Single B cell antibody development method based on B cell immortalization and application thereof

Abstract

The invention particularly relates to a single B-cell antibody development method based on B-cell immortalization and application of the single B-cell antibody development method. The method provided by the invention comprises the following steps: S1, preparing an antigen; s2, antigen immunization; s3, transforming a lentiviral vector; s4, virus infection and cell sorting; s5, screening cells with high binding activity; s6, sequencing an antibody sequence; s7, verifying the activity of the target antibody, and the like. The invention provides a brand new antibody development method, which effectively solves the problems that the development of a single B cell antibody needs a large amount of recombinant expression and the verification cost is high, a single B cell technology is combined with a B cell immortalization technology, and a modified lentivirus is adopted to infect a B cell, so that the cell is immortalized and can be proliferated in vitro and secrete an antibody; high-throughput microfluidic screening and cell immortalization technologies are combined, and cell infection is directly carried out in oil drops, so that high throughput and low cost of antibody development are realized.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a single B cell antibody development method based on B cell immortalization and an application thereof. Background Art

[0002] After an animal is immunized with an antigen, the body will produce specific antibodies against the antigen. The purpose of antibody screening is to screen out specific antibodies from these antibodies. Traditionally, hybridoma monoclonal antibody screening technology has been widely used. This technology achieves B cell immortalization and antibody screening by fusing myeloma cells and B lymphocytes. The fused hybridoma cells can proliferate and secrete antibodies in vitro, and monoclonal antibodies can be screened by spreading them in 96-well plates. However, hybridoma technology is limited by fusion efficiency and screening throughput, and has great limitations for difficult protein screening, thereby reducing the success rate of antibody screening.

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

[0004] In order to overcome these limitations, single B cell technology came into being. This technology directly screens antibodies from B cells produced after immunization. After screening positive B cells, positive antibody sequences are obtained through high-throughput sequencing. These antibody sequences are then recombinantly expressed through genetic engineering to further screen out active antibody sequences. Single B cell technology has the advantage of high screening throughput, but the subsequent expression verification cost is high.

[0005] Therefore, providing a method that can maintain the advantages of high-throughput screening while reducing the cost of subsequent expression verification is of great significance in the field of antibody screening and drug development. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a single B cell antibody development method based on B cell immortalization and its application. The present invention combines high-throughput single B cell technology, B cell immortalization technology and single B cell microfluidics technology to achieve preliminary enrichment of B cells and subsequent high-throughput activity screening of antibodies, which can solve the problems of high cost of expression verification of single B cell technology and low coverage after NGS sequencing, and is an efficient single B cell antibody discovery technology.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The first object of the present invention is to provide a single B cell antibody development method based on B cell immortalization, which achieves preliminary enrichment of cells through single B cell microfluidics technology, and then further screens in combination with B cell immortalization technology, comprising the following steps: S1. Antigen preparation According to the target antigen protein sequence and gene sequence information, the antigen is expressed and fused with a human Fc-tag or His tag at its C-terminus. After expression and purification, it is used for subsequent mouse immunization. Fluorescently label the target antigen protein for microfluidic screening; S2. Antigen Immunization Immunize animals with target antigens to induce the production of specific B cells against the antigen in the animals, which in turn produce antibody-secreting plasma cells; S3. Transformation of lentiviral vector The receptor antibody on the surface of B cells is fused with VSV-G protein and packaged onto the surface of lentivirus, and the BCL and BCL6 genes are packaged into lentivirus through gene recombination and cloning technology to prepare a packaged lentivirus vector; S4. Virus infection and cell sorting The packaged lentiviral vector prepared in step S3 is added to the plasma cells prepared in step S2 at an MOI of 5 to 20, and the plasma cell concentration is 0.5E6 / mL to 1.5E6 / mL. The lentiviral vector and plasma cells are encapsulated into oil droplets through a microfluidic instrument. The oil droplets also contain fluorescently labeled target antigens and anti-mouse fluorescent secondary antibodies. After incubation, antibody-positive plasma cells are sorted out; Antibody-positive cells were sorted into 96-well cell culture plates, and antibody activity in the supernatant was detected after 12 to 16 days of cell culture; S5. Screening of cells with high binding activity Cells with high binding activity were screened through overexpression cell binding assay; S6. Antibody Sequencing The antibody sequence in the cells with high binding activity obtained by screening in step S5 is amplified, and the light and heavy chains are cloned into T vectors respectively, and the antibody sequence is finally obtained after sequencing; S7. Validation of the activity of target antibodies Detect and verify the activity of the target antibody.

[0008] Preferably, the receptor antibodies on the surface of B cells in step S3 include CD138 receptor protein, CD19 protein, B220 protein and BCMA protein.

[0009] Preferably, the lentivirus in step S3 is a viral vector developed based on HIV-1, comprising the G-glycoprotein, nucleocapsid protein GAG and replicase pol of vesicular stomatitis virus.

[0010] Preferably, in step S3, the BCL and BCL6 genes are connected via P2A.

[0011] Preferably, the incubation time in step S4 is 1 to 2 hours.

[0012] Preferably, the microfluidic instrument is the Cyto-Mine® high-throughput microfluidic single-cell analysis and screening system produced by Sphere Fluidics Limited.

[0013] Preferably, the amplifying of the antibody sequence in the high binding activity cells screened and obtained in step S5 in step S6 comprises obtaining the antibody sequence by 5'RACE, reverse transcription, targeted amplification and Sanger sequencing.

[0014] Preferably, the detection and verification of the activity of the target antibody in step S7 includes detecting the antibody activity by ELISA detection, FACS detection, reporter cell detection and primary cell detection.

[0015] Another object of the present invention is to provide the use of the above-mentioned single B cell antibody development method based on B cell immortalization in the preparation or development of antibodies.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a new single B cell antibody development method. Firstly, by combining single B cell technology with B cell immortalization technology, modified lentivirus is used to infect B cells, so that the cells are immortalized and can proliferate in vitro and secrete antibodies. Secondly, high-throughput microfluidic screening and cell immortalization technology are combined to directly infect cells in oil droplets, thereby achieving high-throughput and low-cost antibody development. Therefore, the present invention effectively solves the problem that single B cell antibody development requires a large amount of recombinant expression and high verification cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of ELISA tests for the binding of 6 antibodies to antigens; Figure 2 The FACS test results of the binding activity of 6 antibodies to TIGIT-overexpressing cells. DETAILED DESCRIPTION

[0018] The above contents of the present invention are further described in detail below by means of specific implementation methods in the form of examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples.

[0019] Example 1 Establishment of the method of developing antibodies using single B cell technology of the present invention S1. Antigen preparation Immunize animals with target antigens to induce the production of specific B cells against the antigen in the animals. Specifically include: Based on the protein sequence of Human TIGIT (Genbank No.: AAI01290.1) and gene sequence information, the present invention expressed the antigen AA Met 22-Pro 141 and fused human Fc-tag or His tag at its C-terminus. After transient transfection expression and purification of HEK293 cells, Human TIGIT / hFc and Human TIGIT / his proteins were obtained as antigens with a protein purity greater than >90. The antigen was used in subsequent mouse immunization experiments.

[0020] Among them, the human TIGIT protein was fluorescently labeled with AF488 using the Alexa Fluor 488 Antibody Labeling Kit (purchased from Thermo Fisher, catalog number A20181) for application in microfluidic screening.

[0021] S2. Antigen Immunization The Human TIGIT / hFc protein prepared in the above steps was added with complete Freund's adjuvant for the primary immunization of healthy, 4-~6-week-old mice, and the antigen and adjuvant were mixed at a volume ratio of 1:1. Subsequently, the Human TIGI / his protein was added with incomplete Freund's adjuvant for the second and third immunizations, and the antigen and adjuvant were mixed at a volume ratio of 1:1. Among them, the second immunization was performed on the 14th day after two weeks of the primary immunization, and the third immunization was performed on the 28th day after two weeks of the second immunization.

[0022] The titer of serum isolated from immunized mice was tested. When the serum titer was qualified, spleen and bone marrow cells were collected, and B cells were sorted using the Pan B cell isolation kit (Miltenyi Biotec, catalog number 130-095-813). Then, plasma cells were sorted from B cells using the CD138 kit (Miltenyi Biotec, Germany, catalog number 130-098-257).

[0023] S3. Transformation of lentiviral vector The receptor antibody on the surface of B cells is fused with VSV-G protein and packaged onto the surface of lentivirus, and the BCL and BCL6 genes are packaged into lentivirus through gene recombination and cloning technology to prepare a packaged lentivirus vector.

[0024] Lentivirus packaging: First, construct packaging vector 1, wherein packaging vector 1 is a lentivirus envelope protein VSV-G fused with an anti-mouse CD19 antibody, and its amino acid sequence is shown in SEQ ID NO.1 below. Secondly, construct the BCL and BCL6 genes into shuttle plasmid 2, and BCL and BCL6 are connected by P2A. Thus, shuttle plasmid 2 carries mouse transcription factors BCL and BCL6, and its amino acid sequence is shown in SEQ ID NO.2 below. Packaging vector 3 (pMDL, Addgene) is a packaging vector for expressing gag / pol genes.

[0025] (SEQ ID NO.1); MGTPKQPSLAPAHALGLRKSDPGIRSLGSDAGGRRWRPAAQSMFQIPEFEPSEQEDASATDRGLGPSLTEDQPGPYLAPGLLGSNIHQQGRAATNSHHGGAGAMETRSRHSSYPAGTEEDEGMEEELSPFRGRSRSAPPNLWAAQRYGRELRRMSDEFEGSFKGLPRPKSAGTATQMRQSAGWTRIIQSWWDRNLGKGGSTPSQGSGATNFSLLKQAGDVEENPGPMASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEISPEGFCILLDFMYTSRLNLREGNIMAVMTTAMYLQMEHVVDTCRKFIKASEAEMAPALKPPREEFLNSRMLMPHDIMAYRGREVVENNMPLRNTPGCESRAFAPPLYSGLSTPPASYPMYSHLPLSTFLFSDEELRDAPRMPVANPFPKERALPCDSARQVPNEYSRPAMEVSPSLCHSNIYSPKEAVPEEARSDIHYSVPEGPKPAVPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEGSEQAELGRLSPRAYPAPPACQPPMEPANLDLQSPTKLSASGEDSTIPQASRLNNLVNRSLAGSPRSSSESHSPLYMHPPKCTSCGSQSPQHTEMCLHTAGPTFPEEMGETQSEYSDSSCENGTFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSAADLPPELPKAC(SEQ ID NO.2)。

[0026] One day before virus packaging, 3E6 293T cells were plated on a 10cm plate. The next day, 15ug of plasmid (plasmid 1: plasmid 2: plasmid 3 = 1: 2: 3) was mixed with 45ug of PEI and incubated for 15min. The incubated mixture was dropped into a 10cm cell plate. Fresh DMEM medium (containing 10% FBS) was replaced 6 hours after transfection. After the cells were cultured for 48 hours, the cell supernatant was collected. The cells were removed by high-speed centrifugation, and the supernatant was concentrated with 5×PEG 8000. 2.5mL of 5×PEG 8000 virus concentrate was added to 10mL of the virus supernatant after centrifugation, and the mixture was mixed overnight at 4 degrees. The next day, the supernatant was centrifuged at 4000g for 30min at 4 degrees. After centrifugation, the supernatant was removed and the virus particles were resuspended with 1mL of PBS and stored in a -80 degree refrigerator.

[0027] S4. Virus infection and cell sorting The packaged lentiviral vector prepared in S3 was added to plasma cells at an MOI of 10, and the plasma cell concentration was 1E6 / mL. The lentiviral vector and plasma cells were encapsulated into oil droplets by a microfluidic instrument. The oil droplets also contained detection reagents. After incubation for 1 hour, the antibody-positive plasma cells were sorted out by the instrument. The positive oil droplets were sorted into a 96-well cell culture plate, which was pre-coated with anti-CD40 antibody. The 1640 culture medium contained IL21, and the antibody activity in the supernatant was detected after 14 days of cell culture. Specifically, it includes: Viral infection: (1) Condition 1: The packaged lentivirus was added to 1E6 plasma cells at an MOI of 10, and fluorescently labeled TIGIT protein and anti-mouse fluorescent secondary antibody were added. After the plasma cells, virus, fluorescently labeled TIGIT protein and fluorescent secondary antibody were co-encapsulated in oil droplets, the virus infected the plasma cells in 350pL oil droplets.

[0028] After incubation for 1 hour, positive oil droplets were sorted. The positive oil droplets were sorted into a 96-well cell culture plate, which was pre-coated with 10ug / mL anti-CD40 antibody and 10ng / mL IL21 in 1640 culture medium. Antibody activity in the supernatant was detected after 14 days of cell culture.

[0029] (2) Condition 2: Fluorescently labeled TIGIT protein and anti-mouse fluorescent secondary antibody were added to 1E6 plasma cells. Plasma cells, fluorescently labeled TIGIT protein, and fluorescent secondary antibody were encapsulated in oil droplets, and positive oil droplets were sorted after incubation for 1 hour.

[0030] After the positive oil droplets were sorted into a 96-well plate, lentivirus with an MOI of 10 was added to the 96-well plate. The virus infected plasma cells in the 96-well plate, and the infection occurred in a 200ul system.

[0031] The 96-well plate was pre-coated with 10ug / mL anti-CD40 antibody, and the 1640 culture medium contained 10ng / mL IL21. Antibody activity in the supernatant was detected after 14 days of cell culture.

[0032] S5. Screening of cells with high binding activity Cells with high binding activity were screened out through overexpression cell binding assay, including: (1) CHO-K1 cells overexpressing TIGIT were trypsinized and plated into 96-well V-bottom plates at 3E5 cells per well. (2) Wash the cells once with PBS solution containing 2% FBS; (3) Add culture supernatant, Anti-HEL mouse IgG1-Kappa Isotype, and positive control, respectively, and incubate at room temperature for 30 min; (4) Wash three times with PBS solution containing 2% FBS, add Anti-mouse IgG (Fc specific) APC fluorescent secondary antibody, incubate at room temperature for 30 min; wash three times with PBS solution containing 2% FBS; (5) The binding activity between the culture supernatant and the overexpressing cells was detected by FACS. The flow cytometry detection results are shown in the following table.

[0033] Table 1 Flow cytometry results

[0034] As shown in Table 1, in the flow cytometry results, the virus in condition 1 was infected in oil droplets, the cells were immortalized, and antibodies were successfully secreted. Among them, the cells in 8 wells, including A1, C1, H1, H2, A3, C3, F3, and G3, were positive for cell binding. The positive signal was judged to be more than 10 times that of the negative well (H3), so the fluorescent signal was detected. However, the virus infection in condition 2 was in a 96-well plate, and no fluorescent signal was detected by flow cytometry.

[0035] S6. Antibody Sequencing The cells with high binding activity obtained in step S5 are screened, the antibody sequence in the cells is amplified, the light and heavy chains are cloned into T vectors respectively, and the antibody sequence is finally obtained after sequencing. Specifically, it includes: The cells corresponding to the positive antibodies are amplified by 5' RACE method, and the light and heavy chains are cloned into T vectors respectively, and sent to Sanger sequencing to finally obtain the antibody sequence. The specific steps include: Flow cytometry was used to detect the wells that had good binding with overexpressed cells. The cells and culture supernatant were transferred to a 1.5 EP tube and centrifuged at 1000rpm for 10 minutes. The supernatant was removed and the cells were lysed after resuspending with 50ul of 0.1% TritonX-100 (containing RNase inhibitor). 2ul of cell lysate was reverse transcribed into cDNA, and the light and heavy chain sequences of the antibody were amplified by 5'RACE method after adding TSO adapter. The amplified light and heavy chain sequences were cloned into T vectors, and 10 clones were picked for Sanger sequencing. The 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 light and heavy chain variable regions are shown in SEQ ID NO.3~14.

[0036] Table 2 Antibody sequence information of 6 pairs of light and heavy chain pairs

[0037] The amino acid sequence of the heavy chain variable region of antibody A1 is: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPHNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCASLYYYGSSPLDVWGTGTTVTVSS (SEQ ID NO. 3); The amino acid sequence of the light chain variable region of antibody A1 is: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK (SEQ ID NO. 4).

[0038] The amino acid sequence of the variable region of the heavy chain of antibody H1 is: QVQLQQSGAELVRPGTSVKMSCKASGYTFTNYWIGWTKQRPGHGLEWIGDIYPGGGYTNYNEKFKGKATLTADKSSSTAYMQFSSLTSEDSAIYYCARGGYGSSYGYFDVWGTGTTVTVSS (SEQ ID NO. 5); The amino acid sequence of the antibody H1 light chain variable region is: DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELK (SEQ ID NO.6).

[0039] The amino acid sequence of the variable region of the heavy chain of antibody H2 is: QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSSGYTKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARDGSSYGFAYWGQGTLVTVSA (SEQ ID NO. 7); The amino acid sequence of the antibody H2 light chain variable region is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLPSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPWTFGGGTKLEIK (SEQ ID NO. 8).

[0040] The amino acid sequence of the heavy chain variable region of antibody A3 is: QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREGGYPWYFDVWGTGTTVTVSS (SEQ ID NO.9); The amino acid sequence of the light chain variable region of antibody A3 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSSLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGGGTKLEIK (SEQ ID NO.10).

[0041] The amino acid sequence of the heavy chain variable region of antibody F3 is: QVQLQQSGAELAKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGYTNPNSGYSKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARDGSSYGFAYWGQGTLVTVSA (SEQ ID NO.11); The amino acid sequence of the light chain variable region of antibody F3 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPWTFGGGTKLEIK (SEQ ID NO. 12).

[0042] The amino acid sequence of the heavy chain variable region of antibody G3 is: QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAHMQFNSLTSEDSAVYFCARGYLGRGFDYWGQGTTLTVSS (SEQ ID NO. 13); The amino acid sequence of the light chain variable region of antibody G3 is: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQLYSTPRTFGGGTKLEIK (SEQ ID NO. 14).

[0043] S7. Validation of the activity of target antibodies The activity of the target antibody is verified by ELISA and FACS testing. Specifically including: 1. ELISA test (protein binding test after antibody expression) The binding activity of the six antibodies (A1, H1, H2, A3, F3, G3) obtained in step S6 of the ELISA to the TIGIT antigen was tested by using the ELISA detection method to detect the binding of the antibody to the antigen. Based on the experimental results, the binding ability of the antibody to the antigen was analyzed. The specific steps are as follows: (1) ELISA plate is coated with 2 μg / mL Human TIGIT / his protein. Dilute the Human TIGIT / his protein to 2 ug / mlL, add 100 ul of protein diluent to the ELISA plate, and incubate overnight at 4°C. (2) Add 200ul of PBS, shake for 1 minute, then discard the PBS, wash the ELISA plate once, and then add 200ul of 1% BSA to block at room temperature for 2h; (3) Wash once with 0.05% PBST, add 7 dilutions of 6 antibodies (A1, H1, H2, A3, F3, G3) diluted 5 times in a gradient, add 100ul of diluted antibodies from high to low concentrations to the ELISA plate, and add negative control antibody (Anti-HELHuman IgG1-Kappa Isotype, purchased from Shanghai Bio-Tech Co., Ltd.), and add 100ul of PBS to the last row as a 0 concentration control. The dilution method is the same as that of the detection antibody, and incubate at room temperature for 1 hour; (4) Wash with 0.05% PBST, add 100ul secondary antibody Anti-human IgG (Fc specific) HRP (purchased from Sigma, catalog number A0170), and incubate at room temperature for 40min; wash with 0.05% PBST three times, 5min each time, add 100ul of Beyotime TMB colorimetric solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.), react at room temperature in the dark for 10min, and then add 50ul of Beyotime TMB stop solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to terminate the reaction immediately. After shaking and mixing for 1min, read the OD450 on Envision.

[0044] Antigen binding test results Figure 1 As shown by Figure 1 The results showed that the positive rate of the 6 antibodies was 100% (6 / 6).

[0045] 2. FACS detection (overexpression cell binding experiment after antibody recombinant expression) The binding activity of the six antibodies obtained in the above Example S6 to the cells overexpressing TIGIT was detected by FACS, and the binding of the antibodies to the cells was detected. Based on the experimental results, the binding ability of the antibodies to the cells overexpressing the antigen was analyzed. The specific steps are as follows: CHO-K1 cells overexpressing TIGIT protein were cultured in DMEM containing 10% FBS at 37°C with 10% CO2 Culture for 48 hours. CHO-K1 cells expressing TIGIT protein in T75 culture flasks were diluted once with 10 mL PBS, digested with 10 mL trypsin, terminated with 10 mL DMEM containing 10% FBS, centrifuged and removed supernatant, and plated into 96-well plates at 3E5 per well; (3) Wash cells once with PBS solution containing 2% FBS; (4) 6 antibodies (Sequence 1, Sequence 2, Sequence 3, Sequence 4, Sequence 5, Sequence 6) and negative control (Anti-HEL mouse IgG1-Kappa Isotype, purchased from Shanghai Bio-Tech Co., Ltd.) were serially diluted, starting at a concentration of 200 nM and diluted 4-fold in 7 dilution gradients. 100 μl of the diluted antibodies were added to a 96-well plate and incubated at room temperature for 30 min. (5) Wash three times with 2% FBS PBS solution, 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, add 200 ul of 2% FBS PBS solution each time, mix evenly with a pipette, and centrifuge at 300 g for 5 mins; resuspend the cells with 200 ul of 2% FBS PBS solution.

[0046] (6) Flow cytometry (purchased from thermo manufacturer, model Attune® NxT) was used to detect cell fluorescence signals. The APC dye detection channel was selected, with excitation light of 637 nM, emission light of 670 / 14, FSC voltage of 100, SSC voltage of 300, and APC channel voltage of 400. 100 ul of cells were taken for analysis, and cells without antibody were used as blank controls. The average fluorescence of the sample cells was analyzed; EC50 was calculated using a 4-parameter nonlinear fit, and the software used was GraphPad Prism.

[0047] The experimental results are as follows Figure 2 As shown by Figure 2 It can be seen that the antibody sequence obtained by screening has a good binding ability with the TIGIT protein on the cell membrane.

[0048] In summary, immortalization of cells by infecting B cells in oil droplets has a better effect than virus infection in 96-well plates as shown in Table 1. Combined with microfluidics technology, after antigen enrichment, positive cells bound to cells are quickly and efficiently screened by the immortalization technology in the present invention. Further screening can exclude false positive cells screened by microfluidics or cells that do not bind to cells, as shown in Table 1. Positive sequences are further directly obtained by the 5'RACE method, and the antibody sequence has protein and cell binding activity after recombinant expression verification. The present invention realizes efficient, rapid, low-cost, and multi-dimensional screening for antibody development.

[0049] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall 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: The initial enrichment of cells was achieved through single B cell microfluidics technology, and further screening was performed in combination with B cell immortalization technology, including the following steps: S1. Antigen preparation According to the target antigen protein sequence and gene sequence information, the antigen is expressed and fused with a human Fc-tag or His tag at its C-terminus. After expression and purification, it is used for subsequent mouse immunization. Fluorescently label the target antigen protein for microfluidic screening; S2. Antigen Immunization Immunize animals with target antigens to induce the production of specific B cells against the antigen in the animals, which in turn produce antibody-secreting plasma cells; S3. Transformation of lentiviral vector The receptor antibody on the surface of B cells is fused with VSV-G protein and packaged onto the surface of lentivirus, and the BCL and BCL6 genes are packaged into lentivirus through gene recombination and cloning technology to prepare a packaged lentivirus vector; S4. Virus infection and cell sorting The packaged lentiviral vector prepared in step S3 is added to the plasma cells prepared in step S2 at an MOI of 5 to 20, and the plasma cell concentration is 0.5E6 / mL to 1.5E6 / mL. The lentiviral vector and plasma cells are encapsulated into oil droplets through a microfluidic instrument. The oil droplets also contain fluorescently labeled target antigens and anti-mouse fluorescent secondary antibodies. After incubation, antibody-positive plasma cells are sorted out; Antibody-positive cells were sorted into 96-well cell culture plates, and antibody activity in the supernatant was detected after 12 to 16 days of cell culture; S5. Screening of cells with high binding activity Cells with high binding activity were screened through overexpression cell binding assay; S6. Antibody Sequencing The antibody sequence in the cells with high binding activity obtained by screening in step S5 is amplified, and the light and heavy chains are cloned into T vectors respectively, and the antibody sequence is finally obtained after sequencing; S7. Validation of the activity of target antibodies Detect and verify the activity of the target antibody.

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 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, and includes the G-glycoprotein, nucleocapsid protein GAG and replicase pol of vesicular stomatitis virus.

4. The method for developing single B cell antibodies based on B cell immortalization according to claim 1, characterized in that: In step S3, the BCL and BCL6 genes 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 the Cyto-Mine® high-throughput microfluidic single-cell analysis and screening system produced by Sphere Fluidics Limited.

7. The method for developing single B cell antibodies based on B cell immortalization according to claim 1, characterized in that: The step S6 of amplifying the antibody sequence in the cells with high binding activity obtained by screening in the step S5 includes obtaining the antibody sequence by 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 activity of the target antibody in step S7 includes detecting the antibody activity by ELISA detection, FACS detection, reporter cell detection and primary cell detection.

9. Use of the single B cell antibody development method based on B cell immortalization according to any one of claims 1 to 8 in the preparation or development of antibodies.

Images