GPA33 recombinant monoclonal antibody and application thereof

Abstract

The invention discloses a GPA33 recombinant monoclonal antibody and application thereof. The GPA33 recombinant monoclonal antibody is an antibody A or an antibody B or an antibody C or an antibody D, the amino acid sequence of the light chain variable region of the antibody A is as shown in SEQ ID No: 1, and the amino acid sequence of the heavy chain variable region of the antibody A is as shown in SEQ ID No: 2; the amino acid sequence of the light chain variable region of the antibody B is as shown in SEQ ID No: 5, and the amino acid sequence of the heavy chain variable region of the antibody B is as shown in SEQ ID No: 6; the amino acid sequence of the light chain variable region of the antibody C is as shown in SEQ ID No: 9, and the amino acid sequence of the heavy chain variable region of the antibody C is as shown in SEQ ID No: 10; the amino acid sequence of the light chain variable region of the antibody D is shown as SEQ ID No: 13, and the amino acid sequence of the heavy chain variable region of the antibody D is shown as SEQ ID No: 14. The antibody provided by the invention can recognize and specifically bind to GPA33 protein on the surface of cancer cells in a targeting manner.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of antibody bioengineering, and particularly relates to a GPA33 recombinant monoclonal antibody or fragment thereof, a DNA molecule, a vector, application and a cancer treatment drug. BACKGROUND

[0002] Colorectal cancer is a malignant tumor of the digestive tract that seriously threatens human health, and its global incidence and mortality rates are among the highest. Current drug treatment for colorectal cancer is based on fluorouracil, oxaliplatin, irinotecan and other chemotherapeutic drugs, and a combined drug regimen (such as FOLFOX, FOLFIRI) is often used to improve efficacy, but its significant toxic side effects seriously affect the quality of life and treatment compliance of patients. In the field of targeted therapy, drugs approved for colorectal cancer mainly include pathway inhibitors targeting VEGF (such as bevacizumab) and inhibitors targeting EGFR (such as cetuximab, suitable for RAS wild-type patients). However, EGFR-targeted therapy is only effective for a portion of patients and is prone to secondary drug resistance; and the efficacy of anti-angiogenic drugs has a limited improvement range. Although immune checkpoint inhibitors (such as anti-PD-1 drugs) have brought hope to a small number of patients with high microsatellite instability (MSI-H), their efficacy is very limited in the vast majority of microsatellite stable (MSS) colorectal cancer. Therefore, exploring new therapeutic targets and developing effective therapies for a wider population of colorectal cancer patients is an urgent need in current clinical practice.

[0003] In recent years, adoptive cell immunotherapy represented by chimeric antigen receptor T cell therapy has made breakthrough progress in the treatment of hematological tumors, but still faces many challenges in the field of solid tumors, including immune suppression of the tumor microenvironment, lack of high specificity and uniform tumor targets, and difficulty for CAR-T cells to effectively infiltrate tumor tissues. Macrophages, as key effector cells of the innate immune system, play a complex role in the tumor microenvironment. Compared with T cells, macrophages have natural tumor infiltration ability, strong antigen presentation function and phagocytic ability to remove foreign substances. Chimeric antigen receptor macrophages are genetically engineered to express chimeric antigen receptors that can specifically recognize tumor surface antigens, thereby "reprogramming" macrophages into powerful weapons targeting tumor killing. CAR-M therapy is expected to overcome the bottleneck of CAR-T in the treatment of solid tumors and has great application potential. However, the success of CAR-M therapy is highly dependent on the selection of ideal tumor-specific targets.

[0004] GPA33 (Glycoprotein A33) is a cell surface antigen belonging to the immunoglobulin superfamily. It is a highly glycosylated type I membrane protein that is specifically and highly expressed in normal intestinal epithelial cells and up to 95% of colorectal cancer tissues, while its expression level is extremely low or absent in most other normal tissues. This unique expression pattern makes GPA33 a highly attractive target for targeted therapy in colorectal cancer. GPA33 protein is involved in intercellular adhesion and signal transduction, and its high expression may be associated with the progression and poor prognosis of colorectal cancer. GPA33 has been demonstrated as a potential diagnostic and therapeutic target in numerous colorectal cancer-related studies. Early immunohistochemical studies confirmed the widespread and specific expression of GPA33 in colorectal cancer tissues.

[0005] Currently, although some studies have explored the possibility of antibody drugs targeting GPA33, there is still a gap in the development of novel colorectal cancer treatments by combining this highly effective target with chimeric antigen receptor macrophages, which possess strong tumor infiltration and phagocytic functions. Existing technologies lack a mature protocol for effectively utilizing the antigen-binding region of GPA33 antibodies to construct CAR-M inhibitors specifically targeting colorectal cancer and verifying their highly effective anti-tumor activity. Therefore, there is an urgent need in this field to develop a novel monoclonal antibody based on the GPA33 target. Summary of the Invention

[0006] The purpose of this invention is to provide a novel high-affinity anti-GPA33 monoclonal antibody with excellent performance, so as to overcome the shortcomings of the prior art and meet unmet clinical needs.

[0007] The first objective of this invention is to provide a GPA33 recombinant monoclonal antibody or a fragment thereof, wherein the recombinant monoclonal antibody is antibody A, antibody B, antibody C, or antibody D;

[0008] The amino acid sequence of the light chain variable region of antibody A is shown in SEQ ID No:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:2;

[0009] The amino acid sequence of the light chain variable region of antibody B is shown in SEQ ID No:5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:6.

[0010] The amino acid sequence of the light chain variable region of antibody C is shown in SEQ ID No:9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:10.

[0011] The amino acid sequence of the light chain variable region of antibody D is shown in SEQ ID No:13, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:14.

[0012] Preferably, the nucleotide sequence of the light chain variable region of antibody A is shown in SEQ ID No:3, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:4.

[0013] Preferably, the nucleotide sequence of the light chain variable region of antibody B is shown in SEQ ID No:7, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:8.

[0014] Preferably, the nucleotide sequence of the light chain variable region of antibody C is shown in SEQ ID No:11, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:12.

[0015] Preferably, the nucleotide sequence of the light chain variable region of antibody D is shown in SEQ ID No:15, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:16.

[0016] The second objective of this invention is to provide a method for preparing a GPA33 recombinant monoclonal antibody or a fragment thereof as described in any of the above claims, based on plasma cell separation, single B cell sorting, and single-cell PCR technology.

[0017] A third object of the present invention is to provide a DNA molecule encoding a GPA33 recombinant monoclonal antibody or a fragment thereof as described in any of the preceding claims.

[0018] A fourth object of the present invention is to provide a carrier comprising a DNA molecule as described above.

[0019] The present invention further provides the use of the antibody or fragment thereof described in any of the above claims in the preparation of a cancer treatment drug, wherein the cancer is a cancer associated with high expression of GPA33;

[0020] And / or, the cancers include colorectal cancer, stomach cancer, pancreatic cancer, bile duct cancer, lung cancer, and liver cancer.

[0021] The present invention also provides a medicament for treating cancer, comprising an antibody or a fragment thereof as described in any of the preceding claims.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] 1. The recombinant monoclonal antibody of this invention can recognize and specifically target the GPA33 protein on the surface of cancer cells, providing a precise targeting device for subsequent cell immunotherapy regimens. It has significant meaning and value in disease diagnosis, immunotherapy design, antibody drug design, and research.

[0024] 2. This invention obtains GPA33 monoclonal antibodies, particularly the DNA and amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL), through single-cell PCR technology. These sequences determine the specificity and targeting of the monoclonal antibody, enabling it to effectively recognize the GPA33 antigen on colorectal cancer cell lines without the risk of off-target binding.

[0025] 3. The plasma cell separation and single B cell sorting technologies used in this invention can efficiently and faithfully separate individual B cells that produce natural, high-affinity antibodies, perfectly preserving the natural pairings selected in vivo. This ensures that the final antibody has the same high affinity and specificity as when it is in vivo, greatly improving the success rate of obtaining high-quality candidate antibodies. This technology can capture all antibody types produced in vivo, including rare subtypes or unstable clones that are difficult to obtain using traditional hybridoma techniques. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0027] Figure 1 The image shows the SDS-PAGE electrophoresis result of the purified antigen protein in Example 1.

[0028] Figure 2 This is a statistical graph showing the serum antibody titers of mice after immunization in Example 1.

[0029] Figure 3 This is a statistical chart showing the proportion of CD138 positive cells before and after sorting with CD138 magnetic beads in Example 1.

[0030] Figure 4 This is a visualization of a single positive B cell in Example 1.

[0031] Figure 5 The image shows the effect of sorting a single positive B cell before and after in Example 1.

[0032] Figure 6 The results are obtained by nucleic acid electrophoresis of the antibody light and heavy chain sequence products amplified by PCR in Example 1.

[0033] Figure 7 The results are obtained by SDS-PAGE electrophoresis of the reductive and non-reductive properties of the purified recombinant monoclonal antibody in Example 2.

[0034] Figure 8 The image shows the SEC-HPLC detection results of the purified recombinant monoclonal antibody A33-mh1 in Example 2.

[0035] Figure 9 The image shows the SEC-HPLC detection results of the purified recombinant monoclonal antibody A33-mh6 in Example 2.

[0036] Figure 10 The image shows the SEC-HPLC detection results of the purified recombinant monoclonal antibody A33-mh7 in Example 2.

[0037] Figure 11 The image shows the SEC-HPLC detection results of the purified recombinant monoclonal antibody A33-mh8 in Example 2.

[0038] Figure 12 This is a graph showing the antigen-binding ELISA results of the purified recombinant monoclonal antibody in Example 2.

[0039] Figure 13 This is a flow cytometry result of the binding affinity of the purified recombinant monoclonal antibody A33-mh1 to LS174T colon cancer cells under gradient dilution conditions, as shown in Example 3.

[0040] Figure 14 This is a flow cytometry result of the binding affinity of the purified recombinant monoclonal antibody A33-mh6 to LS174T colon cancer cells under gradient dilution conditions, as shown in Example 3.

[0041] Figure 15 This is a flow cytometry result of the binding affinity of the purified recombinant monoclonal antibody A33-mh7 to LS174T colon cancer cells under gradient dilution conditions, as shown in Example 3.

[0042] Figure 16 This is a flow cytometry result of the binding affinity of the purified recombinant monoclonal antibody A33-mh8 to LS174T colon cancer cells under gradient dilution conditions, as shown in Example 3.

[0043] Figure 17 This is an immunofluorescence diagram showing the specific binding of the purified recombinant monoclonal antibody to LS174T colon cancer cells in Example 3.

[0044] Figure 18This is a schematic diagram of immunofluorescence showing the specific binding of the purified recombinant monoclonal antibody to colon cancer tissue in Example 3. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0046] This invention provides a recombinant monoclonal antibody for GPA33. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve its implementation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0047] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0048] An antibody is a protein composed of one or more polypeptides that specifically bind to antigens. One form of antibody constitutes its basic structural unit. This form is a tetramer, which consists of two pairs of identical antibody chains, each pair containing a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains work together to bind the antigen, while the constant regions are responsible for the antibody's effector function.

[0049] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0050] The present invention will be further illustrated below with reference to the embodiments:

[0051] Example 1:

[0052] This embodiment provides a method for preparing a recombinant monoclonal antibody against GPA33, obtaining the amino acid and nucleotide sequences of the variable region of the monoclonal antibody targeting GPA33.

[0053] The specific steps are as follows:

[0054] 1. First, the extracellular domain (ECD) amino acid sequence (amino acids 22-235) of human GPA33 protein was selected, and a sequence encoding a 6×His tag was fused to its N-terminus. The recombinant gene was obtained through gene synthesis and inserted into the vector plasmid pTT5. Finally, positive clones containing the inserted fragment were screened and verified by sequencing to obtain expression plasmids containing the correct gene sequence. These plasmids were then expanded and cultured, and the amplified expression plasmids were extracted.

[0055] 2. The expression plasmid was transformed into HEK-293F cells for protein expression, and the recombinant His-GPA33 protein was purified by nickel column affinity chromatography to obtain high purity. The purified antigen was analyzed by SDS-PAGE electrophoresis as follows: Figure 1 .

[0056] 3. The purified protein was used as an antigen to immunize three 6-week-old female BALB / c mice.

[0057] The immunization protocol was as follows: Each mouse was subcutaneously injected with an emulsion mixture of 50 μg antigen and Freund's complete adjuvant (for primary immunization) or incomplete adjuvant (for booster immunization), at four injection sites per injection, for a total of four immunizations, with each injection spaced two weeks apart. One week after the last immunization, serum was collected from the mice via orbital blood collection.

[0058] 4. The titer of anti-GPA33 antibodies in serum was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0059] (1) Antigen coating: Dilute the antigen to 1.5 µg / ml with coating solution, add 100 µL / well to a polystyrene 96-well reaction plate, and incubate overnight at 4°C;

[0060] (2) Washing: Discard the liquid in the hole the next day and wash 3 times with washing solution;

[0061] (3) Sealing: Add 200 μL / well sealing solution and let stand at room temperature for 1 hour;

[0062] (4) Washing: Wash 3 times with detergent;

[0063] (5) Add test sample (primary antibody): Add test serum (take blood and incubate overnight at 4°C, then centrifuge at 4000 r / min for 10 min to obtain supernatant), and serially dilute the serum at 1:5000, 1:10000, 1:20000, 1:50000, and 1:100000 (pre-immunization serum is used as a negative control), 100 µl per well, and incubate at room temperature for 1 h;

[0064] (6) Washing: Wash 3 times with detergent;

[0065] (7) Add enzyme-labeled anti-antibody: Add HRP-labeled anti-mouse IgG secondary antibody, 100µl / well, and incubate at 37℃ for 30min;

[0066] (8) Washing: Wash 3 times with detergent;

[0067] (9) Color development: Add 100µl of freshly prepared substrate solution per well and place in the dark at room temperature for 5–30 min;

[0068] (10) Termination of reaction and colorimetric analysis: Add 50 µL of stop solution per well. The color turns yellow; measure the absorbance of each well at 450 nm using a microplate reader. Figure 2 As shown.

[0069] 5. After confirming successful immunization, select mice with the highest immunization titer, sacrifice them, and aseptically remove their spleens. Prepare a single-cell suspension of the spleen by mechanical grinding. Subsequently, use CD138+ plasma cell sorting magnetic beads to positively sort the CD138+ plasma cell population from the spleen cell suspension.

[0070] The specific steps are as follows:

[0071] (1) Tissue disruption: Place the spleen in cold PBS (containing 2% FBS) and gently grind it into a single-cell suspension using a syringe plunger.

[0072] (2) Filter with a 70μm nylon filter to remove tissue debris and cell clumps.

[0073] (3) Centrifugation: Centrifuge at 300 × g for 10 minutes and discard the supernatant.

[0074] (4) Resuspend the cells in cold, recommended buffer solution and adjust the concentration to 1 × 10⁻⁶. 8 Cells / mL, store on ice for later use.

[0075] (5) Using CD138 positive cell sorting magnetic beads, plasma cells that can express antibodies were isolated from spleen cells.

[0076] 6. The CD138 positivity rate of cells before and after sorting was determined using flow cytometry:

[0077] Resuspend cells in 100 µl of cell staining buffer, add 1.25 µl of PE-labeled CD138 antibody, and incubate on ice in the dark for 15–20 minutes. Wash twice with at least 2 mL of cell staining buffer, centrifuge at 350 x g for 5 minutes, resuspend the cell pellet in 0.5 mL of cell staining buffer, and add 0.5 µl of 7-AAD activity staining solution to remove dead cells. Perform flow cytometry analysis. The proportion of CD138-positive spleen cells before and after sorting was statistically analyzed. The results are shown below. Figure 3As shown, from Figure 3 The results show that the CD138 positivity rate of the sorted spleen cells reached over 90%.

[0078] 7. To obtain monoclonal B cells secreting GPA33-targeting antibodies from the plasma cells obtained in step 5, we used Sartorius' CellCelector fully automated cell sorting system for single-cell isolation. Using the CellCelector's fluorescence microscopy system, we automatically identified and visualized the single B cells secreting specific antibodies, directly sorted them into 96-well PCR plates containing cell lysis buffer, and immediately used them for subsequent reverse transcription.

[0079] The specific steps are as follows:

[0080] (1) Nanopore pre-wetting.

[0081] (2) Add 0.5 mL of GPA33 antigen (filtered for sterilization), with a concentration of 50 µg / mL, and incubate overnight at 4°C.

[0082] (3) Use 2% BSA to seal the orifice plate.

[0083] (4) Prepare 1 mL of single-cell suspension per well, and mix the following substances: 15,000 plasma cells and dye-bound secondary antibody (concentration 7.5 µg / mL).

[0084] (5) Incubate overnight at 37°C and 5% CO2.

[0085] (6) The machine automatically scans and detects secretory cells (Hits) to confirm positive results using single-cell analysis. The results of selecting a single positive B cell are shown below. Figure 4 As shown.

[0086] (7) Hits were exported from the nanopores to a 96-well PCR plate for cDNA synthesis. The results before and after sorting are as follows: Figure 5 As shown, the sorting results indicate that the selected positive individual B cells have been successfully isolated.

[0087] 8. Synthesize single-cell cDNA using single-cell PCR technology.

[0088] The sorted individual B cells underwent mRNA reverse transcription and cDNA pre-amplification. The specific steps were as follows:

[0089] (1) Lysis / hybridization reaction: Prepare lysis mixture: 10X lysis buffer: 1 µL; capture oligonucleotide: 1 µL; RNase inhibitor: 0.4 µL; nuclease-free water to bring the total reaction volume to 10 µL. Incubate at 72°C for 1 minute.

[0090] (2) Reverse transcription (RT) reaction: Add 10 µL of RT reaction mixture to the lysed sample: nuclease-free water: 4 µL; 4X cDNA synthesis premix: 5 µL; template switching oligonucleotide: 1 µL. Thermal cycling program: 50°C for 30 minutes, 85°C for 5 minutes. At this point, cDNA from a single B cell has been obtained.

[0091] (3) Pre-amplified cDNA: Add 80 µL of pre-amplification mixture: 29 µL of nuclease-free water; 50 µL of 2X pre-amplification mixture; 1 µL of pre-amplification primers. Thermal cycling program: Initial denaturation: 98°C for 30 seconds; 21 cycles (denaturation / annealing / extension): 98°C for 10 seconds → 65°C for 10 seconds → 67°C for 3 minutes; Final extension: 67°C for 5 minutes.

[0092] 9. Using the pre-amplified products as templates, nested PCR was employed to amplify the variable regions of the antibody light and heavy chains. A mixed primer set of mouse IgG VH and IgG Vκ primers was used. All PCR reactions were performed using high-fidelity DNA polymerase to ensure amplification fidelity. PCR conditions: IgH chain: Round 1 (95°C 3 min → 35 cycles: 95°C 15 s, 56°C 15 s, 72°C 55 s); Round 2 annealing temperature 60°C. Finally, incubation at 72°C for 5 minutes. Igκ chain: Round 1 (95°C 3 min → 35 cycles: 94°C 15 s, 50°C 15 s, 72°C 55 s); Round 2 annealing temperature 45°C. Finally, incubation at 72°C for 5 minutes.

[0093] 10. The PCR products were verified by agarose gel electrophoresis. The electrophoresis results are as follows: Figure 6 As shown. Products with an electrophoresis range of 350bp-450bp are considered correct. Correct product bands are purified by gel extraction and then subjected to Sanger sequencing.

[0094] 11. Sequencing results:

[0095] (1) Antibody A:

[0096] The amino acid sequence of the V region of the A33-mh1 light chain is shown in SEQ ID No:1:

[0097] DIVLTQSPASLAVSLGQRATISCRASKSVSKSGYSYMHWYRQKPGQPPKLLIFLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAAIYYCQHSRELPRTFGGGTKLEIK

[0098] The amino acid sequence of the V region of the A33-mh1 heavy chain is shown in SEQ ID No: 2:

[0099] EVQLQESGPGLVAPSQSLSITCTVSGFSLTNYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAYGSSYGYFDYWGQGTTLTVSS

[0100] The nucleotide sequence of the V region of the A33-mh1 light chain is shown in SEQ ID No: 3: GACATTGTGCTCACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGACAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTAAATCTGGCTATAGTTATATGCACTGGTACCGACAGAAACCAGGACAGCCACCCAAACTCCTCATCTTTCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAATCTATTACTGTCAGCACAGTAGGGAGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0101] The nucleotide sequence of the V region of the A33-mh1 heavy chain is shown in SEQ ID No: 4:

[0102] GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAACTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAATTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCTACGGTAGTAGCTACGGGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0103] (2) Antibody B:

[0104] The amino acid sequence of the V region of the A33-mh6 light chain is shown in SEQ ID No:5:

[0105] DVVVTQTPLSLPVSFGDQVSISCRSSQSLANSYGNTYLSWYLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK

[0106] The amino acid sequence of the V region of the A33-mh6 heavy chain is shown in SEQ ID No:6:

[0107] EVQLQESGGGLVKPGGSLKLSCAASGFTFTTYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGDYGITYLAWFAYWGQGTLVTVSA

[0108] The nucleotide sequence of region V of the A33-mh6 light chain is shown in SEQ ID No:7:

[0109] GATGTTGTGGTGACTCAAACTCCACTCTCCCTGCCTGTCAGCTTTGGAGATCAAGTTTCTATCTCTTGCAGGTCTAGTCAGAGTCTTGCAAACAGTTATGGGAACACCTATTTGTCTTGGTACCTGCACAAGCCTGGCCAGTCTCCACAGCTCCTCATCTATGGGATT TCCAACAGATTTTCTGGGGTGCCAGACAGGTTCAGTGGCAGTGGTTCAGGGACAGATTTCACACTCAAGATCAGCACAATAAAGCCTGAGGACTTGGGAATGTATTACTGCTTACAAGGTACACATCAGCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0110] The nucleotide sequence of region V of the A33-mh6 heavy chain is shown in SEQ ID No:8:

[0111] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGGTTCACTTTCACTACCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAG ACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGGCGACTACGGTATTACCTACCTGGCCTGGTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0112] (3) Antibody C

[0113] The amino acid sequence of the V region of the A33-mh7 light chain is shown in SEQ ID No:9:

[0114] DIVLTQTPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEIPWTFGGGTKLEIK

[0115] The amino acid sequence of the V region of the A33-mh7 heavy chain is shown in SEQ ID No:10:

[0116] EVQLQESGPGLVKPSQSLSLTCTVTGYSFPSDYAWNWIRQFPGNKLEWMGYISYSGRTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARSGGNYVYWGQGTTLTVSS

[0117] The nucleotide sequence of the V region of the A33-mh7 light chain is shown in SEQ ID No:11:

[0118] GACATTGTGCTCACACAGACTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGACACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGATTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0119] The nucleotide sequence of the V region of the A33-mh7 heavy chain is shown in SEQ ID No:12:

[0120] GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCATTCCCCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATTAGTTACAGTGGTAGAACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATCGGGCGGTAACTACGTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0121] (4) Antibody D:

[0122] The amino acid sequence of the V region of the A33-mh8 light chain is shown in SEQ ID No:13:

[0123] DIVMTQTPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLLTFGAGTKLELK

[0124] The amino acid sequence of the V region of the A33-mh8 heavy chain is shown in SEQ ID No:14:

[0125] EVQLQESGAELVRPGASVTLSKASGYTFTDSEIHWVKQTPVHGLEWIGANDPETGGTASNQKFKGKATLTVDKYSSTAYMELRSLTSEDSAVYYCSRGPHVGIVYWGQGTLVTVSA

[0126] The nucleotide sequence of the V region of the A33-mh8 light chain is shown in SEQ ID No:15:

[0127] GACATTTGTGATGACACAGACTCCGTCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTTTGAGCTGCAAATCCAGTCAGAGTCTGCTCACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAATTGTTGATCTACTGG GCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGCAAGCAATCTTATAATCTGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0128] The nucleotide sequence of region V of the A33-mh8 heavy chain is shown in SEQ ID No:16:

[0129] GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTCTGAAATACACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCTAATGATCCTGAAACTGGTGGTACTGC CTCCAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACAAATATTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTTCAAGAGGTCCCCACGTGGGTATTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0130] Example 2: Batch-stable recombinant monoclonal antibodies were prepared based on the above sequencing results.

[0131] 1. Synthesize recombinant monoclonal antibodies using a mammalian expression system.

[0132] Based on the above sequencing results, the recombinant chimeric antibody was expressed. The specific steps are as follows:

[0133] (1) Insert the variable region sequence of the antibody into a human IgG antibody expression vector containing the constant region sequence of all human IgG1 antibodies except for the V region to construct a recombinant antibody expression vector.

[0134] (2) The paired light and heavy chain expression vectors were co-transfected into HEK293F cells for expression. Three days after transfection, the supernatant was collected and replaced with 25 ml of fresh DMEM medium supplemented with 1% Nutridoma-SP. The supernatant was collected again on the sixth day after transfection.

[0135] (3) The culture supernatant was centrifuged at 800×g for 10 minutes to remove cell debris and stored at 4°C. The recombinant chimeric antibody was purified by incubating with 25 µl Protein G beads overnight at 4°C using 25 ml of cell culture supernatant. After centrifugation, the supernatant was removed, the beads were resuspended in PBS, and transferred to a chromatographic column equilibrated with PBS. After two rounds of washing with 1 ml PBS, the antibody was eluted in 0.1 M glycine (pH 3.0).

[0136] (4) The purified antibody was subjected to reducing and non-reducing SDS-PAGE electrophoresis, and the results are as follows: Figure 7As shown in the figure, the detection results indicate that the purified recombinant monoclonal antibody has high purity and its molecular weight is consistent with the prediction.

[0137] (5) The purified recombinant monoclonal antibody was analyzed by SEC-HPLC, and the results are as follows: Figure 8 , 9 As shown in Figures 10 and 11, the purified recombinant monoclonal antibody monomer content is all above 95%, indicating batch stability.

[0138] 2. Antibody affinity detection

[0139] The binding affinity of each recombinant antibody to the antigen was determined using an indirect ELISA method. The recombinant His-GPA33 antigen was diluted to 1 μg / mL with coating buffer, and 100 μL / well was added to a 96-well plate for overnight coating at 4°C. The purified antibody was then serially diluted (starting from 20 nM and performing 3-fold serial dilutions) and added to the corresponding wells for incubation. Results are shown below. Figure 12 .Depend on Figure 12 It can be seen that antibodies A33-mh1, A33-mh7, and A33-mh8 exhibited better affinity, while A33-mh6 also had a certain affinity.

[0140] Example 3: Application of recombinant monoclonal antibodies

[0141] 1. The inventors used flow cytometry to detect the binding ability of the obtained antibody to LS174T colon cancer cells expressing GPA33. One million LS174T cells were incubated with the recombinant antibody at the corresponding dilution concentration at 4°C for one hour, and then Goat anti-Human IgG Fc antibody was used as the secondary antibody for flow cytometry detection. The detection results are as follows: Figure 13 , Figure 14 , Figure 15 , Figure 16 As shown in the figures above, antibody A33-mh1 exhibits the strongest binding ability to cells expressing GPA33, displaying the strongest average fluorescence intensity at the same antibody incubation concentration. A33-mh7 and A33-mh8 also show good binding ability.

[0142] 2. The inventors used live-cell immunofluorescence staining technology to study the binding ability of the prepared recombinant monoclonal antibody to LS174T colon cancer cells. The results are as follows: Figure 17 As shown, the prepared recombinant monoclonal antibodies (such as A33-mh1 and A33-mh8) can effectively and specifically recognize and label live tumor cells.

[0143] 3. The inventors used frozen section immunofluorescence technology to study the binding ability of the prepared recombinant monoclonal antibody to colon cancer tissue. The results are as follows:Figure 18 As shown, the prepared recombinant monoclonal antibody can effectively and specifically recognize tumor sites, providing a foundation for the subsequent development of immune cell-targeted therapies.

[0144] The GPA33 recombinant monoclonal antibody provided by this invention is obtained by combining plasma cell separation technology, visual single B cell sorting technology and single cell PCR technology. It is a recombinant monoclonal antibody that can specifically bind to the GPA33 antigen on the surface of cancer cells. This antibody has high affinity, high specificity, batch stability and high yield, and can provide a precise positioning device for subsequent cell immunotherapy.

[0145] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A GPA33 recombinant monoclonal antibody or a fragment thereof, characterized in that, It may be antibody A, antibody B, antibody C, or antibody D; The amino acid sequence of the light chain variable region of antibody A is shown in SEQ ID No:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:2; The amino acid sequence of the light chain variable region of antibody B is shown in SEQ ID No:5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:

6. The amino acid sequence of the light chain variable region of antibody C is shown in SEQ ID No:9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:

10. The amino acid sequence of the light chain variable region of antibody D is shown in SEQ ID No:13, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:

14.

2. The GPA33 recombinant monoclonal antibody or a fragment thereof according to claim 1, characterized in that, The nucleotide sequence of the light chain variable region of antibody A is shown in SEQ ID No:3, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:

4.

3. A GPA33 recombinant monoclonal antibody or a fragment thereof according to claim 1, characterized in that, The nucleotide sequence of the light chain variable region of antibody B is shown in SEQ ID No:7, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:

8.

4. A GPA33 recombinant monoclonal antibody or a fragment thereof according to claim 1, characterized in that, The nucleotide sequence of the light chain variable region of antibody C is shown in SEQ ID No:11, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:

12.

5. A GPA33 recombinant monoclonal antibody or a fragment thereof according to claim 1, characterized in that, The nucleotide sequence of the light chain variable region of antibody D is shown in SEQ ID No:15, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:

16.

6. A method for preparing a GPA33 recombinant monoclonal antibody or a fragment thereof according to any one of claims 1 to 5, characterized in that, It was prepared based on plasma cell isolation, single B cell sorting and single-cell PCR technology.

7. A DNA molecule encoding a GPA33 recombinant monoclonal antibody or a fragment thereof as described in any one of claims 1 to 5.

8. A vector comprising a DNA molecule as described in claim 7.

9. The use of the antibody or fragment thereof according to any one of claims 1 to 5 in the preparation of a cancer treatment drug, wherein the cancer is a cancer related to high expression of GPA33; And / or, the cancers include colorectal cancer, stomach cancer, pancreatic cancer, bile duct cancer, lung cancer, and liver cancer.

10. A drug for treating cancer, characterized in that, Includes the antibody or fragment thereof as described in any one of claims 1 to 5.

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