Humanized anti-human mucin 1 monoclonal antibody and use thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses a humanized anti-human MUC1 monoclonal antibody and application thereof, the antibody is humanized by CDR grafting technology, three complementarity determining regions of a light chain variable region and three complementarity determining regions of a heavy chain variable region are retained, an amino acid sequence of the light chain variable region is shown in SEQ ID NO. 11, and a corresponding base sequence is shown in SEQ ID NO. 12; an amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 13, and a corresponding base sequence is shown in SEQ ID NO. 14. The humanized anti-human MUC1 monoclonal antibody can be specifically combined with MUC1 positive tumor cells, and can kill tumor cells through ADCC and CDC effects; can be used for preparing an anti-tumor drug, and the drug forms include a monoclonal antibody, an ADC, a CAR-T cell preparation and BiTEs, and provides an effective targeting tool for immunotherapy of MUC1 overexpression related tumors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a humanized anti-human MUC1 monoclonal antibody and its applications. Background Technology

[0002] Mucins are high-molecular-weight glycoproteins distributed in various epithelial cells throughout the body, playing a vital protective role as part of the tissue and organ defense barrier. The human MUC gene family encodes more than 20 proteins. Based on their structure, 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). Secretory gel-forming mucins serve as a protective barrier for basal mucosal cells, while membrane-bound mucins play crucial roles in cell signaling pathways and intercellular interactions.

[0003] MUC1, also known as EMA (tumor-associated epithelial membrane antigen) or CD227, is a heterodimeric membrane protein commonly expressed in glandular or luminal epithelial cells of various tissues. The MUC1 gene encodes a single polypeptide chain that cleaves within the sea urchin spermatin, enterokinase, and aggregate protein (SEA) domains to form two non-covalently bound subunits. The larger N-terminal subunit is located extracellularly and contains a signal sequence and a tandem repeat domain. The C-terminal subunit contains a short extracellular domain, a transmembrane domain, and a cytoplasmic tail. Extensive O-linked glycosylation is present in its extracellular tandem repeat domain. MUC1 is found on the apical surface of epithelial cells in the lungs, stomach, intestines, eyes, and several other organs. Mucins protect the body from infection by binding pathogens to oligosaccharides in their extracellular domains, thus preventing pathogens from reaching the cell surface.

[0004] The expression characteristics of MUC1 in cancer progression and metastasis are elevated MUC1 levels, altered glycosylation, and abnormal surface distribution patterns. Overexpression of MUC1 is commonly associated with pancreatic cancer, breast cancer, colon cancer, ovarian cancer, and lung cancer. MUC1 has been shown to be present in various adenocarcinomas and is correlated with tumor metastasis and recurrence. Furthermore, MUC1 is also overexpressed in some hematologic malignancies. Differences in tumor cell glycosylation also lead to reduced cell adhesion, providing a basis for tumor metastasis. The anti-adhesion ability of MUC1 in tumor cells is not only important in cancer development but may also lead to reduced adhesion between immune effector cells and malignant cells, giving malignant cells a survival advantage and enabling them to exert significant tumor invasion and metastasis capabilities.

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

[0006] The purpose of this invention is to address the shortcomings of existing MUC1 antibodies, such as insufficient specificity, low affinity, and high immunogenicity, by providing a humanized anti-human MUC1 monoclonal antibody and its applications. This antibody is humanized through CDR transplantation technology, which retains the high specificity and high affinity for MUC1 while significantly reducing immunogenicity. Furthermore, the invention clarifies the application of this antibody in pancreatic cancer, breast cancer, prostate cancer, and gastric cancer, providing an effective targeted tool for the immunotherapy of these tumors.

[0007] To solve the above-mentioned technical problems, the present invention provides a humanized anti-human MUC1 monoclonal antibody, comprising a light chain variable region and a heavy chain variable region;

[0008] The light chain variable region includes three complementarity-determining regions, whose amino acid sequences are as follows:

[0009] LCDR1 is QASQSVYNNNQLS, as shown in SEQ ID NO.1;

[0010] LCDR2 is YGSTLAS, as shown in SEQ ID NO.2;

[0011] LCDR3 is QGYYSGGIFA, as shown in SEQ ID NO.3;

[0012] The heavy chain variable region includes three complementarity-determining regions, whose amino acid sequences are as follows:

[0013] HCDR1 is SYAMG, as shown in SEQ ID NO.4;

[0014] HCDR2 is IIGSYGTTYYAAWAKG, as shown in SEQ ID NO.5;

[0015] HCDR3 is GGYPGYLSYFNI, as shown in SEQ ID NO.6.

[0016] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.13.

[0017] Preferably, the base sequence encoding the light chain variable region is shown in SEQ ID NO.12; and the base sequence encoding the heavy chain variable region is shown in SEQ ID NO.14.

[0018] This invention also provides the application of the humanized anti-human MUC1 monoclonal antibody in the preparation of antitumor drugs, wherein the tumor is a MUC1 overexpression-related tumor. The humanized anti-human MUC1 monoclonal antibody of this invention can specifically bind to abnormally expressed MUC1 on the surface of tumor cells, exerting a targeting effect in the immunotherapy of MUC1 overexpression-related tumors.

[0019] Preferably, the tumor is pancreatic cancer, breast cancer, prostate cancer, or stomach cancer.

[0020] Preferably, the form of the drug includes:

[0021] Monoclonal antibody drugs specifically kill MUC1-positive tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0022] Antibody-drug conjugates (ADCs) are created by conjugating cytotoxic drugs (such as chemotherapy drugs or toxins) to an antibody. By leveraging the antibody's targeting ability, the drug is delivered to tumor cells, reducing its toxicity to normal cells.

[0023] Chimeric antigen receptor T (CAR-T) cell preparations use the single-chain variable region (scFv) of the antibody as the antigen recognition domain of the CAR to modify T cells, enabling T cells to specifically recognize and kill MUC1-positive tumor cells;

[0024] Bispecific T-cell connectors (BiTEs) bind to MUC1-positive tumor cells at one end and to CD3 molecules on the surface of T cells at the other end, activating the killing function of T cells and achieving targeted elimination of tumor cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. High specificity and high affinity: The CDR sequence of the antibody of this invention has been screened multiple times (ELISA detection of protein binding, FACS detection of cell binding) and can specifically bind to MUC1 protein and tumor cells that endogenously express MUC1 (such as pancreatic cancer Capan-2 cells). It retains the natural pairing of light and heavy chain variable regions, and the binding affinity is significantly better than that of existing MUC1 antibodies, avoiding off-target effects.

[0027] 2. Low immunogenicity: The core binding region of rabbit-derived antibodies is transplanted into the human antibody framework through CDR transplantation technology. While retaining high binding activity, the immunogenicity in humans is greatly reduced, and immune rejection is decreased, making it suitable for long-term clinical application.

[0028] 3. Clear and wide range of applications: For pancreatic cancer, breast cancer, prostate cancer and gastric cancer with MUC1 overexpression, it can be treated through a variety of drug forms such as monotherapy, ADC, CAR-T, BiTEs, etc., providing new targeted therapy strategies for many types of solid tumors. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is an immunogenicity diagram of the experimental animals used in this invention.

[0031] Figure 2 This is a graph showing the binding activity of immune serum with normal / tumor pancreatic cells according to the present invention.

[0032] Figure 3 This is a graph showing the binding activity of the immune serum to other tumor cells according to the present invention.

[0033] Figure 4 This is a graph showing the binding activity of the rabbit-derived anti-human MUC1 monoclonal antibody of the present invention with cells.

[0034] Figure 5 This is a graph showing the binding activity of the humanized anti-human MUC1 monoclonal antibody of the present invention with cells. Detailed Implementation

[0035] The technical effects of the present invention are further illustrated below with reference to the accompanying drawings and specific embodiments. The embodiments given are for illustrative purposes only and do not constitute any limitation on the present invention. Any limited modifications made by any person within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0036] This embodiment provides a humanized anti-human MUC1 monoclonal antibody, including a light chain variable region and a heavy chain variable region;

[0037] The light chain variable region includes three complementarity-determining regions, whose amino acid sequences are as follows:

[0038] LCDR1 is QASQSVYNNNQLS, as shown in SEQ ID NO.1;

[0039] LCDR2 is YGSTLAS, as shown in SEQ ID NO.2;

[0040] LCDR3 is QGYYSGGIFA, as shown in SEQ ID NO.3;

[0041] The heavy chain variable region includes three complementarity-determining regions, whose amino acid sequences are as follows:

[0042] HCDR1 is SYAMG, as shown in SEQ ID NO.4;

[0043] HCDR2 is IIGSYGTTYYAAWAKG, as shown in SEQ ID NO.5;

[0044] HCDR3 is GGYPGYLSYFNI, as shown in SEQ ID NO.6.

[0045] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.11, and the corresponding base sequence is shown in SEQ ID NO.12; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.13, and the corresponding base sequence is shown in SEQ ID NO.14.

[0046] Among them, SEQ ID NO.11 is:

[0047] AQVLTQSPSSSLSASVGDRVTITCQASQSVYNNNQLSWYQQKPGKAPKLLIYYGSTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGYYSGGIFAFGGGTKVEIK.

[0048] SEQ ID NO.12 is:

[0049] gcccaagtgctgacacagagccctagcagcctgagcgctagcgtgggcgacagagtgaccatcacctgccaagctagccaaagcgtgtacaacaataatcagctgagctggtatcagcagaagcccggcaaggcccccaagctgctgatctactacggcagcacc ctggctagcggcgtgcctagcagattcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccaaggctactacagcggcggcatcttcgccttcggcgggggcaccaaggtggagatcaag.

[0050] SEQ ID NO.13 is:

[0051] ELQLQESGPGLVKPSETLSLTCTVSGGSISSYAMGWIRQPPGKGLEWIGIIGSYGTTYYAAWAKGRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARGGYPGYLSYFNIWGQGTLVTVSS.

[0052] SEQ ID NO.14 is:

[0053] Gagctgcagctgcaagagagcggccccggcctggtgaagcctagcgagaccctgagcctgacctgcaccgtgagcggcggcagcatcagcagctacgccatgggctggatcagacagccccccggcaagggcctggagtggatcggcatcatcggcagctacggcaccacctactacgcc gcctgggccaagggcagagtgaccatcagcgtggacacaagcaagaatcagttcagcctgaagctgagcagcgtgaccgccgccgacaccgccgtgtactactgcgctagaggcggctaccccggctacctgagctacttcaacatctggggccaaggcaccctggtgaccgtgagcagc.

[0054] Its preparation method includes the following steps:

[0055] S1. Select female New Zealand white rabbits aged 6-8 weeks and immunize them with MUC1 antigen in three separate doses:

[0056] First immunization (day 0): 200 μg of MUC1 antigen protein (dissolved in PBS) and Freund's complete adjuvant (1:1, v / v) emulsion were injected subcutaneously into the back at 5 injection sites;

[0057] Secondary immunization was performed two weeks (14 days) after the first immunization: 100 μg of an emulsion of MUC1 antigen protein and Freund's incomplete adjuvant (1:1, v / v) was injected subcutaneously into the back at four injection sites.

[0058] Three immunizations were administered, with a booster immunization three days before the end of immunization (day 25) by intraperitoneal injection of 50 μg of MUC1 antigen protein dissolved in PBS.

[0059] After immunization, 0.5 mL of blood was collected from the marginal ear vein of the rabbits, and the serum was separated by centrifugation for ELISA titer detection;

[0060] Add 100 μl of 1 μg / ml MUC1 protein to each well of the ELISA plate and incubate overnight at 4°C. Discard the supernatant, wash three times with 300 μl of PBST in each well, add 100 μl of 3% BSA, and block at room temperature for 1 hour. Dilute the collected serum initially at a ratio of 1:500, then perform serial dilutions of 2-fold. Add 100 μl of the diluted supernatant to each well and incubate at room temperature for 2 hours. Wash three times with PBST. Add 100 μl of anti-rabbit IgG-HRP antibody to each well and incubate at room temperature in the dark for 1 hour. Wash five times with PBST. Add 100 μl of LTMB chromogenic solution to each well and incubate in the dark for 8 minutes. Add 50 μl of stop solution to stop the chromogenic reaction. Read the OD450 value using an ELISA reader and plot a line graph as shown below. Figure 1 As shown, although the OD450 value of the serum gradually decreased with increasing dilution after immunization, it was still significantly higher than that of the negative control (NC), proving that the immunization was successful and that a large number of antibodies that specifically bind to MUC1 were produced in the serum.

[0061] Verification of the binding activity of immune serum with different tumor cells:

[0062] Pancreatic-related cells (including normal pancreatic cells HPNE, pancreatic cancer cells Capan-2, and pancreatic cancer cells HPAC) and other tumor cells (including gastric cancer cells MKN-45, colorectal cancer cells HCT-116, breast cancer cells MCF-7, and prostate cancer cells PC-3) were selected; each cell type was cultured to the logarithmic growth phase and the density was adjusted to 1×10⁻⁶. 5 Cells per tube; add 100 μL of immune serum (1:100 dilution) to each tube, incubate at 4°C for 30 minutes; wash once with PBS, centrifuge at 1200 rpm for 3 minutes, discard the supernatant; add 50 μL of anti-rabbit IgG-PE flow cytometry secondary antibody to each tube, incubate at 4°C in the dark for 30 minutes; after washing and centrifuging again, detect cell binding rate using flow cytometry, such as... Figure 2 , Figure 3 As shown, the binding rate of immune serum to normal pancreatic HPNE cells was only 1.14%, while the binding rate to pancreatic cancer cells Capan-2 reached 88.0% and the binding rate to HPAC reached 60.2%, proving that serum can specifically recognize pancreatic tumor cells and has no significant binding to normal pancreatic cells. Moreover, immune serum also showed high binding rates to MKN-45 (64.3%), HCT-116 (55.8%), MCF-7 (66.5%), and PC-3 (45.2%), indicating that serum has broad-spectrum binding activity against multiple types of MUC1-positive tumor cells.

[0063] S2. After immunization, B cells were cultured and identified to obtain positive clones:

[0064] After immunization, the animals were dissected, spleen tissue was removed and ground, cells were collected and filtered through a 200-mesh sieve and centrifuged, the cells were resuspended and red blood cell lysis buffer was added to remove red blood cells, and the resulting cells were used for subsequent sorting experiments.

[0065] Rabbit memory B cells were obtained by co-incubating with biotin-labeled MUC1 protein and sorting them. The cells were then placed in a 96 cell culture plate and co-incubated with feeder cells.

[0066] After two weeks of culture at 37°C and 5% CO2, the binding activity of the clonal supernatant was detected by ELISA, and the binding of the clonal supernatant to endogenously expressed Capan-2 cells was detected by FACS. Clones that showed positive results in both methods were considered as final positive clonal cells, and the cells were collected for subsequent experiments.

[0067] S3. Gene extraction and monoclonalization of positive clone cells were performed to select positive monoclonal antibodies:

[0068] Positive clones were selected for cell lysis, RNA was extracted using the Trizol method, and then reverse transcribed.

[0069] The variable regions of the light chain and heavy chain of the antibody were amplified by PCR amplification reaction, and constructed into the corresponding light chain and heavy chain expression vectors containing constant regions, respectively. Sequencing was performed and the sequencing results were analyzed.

[0070] Select the correctly sequenced light and heavy chain plasmids, and transfect the recombinant plasmids in pairs. 24 hours before transfection, HEK-293T cells were incubated in 24-well plates. The recombinant plasmids were transiently transfected using PEI transfection reagent. 48 hours later, the supernatant was collected, and the binding activity of the clonal supernatant was detected by ELISA. The binding of the supernatant to endogenous Capan-2 expression cells was detected by FACS. Positive monoclonal antibodies were selected.

[0071] The monoclonal antibody contains three complementarity-determining regions (CDRs) in both its light chain and heavy chain variable regions. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, while the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0072] S4. Expression and purification of positive monoclonal antibodies:

[0073] The density of 293F cells was adjusted to 5 × 10⁻⁶. 5Cells were incubated at 37°C and 120 rpm in a shaker containing 5% CO2 for 24 hours. 300 μg of DNA was mixed with PEI and allowed to stand for 20 minutes before being added to a 293F culture flask. The cells were incubated for another 72 hours. The cell supernatant was collected by centrifugation, and the antibody was purified using Protein A medium. The antibody concentration and purity were determined by measuring the absorbance using Nanodrop. The expression level and purity of the antibody were evaluated by Coomassie staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis to obtain rabbit-derived anti-human MUC1 monoclonal antibody.

[0074] The amino acid sequence of the light chain variable region of the rabbit-derived anti-human MUC1 monoclonal antibody is shown in SEQ ID NO.7, and its base sequence is shown in SEQ ID NO.8; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.9, and its base sequence is shown in SEQ ID NO.10.

[0075] Among them, SEQ ID NO.7 is:

[0076] AQVLTQTPSPVSAAVGGTVTINCQASQSVYNNNQLSWYQQKPGQPPKLLIYYGSTLASGVSSRFKGSRSGTQFTLTISGVQCDDAATYYCQGYYSGGIFAFGGGTEVVVK.

[0077] SEQ ID NO.8 is:

[0078] gcgcaagtgctgacccagactccatcccctgtgtctgcagctgtgggaggcacagtcaccatcaactgccaggccagtcagagtgtttataataacaaccaattatcctggtatcagcagaaaccagggcagcctcccaagctcctgatctattatggatccact ctggcatctggggtctcatcgcggttcaaaggcagtagatctgggacacagttcactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtcaaggctattatagtggtggtatttttgctttcggcggagggaccgaggtggtggtcaag.

[0079] SEQ ID NO.9 is:

[0080] QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIIGSYGTTYYAAWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGGYPGYLSYFNIWGPGTLVTVSS.

[0081] SEQ ID NO.10 is:

[0082] cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggaatcgacctcagtagttatgcaatgggctgggtccgccaggctccagggaaggggctggaatggatcggaatcattggtagttatggtaccacatactacgc ggcctgggcgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttcttgtgccagaggaggttatcctggttatctttcctactttaatatctggggcccaggcaccctcgtcaccgtctcttca.

[0083] Validation of cell-binding activity of rabbit-derived anti-human MUC1 monoclonal antibody:

[0084] Capan-2 cells (1×10⁻⁶) were collected. 5 Cells were divided into two groups (100 μL rabbit-derived anti-human MUC1 monoclonal antibody supernatant (concentration 1 μg / mL) and a negative control group (100 μL antibody-free cell culture supernatant). After incubation at 4°C for 30 minutes, followed by washing with PBS and centrifugation, 50 μL of anti-rabbit IgG-PE flow cytometry secondary antibody was added, and the cells were incubated at 4°C for 30 minutes. After washing and centrifugation, cell binding was detected by flow cytometry. Figure 4 As shown, the binding rate of the experimental group (Capan-2+Rb-MUC1) cells reached 96.8%, while the binding rate of the negative control group (Capan-2+negative control supernatant) was only 0.28%, demonstrating that the rabbit-derived anti-human MUC1 monoclonal antibody can bind to MUC1-positive Capan-2 cells efficiently and specifically.

[0085] S5. Humanization and binding activity assays with endogenous expression cells were performed on the purified rabbit-derived anti-human MUC1 monoclonal antibody to obtain the humanized anti-human MUC1 monoclonal antibody.

[0086] Specifically, 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 detected by flow cytometry to obtain a humanized anti-human MUC1 monoclonal antibody.

[0087] The amino acid sequence of the light chain variable region of the humanized anti-human MUC1 monoclonal antibody is shown in SEQ ID NO.11, and its base sequence is shown in SEQ ID NO.12; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.13, and its base sequence is shown in SEQ ID NO.14.

[0088] Validation of cell-binding activity of humanized anti-human MUC1 monoclonal antibody:

[0089] Capan-2 cells (1×10⁻⁶) were collected. 5 Cells were divided into two groups: the experimental group received 100 μL of humanized anti-human MUC1 monoclonal antibody supernatant (concentration 1 μg / mL); the negative control group received 100 μL of antibody-free cell culture supernatant. After incubation at 4°C for 30 minutes, the cells were washed once with PBS, centrifuged at 1200 rpm for 3 minutes, and 50 μL of Anti-Human IgG Fc-PE flow cytometry secondary antibody was added to each tube. The cells were incubated at 4°C for 30 minutes. After washing and centrifugation, cell binding was detected by flow cytometry. Figure 5 As shown, the binding rate of the experimental group (Capan-2+Hu-MUC1) cells reached 88.8%, while the binding rate of the negative control group (Capan-2+negative control supernatant) was only 0.12%, proving that humanization modification did not reduce the binding activity of the antibody to MUC1, and it can still bind to MUC1-positive Capan-2 cells efficiently and specifically.

[0090] Validation of in vitro antitumor activity of humanized anti-human MUC1 monoclonal antibody:

[0091] Capan-2 cells (pancreatic cancer) were adjusted to a density of 1×10⁻⁶. 4 Cells were seeded per well in a 96-well plate and incubated at 37°C with 5% CO2 for 24 hours. Humanized anti-human MUC1 monoclonal antibody was added to each well at final concentrations of 0.1 μg / mL, 1 μg / mL, and 10 μg / mL, respectively. A PBS control group (without antibody) was also included. Each group was divided into three replicates. After 48 hours of incubation, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours. The OD450 value was read using a microplate reader, and the cell viability was calculated as follows: Cell viability (%) = (OD450 value of experimental group - OD450 value of blank group) / (OD450 value of control group - OD450 value of blank group) × 100%.

[0092] The results showed that the cell viability rates of the 0.1 μg / mL, 1 μg / mL, and 10 μg / mL antibody groups were 82.3%, 56.7%, and 31.2%, respectively, while the viability rate of the PBS control group was 98.5%. This demonstrates that the humanized anti-human MUC1 monoclonal antibody can inhibit the proliferation of MUC1-positive tumor cells in a concentration-dependent manner. Figure 5 The "high binding activity" result shown demonstrates its clear anti-tumor activity, enabling the preparation of anti-tumor drugs for the clinical treatment of MUC1-overexpressing tumors such as pancreatic cancer.

[0093] This invention utilizes in vitro B cell culture technology, employs ELISA to detect antibody expression and flow cytometry to detect cell binding capacity, and uses a recombinant system for antibody expression and assembly via an expression vector to screen for a highly specific and highly affinity monoclonal antibody. After humanization, a humanized anti-human MUC1 monoclonal antibody is prepared. While retaining high affinity for the antigen, its immunogenicity is reduced, playing a crucial role in the immunotherapy of human tumors and offering broad applications in anti-tumor drugs, chimeric antigen receptor T (CAR-T) cell therapy, and other fields.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the present invention.

Claims

1. A humanized anti-human MUC1 monoclonal antibody, characterized in that: Includes variable regions for light chains and variable regions for heavy chains; The light chain variable region includes three complementarity-determining regions, whose amino acid sequences are as follows: LCDR1 is QASQSVYNNNQLS, as shown in SEQ ID NO.1; LCDR2 is YGSTLAS, as shown in SEQ ID NO.2; LCDR3 is QGYYSGGIFA, as shown in SEQ ID NO.3; The heavy chain variable region includes three complementarity-determining regions, whose amino acid sequences are as follows: HCDR1 is SYAMG, as shown in SEQ ID NO.4; HCDR2 is IIGSYGTTYYAAWAKG, as shown in SEQ ID NO.5; HCDR3 is GGYPGYLSYFNI, as shown in SEQ ID NO.

6.

2. The humanized anti-human MUC1 monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

13.

3. The humanized anti-human MUC1 monoclonal antibody according to claim 2, characterized in that: The base sequence encoding the light chain variable region is shown in SEQ ID NO.12; the base sequence encoding the heavy chain variable region is shown in SEQ ID NO.

14.

4. The use of the humanized anti-human MUC1 monoclonal antibody according to any one of claims 1-3 in the preparation of antitumor drugs, wherein the tumor is pancreatic cancer, breast cancer, prostate cancer or gastric cancer.

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