A pan-tumor antigen antibody D10-3 and its application

Abstract

The present invention relates to a pan-tumor antigen antibody D10-3 and its application, wherein the amino acid sequence of CDR1 of the pan-tumor antigen antibody D10-3 includes the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the pan-tumor antigen antibody D10-3 includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 of the pan-tumor antigen antibody D10-3 includes the sequence shown in SEQ ID NO.3. The present invention uses CD147 protein to immunize alpacas, screens and obtains a pan-tumor antigen antibody D10-3 with high specificity and strong affinity, which can be applied to immunotherapy and is of great significance for the development of tumor treatment drugs.

Description

Technical Field

[0001] The present invention relates to the field of molecular immunology, and in particular to a pan-tumor antigen antibody D10-3 and an application thereof. Background Art

[0002] 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.

[0003] CN108261544A discloses a pharmaceutical preparation comprising a CD147 monoclonal antibody, a buffer, a protein protectant and a surfactant, wherein the CD147 monoclonal antibody comprises a heavy chain and a light chain, and the pharmaceutical preparation can maintain the stability of the CD147 monoclonal antibody for a long time. It also discloses the use of the pharmaceutical preparation in preparing a drug for treating CD147-related diseases, especially non-small cell lung cancer, and a method for preparing the pharmaceutical preparation.

[0004] There is a special antibody lacking light chains in the blood of camelids and cartilaginous fish, namely heavy chain antibodies. Compared with ordinary antibodies, it contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single-domain antibody (also known as a nano-antibody) consisting only of the heavy chain variable region can be obtained, which has the advantages of small molecular weight, strong tissue penetration, high stability, and low immunogenicity. Compared with ordinary antibodies, single-domain antibodies have many advantages: First, they are small in size, one-tenth of ordinary antibodies, which enables them to penetrate into tissues and cells, especially the blood-brain barrier. Second, they are highly stable. Single-domain antibodies can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90°C. Third, they have a simple structure. Single-domain antibodies are easier to transform, such as humanization and multivalent construction, and have low production costs and are suitable for large-scale production. Fourth, they are highly specific. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens, and they are weakly immunogenic to humans, with a low risk of causing immune responses.

[0005] 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.

[0006] In summary, how to provide an anti-CD147 single domain antibody with high specificity and high affinity has become one of the problems to be solved urgently in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a pan-tumor antigen antibody D10-3 and its application. The present invention obtains a single-domain antibody with high specificity and strong affinity for CD147, which can be applied to immunotherapy and is of great significance for the development of tumor treatment drugs.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a pan-tumor antigen antibody D10-3, wherein the amino acid sequence of CDR1 of the pan-tumor antigen antibody D10-3 includes the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the pan-tumor antigen antibody D10-3 includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 of the pan-tumor antigen antibody D10-3 includes the sequence shown in SEQ ID NO.3.

[0010] SEQ ID NO.1: GFTFSSYW.

[0011] SEQ ID NO. 2: INTDGSSTY.

[0012] SEQ ID NO. 3: TKVLPVATPRSIFGS.

[0013] Preferably, the amino acid sequence of the heavy chain variable region of the pan-tumor antigen antibody D10-3 includes the sequence shown in SEQ ID NO.4.

[0014] SEQ ID NO.4: EVQLVESGGDLVQPGGSLRLPCAASGFTFSSYWMYWVRQAPGKGLEWVSAINTDGSSTYYAESVKGRFTISRDNAKNTLYLQMNSLKSEDSAMYYCTKVLPVATPRSIFGSWGQGTQVTVSS.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the pan-tumor antigen antibody D10-3 described in the first aspect.

[0016] In a third aspect, the present invention provides a heavy chain antibody, wherein the heavy chain antibody comprises the pan-tumor antigen antibody D10-3 described in the first aspect and Ig Fc.

[0017] Preferably, the Ig Fc comprises any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

[0018] Preferably, the amino acid sequence of the Fc segment of IgG1 includes the sequence shown in SEQ ID NO.5.

[0019] SEQ ID NO.5: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0020] Preferably, the amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID NO.6.

[0021] SEQ ID NO.6: EVQLVESGGDLVQPGGSLRLPCAASGFTFSSYWMYWVRQAPGKGLEWVSAINTDGSSTYYAESVKGRFTISRDNAKNTLYLQMNSLKSEDSAMYYCTKVLPVATPRSIFGSWGQGTQVTVSSGGCCACGAAGGCCAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0022] 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 D10-3 described in the first aspect.

[0023] In a fifth aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0024] Preferably, the expression vector contains the gene encoding the chimeric antigen receptor described in the fourth aspect.

[0025] 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.

[0026] In a sixth aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the fifth aspect.

[0027] Preferably, the recombinant lentivirus is prepared by transfecting mammalian cells with the expression vector and helper plasmid described in the fifth aspect.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In an eighth aspect, the present invention provides a pharmaceutical composition, comprising the pan-tumor antigen antibody D10-3 described in the first aspect or the chimeric antigen receptor immune cell described in the seventh aspect, and further comprising natural killer cells.

[0032] The present invention uses antibody D10-3 in combination with NK cells to enhance the ability of NK cells to kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0033] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0034] 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.

[0035] In the ninth aspect, the present invention provides the use of any one of the pan-tumor antigen antibody D10-3 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 expression vector described in the fifth aspect, the recombinant lentivirus described in the sixth 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.

[0036] Preferably, the tumor comprises a tumor expressing CD147;

[0037] Preferably, the tumor includes any one of liver cancer, bile duct cancer, lung cancer, nasopharyngeal cancer, colorectal cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, ovarian cancer or breast cancer, or a combination of at least two of them.

[0038] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention provides a pan-tumor antigen antibody D10-3, which uses CD147 protein to immunize alpacas, and obtains a single domain antibody with high specificity and strong affinity for CD147 through separation, screening and picking of monoclonal antibodies. Compared with ordinary antibodies, single domain antibodies have the advantages of small molecular weight, strong tissue penetration, high stability, low immunogenicity, etc., and can be used in immunotherapy, which is of great significance for the development of tumor treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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.

[0042] Figure 2 This is a diagram showing the enrichment of the yeast library before and after sorting detected by flow cytometry.

[0043] Figure 3 This is a graph showing the results of flow cytometry testing of the specificity of recombinant single domain antibodies.

[0044] Figure 4 Detection of EC of recombinant single domain antibodies by flow cytometry 50 Result graph.

[0045] Figure 5 This is a graph showing the concentration-effect curve of alpaca antibodies detected by CHO-S-CD147 cell flow cytometry. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] Reagents used in the following examples:

[0049] PBS (bico, CAT# 14190-250)

[0050] Adjuvant immune adjuvant (GERBU, CAT# 3030)

[0051] HRP-ProteinA (Boster, CAT# BA1080)

[0052] HRP-Streptavidin (Boster, CAT# BA1088)

[0053] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B)

[0054] DNA fragment recovery kit (TakaRa, CAT# 9761)

[0055] Goat anti-Llama IgG (H+L)Secondary Antibody [HRP] (Novus, AT# NB7242)

[0056] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805)

[0057] Streptavidin PE (eBioscience, CAT# 12-4317-87)

[0058] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82)

[0059] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014).

[0060] Example 1 Alpaca immunization and antibody titer determination

[0061] 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; 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; use a microplate reader to read the OD450 value in the well.

[0062] The ratio of the absorbance of the test serum to the blank control was calculated. When the ratio was greater than 2, it was positive, and the positive result with the largest dilution multiple was defined as the titer of the antiserum. The results are shown in Tables 1, 2, and 3. The binding titers of the serum of the three alpacas before immunization with the CD147 protein were 8k, 1k, and 32k, respectively. The binding titers of the serum of the three alpacas after the third immunization with the CD147 protein were all much higher than 64k, indicating that the content of CD147-specific antibodies in the serum of the three alpacas was significantly increased after multiple immunizations, and the immunization was successful, which can be used for the construction of yeast display libraries.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067] Table 3

[0068]

[0069] Example 2 Construction of single domain antibody yeast display library

[0070] (1) VHH antibody fragment cloning

[0071] After confirming that the three alpacas' serum contained CD147-specific antibodies, 100 mL of peripheral blood was collected, PBMCs were separated using lymphocyte separation solution, and RNA was extracted. Reverse transcription was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit to obtain cDNA. Using PBMC cDNA as a template, specific primers (the upstream primer binds to the signal peptide of the VHH antibody ORF, the primer sequence is SEQ ID NO.7 (5'-GTCCTGGCTGCTCTTCTACAAGG-3'), the downstream primer binds to the CH2 region, the primer sequence is SEQ ID NO.8 (5'-GGTACGTGCTGTTGAACTGTTCC-3') PCR amplified the alpaca heavy chain antibody sequence. The results are as follows 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, and specific primers (the upstream primer was combined with the antibody FR1 region, and the 5' end contained the SfiI restriction site GGCCCAGCCGGCC, and the primer sequence was SEQ ID NO.9 (5'-ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTGG-3') and the downstream primer was combined with the anti-Hinge and FR4 regions, and the 5' end contained the SfiI restriction site GGCCACGAAGGCC, and the primer sequence was SEQ ID NO.10 (5'-GATCACTAGTGGGGTCTTCGCTGTGGTGCG-3') to amplify the heavy chain antibody VHH fragment. 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).

[0072] (2) Electroporation of library vectors and detection of library capacity and diversity

[0073] 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 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.

[0074] Example 3 Panning of yeast display library

[0075] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0076] Take 5-10 mL of yeast library (about 2×10 8Yeast 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.

[0077] (2) Yeast display library screening

[0078] 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.

[0079] 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 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 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.

[0080] 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, before and after sorting, the yeast capable of binding to CD147 protein in the yeast antibody display libraries of 73# alpaca, 74# alpaca and 194# alpaca increased by 10%, 41% and 13% respectively, which can be used for subsequent experiments.

[0081] 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-labeled streptavidin, and incubated at 4°C for 60 min. After incubation, flow detection was performed. Finally, a monoclonal clone 74-2-D10-3 with a high positive rate was selected. 74-2-D10-3 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).

[0082] Example 4 Expression of recombinant single domain antibodies and detection of binding to target proteins

[0083] The candidate 74-2-D10-3 VHH sequence, CMV promoter sequence and IgG1 Fc sequence were amplified using a PCR instrument. The amino acid sequence of IgG1 Fc is shown in SEQ ID NO.5.

[0084] SEQ ID NO.5: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0085] 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 74-2-D10-3 VHH sequence, CMV promoter and IgG1 Fc sequence. The amino acid sequence after connection is shown in SEQ ID NO.6.

[0086] SEQ ID NO.6: EVQLVESGGDLVQPGGSLRLPCAASGFTFSSYWMYWVRQAPGKGLEWVSAINTDGSSTYYAESVKGRFTISRDNAKNTLYLQMNSLKSEDSAMYYCTKVLPVATPRSIFGSWGQGTQVTVSSGGCCACGAAGGCCAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0087] 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 74-2-D10-3.

[0088] Detection of the binding specificity of 74-2-D10-3: The culture supernatant containing 74-2-D10-3 was mixed with 3×10 5 CHO-S or CHO-S-CD147 cells were incubated at room temperature for 1 hour. After centrifugation at 800×g for 5 min at room temperature, 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 room temperature for 45 min in the dark. After centrifugation at 800×g for 5 min at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500 μL of PBS for flow cytometry analysis.

[0089] The results are as follows Figure 3As 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. 74-2-D10-3 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 74-2-D10-3 had good affinity and specificity.

[0090] Example 5 Purification and half effective concentration (EC 50 ) Determination

[0091] To determine the EC of 74-2-D10-3 antibody 50 , transiently transfect the relevant expression plasmid into 293F cells, and shake the flask to culture for antibody expression and purification. 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, 0.1 M 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 shake flask according to the required amount of the sample. Incubate at room temperature for 1-4 hours or overnight at 4°C in a shaking incubator at 120 rpm. 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, and resuspend with 1 mL elution buffer. After incubation at room temperature for 5 min, 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, combine the eluates and add neutralization buffer 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 18-25°C for 2 h, then dialyzed at 2-8°C for 14-16 h 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 packaged, and stored in a -80°C refrigerator for later use.

[0092] Serial dilutions of the target antibody were added to 3×10 5CHO-S-CD147 cells were incubated at room temperature for 1 h. 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.

[0093] The results are as follows Figure 4 and Figure 5 As shown, the EC of 74-2-D10-3 on CHO-S-CD147 50 The value is 16.81 μg / mL.

[0094] In summary, the present invention uses CD147 protein to immunize alpacas, and after determining the titer of CD147-specific antibodies in alpacas serum by ELISA, alpacas PBMCs are separated, RNA is extracted and reverse transcribed to obtain cDNA, and alpacas single-domain antibody-specific primers are used to amplify VHH sequences, and cloned into yeast expression plasmids to construct yeast display libraries. Then, biotin-CD147 protein-streptavidin magnetic beads or streptavidin magnetic beads are used to perform positive and negative screening on the yeast display library to enrich yeasts that bind to CD147 protein. Monoclones are picked from the enriched products, and the specific binding of candidate antibodies to CD147 is detected by ELISA. After the positive antibodies are expressed and purified, their affinity for binding to CD147 protein is detected. The results show that alpaca antibodies have high specificity and strong affinity.

[0095] 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 D10-3, characterized in that: The anti-CD147 single-domain antibody D10-3 includes CDR1, CDR2 and CDR3 of the heavy chain variable region, the amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

2. The anti-CD147 single domain antibody D10-3 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-CD147 single-domain antibody D10-3 includes the sequence 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 D10-3 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 D10-3 according to claim 1 or 2 and Ig Fc.

5. The heavy chain antibody according to claim 4, characterized in that The Ig Fc includes any one of the Fc segments of IgG1, IgG2, IgG3 or IgG4.

6. The heavy chain antibody according to claim 5, characterized in that The amino acid sequence of the Fc segment of IgG1 includes the sequence shown in SEQ ID NO.

5.

7. The heavy chain antibody according to claim 4, characterized in that The amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID NO.6.

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