B-cell immortalization-based method for developing single b-cell antibody and use thereof

Abstract

Provided are a B-cell immortalization-based method for developing a single B-cell antibody and the use thereof. The provided method comprises the following steps: S1, antigen preparation; S2, antigen immunization; S3, modification of a lentiviral vector; S4, viral infection and cell sorting; S5, screening for cells with a high binding activity; S6, sequencing of an antibody sequence; S7, activity verification of a target antibody, etc. The provided antibody development method effectively solves the problems that single B-cell antibody development requires extensive recombinant expression and incurs a high verification cost. By combining single B-cell technology with B-cell immortalization technology, a B cell is infected by using a modified lentivirus, so that the cell achieves immortalization and enables in vitro proliferation and antibody secretion. Furthermore, by combining high-throughput microfluidic screening with cell immortalization technology, cell infection is performed directly in an oil droplet, thereby achieving high-throughput and low-cost antibody development.

Description

A method for developing single B-cell antibodies based on B-cell immortalization and its application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for developing single B-cell antibodies based on B-cell immortalization and its application. Background Technology

[0002] After an animal is immunized with an antigen, the body produces specific antibodies against that antigen. The purpose of antibody screening is to select specific antibodies from these antibodies. Traditionally, hybridoma monoclonal antibody screening technology has been widely used. This technology achieves the immortalization of B cells and antibody screening by fusing myeloma cells and B lymphocytes. The fused hybridoma cells can proliferate in vitro and secrete antibodies, which can be spread in a 96-well plate to screen for monoclonal antibodies. However, hybridoma technology is limited by fusion efficiency and screening throughput, and has significant limitations in screening for more challenging proteins, thus reducing the success rate of antibody screening.

[0003] Another technique is phage display, which uses PCR to amplify the variable region of an antibody and display it on the surface of a phage. Through several rounds of binding and elution, specifically bound phages can be enriched. Subsequently, positive monoclonal phages are selected for sequencing to obtain the antibody sequence. However, antibody sequences screened by phage display technology often exhibit non-natural pairing of light and heavy chains, which may affect subsequent drug development.

[0004] To overcome these limitations, single-B-cell technology has emerged. This technology directly screens antibodies from B cells generated after immunization. After identifying positive B cells, high-throughput sequencing is used to obtain positive antibody sequences. These antibody sequences are then recombinantly expressed through genetic engineering to further screen for active antibody sequences. Single-B-cell technology has the advantage of high screening throughput, but the subsequent expression validation cost is high. Therefore, providing a method that maintains the advantages of high-throughput screening while reducing the cost of subsequent expression validation is of great significance for the fields of antibody screening and drug development. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for developing single B-cell antibodies based on B-cell immortalization and its application. This invention combines high-throughput single B-cell technology, B-cell immortalization technology, and single B-cell microfluidic technology to achieve initial enrichment of B cells and subsequent high-throughput antibody activity screening. It solves the problems of high expression validation costs and low coverage after NGS sequencing associated with single B-cell technology, making it a highly efficient single B-cell antibody discovery technology.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first objective of this invention is to provide a method for developing single B-cell antibodies based on B-cell immortalization, which achieves initial enrichment of cells through single B-cell microfluidic technology, followed by further screening using B-cell immortalization technology, including the following steps:

[0008] S1. Preparation of antigen

[0009] Based on the target antigen protein sequence and gene sequence information, the antigen is expressed and fused with human Fc-tag or His tag at its C-terminus. After expression and purification, it is used for subsequent mouse immunization.

[0010] Fluorescent labeling of target antigen proteins for microfluidic screening;

[0011] S2, Antigen Immunization

[0012] Immunizing animals with the target antigen induces the production of specific B cells against the antigen, which in turn produce antibody-secreting plasma cells.

[0013] S3, Modification of Lentiviral Vectors

[0014] By fusing receptor antibodies on the surface of B cells with VSV-G protein and packaging them onto the surface of lentiviruses, and by packaging BCL and BCL6 genes into lentiviruses through gene recombination and cloning technology, packaged lentiviral vectors were obtained.

[0015] S4. Viral infection and cell sorting

[0016] The packaged lentiviral vector prepared in S3 was added to the plasma cells prepared in step S2 at an MOI of 5 to 20, with a plasma cell concentration of 0.5E6 / mL to 1.5E6 / mL. The lentiviral vector and plasma cells were encapsulated into oil droplets using a microfluidic instrument. The oil droplets also contained fluorescently labeled target antigens and anti-mouse fluorescent secondary antibodies. After incubation, antibody-positive plasma cells were sorted out.

[0017] Antibody-positive cells were sorted into 96-well cell culture plates, and the antibody activity in the supernatant was detected after 12–16 days of cell culture.

[0018] S5. Screening for cells with high binding activity

[0019] Cells with high binding activity were screened using overexpression cell binding assays;

[0020] S6, Antibody Sequence Sequencing

[0021] The antibody sequences obtained from the highly binding cells in step S5 were amplified, and the light and heavy chains were cloned into the T vector, respectively. After sequencing, the final antibody sequence was obtained.

[0022] S7. Verification of the activity of the target antibody

[0023] The activity of the target antibody is detected and verified.

[0024] Preferably, the receptor antibodies on the surface of B cells in step S3 include CD138 receptor protein, CD19 protein, B220 protein, and BCMA protein.

[0025] Preferably, the lentivirus described in step S3 is a viral vector developed based on HIV-1, including the G-glycoprotein of vesicular stomatitis virus, the nucleocapsid protein GAG, and the replicase pol.

[0026] Preferably, the BCL and BCL6 genes in step S3 are linked via P2A.

[0027] Preferably, the incubation time in step S4 is 1 to 2 hours.

[0028] Preferably, the microfluidic instrument is from Sphere Fluidics Limited. High-throughput microfluidic single-cell analysis and screening system.

[0029] Preferably, the antibody sequences obtained in step S5 of the screening process for amplification of highly binding cells in step S6 include antibody sequences obtained using 5'RACE, reverse transcription, targeted amplification, and Sanger sequencing.

[0030] Preferably, the detection and verification of the target antibody activity in step S7 includes detecting antibody activity using methods such as ELISA, FACS, reporter cell assay, and primary cell assay.

[0031] Another object of the present invention is to provide the application of the above-described method for developing single B-cell antibodies based on B-cell immortalization in the preparation or development of antibodies.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention provides a novel method for developing single B-cell antibodies. First, by combining single B-cell technology with B-cell immortalization technology, modified lentiviruses are used to infect B cells, enabling them to become immortalized, proliferate in vitro, and secrete antibodies. Second, high-throughput microfluidic screening and cell immortalization technology are combined to directly infect cells in oil droplets, achieving high-throughput and low-cost antibody development. Therefore, this invention effectively solves the problem of high verification costs and the need for large-scale recombinant expression in single B-cell antibody development. Attached Figure Description

[0034] Figure 1 shows the ELISA detection results of the binding of six antibodies to antigens;

[0035] Figure 2 shows the FACS results of the binding activity of the six antibodies to TIGIT-overexpressing cells. Detailed Implementation

[0036] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0037] Example 1: Establishment of the method for developing antibodies using single B cell technology according to the present invention

[0038] S1. Preparation of antigen

[0039] Immunizing animals with a target antigen induces the production of specific B cells against that antigen. Specifically, this includes:

[0040] Based on the protein sequence (Genbank ID: AAI01290.1) and gene sequence information of Human TIGIT, this invention expresses the antigen AAMet 22-Pro 141 and fuses a human Fc-tag or His tag to its C-terminus. After transient transfection expression and purification in HEK293 cells, Human TIGIT / hFc and Human TIGIT / his proteins were prepared as antigens with a protein purity greater than 90%. These antigens were used in subsequent mouse immunization experiments.

[0041] Human TIGIT protein was fluorescently labeled with AF488 using the Alexa Fluor 488 antibody labeling kit (purchased from Thermo Fisher, catalog number A20181) for use in microfluidic screening.

[0042] S2, Antigen Immunization

[0043] Healthy, 4-6 week old mice were initially immunized with the Human TIGIT / hFc protein prepared using the above steps, mixed with complete Freund's adjuvant at a 1:1 volume ratio. Subsequent immunizations were performed with Human TIGI / his protein mixed with incomplete Freund's adjuvant at a 1:1 volume ratio. The second immunization was administered on day 14, two weeks after the initial immunization, and the third immunization was administered on day 28, two weeks after the second immunization.

[0044] Serum from immunized mice was isolated and tested for potency. Once the serum potency was qualified, spleen and bone marrow cells were collected. B cells were sorted using the Pan B cell isolation kit (Medlan, catalog number 130-095-813), and plasma cells were then obtained from the B cells using the CD138 kit (Medlan Biotech GmbH, catalog number 130-098-257).

[0045] S3, Modification of Lentiviral Vectors

[0046] By fusing receptor antibodies on the surface of B cells with VSV-G protein and packaging them onto the surface of lentiviruses, and by packaging the BCL and BCL6 genes into lentiviruses using gene recombination and cloning techniques, a packaged lentiviral vector was prepared.

[0047] Lentiviral Packaging: First, packaging vector 1 was constructed, wherein packaging vector 1 is a lentiviral envelope protein VSV-G fused with an antibody against mouse CD19, and its amino acid sequence is shown in SEQ ID NO.1. Second, the BCL and BCL6 genes were constructed into shuttle plasmid 2, with BCL and BCL6 linked via P2A. Thus, shuttle plasmid 2 carries the mouse transcription factors BCL and BCL6, and its amino acid sequence is shown in SEQ ID NO.2. Packaging vector 3 (pMDL, Addgene) is a packaging vector expressing the gag / pol gene.

[0048] The day before virus packaging, 3E6293T cells were seeded into 10cm plates. The next day, 15ug of plasmid (plasmid 1:plasmid 2:plasmid 3 = 1:2:3) was mixed with 45ug of PEI and incubated for 15 min. The incubated mixture was then dropped into 10cm cell plates. Six hours after transfection, the medium was replaced with fresh DMEM (containing 10% FBS). After culturing the cells for another 48 hours, the cell supernatant was collected. The cells were removed by high-speed centrifugation, and the virus was concentrated in the supernatant using 5×PEG 8000. 2.5mL of 5×PEG 8000 virus concentrate was added to 10mL of the centrifuged virus supernatant, and the mixture was inverted and incubated overnight at 4°C. The next day, the cells were centrifuged at 4000g for 30 min at 4°C. After centrifugation, the supernatant was removed, and the virus particles were resuspended in 1mL of PBS and stored at -80°C.

[0049] S4. Viral infection and cell sorting

[0050] The packaged lentiviral vector prepared by S3 was added to plasma cells at an MOI of 10, with a plasma cell concentration of 1E6 / mL. The lentiviral vector and plasma cells were then encapsulated into oil droplets using a microfluidic instrument. The oil droplets also contained detection reagents. After incubation for 1 hour, the instrument sorted out the antibody-positive plasma cells.

[0051] Positive oil droplets were sorted into 96-well cell culture plates pre-coated with anti-CD40 antibody. The 1640 medium contained IL21. After 14 days of cell culture, antibody activity in the supernatant was detected. Specifically, this included:

[0052] Viral infection:

[0053] (1) Condition 1: The packaged lentivirus was added to 1E6 plasma cells at an MOI of 10, along with fluorescently labeled TIGIT protein and anti-mouse fluorescent secondary antibody. After the plasma cells, virus, fluorescently labeled TIGIT protein and fluorescent secondary antibody were encapsulated together in an oil droplet, the virus infected the plasma cells in a 350 pL oil droplet.

[0054] Positive oil droplets were sorted after 1 hour of incubation. These droplets were then transferred to 96-well cell culture plates pre-coated with 10 μg / mL anti-CD40 antibody in 1640 medium containing 10 ng / mL IL21. Antibody activity in the supernatant was assessed after 14 days of cell culture.

[0055] (2) Condition 2: In 1E6 plasma cells, fluorescently labeled TIGIT protein and anti-mouse fluorescent secondary antibody were added. Plasma cells, fluorescently labeled TIGIT protein and fluorescent secondary antibody were encapsulated in oil droplets, and positive oil droplets were sorted after 1 hour of incubation.

[0056] After the positive oil droplets were sorted into 96-well plates, lentivirus with an MOI of 10 was added to the 96-well plates. The virus infected plasma cells in the 96-well plates in a 200 μL system.

[0057] 96-well plates were pre-coated with 10 μg / mL anti-CD40 antibody, and 1640 medium contained 10 ng / mL IL21. Antibody activity in the supernatant was detected after 14 days of cell culture.

[0058] S5. Screening for cells with high binding activity

[0059] Cells with high binding activity were screened using overexpression cell binding assays, specifically including:

[0060] (1) CHO-K1 cells overexpressing TIGIT were digested with trypsin and seeded into 96-well V plates at a density of 3E5 cells per well.

[0061] (2) Wash the cells once with PBS solution containing 2% FBS;

[0062] (3) Add culture supernatant, Anti-HEL mouse IgG1-Kappa Isotype and positive control respectively, and incubate at room temperature for 30 min;

[0063] (4) Wash three times with PBS solution containing 2% FBS, add Anti-mouse IgG (Fc specific) APC fluorescent secondary antibody, and incubate at room temperature for 30 min; wash three times with 2% FBS PBS solution;

[0064] (5) The binding activity between the culture supernatant and the overexpressing cells was detected by FACS. The flow cytometry results are shown in the table below.

[0065] Table 1. Results of flow cytometry analysis

[0066] As shown in Table 1, in the flow cytometry results, under condition 1, the virus infected the cells in oil droplets, achieving immortalization and successfully secreting antibodies. Cells in eight wells, namely A1, C1, H1, H2, A3, C3, F3, and G3, were positive for binding. The positive signal was more than 10 times that of the negative well (H3), so a fluorescent signal was detected. Under condition 2, the virus infection occurred in a 96-well plate, and no fluorescent signal was detected by flow cytometry.

[0067] S6, Antibody Sequence Sequencing

[0068] The highly binding cells obtained in step S5 were used to amplify the antibody sequences within the cells. The light and heavy chains were cloned into the T vector, respectively, and the final antibody sequences were obtained after sequencing. Specifically, this includes:

[0069] Cells corresponding to positive antibodies were amplified using the 5' RACE method to amplify the antibody sequence. The light and heavy chains were cloned into T vectors, respectively, and then sent for Sanger sequencing to obtain the final antibody sequence. The specific steps include the following:

[0070] Flow cytometry was used to detect wells with good binding to overexpressing cells. Cells and culture supernatant were transferred to 1.5 μL EP tubes and centrifuged at 1000 rpm for 10 minutes. The supernatant was removed, and the cells were resuspended in 50 μL of 0.1% Triton X-100 (containing an RNase inhibitor) and lysed. 2 μL of cell lysate was reverse transcribed into cDNA. A TSO adapter was added, and the light and heavy chain sequences of the antibody were amplified using the 5' RACE method. The amplified light and heavy chain sequences were cloned into T vectors, and 10 clones from each vector were sent for Sanger sequencing. Wells with positive cell binding were selected for sequencing. The cells with the strongest signal among the 8 positive antibodies were selected to obtain 6 pairs of light and heavy chain paired antibody sequences (A1, H1, H2, A3, F3, G3). The light and heavy chain sequences of the antibodies are shown in Table 2. The amino acid sequences of the variable regions of the light and heavy chains are shown in SEQ ID NO. 3-14.

[0071] Table 2. Antibody sequence information for 6 pairs of light and heavy chains.

[0072] The amino acid sequence of the variable region of the heavy chain of antibody A1 is: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPHNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCASLYYYGSSPLDVWGTGTTVTVSS (SEQ ID NO.3);

[0073] The amino acid sequence of the variable region of the light chain of antibody A1 is: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK (SEQ ID NO.4).

[0074] The amino acid sequence of the variable region of the heavy chain of antibody H1 is: QVQLQQSGAELVRPGTSVKMSCKASGYTFTNYWIGWTKQRPGHGLEWIGDIYPGGGYTNYNEKFKGKATLTADKSSSTAYMQFSSLTSEDSAIYYCARGGYGSSYGYFDVWGTGTTVTVSS (SEQ ID NO.5);

[0075] The amino acid sequence of the variable region of the light chain of antibody H1 is: DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELK (SEQ ID NO.6).

[0076] The amino acid sequence of the variable region of the H2 heavy chain of the antibody is: QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSSGYTKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARDGSSYGFAYWGQGTLVTVSA (SEQ ID NO.7);

[0077] The amino acid sequence of the variable region of the light chain of antibody H2 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLPSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPWTFGGGTKLEIK (SEQ ID NO.8).

[0078] The amino acid sequence of the variable region of the antibody A3 heavy chain is: QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREGGYPWYFDVWGTGTTVTVSS (SEQ ID NO.9);

[0079] The amino acid sequence of the variable region of the light chain of antibody A3 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSSLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGGGTKLEIK (SEQ ID NO.10).

[0080] The amino acid sequence of the variable region of the heavy chain of antibody F3 is: QVQLQQSGAELAKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGYTNPNSGYSKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARDGSSYGFAYWGQGTLVTVSA (SEQ ID NO.11);

[0081] The amino acid sequence of the variable region of the light chain of antibody F3 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPWTFGGGTKLEIK (SEQ ID NO.12).

[0082] The amino acid sequence of the variable region of the antibody G3 heavy chain is: QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAHMQFNSLTSEDSAVYFCARGYLGRGFDYWGQGTTLTVSS (SEQ ID NO.13);

[0083] The amino acid sequence of the variable region of the G3 light chain of the antibody is: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQLYSTPRTFGGGTKLEIK (SEQ ID NO.14).

[0084] S7. Verification of the activity of the target antibody

[0085] The activity of the target antibody was validated using ELISA and FACS assays. Specifically, this included:

[0086] 1. ELISA detection (protein binding assay after antibody expression)

[0087] The binding activity of the six antibodies (A1, H1, H2, A3, F3, G3) obtained in step S6 of the enzyme-linked immunosorbent assay (ELISA) with the TIGIT antigen was analyzed using the ELISA detection method. Based on the experimental results, the binding ability of the antibodies to the antigen was analyzed. The specific steps are as follows:

[0088] (1) Pack 2 μg / mL of Human TIGIT / his protein onto an ELISA plate, dilute Human TIGIT / his protein to 2 μg / mL, add 100 μL of protein dilution buffer to the ELISA plate, and incubate overnight at 4°C.

[0089] (2) Add 200 μL of PBS, shake for 1 minute, then remove the PBS, wash the microplate once, and then add 200 μL of 1% BSA to block at room temperature for 2 hours.

[0090] (3) Wash once with 0.05% PBST, and add 6 antibodies (A1, H1, H2, A3, F3, G3) at 7 different concentrations (5-fold serial dilution) to the microplate. Add 100 μL of the diluted antibody concentration from high to low to the microplate. Add negative control antibody (Anti-HELHuman IgG1-Kappa Isotype, purchased from Shanghai Baiying Biotechnology Co., Ltd.) to the last row as 0 concentration control. The dilution method is the same as that for the detection antibody. Incubate at room temperature for 1 h.

[0091] (4) Wash with 0.05% PBST, add 100 μL of secondary antibody Anti-human IgG (Fc specific) HRP (purchased from Sigma, catalog number A0170), and incubate at room temperature for 40 min; wash 3 times with 0.05% PBST, 5 mins each time, add 100 μL of Beyotime TMB chromogenic solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.), react at room temperature in the dark for 10 min, then add 50 μL of Beyotime TMB stop solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to immediately stop the reaction, shake to mix for 1 min, and read OD450 on Envision.

[0092] The results of the antigen binding test are shown in Figure 1. As can be seen from the results in Figure 1, the positive rate of the 6 antibodies is 100% (6 / 6).

[0093] 2. FACS detection (antibody recombinant expression followed by overexpression cell binding assay)

[0094] The binding activity of the six antibodies obtained in Example S6 above with TIGIT-overexpressing cells was detected by FACS. The binding of antibodies to cells was detected, and the binding ability of antibodies to cells overexpressing antigen was analyzed based on the experimental results. The specific steps are as follows:

[0095] (1) CHO-K1 cells overexpressing TIGIT protein were cultured in 10% FBSDMEM at 37°C and 10% CO2 for 48 hours.

[0096] (2) CHO-K1 cells expressing TIGIT protein in T75 culture flasks were diluted once with 10 mL PBS, digested with 10 mL trypsin, and then centrifuged with 10 mL of 10% FBSDMEM to remove the supernatant. The cells were then seeded into 96-well plates at 3E5 wells each.

[0097] (3) Wash the cells once with PBS solution containing 2% FBS;

[0098] (4) The six antibodies (Sequence 1, Sequence 2, Sequence 3, Sequence 4, Sequence 5, Sequence 6) and the negative control (Anti-HEL mouse IgG1-Kappa Isotype, purchased from Shanghai Baiying Biotechnology Co., Ltd.) were serially diluted. The starting concentration was 200 nM and diluted 4 times. There were 7 dilution gradients. 100 μL of the diluted antibody was added to a 96-well plate and incubated at room temperature for 30 min.

[0099] (5) Wash three times with PBS solution containing 2% FBS, add Anti-mouse IgG (Fc specific) APC fluorescent secondary antibody (109-605-098, Jackson), and incubate at room temperature for 30 min; wash three times with 2% FBS PBS solution, each time add 200 μL of 2% FBS PBS solution, mix well with a multi-channel pipette, and centrifuge at 300 g for 5 mins; resuspend the cells in 200 μL of 2% FBS PBS solution.

[0100] (6) Flow cytometer (purchased from Thermo, model number) Cell fluorescence signals were detected using NxT. The APC dye detection channel was selected, with excitation light at 637 nM, emission light at 670 / 14, FSC voltage at 100, SSC voltage at 300, and APC channel voltage at 400. 100 μL of cells were analyzed, with cells without antibody serving as a blank control. The average fluorescence of the sample cells was analyzed. EC50 was calculated using a 4-parameter nonlinear fitting method with GraphPad Prism software.

[0101] The experimental results are shown in Figure 2. As can be seen from Figure 2, the antibody sequences obtained by screening have a good binding ability with the TIGIT protein on the cell membrane.

[0102] In summary, immortalizing B cells by infecting them with oil droplets, as shown in Table 1, demonstrates better results compared to viral infection in 96-well plates. Combining microfluidic technology, antigen enrichment followed by the immortalization technique described in this invention allows for rapid and efficient screening of positive cells that bind to cells. Further screening, as shown in Table 1, eliminates false positives or cells that do not bind to cells identified through microfluidic screening. Positive sequences are then directly obtained via 5' RACE, and recombinant expression verifies that the antibody sequences possess protein-cell binding activity. This invention achieves efficient, rapid, low-cost, and multi-dimensional screening for antibody development.

[0103] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for developing single B-cell antibodies based on B-cell immortalization, characterized in that, Preliminary enrichment of cells was achieved using single B-cell microfluidic technology, followed by further screening using B-cell immortalization technology, including the following steps: S1. Preparation of antigen Based on the target antigen protein sequence and gene sequence information, the antigen is expressed and fused with human Fc-tag or His tag at its C-terminus. After expression and purification, it is used for subsequent mouse immunization. Fluorescent labeling of target antigen proteins for microfluidic screening; S2, Antigen Immunization Immunizing animals with the target antigen induces the production of specific B cells against the antigen, which in turn produce antibody-secreting plasma cells. S3, Modification of Lentiviral Vectors By fusing receptor antibodies on the surface of B cells with VSV-G protein and packaging them onto the surface of lentiviruses, and by packaging BCL and BCL6 genes into lentiviruses through gene recombination and cloning technology, packaged lentiviral vectors were obtained. S4. Viral infection and cell sorting The packaged lentiviral vector prepared in S3 was added to the plasma cells prepared in step S2 at an MOI of 5 to 20, with a plasma cell concentration of 0.5E6 / mL to 1.5E6 / mL. The lentiviral vector and plasma cells were encapsulated into oil droplets using a microfluidic instrument. The oil droplets also contained fluorescently labeled target antigens and anti-mouse fluorescent secondary antibodies. After incubation, antibody-positive plasma cells were sorted out. Antibody-positive cells were sorted into 96-well cell culture plates, and the antibody activity in the supernatant was detected after 12–16 days of cell culture. S5. Screening for cells with high binding activity Cells with high binding activity were screened using overexpression cell binding assays; S6, Antibody Sequence Sequencing The antibody sequences obtained from the highly binding cells in step S5 were amplified, and the light and heavy chains were cloned into the T vector, respectively. After sequencing, the final antibody sequence was obtained. S7. Verification of the activity of the target antibody The activity of the target antibody is detected and verified.

2. The method for developing single B-cell antibodies based on B-cell immortalization according to claim 1, characterized in that, The receptor antibodies on the surface of B cells mentioned in step S3 include CD138 receptor protein, CD19 protein, B220 protein, and BCMA protein.

3. The method for developing single B-cell antibodies based on B-cell immortalization according to claim 1, characterized in that, The lentivirus described in step S3 is a viral vector developed based on HIV-1, including the G-glycoprotein of vesicular stomatitis virus, the nucleocapsid protein GAG, and the replicase pol.

4. The method for developing single B-cell antibodies based on B-cell immortalization according to claim 1, characterized in that, The BCL and BCL6 genes mentioned in step S3 are linked via P2A.

5. The method for developing single B-cell antibodies based on B-cell immortalization according to claim 1, characterized in that, The incubation time in step S4 is 1 to 2 hours.

6. The method for developing single B-cell antibodies based on B-cell immortalization according to claim 1, characterized in that, The microfluidic instrument is from Sphere Fluidics Limited. High-throughput microfluidic single-cell analysis and screening system.

7. The method for developing single B-cell antibodies based on B-cell immortalization according to claim 1, characterized in that, The antibody sequences obtained in step S6 from the highly binding cells screened in step S5 include antibody sequences obtained using 5'RACE, reverse transcription, targeted amplification, and Sanger sequencing.

8. The method for developing single B-cell antibodies based on B-cell immortalization according to claim 1, characterized in that, The detection and verification of the target antibody activity described in step S7 includes detecting antibody activity using methods such as ELISA, FACS, reporter cell assay, and primary cell assay.

9. The application of the method for developing single B-cell antibodies based on B-cell immortalization as described in any one of claims 1 to 8 in the preparation or development of antibodies.

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