and incubated at 4°C for 30 minutes. The cells were washed once with PBS and centrifuged at 1200 rpm for 3 minutes for detection and data recording.

[0039] The use of the anti-human MUC1 antibody prepared by the method for preparing the anti-human MUC1 antibody as described above in pancreatic cancer, breast cancer, prostate cancer and gastric cancer.

[0040] The invention discloses a method for preparing an anti-human MUC1 antibody and its application. The invention utilizes in vitro B cell culture technology, uses ELISA to detect the expression of the antibody and flow cytometry to detect the level of binding ability to cells, and uses an expression vector to express and assemble the antibody in a recombinant system, thereby screening out a monoclonal antibody with high specificity and strong affinity. Furthermore, after humanization, the monoclonal antibody retains the high affinity for binding to the antigen and reduces the immunogenicity, which is beneficial for application in human immunotherapy.

[0041] The anti-human MUC1 antibody prepared by the present invention plays an important role in the immunotherapy of tumors, and has a wide range of application prospects in the fields of anti-tumor drugs, chimeric antigen receptor T (CAR-T) cell therapy, etc., and has the following beneficial effects:

[0042] 1. Natural pairing: Single B cell antibody preparation technology retains the natural pairing of light and heavy chain variable regions;

[0043] 2. High throughput: Single B cell antibody preparation technology can meet the needs of large-scale sample processing and improve the efficiency of antibody discovery;

[0044] 3. High specificity: Single B cell antibody preparation technology can obtain monoclonal antibodies that bind to specific antigens with high specificity;

[0045] 4. Wide application: A humanized monoclonal antibody targeting MUC1 was invented. MUC1 is highly expressed in various tumors such as pancreatic cancer, breast cancer, prostate cancer, and gastric cancer. It is used in immunotherapy such as neutralizing antibodies, chimeric antigen receptors (CARs), bispecific T cell engagers (BiTEs), and antibody-drug conjugates (ADCs). BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 It is a diagram of the immune titer of the experimental animals of the present invention.

[0048] Figure 2 This is a graph showing the binding activity of the immune serum of the present invention to normal / tumor cells of the pancreas.

[0049] Figure 3 This is a graph showing the binding activity of the immune serum of the present invention to other tumor cells.

[0050] Figure 4 This is a graph for detecting the binding activity of the experimental animal monoclonal antibody to cells of the present invention.

[0051] Figure 5 This is a graph showing the detection of the binding activity of the humanized monoclonal antibody of the present invention to cells. DETAILED DESCRIPTION

[0052] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0053] See also Figure 1 to Figure 5 ,in Figure 1 is the immune titer diagram of the experimental animals of the present invention, Figure 2 This is a graph showing the binding activity of the immune serum of the present invention to normal / tumor cells of the pancreas. Figure 3 This is a graph showing the activity of the immune serum of the present invention binding to other tumor cells. Figure 4 This is a graph showing the binding activity of the experimental animal monoclonal antibody to cells of the present invention. Figure 5 This is a diagram for detecting the binding activity of the humanized monoclonal antibody of the present invention to cells.

[0054] The present invention provides a method for preparing an anti-human MUC1 antibody, comprising the following steps:

[0055] S101, first immunize the selected experimental animals, and extract animal serum during the immunization process;

[0056] According to this specific embodiment, animal immunization of the selected experimental animals includes three immunizations;

[0057] At the first immunization, the immunization dose for each animal was: 200 μg protein, the antigen protein was mixed with Freund's complete adjuvant in equal proportions and completely emulsified, and immunized by multiple subcutaneous injections on the back;

[0058] The second immunization should be carried out two weeks after the first immunization. The immunization dose for each animal is: 100 μg antigen protein mixed with Freund's incomplete adjuvant in equal proportions and completely emulsified, and injected subcutaneously on the back for multiple times.

[0059] During the three immunizations, the experimental animals should be boosted with immunization three days before the termination of immunization. The immunization dose for each animal is: 50 μg antigen protein dissolved and injected intraperitoneally.

[0060] S102, performing ELISA titer detection on the extracted animal serum;

[0061] For this specific embodiment, the specific method is as follows: 1 μg / ml MUC1 protein is coated in an ELISA plate, 100 μl is added to each well, and it is kept at 4°C overnight; the supernatant is discarded, and each well is washed 3-5 times with 300 μl PBST; 100 μl of 3% BSA is added to each well, and it is blocked at room temperature for 1 hour; the supernatant is discarded, and each well is washed 3-5 times with 300 μl PBST;

[0062] The collected animal serum was diluted with an initial ratio of 1:500, and then double-fold gradient dilution was performed. 100 μl of the diluted supernatant was added to each well and incubated at room temperature for 2 hours; the supernatant was discarded, and each well was washed 3-5 times with 300 μl PBST; incubated with anti-rabbit IgG-HRP antibody, 100 μl was added to each well, and incubated at room temperature for 1 hour in the dark; the supernatant was discarded, and each well was washed 5-7 times with 300 μl PBST; 100 μL of TMB colorimetric solution was added to each well for color development, and the color development was carried out at room temperature in the dark for 5-10 minutes; 50 μl of stop solution was added to each well to stop the color development, and the OD450 enzyme label reading was performed and the data was recorded.

[0063] S103, after the animal immunization is completed, B cells are cultured and identified to obtain positive clone cells, which are collected for future use;

[0064] For this specific implementation, the specific method is:

[0065] After the animal is immunized, it is dissected, the spleen tissue is removed and ground, the cells are collected and filtered and centrifuged using a 200-mesh sieve, the cells are resuspended and added with red blood cell lysis buffer to remove red blood cells, and the obtained cells are used for subsequent sorting experiments;

[0066] The cells were co-incubated with biotin-labeled MUC1 protein, and the memory B cells of the rabbit were obtained after sorting, and the memory B cells were placed in a 96-cell culture plate and co-incubated with feeder layer cells;

[0067] After two weeks of culture at 37°C and 5% CO2, the binding activity of the clone supernatant was detected by ELISA, and the binding of the clone supernatant to endogenously expressing cells Capan-2 was detected by FACS;

[0068] The clones with positive results from both methods were considered as the final positive clone cells, and the cells were collected for subsequent experiments.

[0069] S104, extracting genes and cloning positive cloned cells, and selecting positive monoclonal antibodies;

[0070] For this specific implementation, the specific method is:

[0071] Positive clones were selected for cell lysis, RNA was extracted using the Trizol method, and reverse transcription was performed;

[0072] Amplify the light chain variable region and heavy chain variable region of the antibody by PCR amplification reaction, construct them into the corresponding light chain and heavy chain expression vectors containing constant regions, perform sequencing and analyze the sequencing results;

[0073] The correctly sequenced light chain and heavy chain plasmids were selected, and the recombinant plasmids were paired for transfection. HEK-293T cells were incubated in a 24-well plate 24 hours before transfection, and the recombinant paired plasmids were transiently transfected with PEI transfection reagent. The supernatant was collected 48 hours later, and the binding activity of the cloned supernatant was detected by ELISA. The binding of the supernatant to the endogenous expression cell Capan-2 was detected by FACS method, and the positive monoclonal antibody was selected.

[0074] The variable region of the monoclonal antibody comprises three CDR light chain hypervariable regions and three CDR heavy chain hypervariable regions, the amino acid sequences of the light chain hypervariable regions CDR1, CDR2, and CDR3 are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the amino acid sequences of the heavy chain hypervariable regions CDR1, CDR2, and CDR3 are SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0075] Among them, SEQ ID NO.1 is QASQSVYNNNQLS.

[0076] SEQ ID NO.2 is: YGSTLAS.

[0077] SEQ ID NO.3 is: QGYYSGGIFA.

[0078] SEQ ID NO.4 is: SYAMG.

[0079] SEQ ID NO.5 is: IIGSYGTTYYAAWAKG.

[0080] SEQ ID NO.6 is: GGYPGYLSYFNI.

[0081] S105, expressing and purifying the positive monoclonal antibody;

[0082] For this specific implementation, the specific method is:

[0083] The density of 293F cells was adjusted to 5 × 10 5 / mL, incubate in a shaking incubator with 37°C, 120rpm and 5% CO2 for 24 hours, mix 300μg of DNA with PEI and let it stand for 20 minutes, then add it to a 293F culture flask and continue incubating for 72 hours. Collect the cell supernatant by centrifugation, purify the antibody with Protein A medium, determine the antibody concentration and purity by measuring the absorbance with Nanodrop, and evaluate the expression level and purity of the antibody by Coomassie staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0084] Thus, a rabbit anti-human MUC1 monoclonal antibody was obtained, wherein the amino acid sequence of the light chain variable region of the rabbit anti-human MUC1 antibody is SEQ ID NO.7, and its base sequence is SEQ ID NO.8. The amino acid sequence of the heavy chain variable region of the rabbit anti-human MUC1 antibody is SEQ ID NO.9, and its base sequence is SEQ ID NO.10.

[0085] Wherein, SEQ ID NO.7 is:

[0086] AQVLTQTPSPVSAAVGGTVTINCQASQSVYNNNQLSWYQQKPGQPPKLL IYYGSTLASGVSSRFKGSRSGTQFTLTISGVQCDDAATYYCQGYYSGGIFAFG GGTEVVVK.

[0087] SEQ ID NO.8 is:

[0088] gcgcaagtgctgacccagactccatcccctgtgtctgcagctgtgggaggcacagtcaccatcaactgccaggccagtcagagtgtttataataacaaccaattatcctggtatcagcagaaaccagggcagcctcccaagctcctgatctattatggatccactctggcatctggggtctcatcgcggttcaaaggcagtagatctgggacacagttcactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtcaaggctattatagtggtggtatttttgctttcggcggagggaccgaggtggtggtcaag。

[0089] SEQ ID NO.9 is:

[0090] QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGII GSYGTTYYAAWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGGYPGYLSY FNIWGPGTLVTVSS。

[0091] SEQ ID NO.10 is:

[0092] cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggaatcgacctcagtagttatgcaatgggctgggtccgccaggctccagggaaggggctggaatggatcggaatcattggtagttatggtaccacatactacgcggcctgggcgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagaggaggttatcctggttatctttcctactttaatatctggggcccaggcaccctcgtcaccgtctcttca。

[0093] S106, humanizing the expressed and purified antibody and detecting its binding activity with endogenous expression cells to obtain an anti-human MUC1 antibody;

[0094] For this specific implementation, the specific method is:

[0095] The rabbit CDR sequence was transplanted into the variable region framework of the human antibody, and the cell binding activity of the humanized antibody was tested by flow cytometry;

[0096] Thus, a humanized anti-human MUC1 monoclonal antibody was obtained, wherein the amino acid sequence of the light chain variable region of the humanized anti-human MUC1 antibody is SEQ ID NO.11, and its base sequence is SEQ ID NO.12. The amino acid sequence of the heavy chain variable region of the humanized anti-human MUC1 antibody is SEQ ID NO.13, and its base sequence is SEQ ID NO.14.

[0097] Wherein, SEQ ID NO.11 is:

[0098] AQVLTQSPSSSLSASVGDRVTITCQASQSVYNNNQLSWYQQKPGKAPKLL IYYGSTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGYYSGGIFAFGG GTKVEIK.

[0099] SEQ ID NO.12 is:

[0100] gcccaagtgctgacacagagccctagcagcctgagcgctagcgtgggcgacagagtgaccatcacctgccaagctagccaaagcgtgtacaacaataatcagctgagctggtatcagcagaagcccggcaaggcccccaagctgctgatctactacggcagcacc ctggctagcggcgtgcctagcagattcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccaaggctactacagcggcggcatcttcgccttcggcgggggcaccaaggtggagatcaag.

[0101] SEQ ID NO.13 is:

[0102] ELQLQESGPGLVKPSETLSLTCTVSGGSISSYAMGWIRQPPGKGLEWIGII GSYGTTYYAAWAKGRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARGGYPGY LSYFNIWGQGTLVTVSS.

[0103] SEQ ID NO.14 is:

[0104] gagctgcagctgcaagagagcggccccggcctggtgaagcctagcgagaccctgagcctgacctgcaccgtgagcggcggcagcatcagcagctacgccatgggctggatcagacagccccccggcaagggcctggagtggatcggcatcatcggcagctacggcaccacctactacgcc gcctgggccaagggcagagtgaccatcagcgtggacacaagcaagaatcagttcagcctgaagctgagcagcgtgaccgccgccgacaccgccgtgtactactgcgctagaggcggctaccccggctacctgagctacttcaacatctggggccaaggcaccctggtgaccgtgagcagc.

[0105] The antibody expression supernatant was incubated with the Capan-2 cells that highly expressed the antigen at 4°C for 30 minutes, then washed once with PBS and centrifuged at 1200 rpm for 3 minutes. Then, Anti-Human IgG Fc secondary antibody was added and incubated at 4°C for 30 minutes. The cells were washed once with PBS and centrifuged at 1200 rpm for 3 minutes for detection and data recording.

[0106] The use of the anti-human MUC1 antibody prepared by the method for preparing the anti-human MUC1 antibody as described above in pancreatic cancer, breast cancer, prostate cancer and gastric cancer.

[0107] The preparation method and application of an anti-human MUC1 antibody of the present invention utilizes in vitro B cell culture technology, uses ELISA to detect the expression of the antibody and flow cytometry to detect the level of binding ability to cells, and uses an expression vector to express and assemble the antibody in a recombinant system to screen out a monoclonal antibody with high specificity and strong affinity, and after humanization, while retaining the high affinity for binding to the antigen, the immunogenicity is reduced, which is conducive to the application in human immunotherapy;

[0108] The anti-human MUC1 antibody prepared by the present invention plays an important role in the immunotherapy of tumors, and has a wide range of application prospects in the fields of anti-tumor drugs, chimeric antigen receptor T (CAR-T) cell therapy, etc., and has the following beneficial effects:

[0109] 1. Natural pairing: Single B cell antibody preparation technology retains the natural pairing of light and heavy chain variable regions;

[0110] 2. High throughput: Single B cell antibody preparation technology can meet the needs of large-scale sample processing and improve the efficiency of antibody discovery;

[0111] 3. High specificity: Single B cell antibody preparation technology can obtain monoclonal antibodies that bind to specific antigens with high specificity;

[0112] 4. Wide application: A humanized monoclonal antibody targeting MUC1 was invented. MUC1 is highly expressed in various tumors such as pancreatic cancer, breast cancer, prostate cancer, and gastric cancer. It is used in immunotherapy such as neutralizing antibodies, chimeric antigen receptors (CARs), bispecific T cell engagers (BiTEs), and antibody-drug conjugates (ADCs).

[0113] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for preparing an anti-human MUC1 antibody, characterized in that: The following steps are involved: Step 1: First, immunize the test object and extract serum during the immunization process; Step 2: Perform ELISA titer test on the extracted serum; Step 3: After the immunization of the experimental object is completed, B cells are cultured and identified to obtain positive clone cells, which are then collected for later use; Step 4: Gene extraction and monoclonal cloning of positive cloned cells to select positive monoclonal antibodies; Step 5: Express and purify the positive monoclonal antibody; Step 6: Humanize the expressed and purified antibody and detect its binding activity with endogenous expression cells to obtain the anti-human MUC1 antibody.

2. The method for preparing an anti-human MUC1 antibody according to claim 1, characterized in that: Immunization of the experimental articles consisted of three immunizations; At the first immunization, the immunization dose for each experimental object was: 200 μg protein, the antigen protein was mixed with Freund's complete adjuvant in equal proportions and completely emulsified, and immunized by multiple subcutaneous injections on the back; The second immunization should be carried out two weeks after the first immunization. The immunization dose for each experimental object is: 100 μg antigen protein mixed with Freund's incomplete adjuvant in equal proportions and completely emulsified, and injected subcutaneously on the back for multiple times; In the third immunization, the test subjects should be boosted three days before the termination of immunization. The immunization dose for each test subject is: 50 μg antigen protein dissolved and injected intraperitoneally.

3. The method for preparing an anti-human MUC1 antibody according to claim 2, characterized in that: The specific method of ELISA titer detection of the extracted serum is as follows: 1 μg / ml MUC1 protein was coated in the ELISA plate, 100 μl was added to each well, and the plate was incubated at 4°C overnight; the supernatant was discarded, and each well was washed 3-5 times with 300 μl PBST; 100 μl of 3% BSA was added to each well, and the plate was blocked for 1 hour at room temperature; the supernatant was discarded, and each well was washed 3-5 times with 300 μl PBST; The collected serum was diluted with an initial ratio of 1:500, followed by two-fold gradient dilution, 100 μl of the diluted supernatant was added to each well and incubated at room temperature for 2 hours; the supernatant was discarded, and each well was washed 3-5 times with 300 μl PBST; incubated with anti-rabbit IgG-HRP antibody, 100 μl was added to each well, and incubated at room temperature for 1 hour in the dark; the supernatant was discarded, and each well was washed 5-7 times with 300 μl PBST; 100 μL of TMB colorimetric solution was added to each well for color development, and the color development was carried out at room temperature in the dark for 5-10 minutes; 50 μl of stop solution was added to each well to stop the color development, and the OD450 enzyme label reading was performed and the data was recorded.

4. The method for preparing an anti-human MUC1 antibody according to claim 3, characterized in that: The specific method for B cell culture and identification after immunization of the experimental object is as follows: After the immunization of the experimental object was completed, the spleen tissue was removed and ground, and the cells were collected and filtered and centrifuged using a 200-mesh sieve. The cells were resuspended and added with red blood cell lysis buffer to remove red blood cells, and the obtained cells were used for subsequent sorting experiments; The cells were co-incubated with biotin-labeled MUC1 protein, and the memory B cells of the rabbit were obtained after sorting, and the memory B cells were placed in a 96-cell culture plate and co-incubated with feeder layer cells; After two weeks of culture at 37°C and 5% CO2, the binding activity of the clone supernatant was detected by ELISA, and the binding of the clone supernatant to endogenously expressing cells Capan-2 was detected by FACS; The clones with positive results from both methods were considered as the final positive clone cells, and the cells were collected for subsequent experiments.

5. The method for preparing an anti-human MUC1 antibody according to claim 4, characterized in that: The specific method for gene extraction and monocloning of positive clone cells is as follows: Positive clones were selected for cell lysis, RNA was extracted using the Trizol method, and reverse transcription was performed; Amplify the light chain variable region and heavy chain variable region of the antibody by PCR amplification reaction, construct them into the corresponding light chain and heavy chain expression vectors containing constant regions, perform sequencing and analyze the sequencing results; The correctly sequenced light chain and heavy chain plasmids were selected, and the recombinant plasmids were paired for transfection. HEK-293T cells were incubated in a 24-well plate 24 hours before transfection, and the recombinant paired plasmids were transiently transfected with PEI transfection reagent. The supernatant was collected 48 hours later, and the binding activity of the cloned supernatant was detected by ELISA. The binding of the supernatant to the endogenous expression cell Capan-2 was detected by FACS method, and the positive monoclonal antibody was selected.

6. The method for preparing an anti-human MUC1 antibody according to claim 5, characterized in that: The specific method for expressing and purifying positive monoclonal antibodies is as follows: The density of 293F cells was adjusted to 5 × 10 5 / mL, incubate in a shaking incubator with 37°C, 120rpm and 5% CO2 for 24 hours, mix 300μg of DNA with PEI and let it stand for 20 minutes, then add it to a 293F culture flask and continue incubating for 72 hours. Collect the cell supernatant by centrifugation, purify the antibody with Protein A medium, determine the antibody concentration and purity by measuring the absorbance with Nanodrop, and evaluate the expression level and purity of the antibody by Coomassie staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

7. The method for preparing an anti-human MUC1 antibody according to claim 6, characterized in that: The specific method of obtaining the anti-human MUC1 antibody after humanization of the expressed and purified antibody and detection of binding activity with endogenous expression cells is as follows: The rabbit CDR sequence was transplanted into the variable region framework of the human antibody, and the cell binding activity of the humanized antibody was tested by flow cytometry; The antibody expression supernatant was incubated with the Capan-2 cells that highly expressed the antigen at 4°C for 30 minutes, then washed once with PBS and centrifuged at 1200 rpm for 3 minutes. Then, Anti-Human IgG Fc secondary antibody was added and incubated at 4°C for 30 minutes. The cells were washed once with PBS and centrifuged at 1200 rpm for 3 minutes for detection and data recording.

8. Use of the anti-human MUC1 antibody prepared by the method for preparing the anti-human MUC1 antibody according to claim 7 in pancreatic cancer, breast cancer, prostate cancer and gastric cancer.

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