A method for developing single B cell antibodies based on B cell immortalization and its applications

Abstract

The present invention specifically relates to a method for developing single B cell antibodies based on B cell immortalization and its application. The method provided by the present invention includes the following steps: S1, preparation of antigens; S2, antigen immunization; S3, modification of lentiviral vectors; S4, virus infection and cell sorting; S5, screening of cells with high binding activity; S6, antibody sequence sequencing; S7, activity verification of target antibodies and other steps. The present invention provides a brand-new method for antibody development, effectively solving the problems of the need for a large amount of recombinant expression and high verification cost in the development of single B cell antibodies. By combining single B cell technology with B cell immortalization technology, the modified lentivirus is used to infect B cells to immortalize the cells, enabling them to proliferate in vitro and secrete antibodies. Secondly, by combining high-throughput microfluidic screening and cell immortalization technology, cell infection is directly carried out in oil droplets, achieving high-throughput and low-cost antibody development.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for developing single B cell antibodies based on B cell immortalization and its application. Background Art

[0002] After immunizing an animal 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, the hybridoma monoclonal antibody screening technology has been widely used. This technology fuses myeloma cells and B lymphocytes to achieve the immortalization of B cells and the screening of antibodies. The fused hybridoma cells can proliferate in vitro and secrete antibodies. Spreading them on a 96-well plate can screen out monoclonal antibodies. However, the hybridoma technology is limited by the fusion efficiency and screening throughput, and has great limitations for the screening of proteins with high difficulty, thus reducing the success rate of antibody screening.

[0003] Another technology is phage display technology, 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 processes, phages with specific binding can be enriched. Subsequently, positive monoclonal phages are 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 heavy and light 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 screening out 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 antibody sequences with activity. Single B cell technology has the advantage of a large screening throughput, but the subsequent expression verification cost is relatively high.

[0005] Therefore, providing a method that can both maintain the advantage of high-throughput screening and reduce the subsequent expression verification cost is of great significance for the fields of antibody screening and drug development. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a method for developing single B cell antibodies 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 the preliminary enrichment of B cells and subsequent high-throughput activity screening of antibodies, and can solve the problems of high expression verification cost and low coverage rate after NGS sequencing in single B cell technology, and is an efficient single B cell antibody discovery technology.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide a method for developing single B cell antibodies based on B cell immortalization, which realizes the preliminary enrichment of cells through single B cell microfluidic technology and further screens by combining B cell immortalization technology, including the following steps:

[0009] S1. Preparation of antigen

[0010] According to the target antigen protein sequence and gene sequence information, express the antigen and fuse and express human Fc-tag or His tag at its C-terminus. After expression and purification, it is used for subsequent mouse immunization;

[0011] Fluorescently label the target antigen protein for microfluidic screening;

[0012] S2. Antigen immunization

[0013] Immunize an animal with the target antigen to induce the generation of specific B cells against the antigen in the animal body, and then generate antibody-secreting plasma cells;

[0014] S3. Modification of lentiviral vector

[0015] By fusing the receptor antibody on the B cell surface with the VSV-G protein and packaging it on the surface of the lentivirus, and packaging the BCL and BCL6 genes into the lentivirus through gene recombination and cloning techniques, a packaged lentiviral vector is prepared;

[0016] S4. Infection of virus and sorting of cells

[0017] Add the packaged lentiviral vector prepared in S3 to the plasma cells prepared in step S2 according to MOI = 5-20. The concentration of plasma cells is 0.5E6 / mL-1.5E6 / mL. The lentiviral vector and plasma cells are encapsulated into oil droplets through a microfluidic instrument. At the same time, there are also fluorescently labeled target antigen and anti-mouse fluorescent secondary antibody in the oil droplets. After incubation, antibody-positive plasma cells are sorted out;

[0018] Sort the antibody-positive cells into a 96-well cell culture plate, and detect the antibody activity in the supernatant after culturing the cells for 12-16 days;

[0019] S5. Screening of cells with high binding activity

[0020] Screen out cells with high binding activity through overexpression cell binding assay;

[0021] S6. Antibody sequence sequencing

[0022] Amplify the antibody sequences in the cells with high binding activity screened in step S5, clone the heavy and light chains into the T vector respectively, and finally obtain the antibody sequences after sequencing;

[0023] S7. Activity verification of the target antibody

[0024] Detect and verify the activity of the target antibody.

[0025] Preferably, the receptor antibodies on the surface of B cells described in step S3 include CD138 receptor protein, CD19 protein, B220 protein, and BCMA protein.

[0026] 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.

[0027] Preferably, the BCL and BCL6 genes in step S3 are linked by P2A.

[0028] Preferably, the incubation time in step S4 is 1 to 2 hours.

[0029] Preferably, the microfluidic instrument is the Cyto-Mine® high-throughput microfluidic single-cell analysis and screening system of Sphere Fluidics Limited.

[0030] Preferably, the method for amplifying the antibody sequence in the highly binding active cells screened in step S5 in step S6 includes obtaining the antibody sequence by methods such as 5' RACE, reverse transcription, targeted amplification, and Sanger sequencing.

[0031] Preferably, the detection and verification of the activity of the target antibody in step S7 include detecting the antibody activity by methods such as ELISA detection, FACS detection, reporter cell detection, and primary cell detection.

[0032] Another object of the present invention is to provide the application of the single B cell antibody development method based on B cell immortalization as described above in the preparation or development of antibodies.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a brand-new single B cell antibody development method. First, by combining single B cell technology with B cell immortalization technology, the modified lentivirus is used to infect B cells to make the cells immortalized, enabling in vitro proliferation and antibody secretion. Secondly, by combining high-throughput microfluidic screening and cell immortalization technology, cell infection is directly carried out in oil droplets, achieving high-throughput and low-cost antibody development. Therefore, the present invention effectively solves the problems of large-scale recombinant expression required for single B cell antibody development and high verification cost. Description of the Drawings

[0035] Figure 1ELISA test results for the binding of 6 antibodies to the antigen

[0036] Figure 2 FACS test results for the binding activity of 6 antibodies to TIGIT-overexpressing cells Detailed implementation manners

[0037] The above content of the present invention will be further described in detail below through specific implementation manners in the form of examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only.

[0038] Example 1 Establishment of the method for developing antibodies using single B cell technology in the present invention

[0039] S1. Preparation of antigen

[0040] The target antigen is used to immunize an animal to induce the generation of specific B cells against the antigen in the animal body. Specifically, it includes:

[0041] Based on the protein sequence (Genbank accession number: AAI01290.1) and gene sequence information of Human TIGIT, the antigen AA Met 22 - Pro 141 was expressed, and human Fc-tag or His tag was fused to its C-terminus. After transient transfection expression and purification in HEK293 cells, the Human TIGIT / hFc and Human TIGIT / his proteins were prepared as antigens, and the protein purity was greater than 90%. This antigen was used in subsequent mouse immunization experiments.

[0042] Among them, the Alexa Fluor 488 antibody labeling kit (purchased from Thermo Fisher, catalog number A20181) was used to perform AF488 fluorescence labeling on the human TIGIT protein for application in microfluidic screening.

[0043] S2. Antigen immunization

[0044] The healthy 4 - 6-week-old mice were initially immunized with the Human TIGIT / hFc protein prepared in the above steps plus complete Freund's adjuvant, and the antigen and adjuvant were mixed at a volume ratio of 1:1. Subsequently, the second and third immunizations were continued with the Human TIGI / his protein plus incomplete Freund's adjuvant, and the antigen and adjuvant were mixed at a volume ratio of 1:1. Among them, two weeks after the initial immunization, the second immunization was carried out on the 14th day, and two weeks after the second immunization, the third immunization was carried out on the 28th day.

[0045] After detecting the titer of the serum isolated from the immunized mice, and collecting the spleen and bone marrow cells after the serum titer passed the standard, B cells were sorted using a Pan B cell isolation kit (Miltenyi Biotec, catalog number 130-095-813), and then plasma cells were sorted from the B cells using a CD138 kit (Miltenyi Biotec GmbH, catalog number 130-098-257).

[0046] S3. Modification of the lentiviral vector

[0047] By fusing the receptor antibody on the surface of B cells with the VSV-G protein and packaging it on the surface of the lentivirus, and packaging the BCL and BCL6 genes into the lentivirus through gene recombination and cloning techniques, the packaged lentiviral vector was prepared.

[0048] Packaging of the lentivirus: First, a packaging vector 1 was constructed. The packaging vector 1 was the lentiviral envelope protein VSV-G fused with the anti-mouse CD19 antibody, and its amino acid sequence was as shown in SEQ ID NO.1 below. Second, the BCL and BCL6 genes were constructed into the shuttle plasmid 2. BCL and BCL6 were linked by P2A. Thus, the shuttle plasmid 2 carried the mouse transcription factors BCL and BCL6, and its amino acid sequence was as shown in SEQ ID NO.2 below. The packaging vector 3 (pMDL, Addgene) was a packaging vector expressing the gag / pol gene.

[0049] MKCLLYLAFLFIGVNCKFDIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLELKGGGGSGGGGSGGGGSEVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSSASGGGGTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO.1);

[0050] MGTPKQPSLAPAHALGLRKSDPGIRSLGSDAGGRRWRPAAQSMFQIPEFEPSEQEDASATDRGLGPSLTEDQPGPYLAPGLLGSNIHQQGRAATNSHHGGAGAMETRSRHSSYPAGTEEDEGMEEELSPFRGRSRSAPPNLWAAQRYGRELRRMSDEFEGSFKGLPRPKSAGTATQMRQSAGWTRIIQSWWDRNLGKGGSTPSQGSGATNFSLLKQAGDVEENPGPMASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEISPEGFCILLDFMYTSRLNLREGNIMAVMTTAMYLQMEHVVDTCRKFIKASEAEMAPALKPPREEFLNSRMLMPHDIMAYRGREVVENNMPLRNTPGCESRAFAPPLYSGLSTPPASYPMYSHLPLSTFLFSDEELRDAPRMPVANPFPKERALPCDSARQVPNEYSRPAMEVSPSLCHSNIYSPKEAVPEEARSDIHYSVPEGPKPAVPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEGSEQAELGRLSPRAYPAPPACQPPMEPANLDLQSPTKLSASGEDSTIPQASRLNNLVNRSLAGSPRSSSESHSPLYMHPPKCTSCGSQSPQHTEMCLHTAGPTFPEEMGETQSEYSDSSCENGTFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSAADLPPELPKAC (SEQ ID NO.2).

[0051] One day before virus packaging, seed 3E6 293T cells into a 10-cm plate. The next day, mix 15 μg of plasmids (plasmid 1: plasmid 2: plasmid 3 = 1:2:3) with 45 μg of PEI and incubate for 15 min. Drop the incubated mixture onto the 10-cm cell plate. Six hours after transfection, replace the medium with fresh DMEM medium (containing 10% FBS). After culturing the cells for another 48 hours, collect the cell supernatant. Remove the cells by high-speed centrifugation, and concentrate the virus in the supernatant with 5× PEG 8000. Add 2.5 mL of 5× PEG 8000 virus concentrate to 10 mL of the centrifuged virus supernatant, and mix by inverting at 4°C overnight. The next day, centrifuge at 4000g for 30 min at 4°C. After centrifugation, remove the supernatant and resuspend the virus particles in 1 mL of PBS, and store at -80°C in the refrigerator.

[0052] S4. Infection of virus and sorting of cells

[0053] Add the packaged lentiviral vector prepared in S3 to plasmacytes at an MOI of 10. The concentration of plasmacytes is 1E6 / mL. The lentiviral vector and plasmacytes are encapsulated into oil droplets by a microfluidic instrument. Detection reagents are also present in the oil droplets. After incubating for 1 hour, the instrument sorts out antibody-positive plasmacytes;

[0054] Sort the positive oil droplets into a 96-well cell culture plate. The 96-well plate is pre-coated with anti-CD40 antibody, and the 1640 medium contains IL21. Detect the antibody activity in the supernatant after culturing the cells for 14 days. Specifically include:

[0055] Infection of virus:

[0056] (1) Condition 1: Add the packaged lentivirus to 1E6 plasmacytes at a ratio of MOI = 10, and add fluorescently labeled TIGIT protein and anti-mouse fluorescent secondary antibody. After encapsulating the plasmacytes, virus, fluorescently labeled TIGIT protein, and fluorescent secondary antibody together in oil droplets, the virus infects the plasmacytes in 350 pL of oil droplets.

[0057] Sort the positive oil droplets after incubating for 1 hour. Sort the positive oil droplets into a 96-well cell culture plate. The 96-well plate is pre-coated with 10 μg / mL anti-CD40 antibody, and the 1640 medium contains 10 ng / mL IL21. Detect the antibody activity in the supernatant after culturing the cells for 14 days.

[0058] (2) Condition 2: In 1E6 plasmacytes, add fluorescently labeled TIGIT protein and anti-mouse fluorescent secondary antibody. Encapsulate the plasmacytes, fluorescently labeled TIGIT protein, and fluorescent secondary antibody in oil droplets, and sort the positive oil droplets after incubating for 1 hour.

[0059] 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, and the virus infected plasma cells in the 96-well plate. The infection occurred in a 200 μL system.

[0060] The 96-well plate was pre-coated with anti-CD40 antibody at 10 μg / mL, and the 1640 medium contained IL21 at 10 ng / mL. The antibody activity in the supernatant was detected 14 days after cell culture.

[0061] S5. Screening for cells with high binding activity

[0062] Cells with relatively high binding activity were screened through an overexpressed cell binding assay, specifically including:

[0063] (1) After trypsin digestion, CHO-K1 cells overexpressing TIGIT were seeded into a 96-well V-bottom plate at a density of 3×10⁵ cells per well;

[0064] (2) The cells were washed once with PBS solution containing 2% FBS;

[0065] (3) Culture supernatant, Anti-HEL mouse IgG1-Kappa Isotype, and positive control were added respectively, and incubated at room temperature for 30 min;

[0066] (4) The cells were washed 3 times with PBS solution containing 2% FBS, and Anti-mouse IgG (Fc specific) APC fluorescent secondary antibody was added, and incubated at room temperature for 30 min; then washed 3 times with 2% FBS PBS solution;

[0067] (5) The binding activity between the culture supernatant and the overexpressed cells was detected by FACS. The flow cytometry detection results are shown in the following table.

[0068] Table 1 Flow cytometry detection results

[0069]

[0070] As shown in Table 1, in the flow cytometry detection results, in Condition 1, the virus infected in the oil droplets, the cells were immortalized and successfully secreted antibodies. Among them, the cells in 8 wells such as A1, C1, H1, H2, A3, C3, F3, and G3 showed positive cell binding. The positive signal was judged to be more than 10 times that of the negative well (H3), so fluorescent signals were detected; while in Condition 2, the virus infection was in the 96-well plate, and no fluorescent signals were detected by flow cytometry.

[0071] S6. Antibody sequence sequencing

[0072] Amplify the antibody sequences in the highly binding - active cells obtained by screening in step S5. Clone the heavy and light chains into T - vectors respectively, and finally obtain the antibody sequences after sequencing. Specifically, it includes:

[0073] For the cells corresponding to the positive antibodies, amplify the antibody sequences in the cells by the method of 5’ RACE. Clone the heavy and light chains into T - vectors respectively, and send them for Sanger sequencing to finally obtain the antibody sequences. The specific steps are as follows:

[0074] Flow - cytometry detect the wells with good binding to the over - expressed cells. Transfer the cells and the culture medium supernatant to a 1.5 - mL EP tube, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, resuspend the cells with 50 μL of 0.1% TritonX - 100 (containing RNase inhibitor) and then lyse the cells. Take 2 μL of the cell lysate to reverse - transcribe into cDNA, add TSO adapters, and amplify the heavy and light chain sequences of the antibody by the method of 5’ RACE. Clone the amplified heavy and light chain sequences into T - vectors, pick 10 clones for each and send them for Sanger sequencing. Select the wells with positive cell binding for sequencing. Select the cells with the strongest signal among the 8 positive antibodies, and obtain 6 pairs of heavy - and - light - chain paired antibody sequences (A1, H1, H2, A3, F3, G3). The heavy and light 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 as SEQ ID NO.3 - 14 below.

[0075] Table 2 Information of 6 pairs of heavy - and - light - chain paired antibody sequences

[0076]

[0077] The amino acid sequence of the variable region of antibody A1 heavy chain is: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPHNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCASLYYYGSSPLDVWGTGTTVTVSS (SEQ ID NO.3);

[0078] The amino acid sequence of the variable region of antibody A1 light chain is: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK (SEQ IDNO.4).

[0079] The amino acid sequence of the heavy chain variable region of antibody H1 is: QVQLQQSGAELVRPGTSVKMSCKASGYTFTNYWIGWTKQRPGHGLEWIGDIYPGGGYTNYNEKFKGKATLTADKSSSTAYMQFSSLTSEDSAIYYCARGGYGSSYGYFDVWGTGTTVTVSS (SEQ ID NO.5);

[0080] The amino acid sequence of the light chain variable region of antibody H1 is: DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELK (SEQ ID NO.6).

[0081] The amino acid sequence of the heavy chain variable region of antibody H2 is: QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSSGYTKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARDGSSYGFAYWGQGTLVTVSA (SEQ ID NO.7);

[0082] The amino acid sequence of the light chain variable region of antibody H2 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLPSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPWTFGGGTKLEIK (SEQ ID NO.8).

[0083] The amino acid sequence of the heavy chain variable region of antibody A3 is: QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREGGYPWYFDVWGTGTTVTVSS (SEQ ID NO.9);

[0084] The amino acid sequence of the light chain variable region of antibody A3 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSSLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGGGTKLEIK (SEQ ID NO.10).

[0085] The amino acid sequence of the heavy chain variable region of antibody F3 is: QVQLQQSGAELAKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGYTNPNSGYSKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARDGSSYGFAYWGQGTLVTVSA (SEQ ID NO.11);

[0086] The amino acid sequence of the light chain variable region of antibody F3 is: QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPWTFGGGTKLEIK (SEQ IDNO.12).

[0087] The amino acid sequence of the heavy chain variable region of antibody G3 is: QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAHMQFNSLTSEDSAVYFCARGYLGRGFDYWGQGTTLTVSS (SEQ ID NO.13);

[0088] The amino acid sequence of the light chain variable region of antibody G3 is: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQLYSTPRTFGGGTKLEIK (SEQ IDNO.14).

[0089] S7. Activity verification of the target antibody

[0090] The activity of the target antibody was verified by ELISA and FACS assays. Specifically, it includes:

[0091] 1. ELISA assay (protein binding experiment after antibody expression)

[0092] The binding activities of the 6 antibodies (A1, H1, H2, A3, F3, G3) obtained in step S6 to the TIGIT antigen were detected by enzyme-linked immunosorbent assay. The binding of the antibody to the antigen was detected by the detection method of enzyme-linked immunosorbent assay. According to the experimental results, the binding ability of the antibody to the antigen was analyzed. The specific steps are as follows:

[0093] (1) Coat the ELISA plate with 2 μg / mL of Human TIGIT / his protein. Dilute the Human TIGIT / his protein to 2 μg / mL. Add 100 μL of the protein dilution to the ELISA plate and incubate overnight at 4°C.

[0094] (2) Add 200 μL of PBS, shake for 1 minute, then discard the PBS. Wash the ELISA plate once, and then add 200 μL of 1% BSA to block at room temperature for 2 hours.

[0095] (3) Wash once with 0.05% PBST. Add 7 dilution concentrations of 6 antibodies (A1, H1, H2, A3, F3, G3) diluted in 5-fold gradients, 100 μL of the diluted antibodies from high to low concentration to the ELISA plate, and a negative control antibody (Anti-HEL Human IgG1-Kappa Isotype, purchased from Shanghai Beyotime Biotechnology Co., Ltd.). Add 100 μL of PBS to the last row as a 0-concentration control. The dilution method is the same as that of the detection antibody. Incubate at room temperature for 1 hour.

[0096] (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 minutes. Wash 3 times with 0.05% PBST, 5 minutes each time. Add 100 μL of Beyotime TMB chromogenic solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) and react at room temperature in the dark for 10 minutes. Then add 50 μL of Beyotime TMB stop solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to terminate the reaction immediately. Shake and mix for 1 minute, and then read OD450 on Envision.

[0097] The detection results of the antigen-binding assay are as Figure 1 shown. From the Figure 1 results, it can be seen that the positive rate of the 6 antibodies is 100% (6 / 6).

[0098] 2. FACS detection (binding experiment of overexpressed cells after recombinant expression of antibodies)

[0099] Detect the binding activity of the 6 antibodies obtained in Example S6 above to TIGIT-overexpressing cells by FACS, detect the binding of the antibodies to the cells, and analyze the binding ability of the antibodies to the antigen-overexpressing cells according to the experimental results. The specific steps are as follows:

[0100] CHO-K1 cells overexpressing TIGIT protein are cultured in DMEM containing 10% FBS and placed at 37°C with 10% CO2.

[0101] Incubate for 48 hours. The CHO-K1 cells expressing TIGIT protein in a T75 culture flask were diluted once with 10 mL of PBS, added with 10 mL of trypsin for digestion, and the trypsin digestion was terminated with 10 mL of DMEM containing 10% FBS. After centrifugation to remove the supernatant, the cells were seeded into a 96-well plate at a density of 3E5 cells per well; (3) The cells were washed once with PBS solution containing 2% FBS;

[0102] (4) Six antibodies (Sequence 1, Sequence 2, Sequence 3, Sequence 4, Sequence 5, Sequence 6) and a negative control (Anti-HEL mouse IgG1-Kappa Isotype, purchased from Shanghai Beying Biotechnology Co., Ltd.) were serially diluted. The initial concentration was 200 nM and diluted 4-fold for 7 dilution gradients. 100 μL of the diluted antibody was added to the 96-well plate and incubated at room temperature for 30 min;

[0103] (5) The cells were washed 3 times with PBS solution containing 2% FBS, and Anti-mouse IgG(Fc specific) APC fluorescent secondary antibody (109-605-098, Jackson) was added and incubated at room temperature for 30 min; The cells were washed 3 times with 2% FBS PBS solution. Each time, 200 μL of 2% FBS PBS solution was added, mixed well with a multi-channel pipette, and then centrifuged at 300 g for 5 min; The cells were resuspended with 200 μL of 2% FBS PBS solution.

[0104] (6) The fluorescence signal of the cells was detected by a flow cytometer (purchased from Thermo, model Attune® NxT). The APC dye detection channel was selected, with an excitation light of 637 nM, an emission light of 670 / 14, an FSC voltage of 100, an SSC voltage of 300, and an APC channel voltage of 400. 100 μL of the cells were analyzed, and the cells without antibody were used as a blank control to analyze the mean fluorescence of the sample cells; The EC50 was calculated using 4-parameter non-linear fitting, and the software used was GraphPad Prism.

[0105] The experimental results are as Figure 2 shown, and it can be Figure 2 seen that the selected antibody sequence has a good binding ability to the TIGIT protein on the cell membrane.

[0106] In summary, immortalizing cells by infecting B cells in oil droplets shows better results in virus infection in a 96-well plate as shown in Table 1. Combining with microfluidic technology, after antigen enrichment, positive cells that bind to cells can be rapidly and efficiently screened through the immortalization technology in the present invention. Further screening as shown in Table 1 can exclude false positive cells screened by microfluidics or cells that do not bind to cells. Further, the positive sequence can be directly obtained by the method of 5’RACE. After recombinant expression verification, the antibody sequence has the binding activity of protein and cells. The present invention realizes the high-efficiency, rapid, low-cost and multi-dimensional screening of antibody development.

[0107] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by 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: 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 target antibodies; The step S3 of packaging the BCL and BCL6 genes into lentivirus by gene recombination and cloning technology includes: Packaging of lentivirus: 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 BCL and BCL6 genes into shuttle plasmid 2, and BCL and BCL6 are connected by P2A, thereby, 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 is a packaging vector for expressing gag / pol genes.

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.

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