A pan-tumor antigen antibody D6 and its application

Abstract

The present invention relates to a pan-tumor antigen antibody D6 and its application, wherein the pan-tumor antigen antibody D6 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 D6 of the present invention has strong affinity and strong killing ability to 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 a pan-tumor antigen antibody D6 and an application thereof. Background Art

[0002] Antibodies are proteins with protective effects produced by the body due to the stimulation of antigens. They are large Y-shaped proteins secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. They are only found in the blood and other body fluids of vertebrates and on the cell membrane surface of their B cells. Antibodies can recognize a unique feature of a specific foreign object, which is called an antigen.

[0003] CD147, also known as Basigin or EMMPRIN, is a highly glycosylated single-pass transmembrane protein with a relative molecular mass of 50kDa-60kDa, belonging to the immunoglobulin superfamily. CD147 is expressed in almost all tumor cells and is highly expressed in a variety of malignant tumors, playing an important role in the occurrence and development of tumors. CD147 promotes tumor growth, invasion and metastasis by inducing the production of matrix metalloproteins (MMPs). The occurrence and development of tumors is a dynamic process involving interactions between different cells and non-cellular components in the tumor microenvironment (TM). The infiltration and metastasis of malignant tumor cells require crossing the basement membrane and tissue gaps, and the high expression of CD147 can increase the expression and activity of MMPs, thereby degrading the basement membrane, destroying the mechanical barrier of the tissue, and promoting tumor invasion and metastasis. CD147 can also participate in the adhesion of cells to the matrix by forming complexes with α3β1 and α6β1, promoting the spread and metastasis of malignant tumor cells. In addition, changes in the intracellular C-terminal structure of CD147 affect the aggregation and disaggregation of intracellular skeletal proteins, participate in cell movement and pseudopodia formation, and promote the metastasis of tumor cells.

[0004] CD147 is highly expressed in a variety of malignant tumors, 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., and plays an important role in the occurrence and development of tumors. 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

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a pan-tumor antigen antibody D6 and its application. The present invention uses CD147 protein to immunize alpacas, and obtains a single domain antibody with strong affinity to CD147 through serum titer detection, yeast library construction, yeast library screening and monoclonal antibody picking. The single domain antibody sequence is cloned into a chimeric antigen receptor expression vector to construct a chimeric antigen receptor T cell with CD147 single domain antibody as the antigen binding domain, which has a good tumor killing effect.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a pan-tumor antigen antibody D6, wherein the pan-tumor antigen antibody D6 comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3;

[0008] The amino acid sequence of the CDR1 is shown in SEQ ID NO.1;

[0009] The amino acid sequence of the CDR2 is shown in SEQ ID NO.2;

[0010] The amino acid sequence of the CDR3 is shown in SEQ ID NO.3.

[0011] SEQ ID NO.1: CAASGFVFDDYA.

[0012] SEQ ID NO. 2: ISASDGRP.

[0013] SEQ ID NO.3:ATACPYSGSYYYTEGLDY.

[0014] This application uses CD147 protein to immunize alpacas, and obtains a single-domain antibody D6 with strong affinity for CD147 through serum titer detection, yeast library construction, yeast library screening and monoclonal antibody picking. D6 then detects its affinity for binding to CD147 protein. D6 was mixed and incubated with NK cells and ovarian cancer cells SK-OV-3 cells to detect its mediated antibody-dependent cytotoxicity (ADCC). At the same time, the CD147 single-domain antibody sequence was cloned into the CAR expression vector to construct CAR-T cells with D6 antibody as the antigen binding domain, and its ability to kill CD147-positive HCT116 cells in vitro was detected. The results showed that the alpaca antibody has high specificity, and the ADCC and CAR-T mediated by it have good tumor killing effects.

[0015] Preferably, the amino acid sequence of the pan-tumor antigen antibody D6 is as shown in SEQ ID NO.4.

[0016] SEQ ID NO.4:

[0017] QLQLVESGGGLVQAGGSLRLSCAASGFVFDDYAIGWFRQAPGKEREGVSCISASDGRPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACPYSGSYYYTEGLDYWGQGTQVTVSS.

[0018] In a second aspect, the present invention provides a heavy chain antibody, which includes the entire amino acid sequence or a portion of the amino acid sequence of the pan-tumor antigen antibody D6 and the IgG antibody Fc segment described in the first aspect.

[0019] Preferably, the IgG antibody comprises any one of IgG1, IgG2, IgG3 or IgG4, or a combination of at least two of them.

[0020] Preferably, the amino acid sequence of the Fc segment of the IgG1 antibody is as shown in SEQ ID NO.5.

[0021] SEQ ID NO.5:

[0022] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] Preferably, the amino acid sequence of the heavy chain antibody is as shown in SEQ ID NO.6.

[0024] SEQ ID NO.6:

[0025] Question VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0026] In a third aspect, the present invention provides a nucleic acid molecule encoding the pan-tumor antigen antibody D6 described in the first aspect.

[0027] In a fourth aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the third aspect.

[0028] In a fifth aspect, the present invention provides a host cell, wherein the host cell contains the expression vector described in the fourth aspect.

[0029] Preferably, the host cell is an immune cell.

[0030] Preferably, the immune cells include one or a combination of at least two of T cells, TIL cells, B cells, natural killer cells or macrophages.

[0031] In a sixth aspect, the present invention provides a chimeric antigen receptor, comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

[0032] Preferably, the antigen binding domain includes the pan-tumor antigen antibody D6 described in the first aspect.

[0033] 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.

[0034] Preferably, the hinge region is selected from the hinge regions of the following proteins: CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ.

[0035] 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.

[0036] 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.

[0037] Preferably, the intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following proteins: CD3ζ, CD3ε, CD3γ or CD3δ.

[0038] Preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.7.

[0039] SEQ ID NO.7: MALPVTALLLPLALLLHAARPS.

[0040] Preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO.8.

[0041] SEQ ID NO.8:

[0042] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0043] Preferably, the amino acid sequence of the transmembrane domain is as shown in SEQ ID NO.9.

[0044] SEQ ID NO.9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0045] Preferably, the amino acid sequence of the intracellular co-stimulatory domain is as shown in SEQ ID NO.10.

[0046] SEQ ID NO.10:

[0047] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0048] Preferably, the amino acid sequence of the intracellular signal transduction domain is as shown in SEQ ID NO.11.

[0049] SEQ ID NO.11:

[0050] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0051] In a seventh aspect, the present invention provides a use of the pan-tumor antigen antibody D6 according to the first aspect in the preparation of a drug for treating colon cancer or ovarian cancer.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) Antibodies are small in size, one-tenth the size of ordinary antibodies, and can penetrate into tissues and cells, especially the blood-brain barrier.

[0054] (2) Due to their simple structure, nanoantibodies are easier to modify, such as humanization and multivalent construction, and have low production costs, making them suitable for large-scale production.

[0055] (3) High specificity. The alpaca's immune system can produce antibodies with high specificity and affinity for specific antigens. The ADCC and CAR-T mediated by them have good tumor killing effects and weak immunogenicity to humans. Nano-antibodies have a low risk of causing immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a graph showing the results of flow cytometry testing of the specificity of the recombinant single domain antibody.

[0057] Figure 2 This is a graph showing the results of flow cytometry detection of the EC50 of the recombinant single domain antibody.

[0058] Figure 3 This is a graph showing the results of flow cytometry detection of the EC50 of the recombinant single domain antibody.

[0059] 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.

[0060] Figure 5 This is the result of IncuCyte real-time quantitative live cell imaging analysis of recombinant single-domain antibody-mediated ADCC function, where Figure A is a 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.

[0061] 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.

[0062] 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 Statistical graph of relative fluorescence area values ​​at the last time point in Figure A. DETAILED DESCRIPTION

[0063] 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.

[0064] Reagents used in the following examples:

[0065] Adjuvant Immunoadjuvant (GERBU, CAT# 3030)

[0066] HRP-ProteinA(Boster, CAT# BA1080)

[0067] HRP-Streptavidin(Boster, CAT# BA1088)

[0068] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B)

[0069] DNA Fragment Recovery Kit (TakaRa, CAT# 9761)

[0070] Goat anti-Llama IgG (H+L))Secondary Antibody [HRP] (Novus, AT#NB7242)

[0071] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805)

[0072] Streptavidin PE (eBioscience, CAT# 12-4317-87)

[0073] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82)

[0074] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014)

[0075] Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CAT#L3000001)

[0076] Example 1

[0077] (1) Alpaca immunization and serum titer testing

[0078] Three alpacas (73#, 74# and 194#) were immunized with CD147 protein, and the immunization experiment was started after recording the ear number of the blank alpaca. 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 and had no discomfort symptoms. Immunization was performed once every 3 weeks, and at least 3 immunizations were performed. 5 mL of peripheral blood was collected before immunization, two weeks after the second immunization, and the third immunization, respectively. The centrifuge tubes containing blood samples were placed in a centrifuge 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, take a new 96-well ELISA plate, add 1 µg / mL CD147-His recombinant protein to 100 µL / well, and coat overnight at 4°C; then shake off the antigen coating solution, wash 5 times with PBST (containing 0.05% Tween 20); add 200 µL / well of blocking solution and block at 37°C for 2 hours; after shaking off the blocking buffer, wash the plate 5 times with PBST; dilute the above-collected serum with PBS gradient, add 100 µL of the gradient diluted serum to the 96-well ELISA plate, incubate at room temperature for 1 hour, and the control well is PBS; shake off the liquid in the well and wash 5 times with PBST; add 100 µL HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour; after shaking off the liquid in the well, wash the plate 5 times with PBST; add 100 µL / well TMB colorimetric solution; incubate at room temperature in the dark for 15 minutes; add 50 µL / well stop solution; read the OD450 value in the well using a microplate reader.

[0079] 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 antibody in the serum of the three alpacas increased significantly and can be used to construct a yeast display library.

[0080] Example 2

[0081] Construction of a single domain antibody yeast display library

[0082] (1) VHH antibody fragment cloning

[0083] After confirming that the three alpacas’ sera contained CD147-specific antibodies, 100 mL of peripheral blood was collected, PBMCs were separated using lymphocyte separation buffer, and RNA was extracted. stStrand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. Using PBMC cDNA as a template, specific primers were used for PCR amplification of 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).

[0084] SEQ ID NO. 12: GTCCTGGCTGCTCTTCTACAAGG.

[0085] SEQ ID NO. 13: GGTACGTGCTGTTGAACTGTTCC.

[0086] 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).

[0087] SEQ ID NO. 14: GGCCCAGCCGGCC.

[0088] SEQ ID NO. 15: AGKTGCAGCTCGTGGAGTCNGGNGG.

[0089] SEQ ID NO. 16: GGCCACGAAGGCC.

[0090] SEQ ID NO. 17: GATCACTAGTGGGGTCTTCGCTGTGGTGCG.

[0091] (2) Electroporation of library vectors and detection of library capacity and diversity

[0092] 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 shaken 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 yeast competent strain after electroporation was transferred to a culture bottle and shaken 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.

[0093] 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.

[0094] Example 3

[0095] Panning of yeast display libraries

[0096] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0097] 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 discard the supernatant; at the same time, dilute sterile 5% PBSA (PBS+5% BSA) to 0.5% PBSA; resuspend the yeast library with 1 mL 0.5% 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. Prepare three 1.5 mL centrifuge tubes (two for negative panning with empty magnetic beads and one for positive panning), add 1 mL of 0.5% PBSA to each tube; use a pipette to fully resuspend the aliquoted streptavidin magnetic beads, and pipette 10 μL of magnetic beads into a 1.5 mL centrifuge tube; put the tubes in a bag, fix them on a rotating mixer, and rotate and incubate at 4°C for 5 min; place the tubes on a magnetic stand for 5 min, and remove the supernatant with a pipette; then add 1 mL of 0.5% PBSA again, rotate and incubate at 4°C for another 5 min, and remove the supernatant.

[0098] (2) Yeast display library screening

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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 the incubation, flow detection was performed. Finally, the monoclonal 194-3-D6 with a high positive rate was selected. 194-3-D6 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).

[0103] Example 4

[0104] Expression of recombinant single domain antibodies and detection of binding to target proteins

[0105] The candidate 194-3-D6 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-D6 VHH sequence, CMV promoter and IgG1 Fc sequence were connected using a PCR instrument. 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 194-3-D6.

[0106] The binding specificity of 194-3-D6 was tested. The culture supernatant containing 194-3-D6 was mixed with 3×10 5 CHO-S cells or 3×10 5CHO-S-CD147 cells were incubated at 25°C for 1 h. After incubation, the cells were centrifuged at 25°C at 800×g for 5 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. 100 μL of PE-labeled Anti-human IgG antibody (1:500 dilution) was added and incubated at 25°C for 45 minutes. The cells were protected from light during incubation. After incubation, the cells were centrifuged at 25°C at 800×g for 5 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. After washing, the cells were resuspended in 500 μL of PBS for flow cytometry analysis.

[0107] The results are as follows Figure 1 As shown, the expression supernatant of the negative control group had no significant binding to CHO-S or CHO-S-CD147; the positive control antibody had significant binding 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. 194-3-D6 did not bind to CHO-S cells, but had significant binding to CHO-S-CD147, and most CHO-S-CD147 cells expressing CD147 protein could be detected, indicating that 194-3-D6 had good affinity and specificity.

[0108] Example 5

[0109] Purification of recombinant antibodies and determination of half effective concentration (EC50)

[0110] To determine the EC50 value of the 194-3-D6 antibody, the relevant expression plasmid was transiently transfected into 293F cells, and the cells were cultured in a shaking flask to promote the expression of the antibody, followed by purification of the antibody. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30 mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20 mg IgG / mL Protein A magnetic beads) to the 293F cell shaking flask according to the required amount of the sample. Incubate at 120 rpm and 25°C for 4 h in a shaking incubator. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50 ml centrifuge tube. Wash twice with 30 ml PBS buffer and deionized water, respectively, and resuspend with 1 ml elution buffer. After incubation at 25°C for 5 minutes, collect the magnetic beads with a magnetic separation rack, and transfer the supernatant containing the target antibody to a 15 ml centrifuge tube. After eluting the Protein A magnetic beads twice, the eluates were combined and the neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with PBS at least 100 times the volume of the sample, first dialyzed at 25°C for 2 hours, and then dialyzed at 8°C for 16 hours after changing the solution once. Finally, the protein concentration was determined, and the sample was filtered with a 0.22 μm sterile filter membrane and aliquoted, and stored in a -80°C refrigerator for later use.

[0111] The 194-3-D6 antibody was serially diluted and mixed with 3×10 5 CHO-S-CD147 cells were incubated at 25°C for 1 h. After incubation, the cells were centrifuged at 25°C at 600×g for 7 minutes. The supernatant was discarded and the cells were washed 3 times with PBS. 100 μL of PE-labeled Anti-human IgG antibody diluted 1:500 was added, mixed thoroughly, and incubated at 25°C for 45 minutes. The cells were protected from light during incubation. Centrifuged at 800×g at 25°C for 5 minutes, the supernatant was discarded, the cells were washed 3 times with PBS, and resuspended with 200 μL PBS for flow cytometry analysis.

[0112] The results are as follows Figure 2 and Figure 3 As shown, the EC of 194-3-D6 on CHO-S-CD147 50 The value was 0.74 μg / mL, indicating that 194-3-D6 had a higher affinity.

[0113] Example 6

[0114] Binding detection of 194-3-D6 antibody to colorectal cancer cells

[0115] In order to detect the binding ability of 194-3-D6 antibody to tumor cells, the intestinal cancer cell HCT116 was incubated with 194-3-D6 antibody and positive control antibody at 25℃ for 1 h, and the binding test was performed. After 1 h, the cells were centrifuged at 25℃ at a speed of 800×g for 5 minutes, and the supernatant was discarded and the cells were washed 3 times with PBS. Then 100 μL APC-labeled Anti-human IgG antibody was added, mixed thoroughly, incubated at 25℃ in the dark for 45 minutes, centrifuged at 800×g for 5 minutes at 25℃, the supernatant was discarded, the cells were washed 3 times with PBS, and resuspended with 200 μL PBS for flow cytometry analysis.

[0116] The results of flow cytometry analysis are as follows Figure 4 As shown, the positive control antibody significantly binds to HCT116, indicating that HCT116 expresses CD147 protein and can be used as a detection cell for alpaca antibodies; 194-3-D6 significantly binds to HCT116 and can detect 99.6% of HCT116 cells expressing CD147 protein, indicating that 194-3-D6 has a high affinity for the CD147 protein of HCT116.

[0117] Example 7

[0118] 194-3-D6 antibody ADCC function test

[0119] In order to detect the ability of 194-3-D6 antibody to mediate NK cell killing of tumor cells, ovarian cancer cells SK-OV-3 (expressing red fluorescent reporter protein mCherry) were selected to detect the ADCC function of the target antibody. After washing the target cells three times with OptiVitro NK cell expansion medium, 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 194-3-D6 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 IncuCyte S×5 software. The lower the signal value, the fewer cells in the group and the better the NK cell killing effect. Figure 5 As shown, compared with the NK cell group without antibody, 194-3-D6 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 194-3-D6 can enhance the killing function of NK cells against SK-OV-3 cells 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] BVH147-005 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CD147 single-domain antibody 194-3-D6 [VHH (194-3-D6) 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 molecule expression sequence was fully synthesized and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning, so that it was expressed under the regulation of the human EF-1α promoter and Kozak sequence. The above lentiviral vector expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G using the transfection reagent Lipofectamine 3000 according to the relevant instructions. The viral supernatant was collected 48 h after transfection, centrifuged at 4°C at a speed of 3000 rpm for 15 minutes, and then filtered through a 0.45 μm pore size filter membrane, and finally ultracentrifuged at 4°C at a speed of 25000 rpm for 3 h. The obtained virus concentrate was stored at -80°C and named BVH147-005. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0133] (3) CAR-T cell preparation

[0134] PBMCs from healthy donors were resuscitated 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, and lentivirus was added at an MOI of 5 TU / mL for transduction. At the same time, 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, and 300 IU / mL recombinant hIL-2 were supplemented and cultured in a cell culture incubator (culture temperature was 37°C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 2×10 6 / mL, and supplemented with 300 IU / mL of hIL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for 5 days (culture temperature was 37°C, carbon dioxide concentration was 5%), during which the cell density was maintained at 2×10 6 / mL. The cells were collected on the 10th day after transduction and frozen in liquid nitrogen for future use. The obtained CAR-T cells were named after the corresponding CAR molecules, and the T cells not transduced with lentivirus were named Ctrl T.

[0135] (4) Detection of CAR molecule expression

[0136] The BVH147-005 CAR-T cells to be tested were washed twice with PBS and resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, the FITC-labeled anti-VHH antibody was incubated with the CAR-T cells for 1 h. The supernatant was then removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Ctrl T cells as a negative control, the CAR molecule expression rate of BVH147-005 cells was detected by flow cytometry. The results are shown in Figure 6 As shown in Figure B, the expression rate of BVH147-005 CAR was 25.2%.

[0137] Example 9

[0138] In vitro killing function detection of CAR-T cells

[0139] In order to detect the killing function of CAR-T cells in vitro, 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 S×5 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 by the IncuCyte S×5 software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells.

[0140] The results are as follows Figure 7 As shown in the figure, compared with Mock-T cells, BVH147-005 cells have a more significant killing effect on HCT116, and the relative fluorescence area at the last time point in the curve is statistically significant P<0.05. This result shows that CAR-T cells constructed based on 194-3-D6 antibodies have a good killing function on CD147-positive tumor cells.

[0141] The applicant declares that the present invention illustrates a pan-tumor antigen antibody D6 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.

[0142] 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.

[0143] 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. A CD147 single domain antibody D6, characterized in that: The CD147 single-domain antibody D6 includes a heavy chain variable region, and the heavy chain variable region includes 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 CD147 single domain antibody D6 according to claim 1, characterized in that: The amino acid sequence of the CD147 single-domain antibody D6 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 CD147 single domain antibody D6 or the IgG antibody Fc segment as described in claim 1 or 2.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CD147 single-domain antibody D6 according to claim 1 or 2.

5. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector according to claim 5.

7. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a signal peptide, the CD147 single-domain antibody D6 according to claim 1 or 2, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

8. The chimeric antigen receptor according to claim 7, characterized in that 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.

9. Use of the CD147 single-domain antibody D6 according to claim 1 or 2 in the preparation of a drug for treating colon cancer or ovarian cancer.

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