Anti-CD147 antibody A5 and its application

Abstract

The present invention relates to an anti-CD147 antibody A5 and its application, wherein the anti-CD147 antibody A5 comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO.1; the amino acid sequence of the CDR2 is shown in SEQ ID NO.2; the amino acid sequence of the CDR3 is shown in SEQ ID NO.3. The pan-tumor antigen antibody A5 of the present invention has a strong affinity, and the chimeric antigen receptor T cell with the single domain antibody as the antigen binding domain has a strong ability to kill tumor cells, and the antibody can enhance the ability of NK cells to kill tumor cells through ADCC.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies and relates to an anti-CD147 antibody A5 and an application thereof. Background Art

[0002] There is a special antibody lacking light chains in the blood of camelids and cartilaginous fish, namely heavy chain antibodies. Compared with ordinary antibodies, it contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single-domain antibody (also known as a nano-antibody) consisting only of the heavy chain variable region can be obtained, which has the advantages of small molecular weight, strong tissue penetration, high stability, and low immunogenicity. Compared with ordinary antibodies, single-domain antibodies have many advantages: First, they are small in size, one-tenth of ordinary antibodies, which enables them to penetrate into tissues and cells, especially the blood-brain barrier. Second, they are highly stable. Single-domain antibodies can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90°C. Third, they have a simple structure. Single-domain antibodies are easier to transform, such as humanization and multivalent construction, and have low production costs and are suitable for large-scale production. Fourth, they are highly specific. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens, and they are weakly immunogenic to humans, with a low risk of causing immune responses.

[0003] CD147 plays an important role in the growth, infiltration and metastasis of tumor cells, and is expressed in various tumor cells, such as bone cancer, ovarian cancer, breast cancer, oral cancer, gastric cancer, bladder cancer, lung cancer, prostate cancer, kidney cancer, leukemia, colorectal cancer, glioblastoma, melanoma, esophageal cancer, liver cancer, pancreatic cancer, etc. Preclinical and clinical studies on CD147 have found that antibody drugs and engineered immune cell drugs targeting CD147 can effectively kill a variety of tumor cells, including liver cancer, lung cancer, cervical cancer, pancreatic cancer, etc. Therefore, it is urgent to provide an antibody with CD147 as a therapeutic target for the preparation of antibody drugs or cell drugs. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide an anti-CD147 antibody A5 and its application.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-CD147 antibody A5, wherein the anti-CD147 antibody A5 comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3;

[0007] The amino acid sequence of the CDR1 is shown in SEQ ID NO.1;

[0008] The amino acid sequence of the CDR2 is shown in SEQ ID NO.2;

[0009] The amino acid sequence of the CDR3 is shown in SEQ ID NO.3.

[0010] SEQ ID NO.1: GFTLDEYA.

[0011] SEQ ID NO.2: ISSSGGNTN.

[0012] SEQ ID NO. 3: AAVREVRSGYVCYTSVRISYDY.

[0013] This application uses CD147 protein to immunize alpacas, and after determining the titer of CD147-specific antibodies in alpacas serum by ELISA, alpacas peripheral blood mononuclear cells are isolated, RNA is extracted and reverse transcribed to obtain cDNA. Alpaca single-domain antibody-specific primers are used to amplify the VHH sequence and clone it into a yeast expression plasmid to construct a yeast display library. The yeast display library is then positively and negatively screened with biotin-CD147 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich yeast that binds to CD147 protein. Monoclones are picked from the enriched products, and the specific binding of candidate antibodies to CD147 is detected by ELISA. Positive antibodies with better effects are selected for expression and purification. After the positive antibodies are expressed and purified, their affinity for binding to CD147 protein is detected. Antibody clones with strong affinity for CD147 are selected, co-cultured with NK cells and SK-OV-3 ovarian cancer cells, and antibody-dependent cell-mediated cytotoxicity (ADCC) of each clone is detected. At the same time, the CD147 single domain antibody sequence was cloned into the CAR expression vector to construct CAR-T cells with CD147 single domain antibody as the antigen binding domain, and its ability to kill CD147-positive HCT116 cells in vitro was tested. The results showed that alpaca antibodies have high specificity and good tumor killing effects of ADCC and CAR-T mediated by them.

[0014] Preferably, the amino acid sequence of the anti-CD147 antibody A5 is as shown in SEQ ID NO.4.

[0015] SEQ ID NO.4:

[0016] QLQLVESGGGLVQPGGSLRLSCAASGFTLDEYAIGWFRQAPGKEREGVSCISSSGGNTNYASSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAAVREVRSGYVCYTSVRISYDYWGQGTQVTVSS.

[0017] In a second aspect, the present invention provides a heavy chain antibody, which comprises the entire amino acid sequence or a portion of the amino acid sequence of the anti-CD147 antibody A5 and the IgG antibody Fc segment described in the first aspect.

[0018] Preferably, the IgG antibody comprises any one of IgG1, IgG2, IgG3 or IgG4, or a combination of at least two of them.

[0019] Preferably, the amino acid sequence of the Fc segment of the IgG1 antibody is as shown in SEQ ID NO.5.

[0020] SEQ ID NO.5:

[0021] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0022] Preferably, the amino acid sequence of the heavy chain antibody is as shown in SEQ ID NO.6.

[0023] SEQ ID NO.6:

[0024] Question VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0025] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-CD147 antibody A5 described in the first aspect.

[0026] In a fourth aspect, the present invention provides a chimeric antigen receptor, which comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

[0027] Preferably, the antigen binding domain comprises the anti-CD147 antibody A5 described in the first aspect.

[0028] Preferably, the signal peptide is selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ.

[0029] Preferably, the hinge region is selected from the hinge regions of the following proteins: CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ.

[0030] Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233 or CD314.

[0031] Preferably, the intracellular co-stimulatory domain is selected from the co-stimulatory domains of the following proteins: CD3ε, CD3γ, CD3δ, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, PD1, Dap10, CDS, ICAM-1, LFA-1 / CD11a / CD18, ICOS, NKG2D, GITR or OX40L.

[0032] Preferably, the intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following proteins: CD3ζ, CD3ε, CD3γ or CD3δ.

[0033] Preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.7.

[0034] SEQ ID NO.7: MALPVTALLLPLALLLHAARPS.

[0035] Preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO.8.

[0036] SEQ ID NO.8:

[0037] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0038] Preferably, the amino acid sequence of the transmembrane domain is as shown in SEQ ID NO.9.

[0039] SEQ ID NO.9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0040] Preferably, the amino acid sequence of the intracellular co-stimulatory domain is as shown in SEQ ID NO.10.

[0041] SEQ ID NO.10:

[0042] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0043] Preferably, the amino acid sequence of the intracellular signal transduction domain is as shown in SEQ ID NO.11.

[0044] SEQ ID NO.11:

[0045] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0046] In a fifth aspect, the present invention provides an expression vector, which comprises the gene encoding the anti-CD147 antibody A5 described in the first aspect, the nucleic acid molecule described in the third aspect, or the gene encoding the chimeric antigen receptor described in the fourth aspect.

[0047] In a sixth aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the fifth aspect.

[0048] In a seventh aspect, the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor described in the fourth aspect.

[0049] In an eighth aspect, the present invention provides a pharmaceutical composition, comprising the anti-CD147 antibody A5 described in the first aspect, the chimeric antigen receptor described in the fourth aspect, or the chimeric antigen receptor immune cell described in the seventh aspect.

[0050] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0051] Preferably, the auxiliary materials include any one or a combination of at least two of a carrier, a surfactant, a disintegrant, a coating material, an excipient, a solubilizer, a diluent, a pH regulator, a binder, a wetting agent, a colorant, an emulsifier, an antibacterial agent, a solubilizer, an osmotic pressure regulator, a filler, an antioxidant or a buffer.

[0052] In a ninth aspect, the present invention provides a use of the anti-CD147 antibody A5 according to the first aspect in the preparation of a drug for treating colon cancer or ovarian cancer.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) The antibodies prepared in the present application have the advantage of being small in size and can penetrate into tissues and cells, especially the blood-brain barrier.

[0055] (2) Antibodies have a simple structure and are easier to modify.

[0056] (3) The antibodies prepared in the present application are highly specific and can produce antibodies with high specificity and affinity for specific antigens, have good tumor-killing effects, and have weak immunogenicity to humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a graph showing the results of flow cytometry testing of the specificity of the recombinant single domain antibody.

[0058] Figure 2 EC of recombinant single domain antibodies detected by flow cytometry 50 Result graph.

[0059] Figure 3 EC of a recombinant single domain antibody 50 Graph of the curve.

[0060] Figure 4 This is a graph showing the results of flow cytometry detection of the binding of recombinant single domain antibodies to colorectal cancer HCT116 cells.

[0061] Figure 5 This is the result of real-time quantitative live cell imaging analysis of recombinant single-domain antibody-mediated ADCC function by IncuCyte, where Figure A is the curve of the change of relative fluorescence area over time, and Figure B is Figure 5 Figure A shows the statistical graph of the relative fluorescence area values ​​at the last time point recorded.

[0062] Figure 6 Figure 1 is the expression rate test result of recombinant single-domain antibody CAR-T cells, where Figure A is a schematic diagram of the chimeric antigen receptor structure, and Figure B is the expression rate test result of the chimeric antigen receptor in CAR-T cells.

[0063] Figure 7 IncuCyte real-time quantitative live cell imaging analysis of the killing function of CAR-T cells targeting CD147, where Figure A is a curve of the change of relative fluorescence area over time, and Figure B is Figure 7 Figure A shows the statistical graph of the relative fluorescence area values ​​at the last time point recorded. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0065] Reagents used in the following examples:

[0066] PBS (bico, CAT# 14190-250)

[0067] Adjuvant Immunoadjuvant (GERBU, CAT# 3030)

[0068] HRP-ProteinA(Boster, CAT# BA1080)

[0069] HRP-Streptavidin(Boster, CAT# BA1088)

[0070] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B)

[0071] DNA Fragment Recovery Kit (TakaRa, CAT# 9761)

[0072] Goat anti-Llama IgG (H+L))Secondary Antibody [HRP] (Novus, AT#NB7242)

[0073] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805)

[0074] Streptavidin PE (eBioscience, CAT# 12-4317-87)

[0075] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82)

[0076] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014)

[0077] Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CAT#L3000001)

[0078] Example 1

[0079] Three alpacas were immunized with CD147 protein. Each time, the alpacas were injected on both sides near the cervical lymph nodes, with 2 injections on each side, and about 500 μg of emulsified antigen was injected at each point. After immunization, the alpacas were observed for half an hour to confirm that they were in good condition. Immunization was performed once every 3 weeks, for a total of 3 immunizations. 5 mL of peripheral blood was collected before immunization, two weeks after the second immunization, and the third immunization, and centrifuged at 800×g for 10 min to collect the upper serum. Dilute CD147-His recombinant protein with sterile PBS to a final concentration of 1 µg / mL, add 1 µg / mL CD147-His recombinant protein at 100 µL / well, and coat overnight at 4°C; then shake off the antigen coating solution and wash 5 times with PBST; add 200 µL / well of blocking solution and block at 37°C for 2 hours; shake off the blocking buffer and wash the plate 5 times with PBST; use PBS to gradient dilute the above-collected serum, add 100 µL of the gradient diluted serum to the 96-well ELISA plate, and incubate at room temperature for 1 hour, with PBS as the control well; shake off the liquid in the well and wash 5 times with PBST; add 100 µL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour; shake off the liquid in the well and wash the plate 5 times with PBST; add 100 µL / well of TMB colorimetric solution; incubate at room temperature in the dark for 15 min; add 50 µL / well stop solution; use a microplate reader to read the OD450 value in the well.

[0080] The CD147-specific antibody content in the serum of the three alpacas before immunization was extremely low. After multiple immunizations, the content of CD147-specific antibodies in the serum of the three alpacas increased significantly. The immunization was successful and can be used for the construction of a yeast display library.

[0081] Example 2

[0082] Construction of a single domain antibody yeast display library

[0083] (1) VHH antibody fragment cloning

[0084] After confirming that the three alpacas’ serum contained CD147-specific antibodies, 100 mL of alpaca peripheral blood was collected, PBMCs were separated using lymphocyte separation solution, and RNA was extracted. Reverse transcription was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit to obtain cDNA. Using PBMC cDNA as a template, specific primers were used to PCR amplify the alpaca heavy chain antibody sequence (the upstream primer binds to the signal peptide of the VHH antibody ORF, the primer sequence is shown in SEQ ID NO.12, and the downstream primer binds to the CH2 region, the primer sequence is shown in SEQ ID NO.13).

[0085] SEQ ID NO. 12: GTCCTGGCTGCTCTTCTACAAGG.

[0086] SEQ ID NO. 13: GGTACGTGCTGTTGAACTGTTCC.

[0087] 1% agarose was used for electrophoresis analysis of PCR products, and the target fragment with a molecular weight of about 750 bp was recovered and separated. The first-round PCR product was then used as a template to amplify the heavy chain antibody VHH fragment with specific primers (the upstream primer was bound to the antibody FR1 region, and the 5' end contained an SfiI restriction site, the nucleotide sequence of the restriction site was shown in SEQ ID NO.14, and the primer sequence was shown in SEQ ID NO.15; the downstream primer was bound to the anti-Hinge and FR4 regions, and the 5' end contained an SfiI restriction site, the nucleotide sequence of the restriction site was shown in SEQ ID NO.16, and the primer sequence was shown in SEQ ID NO.17). 1% agarose was used for electrophoresis analysis of PCR products, and the target fragment with a molecular weight of about 450 bp was recovered and separated. The target fragment was stored at -80°C (the storage solution contained 1 / 10 volume of 3 M sodium acetate, 1 μg / μL glycogen, and 80% anhydrous ethanol).

[0088] SEQ ID NO. 14: GGCCCAGCCGGCC.

[0089] SEQ ID NO. 15: AGKTGCAGCTCGTGGAGTCNGGNGG.

[0090] SEQ ID NO. 16: GGCCACGAAGGCC.

[0091] SEQ ID NO. 17: GATCACTAGTGGGGTCTTCGCTGTGGTGCG.

[0092] (2) Electroporation of library vectors and detection of library capacity and diversity

[0093] The yeast surface display vector pYDisplay and the VHH fragment library obtained above were digested with SfiI endonuclease. The pYDisplay vector fragment was separated using 1% agarose gel, and a 5000 bp vector fragment was cut for gel recovery. The yeast competent strain frozen at -80°C was streaked onto a YPD solid medium plate in advance and activated at 30°C for 5 days. A single colony of yeast competent strain was inoculated into 50 mL YPD medium and cultured at 250 rpm and 30°C for 2 days. The linearized vector fragment and PCR product were mixed and added to the electroporation cup for electroporation; the electroporated yeast competent strain was transferred to a culture bottle and cultured at 220 rpm and 30°C for 1 h. Take 20 μL of the resuspension, dilute 5000 times with SDCAA, take 100 μL, apply it to the SDCAA plate, culture for 3 days, calculate the reservoir capacity, and continue to culture the rest of the bacterial solution for 24 h. Collect the remaining bacterial liquid into a 50 mL centrifuge tube, centrifuge at 3000×g for 5 min, discard the supernatant, add 10 mL SDCAA to resuspend, mix with 50% glycerol: resuspension solution = 1:1, and freeze at -80 °C.

[0094] The results showed that the single domain antibody yeast display library obtained from 73# alpaca PBMC had a library capacity of 1.6×10 9 The single domain antibody yeast display library obtained from 74# alpaca PBMC has a capacity of 1.3×10 9 The single domain antibody yeast display library obtained from 194# alpaca PBMC has a capacity of 1.1×10 9 After Sanger sequencing, the results showed that the yeast library had large sequence differences, no repeated sequences, good diversity, and could be used for subsequent experiments.

[0095] Example 3

[0096] Panning of yeast display libraries

[0097] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0098] Take 10 mL of yeast library (about 2×10 8Yeast cells) to a 50 mL centrifuge tube, centrifuge at 3000×g for 5 min, and remove the supernatant; at the same time, dilute sterile 5% PBSA (PBS+5% BSA) to 0.5% PBSA; resuspend the yeast library with 1 mL0.5% PBSA, add it to a 1.5 mL centrifuge tube, centrifuge at 3000×g for 5 min, and discard the supernatant; wash again with 0.5% PBSA. Add 1 mL 0.5% PBSA to the centrifuge tube; resuspend the aliquoted streptavidin magnetic beads with a pipette, and pipette 10 μL of magnetic beads into a 1.5 mL centrifuge tube; rotate and incubate at 4°C for 5 min; place the centrifuge tube on a magnetic stand for 5 min, and remove the supernatant with a pipette; then add 1 mL 0.5% PBSA again, rotate and incubate at 4°C for another 5 min, and remove the supernatant.

[0099] (2) Yeast display library screening

[0100] Negative selection with empty magnetic beads: Place the centrifuge tube on the magnetic rack, add the washed yeast solution to the centrifuge tube containing the empty magnetic beads, put it in a bag, and rotate and incubate at 4°C for 60 min; Place the empty magnetic bead centrifuge tube with yeast on the magnetic rack for 10 min, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube, being careful not to aspirate the old magnetic beads, and rotate and incubate at 4°C for 30 min. After the incubation, place it on the magnetic rack for 15 min, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube.

[0101] Biotin-CD147-His magnetic separation: add 100 μL of 50 μg / mL Biotin-CD147-His protein solution (biotin-coupled, diluted with 0.5% PBSA) to a centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and rotate and incubate at 4°C for 60 min; then add 1 mL of 0.5% PBSA, let it stand for 5 min, keep the centrifuge tube on the magnetic rack, and remove the supernatant with a pipette, then add 1 mL of 0.5% PBSA, remove the centrifuge tube from the magnetic rack, mix it with a pipette, place the centrifuge tube on the magnetic rack again, let it stand for 5 min, keep the centrifuge tube on the magnetic rack, and remove the supernatant with a pipette; repeat the above washing steps once more to obtain positive selection magnetic beads coated with Biotin-CD147-His. Add the positive selection magnetic beads bound to Biotin-CD147-His to the yeast cells that have completed negative selection, put them into a bag and place them on a rotating mixer, incubate them at 4°C for 60 min, and after the incubation, place them on a magnetic rack and let them stand at 25°C for 15 min; keep the centrifuge tube on the magnetic rack and use a pipette to absorb the unbound yeast solution; remove the 1.5 mL centrifuge tube from the magnetic rack, and add 1 mL of sterile 0.5% PBSA buffer to the centrifuge tube with a pipette, gently blow the magnetic beads, and then transfer them to a sterile 1.5 mL centrifuge tube, place the centrifuge tube on the magnetic rack, let it stand at 25°C for 5 min, and discard the supernatant; repeat the washing twice, and the whole operation is performed in accordance with the sterile operation requirements. After washing, resuspend the magnetic beads and adhered yeast cells with 1 mL SDCAA medium, pipette 20 μL of the resuspension into 180 μL SDCAA medium and coat two plates, each with a volume of 100 μL, then pipette 5 μL of the resuspension into 95 μL SDCAA medium and coat one plate, i.e. a total of 3 plates need to be coated.

[0102] Yeast flow cytometry after selection: The yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into yeast. The yeast that has undergone negative selection with empty magnetic beads and Biotin-CD147-His magnetic selection was incubated with CD147 protein containing His tag at 4°C for 60 min, then centrifuged to remove the supernatant, added flow antibodies with anti-His tag and anti-V5 tag, incubated at 4°C for 60 min, then centrifuged to remove the supernatant, and resuspended the cells in 500 μL PBS for flow analysis. Before and after sorting, the number of yeast that can bind to CD147 protein in the yeast antibody display library of 73# alpaca, 74# alpaca and 194# alpaca increased by 10%, 41% and 13% respectively, and subsequent experiments can be carried out.

[0103] After sorting, the yeast solution was spread on the SDCAA plate, and a single clone was picked after overnight culture, and the single clone was inoculated into the culture medium to induce expression. After 48 hours, the bacterial solution was incubated with CD147 protein containing His tag at 4°C for 60 min, and then the supernatant was removed by centrifugation. The precipitate was resuspended with a solution containing biotin-conjugated anti-His tag flow antibody, and incubated at 4°C for 60 min. The supernatant was removed by centrifugation, and the precipitate was resuspended with a solution containing PE-Streptavidin, and incubated at 4°C for 60 min. After incubation, flow detection was performed. Finally, the monoclonal clone 74-1-A5 with a high positive rate was selected. 74-1-A5 was lysed with 0.2% SDS (incubated at 95°C for 10 min), centrifuged, and 0.5 μL of the bacterial supernatant was taken as a template for PCR amplification and sent for testing (the remaining bacterial solution was stored at -20°C).

[0104] Example 4

[0105] Expression of recombinant single domain antibodies and detection of binding to target proteins

[0106] The candidate antibody 74-1-A5 VHH sequence, CMV promoter sequence and IgG1 Fc sequence were amplified using a PCR instrument. 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The band size of CMV was 750 bp, the band size of Fc was 1400 bp, and the band size of VHH was 450 bp. The target band was cut from the gel and the PCR product was purified. The 74-1-A5 VHH sequence, CMV promoter and IgG1 Fc sequence were connected using a PCR instrument, and then 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The target band was cut from the gel and the PCR product was purified. After the obtained PCR product was transiently transfected into HEK293 cells, the harvested cell culture supernatant contained the recombinant single domain antibody 74-1-A5.

[0107] The binding specificity of 74-1-A5 was tested. The culture supernatant containing 74-1-A5 was mixed with 3×10 5 CHO-S or CHO-S-CD147 cells were incubated at 25℃ for 1 h. After centrifugation at 800×g at 25℃ for 5 min, the supernatant was discarded and the cells were washed 3 times with PBS. 100 μL PE-labeled Anti-human IgG antibody (1:500 dilution) was added and incubated at 25℃ in the dark for 45 min. After centrifugation at 800×g at 25℃ for 5 min, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500 μL PBS for flow cytometry analysis.

[0108] The results are as follows Figure 1 As shown, the expression supernatant of the negative control group did not bind significantly to CHO-S or CHO-S-CD147; the positive control antibody significantly bound to CHO-S-CD147 but not to CHO-S cells, indicating that CHO-S cells did not express CD147 protein, and CHO-S-CD147 cells successfully overexpressed CD147 protein and could be used as detection cells for alpaca antibodies. 74-1-A5 did not bind to CHO-S cells, but significantly bound to CHO-S-CD147, and most CHO-S-CD147 cells expressing CD147 protein could be detected, indicating that 74-1-A5 had good affinity and specificity.

[0109] Example 5

[0110] Purification of recombinant antibodies and determination of half effective concentration (EC50)

[0111] To determine the EC50 of 74-1-A5 antibody, the relevant expression plasmids were transiently transfected into 293F cells for antibody expression and purification. Protein A beads were washed twice with 30 mL PBS buffer, 0.1 M sodium hydroxide, and PBS buffer, respectively. The corresponding volume of Protein A beads (calculated based on 20 mg IgG / mL Protein A beads) was added to the 293F cell shaker. Incubate at 120 rpm and 25°C for 4 h. Protein A beads were collected with a magnetic separation rack and transferred to a 50 mL centrifuge tube. After washing twice with 30 mL PBS buffer and deionized water, the beads were resuspended with 1 mL elution buffer. After incubation at 25°C for 5 min, the beads were collected with a magnetic separation rack, and the supernatant containing the target antibody was transferred to a 15 mL centrifuge tube. After eluting the Protein A beads twice, the eluates were combined and the neutralization buffer was added to adjust the pH of the solution. The eluted samples were dialyzed with PBS at 25°C for 2 h, and then dialyzed at 8°C for 16 h after changing the solution once. Finally, the protein concentration was determined, and the samples were filtered with a 0.22 μm sterile filter membrane and aliquoted and stored in a -80°C refrigerator for later use.

[0112] Serial dilutions of the target antibody were added to 3×10 5 CHO-S-CD147 cells were incubated at 25℃ for 1 h. Centrifuged at 800×g for 5 min at 25℃, discarded the supernatant, and washed the cells 3 times with PBS. Added 100 μL PE-labeled Anti-human IgG antibody (1:500 dilution), mixed thoroughly, and incubated at 25℃ in the dark for 45 min. Centrifuged at 800×g for 5 min at 25℃, discarded the supernatant, washed the cells 3 times with PBS, and resuspended the cells with 200 μL PBS for flow cytometry analysis.

[0113] The results of flow cytometry analysis are as follows Figure 2 and Figure 3 As shown in the figure, the EC of 74-1-A5 on CHO-S-CD147 50 The value was 0.23 μg / mL, indicating that 74-1-A5 had a higher affinity.

[0114] Example 6

[0115] Binding detection of recombinant antibodies to colorectal cancer cells

[0116] Incubate the HCT116 colorectal cancer cells with 74-1-A5 antibody and positive control antibody at 25℃ for 1 h. Centrifuge at 800×g at 25℃ for 5 min, discard the supernatant, and wash the cells three times with PBS. Add 100 μL of APC-labeled Anti-human IgG antibody, mix thoroughly, and incubate at 25℃ in the dark for 45 min. Centrifuge at 800×g at 25℃ for 5 min, discard the supernatant, wash the cells three times with PBS, resuspend the cells with 200 μL PBS, and perform flow cytometric analysis. The results are shown in Figure 2. Figure 4 As shown by Figure 4 It can be seen that the positive control antibody has significant binding to HCT116, indicating that HCT116 expresses CD147 protein and can be used as a detection cell for alpaca antibodies; 74-1-A5 significantly binds to HCT116 and can detect 99.7% of HCT116 cells expressing CD147 protein, indicating that 74-1-A5 has a high affinity for the CD147 protein of HCT116.

[0117] Example 7

[0118] ADCC functional detection

[0119] Ovarian cancer cells SK-OV-3 (expressing the red fluorescent reporter protein mCherry) were selected and the target cells were washed three times with OptiVitro NK cell expansion medium and then cultured at 1×10 4 The target cells were seeded in a 96-well plate at a density of 100 cells / well and cultured overnight. NK cells were added at an effector-target ratio of 8:1, and the target antibody 74-1-A5 was added at a final concentration of 5 μg / mL.

[0120] The real-time quantitative live cell imaging and analysis platform IncuCyte was used for detection. The changes in the fluorescence signals of target cells in each group were calculated using the IncuCyte software. The results are shown in the figure below. Figure 5As shown, compared with the NK cell group without antibody, 74-1-A5 antibody can significantly enhance the killing effect of NK cells on tumor cells (the relative fluorescence area at the last time point was statistically significant P<0.05), indicating that 74-1-A5 can enhance the killing function of NK cells against SK-OV-3 through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0121] Example 8

[0122] Construction of CAR-T cells based on target antibodies

[0123] (1) Design of CAR molecules

[0124] The target gene structure of the lentiviral vector involved in this example is as follows Figure 6 As shown in Figure A.

[0125] BVH147-004 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CD147 single-domain antibody 74-1-A5 [VHH (74-1-A5) for short], human IgG4 hinge region (IgG4 hinge for short), human CD8 transmembrane domain (human CD8 TM for short), human 4-1BB intracellular co-stimulatory domain (4-1BB ICD for short), and human CD3ζ intracellular signal transduction domain (CD3ζICD for short).

[0126] The amino acid sequence of the signal peptide is shown in SEQ ID NO.7.

[0127] The amino acid sequence of the hinge region of human IgG4 is shown in SEQ ID NO.8.

[0128] The amino acid sequence of the human CD8 transmembrane domain is shown in SEQ ID NO.9.

[0129] The amino acid sequence of the human 4-1BB intracellular co-stimulatory domain is shown in SEQ ID NO.10.

[0130] The amino acid sequence of the human CD3ζ intracellular signal transduction domain is shown in SEQ ID NO.11.

[0131] (2) Lentivirus preparation

[0132] The above CAR molecules were synthesized and connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning. The above lentiviral vector expression plasmids were co-transfected with lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G into 293T cells using the transfection reagent Lipofectamine 3000. The viral supernatant was collected 48 h after transfection, centrifuged at 3000 rpm for 15 min at 4°C, filtered through a 0.45 μm pore size filter membrane, and ultracentrifuged at 25,000 rpm for 3 h at 4°C. The activity titer of the above lentivirus was detected using Jurkat cells as the material.

[0133] (3) CAR-T cell preparation

[0134] PBMCs from healthy donors were revived in AIM V medium, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, and 300 IU / mL recombinant hIL-2 were added, and cultured in a cell culture incubator for 24 h (culture temperature was 37°C, carbon dioxide concentration was 5%). The obtained T cells were washed, lentiviral transduction was added at an MOI of 5 TU / mL, 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, and 300 IU / mL recombinant hIL-2 were added, and cultured at 37°C, carbon dioxide concentration was 5% for 24 h, and the cell density was adjusted to 2×10 6 / mL, add 300 IU / mL hIL-2. On Day 4 after transduction, wash the cells and continue to culture at 37°C, 5% CO2, maintaining the cell density at 2×10 6 / mL. On Day 10 after transduction, cells were harvested and T cells not transduced with lentivirus were named Ctrl T.

[0135] (4) Detection of CAR molecule expression

[0136] The BVH147-004 CAR-T cells to be tested were washed with PBS and resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). FITC-labeled anti-VHH antibody was incubated with CAR-T cells for 1 h. The supernatant was removed by centrifugation, and the cells were washed with FACS buffer and resuspended. Ctrl T cells were used as negative control, and the expression rate of CAR molecules in BVH147-004 cells was detected by flow cytometry. The results are shown in Figure 2. Figure 6 As shown in Figure B, the results show that the expression rate of BVH147-004 CAR is 37.3%.

[0137] Example 9

[0138] Study on the function of CAR-T cells in vivo and in vitro

[0139] Use culture medium at 1×10 5 HCT116-mCherry cells were resuspended at a density of 100 μL / mL and inoculated in a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte live cell imaging analysis system, CAR-T cells were added for co-culture at an effect-target ratio of 3:1, and the killing effect of CAR-T cells on tumor cells was recorded in real time. After the co-culture, the changes in the mCherry fluorescence signal of the target cells in each group were calculated using the IncuCyte software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells. The results are shown in Figure 7 As shown in the figure, compared with Mock-T cells, BVH147-004 cells have a significant killing effect on HCT116. This result shows that CAR-T cells constructed based on 74-1-A5 antibody have a good killing function on CD147-positive tumor cells.

[0140] The applicant declares that the present invention illustrates an anti-CD147 antibody A5 and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0141] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0142] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An anti-CD147 single-domain antibody A5, characterized in that: The anti-CD147 single-domain antibody A5 comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3; The amino acid sequence of the CDR1 is shown in SEQ ID NO.1; The amino acid sequence of the CDR2 is shown in SEQ ID NO.2; The amino acid sequence of the CDR3 is shown in SEQ ID NO.

3.

2. The anti-CD147 single-domain antibody A5 according to claim 1, characterized in that: The amino acid sequence of the anti-CD147 single-domain antibody A5 is shown in SEQ ID NO.

4.

3. A heavy chain antibody, characterized in that The heavy chain antibody comprises the entire amino acid sequence or a portion of the amino acid sequence of the anti-CD147 single domain antibody A5 and the IgG antibody Fc segment according to claim 1 or 2.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD147 single-domain antibody A5 according to claim 1 or 2.

5. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain; The antigen binding domain is the anti-CD147 single domain antibody A5 according to claim 1 or 2; The amino acid sequence of the signal peptide is shown in SEQ ID NO.7; The amino acid sequence of the hinge region is shown in SEQ ID NO.8; The amino acid sequence of the transmembrane domain is shown in SEQ ID NO.9; The amino acid sequence of the intracellular co-stimulatory domain is shown in SEQ ID NO.10; The amino acid sequence of the intracellular signal transduction domain is shown in SEQ ID NO.

11.

6. An expression vector, characterized in that: The expression vector comprises the coding gene of the anti-CD147 single-domain antibody A5 according to claim 1 or 2, or the nucleic acid molecule according to claim 4, or the coding gene of the chimeric antigen receptor according to claim 5.

7. A recombinant lentivirus, characterized in that: The recombinant lentivirus contains the expression vector according to claim 6.

8. A chimeric antigen receptor immune cell, characterized in that The chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 5.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-CD147 single-domain antibody A5 according to claim 1 or 2, the chimeric antigen receptor according to claim 5, or the chimeric antigen receptor immune cell according to claim 8.

10. Use of the anti-CD147 single-domain antibody A5 according to claim 1 or 2 in the preparation of a drug for treating colon cancer or ovarian cancer.

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