A pan-tumor antigen antibody D2 and its application

Abstract

The present invention relates to a pan-tumor antigen antibody D2 and its application, wherein the amino acid sequence of CDR1 of the pan-tumor antigen antibody D2 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the pan-tumor antigen antibody D2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 of the pan-tumor antigen antibody D2 is shown in SEQ ID NO.3. The present invention obtains a pan-tumor antigen antibody with strong affinity to CD147, and clones the pan-tumor antigen antibody sequence into a chimeric antigen receptor expression vector to construct a chimeric antigen receptor T cell with a CD147 single domain antibody as an antigen binding domain, which has a strong ability to kill tumor cells, and the antibody can enhance NK cells to kill tumor cells through ADCC.

Description

Technical Field

[0001] The present invention relates to the field of molecular immunology, and in particular to a pan-tumor antigen antibody D2 and applications thereof. Background Art

[0002] The immunoglobulin superfamily (IgSF) consists of at least one protein with an immunoglobulin domain and plays an important role in intercellular communication. 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. The CD147 molecule is a single-pass transmembrane glycoprotein, and the gene is located at 19p13.3, consisting of 10 exons spanning approximately 12 kb. From the cloned cDNA sequence, it is known that the mRNA length of the CD147 molecule is approximately 1.7 kb, and there are about 115 nucleotides before the N-terminal start as a non-coding region, the coding region encodes 269 amino acids, 21 amino acids are signal peptides, and the middle 185 amino acids constitute the extracellular domain, and a total of 24 amino acids from 206-229 are transmembrane regions.

[0003] CD147 is expressed in almost all tumor cells and is highly expressed in many malignant tumors. It plays 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 the interaction between different cells and non-cellular components in the tumor microenvironment (TM). The invasion and metastasis of malignant tumor cells need to cross 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, and promote the spread and metastasis of malignant tumor cells. In addition, changes in the intracellular C-terminal structure of CD147 affect the aggregation and depolymerization of intracellular skeletal proteins, participate in cell movement and pseudopodia formation, and promote tumor cell metastasis.

[0004] CN112341544A discloses an anti-CD147 antibody and a preparation method and application thereof. The anti-CD147 antibody is formed by connecting the variable region of the antibody heavy chain and the variable region of the light chain through a short peptide, can better retain its affinity activity for the antigen, and has the characteristics of small molecular weight, strong penetration, short half-life and weak antigenicity. It plays an important role and has broad application prospects in the clinical diagnosis, treatment and prevention of diseases.

[0005] The research carriers of ordinary antibodies are mainly mice and rabbits, while 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 only contains 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, multivalent construction, etc., and have low production costs and are suitable for large-scale production. Fourth, it is highly specific. The alpaca’s immune system can produce antibodies that are highly specific and have affinity for specific antigens, and have weak immunogenicity to humans, so the risk of causing an immune response is low.

[0006] 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, bile duct cancer and nasopharyngeal carcinoma (CD147 Is a Promising Target of Tumor Progression and a Prognostic Biomarker, Cancers (Basel), 2019 Nov 16;11(11):1803). This shows that CD147 is a suitable therapeutic target. 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.) (CD147: an integral and potential molecule to abrogate hallmarks of cancer, "Front Oncol", 2023 Nov 7;13:1238051). At the same time, alpaca antibodies have many advantages over ordinary antibodies, so the application of relevant technologies to prepare CD147 alpaca antibodies, made into antibody drugs or cell drugs, can be used for the treatment of cancer.

[0007] In summary, how to provide an anti-CD147 single domain antibody with high specificity and high affinity, and apply it to the preparation of chimeric antigen receptor cells targeting CD147 to enhance the ability of NK cells to kill tumor cells through ADCC has become one of the urgent problems to be solved in this field. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a pan-tumor antigen antibody D2 and its application. The present invention uses CD147 protein to immunize alpacas, and obtains a single-domain antibody with strong affinity for 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.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In the first aspect, the present invention provides a pan-tumor antigen antibody D2, the amino acid sequence of CDR1 of the pan-tumor antigen antibody D2 is shown as SEQ ID NO.1, the amino acid sequence of CDR2 of the pan-tumor antigen antibody D2 is shown as SEQ ID NO.2, and the amino acid sequence of CDR3 of the pan-tumor antigen antibody D2 is shown as SEQ ID NO.3.

[0011] SEQ ID NO. 1: GGIFSTSA.

[0012] SEQ ID NO. 2: INWSGIAT.

[0013] SEQ ID NO. 3: AAHSGAKWLRPPSYDY.

[0014] Preferably, the amino acid sequence of the heavy chain variable region of the pan-tumor antigen antibody D2 is as shown in SEQ ID NO.4.

[0015] SEQ ID NO.4: EVQLVESGGGLVQPGGSLRLSCAASGGIFSTSAMAWFRQAPGKEREFVAAINWSGIATTYGDSVKGRFTISRDDTKKTLYLQMNNLKPEDAAVYYCAAHSGAKWLRPPSYDYWGQGTQVTVSS.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the pan-tumor antigen antibody D2 described in the first aspect.

[0017] In a third aspect, the present invention provides a heavy chain antibody, wherein the heavy chain antibody comprises the pan-tumor antigen antibody D2 described in the first aspect and Ig Fc.

[0018] Preferably, the Ig Fc comprises any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

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

[0020] SEQ ID NO.5: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0021] Preferably, the amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID NO.6.

[0022] SEQ ID NO.6: EVQLVESGGGLVQPGGSLRLSCAASGGIFSTSAMAWFRQAPGKEREFVAAINWSGIATTYGDSVKGRFTISRDDTKKTLYLQMNNLKPEDAAVYYCAAHSGAKWLRPPSYDYWGQGTQVTVSSGGCCACGAAGGCCAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] In a fourth aspect, the present invention provides a chimeric antigen receptor, comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain, wherein the antigen binding domain comprises the pan-tumor antigen antibody D2 described in the first aspect.

[0024] Preferably, the signal peptide comprises any one of the signal peptides of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ protein.

[0025] Preferably, the hinge region comprises any one of the hinge regions of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ proteins.

[0026] Preferably, the transmembrane domain includes any one of the transmembrane domains of 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 protein.

[0027] Preferably, the signal transduction domain comprises an intracellular co-stimulatory domain and a CD3 intracellular signal transduction domain.

[0028] Preferably, the intracellular co-stimulatory domain includes any one of the intracellular co-stimulatory domains of 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 protein.

[0029] Preferably, the CD3 intracellular signal transduction domain comprises any one of the intracellular signal transduction domains of CD3ζ, CD3ε, CD3γ or CD3δ proteins.

[0030] Preferably, the signal peptide comprises a CD8 signal peptide, and the amino acid sequence of the CD8 signal peptide comprises the sequence shown in SEQ ID NO.7.

[0031] SEQ ID NO.7: MALPVTALLLPLALLLHAARPS.

[0032] Preferably, the hinge region comprises an IgG4 hinge region, and the amino acid sequence of the IgG4 hinge region comprises the sequence shown in SEQ ID NO.8.

[0033] SEQ ID NO.8: ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0034] Preferably, the transmembrane domain includes a CD8 transmembrane domain, and the amino acid sequence of the CD8 transmembrane domain includes the sequence shown in SEQ ID NO.9.

[0035] SEQ ID NO.9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0036] Preferably, the signal transduction domain includes a 4-1BB intracellular co-stimulatory domain and a CD3ζ intracellular signal transduction domain, the 4-1BB intracellular co-stimulatory domain includes the sequence shown in SEQ ID NO.10, and the CD3ζ intracellular signal transduction domain includes the sequence shown in SEQ ID NO.11.

[0037] SEQ ID NO. 10: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0038] SEQ ID NO. 11: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0039] Preferably, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO.12.

[0040] SEQ ID NO.12: AATMALPVTALLLPLALLLHAARPSAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGGIFSTSAMAWFRQAPGKEREFVAAINWSGIATTYGDSVKGRFTISRDDTKKTLYLQM NNLKPEDAAVYYCAAHSGAKWLRPPSYDYWGQGTQVTVSSEPKTPKPQPAAASGHEWESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0041] In a fifth aspect, the present invention provides an expression vector, wherein the expression vector contains the gene encoding the chimeric antigen receptor described in the fourth aspect.

[0042] Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor described in the fourth aspect, preferably a lentiviral vector.

[0043] In a sixth aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the fifth aspect.

[0044] Preferably, the recombinant lentivirus is prepared by transfecting mammalian cells with the expression vector and helper plasmid described in the fifth aspect.

[0045] 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.

[0046] Preferably, the chimeric antigen receptor immune cell comprises the expression vector described in the fifth aspect and / or the recombinant lentivirus described in the sixth aspect.

[0047] Preferably, the chimeric antigen receptor immune cell comprises any one or a combination of at least two of T cells, TIL cells, B cells, natural killer cells, mast cells or macrophages.

[0048] In an eighth aspect, the present invention provides a pharmaceutical composition, comprising the pan-tumor antigen antibody D2 described in the first aspect or the chimeric antigen receptor immune cell described in the seventh aspect, and further comprising natural killer cells.

[0049] The present invention combines pan-tumor antigen antibodies with NK cells to enhance the ability of NK cells to kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC).

[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 carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0052] In the ninth aspect, the present invention provides the use of any one of the pan-tumor antigen antibody D2 described in the first aspect, the nucleic acid molecule described in the second aspect, the heavy chain antibody described in the third aspect, the chimeric antigen receptor described in the fourth aspect, the chimeric antigen receptor immune cell described in the seventh aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of tumor treatment drugs.

[0053] Preferably, the tumor comprises a tumor expressing CD147;

[0054] Preferably, the tumor includes any one of colorectal cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, bile duct cancer, lung cancer or nasopharyngeal cancer, or a combination of at least two of them.

[0055] Preferably, the present invention provides the use of any one of the pan-tumor antigen antibody D2 described in the first aspect, the nucleic acid molecule described in the second aspect, the heavy chain antibody described in the third aspect, the chimeric antigen receptor described in the fourth aspect, the chimeric antigen receptor immune cell described in the seventh aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of a drug for killing CD147-positive tumor cells.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The present invention uses CD147 protein to immunize alpacas, and obtains a single-domain antibody with strong affinity for CD147 through the steps of separation, screening and picking monoclonal antibodies, and clones the single-domain antibody sequence into a chimeric antigen receptor expression vector to construct a chimeric antigen receptor T cell with the CD147 single-domain antibody as the antigen binding domain, which has good tumor killing effect and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 These are the amplification results of the alpaca antibody VHH gene, where Figure A is the first round of PCR amplification of the heavy chain antibody fragment, and Figure B is the second round of PCR amplification of the VHH region gene fragment.

[0059] Figure 2 This is a diagram showing the enrichment of the yeast library before and after sorting detected by flow cytometry.

[0060] Figure 3 This is a graph showing the results of flow cytometry testing of the specificity of recombinant single domain antibodies.

[0061] Figure 4 Detection of EC of recombinant single domain antibodies by flow cytometry 50 Result graph.

[0062] Figure 5 This is a graph showing the concentration-effect curve of alpaca antibodies detected by flow cytometry.

[0063] Figure 6 This is a graph showing the results of flow cytometry detection of the binding of recombinant single domain antibodies to colorectal cancer HCT116 cells.

[0064] Figure 7 This is the result graph of the IncuCyte real-time quantitative living cell imaging analysis of recombinant single-domain antibody-mediated ADCC function, where Figure A is a curve graph of the change of relative fluorescence area over time, and Figure B is a statistical graph of the relative fluorescence area value at the last time point recorded in Figure A.

[0065] Figure 8 Figure 1 is a diagram showing the structure of the chimeric antigen receptor and the expression of the chimeric antigen receptor in CAR-T cells, where Figure A is a schematic diagram of the chimeric antigen receptor structure, and Figure B is a diagram showing the expression rate detection results of the chimeric antigen receptor in CAR-T cells.

[0066] Fig. 9 This is the result of IncuCyte real-time quantitative living cell imaging analysis of the killing function of CAR-T cells targeting CD147, where Figure A is a curve chart of the change of relative fluorescence area over time, and Figure B is a statistical chart of the relative fluorescence area value at the last time point recorded in Figure A. DETAILED DESCRIPTION

[0067] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0068] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0069] Reagents used in the following examples:

[0070] PBS (bico, CAT# 14190-250);

[0071] Adjuvant immune adjuvant (GERBU, CAT# 3030);

[0072] HRP-ProteinA(Boster, CAT# BA1080);

[0073] HRP-Streptavidin(Boster, CAT# BA1088);

[0074] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B);

[0075] DNA fragment recovery kit (TakaRa, CAT# 9761);

[0076] Goat anti-Llama IgG (H+L)Secondary Antibody [HRP] (Novus, AT#NB7242);

[0077] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805);

[0078] Streptavidin PE (eBioscience, CAT# 12-4317-87);

[0079] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82);

[0080] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014);

[0081] Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CAT#L3000001).

[0082] Example 1 Alpaca immunization and antibody titer determination

[0083] Alpaca 74# was immunized with CD147 protein, and the immunization experiment was started after recording the ear number of the blank alpaca. Each time, the alpaca was 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, observe for half an hour to confirm that the alpaca is in good condition and has no discomfort symptoms. Immunization is performed once every 3 weeks, and at least 3 immunizations are performed. 5 mL of peripheral blood was collected before immunization, two weeks after the second immunization, and the third immunization, respectively. The centrifuge tube containing the blood sample was placed in a centrifuge, centrifuged at 800×g for 10 min, and the upper serum was collected. 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; use PBS to graded dilutions of the above-collected serum, add 100 µL of graded diluted serum to the 96-well ELISA plate, incubate at room temperature for 1 hour, and use PBS as the control well; 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; shake off the liquid in the well and wash the plate 5 times with PBST; add 100 µL / well TMB colorimetric solution; incubate at room temperature in the dark for 10-15 minutes; add 50 µL / well stop solution; read the OD in the well using a microplate reader 450 value.

[0084] The titer test results of the serum after immunization of 74# alpaca are shown in Table 1. The binding titer of the serum before immunization of alpaca with CD147 protein is lower than 1000, indicating that the content of CD147 specific antibody contained therein is extremely low. The binding titer of the serum after the third immunization of 74# alpaca with CD147 protein is higher than 64k, indicating that the content of CD147 specific antibody in the serum of alpaca has increased significantly after multiple immunizations, and the immunization is successful, which can be used for the construction of yeast display library.

[0085] Table 1

[0086]

[0087] Example 2 Construction of single domain antibody yeast display library

[0088] (1) VHH antibody fragment cloning

[0089] After confirming that the alpaca serum contained CD147-specific antibodies, 100 mL of peripheral blood was collected, PBMC was 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, the alpaca heavy chain antibody sequence was amplified by PCR using specific primers. The upstream primer was bound to the signal peptide of the VHH antibody ORF, and the downstream primer was bound to the CH2 region. The results are shown in Figure 2. Figure 1 As shown, 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 the SfiI restriction site GGCCCAGCCGGCC; the downstream primer was bound to the antibody hinge region and FR4 region, and the 5' end contained the SfiI restriction site GGCCACGAAGGCC. 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 ( Figure 1 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).

[0090] (2) Electroporation of library vectors and detection of library capacity and diversity

[0091] The yeast surface display vector pYDisplay and the VHH fragment library obtained above were digested with SfiI endonuclease. The pYDisplay vector fragment was separated by 1% agarose gel, and the 5000 bp vector fragment was cut for gel recovery. The yeast competent strain frozen at -80℃ was streaked onto a YPD solid medium plate in advance and activated at 30℃ for 3-5 days. A single colony of yeast competent strain was inoculated into 50 mL YPD medium and shaken at 250 rpm and 30℃ for 1-2 days. After mixing the linearized vector fragment and PCR product, add them to the electroporation cup and electroporate; the yeast competent strain after electroporation was transferred to a culture bottle and shaken at 220 rpm and 30℃ for 1 h. Take 20 μL of the resuspension, dilute 5000 times with SDCAA medium, take 100 μL, apply it to the SDCAA plate, culture for 2-3 days, calculate the reservoir capacity, and continue to culture the rest of the bacterial solution for 24 h. The remaining bacterial liquid was collected into a 50 mL centrifuge tube, centrifuged at 3000×g for 5 min, the supernatant was discarded, 10 mL SDCAA was added for resuspending, 50% glycerol: resuspending liquid was mixed at a ratio of 1:1, and stored at -80°C. The results showed that the single domain antibody yeast display library obtained from 74# alpaca PBMC had a library capacity of 1.3×10 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.

[0092] Example 3 Panning of yeast display library

[0093] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0094] Take 5-10 mL of yeast library (about 2×10 8 Yeast cells) were transferred to a 50 mL centrifuge tube, centrifuged at 3000 × g for 5 min, and the supernatant was discarded; at the same time, sterile 5% PBSA (PBS + 5% BSA) was diluted to 0.5% PBSA; the yeast library was resuspended with 1 mL 0.5% PBSA, added to a 1.5 mL centrifuge tube, centrifuged at 3000 × g for 5 min, and the supernatant was discarded; and it was washed 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 rack 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.

[0095] (2) Yeast display library screening

[0096] 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 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 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.

[0097] Biotin-CD147-His magnetic separation: add 100 μL of 50 μg / mL CD147-His protein solution (biotin-coupled, diluted with 0.5% PBSA) to the centrifuge tube containing streptavidin magnetic beads, put 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, and put them into a bag and place them on a rotating mixer. Incubate them at 4°C for 60 min. After the incubation, place them on a magnetic rack and let them stand at room temperature 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 and let it stand at room temperature for 5 min. Discard the supernatant. Repeat the washing twice. The entire operation is performed in accordance with the aseptic operation requirements. After washing, resuspend the magnetic beads and adhered yeast cells with 1 mL of SDCAA medium, pipette 20 μL of the resuspended solution into 180 μL of SDCAA medium and coat two plates, each with a coating volume of 100 μL, then pipette 5 μL of the resuspended solution into 95 μL of SDCAA medium and coat one plate, i.e. a total of 3 plates need to be coated.

[0098] Yeast flow cytometry after panning: The yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into the yeast. The yeast that has undergone negative panning with empty magnetic beads and biotin-CD147-His magnetic separation was incubated with CD147 protein containing the His tag at 4°C for 60 min, then centrifuged to remove the supernatant, added anti-His tag and anti-V5 tag flow cytometry antibodies, incubated at 4°C for 60 min, then centrifuged to remove the supernatant, resuspended the cells in 500 μL PBS, and analyzed by flow cytometry. The results are shown in Figure 2 As shown, the number of yeasts that can bind to CD147 protein in the yeast antibody display library of 74# alpaca increased by 41% before and after sorting, and subsequent experiments can be carried out.

[0099] 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, incubated at 4°C for 60 min, and then the supernatant was removed by centrifugation. The precipitate was resuspended with a solution containing PE-labeled streptavidin, incubated at 4°C for 60 min, and flow detection was performed after the incubation was completed. Finally, the monoclonal 74-1-D2 with a high positive rate was selected. 74-1-D2 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).

[0100] Example 4 Expression of recombinant single domain antibodies and detection of binding to target proteins

[0101] Alpaca was immunized with CD147 protein, and an alpaca antibody 74-1-D2 was obtained through serum titer detection, alpaca antibody yeast library construction, yeast library screening and monoclonal antibody picking. The candidate 74-1-D2 VHH sequence, CMV promoter sequence and IgG1 Fc sequence were amplified by PCR instrument, and the amino acid sequence of IgG1 Fc is shown in SEQ ID NO.5.

[0102] SEQ ID NO.5: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0103] Take 50 μL of PCR product, add 1 / 10 volume of 10× loading buffer, and use 1% agarose for electrophoresis analysis. The band size of CMV is 750 bp, the band size of Fc is 1400 bp, and the band size of VHH is 500 bp. Cut the target band from the gel and purify the PCR product. Use a PCR instrument to connect the 74-1-D2 VHH sequence, CMV promoter and IgG1 Fc sequence. The amino acid sequence after connection is shown in SEQ ID NO.6.

[0104] SEQ ID NO.6: EVQLVESGGGLVQPGGSLRLSCAASGGIFSTSAMAWFRQAPGKEREFVAAINWSGIATTYGDSVKGRFTISRDDTKKTLYLQMNNLKPEDAAVYYCAAHSGAKWLRPPSYDYWGQGTQVTVSSGGCCACGAAGGCCAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0105] Then, 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and electrophoresis analysis was performed using 1% agarose. The target band was cut out 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-D2.

[0106] Detection of the binding specificity of 74-1-D2: The culture supernatant of 74-1-D2 was mixed with 3×10 5 CHO-S or CHO-S-CD147 cells (CHO-S cells overexpressing CD147 protein) were incubated at room temperature for 1.5 hours. After centrifugation at 800×g for 10 min at room temperature, the supernatant was discarded and the cells were washed 4 times with PBS. 100 μL of PE-labeled anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature in the dark for 60 min. After centrifugation at 800×g for 10 min at room temperature, the supernatant was discarded and the cells were washed 4 times with PBS. The cells were resuspended in 200 μL of PBS for flow cytometry analysis.

[0107] The results are as follows Figure 3 As shown, the expression supernatant of the control antibody in the negative control group did not significantly bind 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-D2 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-D2 had good affinity and specificity.

[0108] Example 5 Purification and half effective concentration (EC 50 ) Determination

[0109] To determine the half effective concentration (EC) of the 74-1-D2 antibody 50, the relevant expression plasmid (VHH antibody coupled with human IgG fragment) was transiently transfected into 293F cells, and the cells were cultured in a shaking flask to express and purify the antibody. The protein A magnetic beads were washed twice with 30 mL PBS buffer, 0.1 M sodium hydroxide, and PBS buffer in sequence. According to the required amount of the sample, the corresponding volume of protein A magnetic beads (calculated as 20 mg IgG / mL protein A magnetic beads) was added to the 293F cell shaking flask. Shake at 120 rpm in a shaking incubator and incubate at room temperature for 4 hours. The protein A magnetic beads were collected with a magnetic separation rack and transferred to a 50 mL centrifuge tube. After washing twice with 40 mL PBS buffer and deionized water, the beads were resuspended with 1 mL elution buffer. After incubation at room temperature for 10 min, the beads were placed in a magnetic separation rack for 10 min, the magnetic beads containing the target antibody were collected, and the magnetic beads containing the target antibody were transferred to a 15 mL centrifuge tube. After eluting the protein A magnetic beads twice, the eluate was combined and the neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with 100 times the volume of PBS, first at 18-25°C for 2 h, then the solution was changed once and then dialyzed at 2-8°C for 14-16 h. Finally, the protein concentration was determined and the sample was filtered with a 0.22 μm sterile filter membrane and packaged, and stored in a -80°C refrigerator for later use.

[0110] The 74-1-D2 antibody was serially diluted and then mixed with 3×10 5 CHO-S-CD147 cells were incubated at room temperature for 1 hour. Centrifuged at 800×g for 5 min at room temperature, discarded the supernatant, and washed the cells three times with PBS. 100 μL of PE-labeled anti-human IgG antibody (1:500 dilution) was added, mixed thoroughly, and incubated at room temperature in the dark for 45 min. Centrifuged at 800×g for 5 min at room temperature, discarded the supernatant, washed the cells three times with PBS, and resuspended the cells with 200 μL PBS for flow cytometry analysis.

[0111] The results are as follows Figure 4 and Figure 5 As shown, the EC of 74-1-D2 on CHO-S-CD147 50 The value was 0.66 μg / mL, indicating that 74-1-D2 had a higher affinity.

[0112] Example 6 Detection of the binding of recombinant antibodies to colorectal cancer cells

[0113] In order to detect the binding of 74-1-D2 antibody to tumor cells, intestinal cancer cells HCT116 were incubated with 74-1-D2 antibody and positive control antibody at room temperature for 1 h. Then centrifuged at 800×g for 5 min at room temperature, discarded the supernatant, and washed the cells 3 times with PBS. Add 100 μL APC-labeled anti-human IgG antibody, mix well, and incubate at room temperature in the dark for 50 min. Then centrifuged at 800×g for 5 min at room temperature, discarded the supernatant, washed the cells 3 times with PBS, resuspended the cells with 200 μL PBS, and analyzed by flow cytometry. The results are shown in Figure 2. Figure 6 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; 74-1-D2 significantly binds to HCT116 and can detect 99.8% of HCT116 cells expressing CD147 protein, indicating that 74-1-D2 has a high affinity for the CD147 protein of HCT116.

[0114] Example 7 ADCC functional detection

[0115] In order to detect the cytotoxicity (ADCC) function of 74-1-D2 antibody, SK-OV-3 cells (ovarian cancer cells overexpressing red fluorescent protein) were used as target cells and NK cells were used as effector cells for relevant experiments. After washing the target cells SK-OV-3 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 / well and cultured overnight in the real-time quantitative live cell imaging and analysis platform IncuCyte instrument. NK cells were added at an effector-target ratio of 8:1, and the target antibody 74-1-D2 was added at a final concentration of 5 μg / mL.

[0116] The data were recorded throughout the process using the real-time quantitative live cell imaging and analysis platform IncuCyte. The changes in the fluorescence signals of target cells in each group were calculated using the IncuCyte S×5 software. The lower the signal value, the fewer cells in the group and the better the NK cell killing effect. Figure 7 As shown in Figures A and B, compared with the NK cell group without antibody, the 74-1-D2 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-D2 can enhance the killing function of NK cells against SK-OV-3 ovarian cancer cells through antibody-dependent cell-mediated ADCC.

[0117] Example 8 Construction of CAR-T cells based on target antibodies

[0118] (1) Design of CAR molecules

[0119] The target gene structure of the lentiviral vector of the CAR molecule involved in this example is as follows Figure 8 As shown in Figure A, BVH147-008 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CD147 single-domain antibody 74-1-D2 [VHH (74-1-D2) for short], human IgG4 hinge region (IgG4 hinge for short), human CD8 transmembrane domain (human CD8TM for short), human 4-1BB intracellular co-stimulatory domain (4-1BB ICD for short), and human CD3ζ intracellular signal transduction domain (CD3ζICD for short).

[0120] (2) Lentivirus preparation

[0121] 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 3000 rpm for 10-15 min at 4°C, filtered through a 0.45 μm pore size filter membrane, and finally ultracentrifuged at 25000 rpm at 4°C for 2-3 h. The obtained virus concentrate was stored at -80°C and named BVH147-008. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0122] (3) CAR-T cell preparation

[0123] PBMCs from healthy donors were revived with 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 placed in a cell culture incubator for 24 h (culture temperature was 37°C, carbon dioxide concentration was 5%). After 24 h, T cells were washed with AIM V medium, 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 placed in a cell culture incubator (culture temperature was 37°C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 1.5×10 6 -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 1×10 6 -2×10 6 Cells were collected on the 12th day after transduction and frozen in liquid nitrogen for later 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.

[0124] (4) Detection of CAR molecule expression

[0125] The BVH147-008 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 BVH147-008 cells was detected by flow cytometry. The results are shown in Figure 8 As shown in Figure B, the expression rate of BVH147-008 CAR was 24.4%.

[0126] Example 9 Study on the function of CAR-T cells in vivo and in vitro

[0127] In order to detect the killing function of BVH147-008 CAR-T cells, HCT116-Mcherry cells (colon cancer cells overexpressing red fluorescent protein) were used as target cells. 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 using 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. The results are shown in Fig. 9As shown, Figure A is the curve of the change of relative fluorescence area over time, and Figure B is the total fluorescence intensity of the last point recorded in Figure A. Compared with Mock-T cells, BVH147-008 cells have a 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 74-1-D2 antibodies have a good killing function on CD147-positive tumor cells.

[0128] In summary, the present invention uses CD147 protein to immunize alpacas, and obtains a single-domain candidate antibody 74-1-D2 with strong affinity for CD147 through serum titer detection, yeast library construction, yeast library screening and monoclonal antibody picking. The specific binding of 74-1-D2 to CD147 is detected by ELISA. After the 74-1-D2 antibody is expressed and purified, its EC binding to CD147 protein is detected. 50 Value and binding ability to tumor cells. 74-1-D2 antibody was co-cultured with NK cells and SK-OV-3 ovarian cancer cells to detect antibody-dependent cell-mediated ADCC of each clone. At the same time, 74-1-D2 antibody was cloned into the CAR expression vector to construct CAR-T cells with 74-1-D2 antibody as antigen binding domain, and its killing ability against CD147-positive HCT116 cells in vitro was detected. The results showed that alpaca antibodies have high specificity, and the ADCC and CAR-T mediated by them have good tumor killing effects.

[0129] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-CD147 single domain antibody D2, characterized in that: The anti-CD147 single-domain antibody D2 includes CDR1, CDR2 and CDR3 of the heavy chain variable region, 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, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.

3.

2. The anti-CD147 single domain antibody D2 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-CD147 single-domain antibody D2 is shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD147 single-domain antibody D2 according to claim 1 or 2.

4. A heavy chain antibody, characterized in that: The heavy chain antibody comprises the anti-CD147 single domain antibody D2 and Ig Fc according to claim 1 or 2; The Ig Fc includes any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

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 and a signal transduction domain, and the antigen binding domain comprises the anti-CD147 single-domain antibody D2 according to claim 1 or 2.

6. The chimeric antigen receptor according to claim 5, characterized in that The signal peptide includes any one of the signal peptides of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ proteins; The hinge region includes any one of the hinge regions of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ proteins; The transmembrane domain includes any one of the transmembrane domains of 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 protein; The signal transduction domain includes an intracellular co-stimulatory domain and a CD3 intracellular signal transduction domain; The intracellular co-stimulatory domain includes any one of the intracellular co-stimulatory domains of 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 protein; The CD3 intracellular signal transduction domain includes any one of the intracellular signal transduction domains of CD3ζ, CD3ε, CD3γ or CD3δ proteins.

7. The chimeric antigen receptor according to claim 5, characterized in that The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

12.

8. A chimeric antigen receptor immune cell, characterized in that The chimeric antigen receptor immune cell expresses the chimeric antigen receptor according to any one of claims 5 to 7; The chimeric antigen receptor immune cells include any one of T cells, B cells, natural killer cells, mast cells or macrophages, or a combination of at least two of them.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-CD147 single domain antibody D2 according to claim 1 or 2 or the chimeric antigen receptor immune cell according to claim 8, and the pharmaceutical composition further comprises natural killer cells.

10. Use of any one of the anti-CD147 single domain antibody D2 according to claim 1 or 2, the nucleic acid molecule according to claim 3, the heavy chain antibody according to claim 4, the chimeric antigen receptor according to any one of claims 5 to 7, the chimeric antigen receptor immune cell according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of a drug for killing colon cancer cells or ovarian cancer cells.

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