An anti-CD147 single domain antibody and its application

Abstract

The present invention relates to an anti-CD147 single-domain antibody and its application, wherein the amino acid sequence of the single-domain antibody comprises CDR1 shown in SEQ ID No. 2, CDR2 shown in SEQ ID No. 3 and CDR3 shown in SEQ ID No. 4. The single-domain antibody of the present invention has strong affinity, and the chimeric antigen receptor T cell with the single-domain antibody as the antigen binding domain has strong killing ability against 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 relates to the field of antibody technology, and in particular to an anti-CD147 single-domain antibody and applications thereof. Background Art

[0002] Single-domain antibodies, also known as nanobodies, VHH antibodies or camelid antibodies, are heavy chain antibodies that naturally lack light chains and are found in camelids such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays. In 1993, Belgian scientists discovered a heavy chain antibody with a natural light chain deficiency in camel serum, with a molecular weight of about 95 kDa, which included two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region. They then cloned a single-domain antibody containing only one heavy chain variable region, namely the VHH antibody. The crystal structure of the VHH antibody is an oval of 4 nm×2.5 nm×3 nm, with a molecular weight of only 1 / 10 of that of an ordinary antibody, about 12-14 kDa. It is the smallest complete antigen-binding fragment, so it is also called a nanobody.

[0003] 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, bile duct cancer, nasopharyngeal carcinoma, etc.), and 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 interactions between different cells and non-cellular components in the tumor microenvironment (TM). The infiltration 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 the invasion and metastasis of tumors. 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 tumor cell metastasis. 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.).

[0004] The research carriers of ordinary antibodies are mainly mice and rabbits. Compared with ordinary antibodies, alpaca antibodies have many advantages: First, they are small in size, one-tenth of ordinary antibodies, which allows them to penetrate into tissues and cells, especially the blood-brain barrier. Second, they are highly stable. Nano antibodies can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90 degrees. Third, they are simple in structure. Nano antibodies are easier to transform, such as humanization and multivalent construction, and have low production costs, making them suitable for large-scale production. Fourth, they are highly specific. The alpaca's immune system can produce antibodies that are highly specific and affinity for specific antigens, and have weak immunogenicity to humans. Nano antibodies have a low risk of causing an immune response.

[0005] CD147 plays an important role in the growth, infiltration and metastasis of tumor cells, and is expressed in various tumor cells, which shows that CD147 is a suitable therapeutic target. 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 cancer treatment.

[0006] Therefore, how to prepare anti-CD147 nanoantibodies has become a problem that needs to be solved urgently. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an anti-CD147 single-domain antibody and its application. The antibody has high affinity and specificity to the CD147 protein. Experiments have shown that the antibody can effectively enhance the killing function of NK cells against cells expressing CD147 protein through antibody-dependent cell-mediated cytotoxicity (ADCC).

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

[0009] In a first aspect, the present invention provides an anti-CD147 single-domain antibody, wherein the amino acid sequence of the single-domain antibody comprises CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.3, and CDR3 shown in SEQ ID No.4.

[0010] In the present invention, CD147 protein is used to immunize alpacas. There is a special antibody lacking light chains, namely heavy chain antibodies, in the blood of camelids and cartilaginous fish. 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 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. The prepared G9 antibody has high specificity, and the tumor killing effect of ADCC and CAR-T mediated by it is good.

[0011] The present invention creatively constructs a single-domain antibody G9 against pan-tumor antigen CD147, which includes three complementary determining regions. Antibody G9 is obtained by amplifying the VHH fragment of the CD147 antibody in alpaca serum. It is a single-domain antibody. The antibody fragment is smaller and easier to penetrate into tissues and cells, has higher stability, and has been proven to have high specificity for CD147 protein through experiments, and can be used for subsequent research on drugs and other aspects.

[0012] SEQ ID No. 2: GRTFNGYF.

[0013] SEQ ID No. 3: VSWNGV.

[0014] SEQ ID No. 4: AADTSIPSIRGSGGDMDY.

[0015] Preferably, the amino acid sequence of the single-domain antibody includes the sequence shown in SEQ ID No.1.

[0016] SEQ ID No.1:

[0017] AVQLVESGGGLVQAGGSLRLACAASGRTFNGYFIHWFRQAPGKEREFVAAVSWNGVNTFIGRSMRGRISISRDVNKNTVYLQMISLKPEDTAVYYCAADTSIPSIRGSGGDMDYWGKGTLVTVSS.

[0018] In a second aspect, the present invention provides a heavy chain antibody, comprising the variable region sequence of the anti-CD147 single domain antibody according to the first aspect and the sequence of the crystallizable region Ig Fc of human immunoglobulin.

[0019] Preferably, the sequence of the crystallizable fragment Ig Fc of human immunoglobulin includes the full-length or partial amino acid sequence of Ig Fc.

[0020] Preferably, the Ig Fc comprises any one or a combination of at least two of IgG1, IgG2, IgG3 or IgG4.

[0021] Preferably, the sequence of the IgG1 Fc fragment is shown as SEQ ID No.5.

[0022] SEQ ID No.5:

[0023] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0024] Preferably, the amino acid sequence of the heavy chain antibody comprises that shown in SEQ ID No.6.

[0025] SEQ ID No.6:

[0026] AVQLVESGGGLVQAGGSLRLACAASGRTFNGYFIHWFRQAPGKEREFVAAVSWNGVNTFIGRSMRGRISISRDVNKNTVYLQMISLKPEDTAVYYCAADTSIPSIRGSGGDMDYWGKGTLVTVSSGGCCACGAAGGCCAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0027] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-CD147 single domain antibody described in the first aspect or the anti-CD147 heavy chain antibody described in the second aspect.

[0028] In a fourth aspect, the present invention provides a chimeric antigen receptor molecule, wherein the sequence of the chimeric antigen receptor molecule comprises a signal peptide, the anti-CD147 single domain antibody according to the first aspect, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signal transduction domain connected in series;

[0029] The signal peptide includes any one or a combination of at least two of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ;

[0030] The hinge region includes any one or a combination of at least two of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ;

[0031] The transmembrane domain includes any one or a combination of at least two 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;

[0032] The intracellular co-stimulatory domain includes any one or a combination of at least two 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;

[0033] The intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, CD3ε, CD3γ or CD3δ.

[0034] The present invention creatively constructs a chimeric antigen receptor molecule containing antibody G9. Tests show that the molecule has a strong killing effect on cells expressing CD147.

[0035] Preferably, the amino acid sequence of the signal peptide includes that shown in SEQ ID No.7.

[0036] SEQ ID No. 7: MALPVTALLLPLALLLHAARPS.

[0037] Preferably, the amino acid sequence of the hinge region includes that shown in SEQ ID No.8.

[0038] SEQ ID No.8:

[0039] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0040] Preferably, the amino acid sequence of the transmembrane domain includes that shown in SEQ ID No.9.

[0041] SEQ ID No. 9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0042] Preferably, the amino acid sequence of the intracellular co-stimulatory domain includes that shown in SEQ ID No.10.

[0043] SEQ ID No.10:

[0044] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0045] Preferably, the amino acid sequence of the intracellular signal transduction domain includes that shown in SEQ ID No.11.

[0046] SEQ ID No.11:

[0047] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0048] Preferably, the coding sequence of the chimeric antigen receptor molecule includes that shown in SEQ ID No.12.

[0049] SEQ ID No.12:

[0050] AATMALPVTALLLPLALLLHAARPSAQPAMAAVQLVESGGGLVQAGGSLRLACAASGRTFNGYFIHWFRQAPGKEREFVAAVSWNGVNTFIGRSMRGRISISRDVNKNTVYLQMISLKP EDTAVYYCAADTSIPSIRGSGGDMDYWGKGTLVTVSSEPKTPKPQPAAASGHEWESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0051] In a fifth aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus comprises a coding sequence of the chimeric antigen receptor molecule described in the fourth aspect.

[0052] In a sixth aspect, the present invention provides an immune cell, wherein the immune cell comprises a chimeric antigen receptor molecule expressing the fourth aspect;

[0053] Preferably, the types of immune cells include any one or a combination of at least two of T cells, TIL cells, B cells, NK cells, and macrophages.

[0054] In the seventh aspect, a use of the anti-CD147 single domain antibody according to the first aspect, the heavy chain antibody according to the second aspect, the nucleic acid molecule according to the third aspect, the chimeric antigen receptor molecule according to the fourth aspect, the recombinant lentivirus according to the fifth aspect, or the immune cell according to the sixth aspect in the preparation of a drug for treating a disease associated with high expression of CD147;

[0055] Preferably, the diseases associated with high CD147 expression include solid tumors;

[0056] Preferably, the solid tumor comprises any one or a combination of at least two of liver cancer, bile duct cancer, colorectal cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, lung cancer or nasopharyngeal cancer.

[0057] Compared with the prior art, the present invention has at least the following beneficial effects:

[0058] 1. In the present invention, the single-domain antibody against CD147 includes three complementary determining regions. Experimental verification shows that the antibody has high affinity and specificity for CD147 protein. Experimental results show that the antibody can effectively enhance the killing function of NK cells against cells expressing CD147 protein through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0059] 2. In the present invention, the single-domain antibody against CD147 is obtained by amplifying the VHH fragment of the CD147 antibody in alpaca serum. It is a single-domain antibody. The antibody fragment is smaller and easier to penetrate into tissues and cells, has higher stability, and has been proven to have higher specificity for CD147 protein through experiments.

[0060] 3. In the present invention, the structure of antibody G9 is simple and easier to modify, and the production cost is low, which is suitable for large-scale production. Alpaca-derived antibodies have a low risk of causing immune response because the alpaca's immune system can produce antibodies with high specificity and affinity for specific antigens and have weak immunogenicity to humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a flow cytometry assay to detect the specificity of the recombinant single domain antibody.

[0062] Figure 2 This is a flow cytometry assay for detecting the EC50 of a recombinant single domain antibody.

[0063] Figure 3 This is the alpaca antibody concentration-effect curve.

[0064] Figure 4 This is a flow cytometry detection chart of the binding of recombinant single domain antibody to colorectal cancer HCT116 cells.

[0065] Figure 5 This is the result of real-time quantitative live cell imaging analysis of recombinant single-domain antibody-mediated ADCC function by IncuCyte. Figure A is a curve chart showing the change of relative fluorescence area over time; Figure B is a statistical chart of the relative fluorescence area value at the last time point recorded in Figure A.

[0066] Figure 6 The following are the results of the structure of chimeric antigen receptor and the expression of chimeric antigen receptor in CAR-T cells. Figure A is a schematic diagram of the structure of chimeric antigen receptor; Figure B is the result of the expression rate detection of chimeric antigen receptor in CAR-T cells.

[0067] Figure 7This is the result of IncuCyte real-time quantitative live cell imaging analysis of the killing function of CAR-T cells targeting CD147. 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.

[0068] Reagents used in the following examples:

[0069] PBS (bico, CAT# 14190-250).

[0070] Adjuvant immune adjuvant (GERBU, CAT# 3030).

[0071] HRP-ProteinA (Boster, CAT# BA1080).

[0072] HRP-Streptavidin (Boster, CAT# BA1088).

[0073] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B).

[0074] DNA fragment recovery kit (TakaRa, CAT# 9761).

[0075] Goat anti-Llama IgG (H+L)) Secondary Antibody [HRP] (Novus, AT#NB7242).

[0076] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805).

[0077] Streptavidin PE (eBioscience, CAT# 12-4317-87).

[0078] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82).

[0079] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014).

[0080] Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CAT#L3000001). DETAILED DESCRIPTION

[0081] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0082] Example 1

[0083] This example is used for alpaca immunization and antibody titer determination

[0084] Three alpacas (73#, 74# and 194#) were immunized with CD147 protein, once every three weeks, for a total of three times. Each time, the alpacas were injected near the cervical lymph nodes, and about 800μg of emulsified antigen protein was injected at each point. 5mL 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 800g for 10min to collect the upper serum. CD147-His recombinant protein was diluted with PBS to a final concentration of 1µg / mL, and 100µL of the above CD147-His recombinant protein was added to each well of the 96-well ELISA plate, and coated at 4℃ overnight; then the antigen coating solution was shaken off and washed 5 times with PBST solution; 200µL of blocking solution was added to each well and blocked at 37℃ for 2h.

[0085] After shaking off the blocking buffer, wash the plate with PBST for 5 times; dilute the above serum with PBS, add 100µL of the gradient diluted serum to the 96-well ELISA plate, and incubate at room temperature for 1.5h. The control well is PBS; shake off the liquid in the well and wash with PBST for 5 times; add 100µL HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1h; shake off the liquid in the well and wash the plate with PBST for 6 times; add 100µL TMB colorimetric solution to each well; incubate at room temperature in the dark for 15min; add 50µL stop solution to each well; read the OD450 value in the well using an ELISA reader.

[0086] The results are shown in Tables 1, 2 and 3. The binding titers of the serum of the three alpacas before immunization to CD147 protein were all at the background level. On the contrary, the binding titers of the serum of 73# alpaca, 74# alpaca and 194# alpaca after the third immunization to CD147 protein were greatly increased, 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.

[0087] Table 1

[0088]

[0089] Table 2

[0090]

[0091] Table 3

[0092]

[0093] Example 2

[0094] This example is used to construct a single domain antibody yeast display library

[0095] (1) VHH antibody fragment cloning

[0096] After confirming that the three alpacas' serum contained CD147-specific antibodies, 100 mL of peripheral blood was collected, PBMCs were separated using Ficoll lymphocyte separation medium, and RNA was extracted. cDNA was obtained by reverse transcription. Using PBMC cDNA as a template, the alpaca heavy chain antibody sequence was PCR amplified using specific primer 1 (the upstream primer binds to the signal peptide of the VHH antibody ORF, and the downstream primer binds to the CH2 region) as shown in Table 4. The PCR product was analyzed by electrophoresis using 1% agarose, 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 the heavy chain antibody VHH fragment was amplified using specific primer 2 (the upstream primer binds to the antibody FR1 region, and the 5' end contains the SfiI restriction site. The downstream primer binds to the anti-Hinge and FR4 regions, and the 5' end contains the SfiI restriction site). The PCR product was analyzed by electrophoresis using 1% agarose, 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).

[0097] Table 4

[0098]

[0099] (2) Electroporation of library vectors and detection of library capacity and diversity

[0100] The yeast surface display vector pYDisplay and the VHH fragment library obtained above were digested with SfiI endonuclease. The pYDisplay vector fragment was separated and recovered using 1% agarose gel. The yeast competent strain frozen at -80℃ was streaked onto a YPD solid medium plate in advance and activated at 28℃ for 3 days. A single colony of yeast competent strain was inoculated into 50mL of YPD medium and shaken at 250rpm and 30℃ for 3 days.

[0101] After mixing the linearized vector fragment and PCR product, add them to the electroporation cup and electroporate; transfer the yeast competent cells after electroporation to the culture bottle and shake at 220rpm and 30℃ for 1h. Take 20μL of the resuspension, dilute it 5000 times with SDCAA medium, take 100μL, apply it to the SDCAA plate, culture it for 3 days, calculate the library capacity, and continue to culture the rest of the bacterial solution for 24h. Collect the remaining bacterial solution into a 50mL centrifuge tube, centrifuge it at 3000×g for 5min, discard the supernatant, and add 10mL of SDCAA medium to resuspend it. Mix it with 50% glycerol: resuspension = 1:1 (volume ratio) and freeze it at -80℃.

[0102] 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 library sequences were highly different, with no repeated sequences and good diversity.

[0103] Example 3

[0104] This example is used for panning of yeast display libraries

[0105] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0106] Take 2×10 8 Yeast cell library, centrifuge at 3000×g for 5min, and remove the supernatant. Resuspend the yeast library with 1mL of 0.5% PBSA, centrifuge at 3000×g for 5min, and discard the supernatant; wash again with 0.5% PBSA. Prepare 3 centrifuge tubes containing 1mL of 0.5% PBSA, add 10 μL of streptavidin magnetic beads, fix them on a rotating mixer, and rotate and incubate at 4℃ for 5min; place the centrifuge tube on a magnetic stand for 5min, and remove the supernatant with a pipette; then add 1mL of 0.5% PBSA again, rotate and incubate at 4℃ for another 5min, and remove the supernatant.

[0107] (2) Yeast display library screening

[0108] Negative selection with empty magnetic beads: Add the washed yeast solution to the centrifuge tube containing empty magnetic beads, and incubate at 4°C for 60 minutes; place the empty magnetic bead centrifuge tube containing yeast on a magnetic rack for 10 minutes, pipette the yeast solution into a new empty magnetic bead centrifuge tube, being careful not to suck up the old magnetic beads, and incubate at 4°C for 30 minutes. After the incubation, place it on a magnetic rack for 15 minutes, and pipette the yeast solution for later use.

[0109] Biotin-CD147-His magnetic separation: add 100 μL of 50 μg / mL Biotin-CD147-His protein solution (0.5% PBSA dilution) to the centrifuge tube containing streptavidin magnetic beads, put the centrifuge tube in a bag, and rotate and incubate at 4°C for 60 minutes; then add 1 mL of 0.5% PBSA, let it stand for 5 minutes, keep the centrifuge tube on the magnetic rack, and use a pipette to remove the supernatant, then add 1 mL of 0.5% PBSA, mix it by pipetting, place the centrifuge tube on the magnetic rack again, let it stand for 5 minutes, keep the centrifuge tube on the magnetic rack, and use a pipette to remove the supernatant; repeat the above cleaning steps once more to obtain positive selection magnetic beads coated with Biotin-CD147-His.

[0110] Add the positive selection magnetic beads bound to Biotin-CD147-His to the yeast cells that have completed negative selection, and place them on a rotating mixer, rotate and incubate at 4°C for 60 minutes. After the incubation, place them on a magnetic rack and let them stand at room temperature for 15 minutes; keep the centrifuge tube on the magnetic rack and use a pipette to absorb the unbound yeast solution; remove the 1.5mL centrifuge tube from the magnetic rack and wash it twice with 1mL of sterile 0.5% PBSA buffer. After washing, resuspend the magnetic beads and adhered yeast cells with 1mL of SDCAA medium, draw 20μL of the resuspension into 180μL of SDCAA medium and apply two plates, each with a volume of 100μL, and then draw 5μL of the resuspension into 95μL of SDCAA medium and apply one plate, that is, a total of 3 plates are required.

[0111] Yeast flow cytometry after selection: 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 selection with empty magnetic beads and magnetic selection with Biotin-CD147-His was incubated with CD147 protein containing His tag at 4°C for 60 minutes, then centrifuged to remove the supernatant, added anti-His tag and anti-V5 tag flow antibodies, incubated at 4°C for 60 minutes, then centrifuged to remove the supernatant, and resuspended the cells in 500μL PBS. After flow analysis, the yeast that can bind to CD147 protein in the yeast antibody display library of 73#, 74# and 194# alpacas before and after sorting increased by 10%, 41% and 13% respectively.

[0112] Example 4

[0113] This example is used for screening of recombinant antibodies

[0114] 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 the CD147 protein containing the His tag at 4°C for 60 minutes, and then the supernatant was removed by centrifugation. The precipitate was resuspended with a solution containing a biotin-coupled anti-His tag flow antibody, and incubated at 4°C for 60 minutes. The supernatant was then removed by centrifugation, and the precipitate was resuspended with a solution containing PE-Streptavidin, and incubated at 4°C for 60 minutes. After the incubation, flow detection was performed. Finally, a monoclonal clone 74-2-G9 with a high positive rate was selected. 74-2-G9 was lysed with 0.2% SDS (incubated at 95°C for 10 minutes), centrifuged, and 0.5μL of the bacterial supernatant was taken as a template for PCR amplification and sent to Sanger sequencing (the remaining bacterial solution was stored at -20°C).

[0115] Example 5

[0116] This example is used for the whole gene analysis of recombinant antibodies

[0117] After the whole gene detection of monoclonal 74-2-G9, it was found that its CDR1, CDR2 and CDR3 sequences were SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4 respectively. After NCBI Blast search and comparison analysis, the closest amino acid similarity rate to 74-2-G9 antibody in the published sequence was only 72.58%, and the similarity of CDR1, CDR2 and CDR3 regions further decreased to 62.5%, 50.0% and 38.9%, indicating that 74-2-G9 antibody is a new antibody clone that has not yet been discovered.

[0118] Example 6

[0119] This example is used for the expression of recombinant single domain antibodies and the detection of binding to target proteins

[0120] A PCR instrument was used to amplify the candidate 74-2-G9 VHH sequence, CMV promoter sequence and IgG1 Fc sequence, 50 μL of PCR product was taken, 1% agarose was used for electrophoresis analysis, and the PCR product was purified. The nucleic acid sequence of the CMV promoter is shown in SEQ ID No. 17.

[0121] SEQ ID No.17:

[0122] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGG ACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT.

[0123] The 74-2-G9 VHH sequence, CMV promoter and IgG1 Fc sequence were connected using a PCR instrument, and then 50 μL of the PCR product was taken and analyzed by electrophoresis using 1% agarose. The PCR product was purified and transiently transfected into HEK293 cells. The harvested cell culture supernatant contained the recombinant single-domain antibody 74-2-G9.

[0124] The binding specificity of 74-2-G9 was tested. The culture supernatant containing 74-2-G9 was mixed with 3×10 5CHO-S or CHO-S-CD147 cells were incubated at room temperature for 1 hour. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. 100μL of PE-labeled anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature in the dark for 45 minutes. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500μL of PBS for flow cytometry analysis.

[0125] 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 could be used as negative control cells for alpaca antibody detection; CHO-S-CD147 cells could be used as positive control cells for alpaca antibody detection. 74-2-G9 did not bind to CHO-S cells, but had significant binding to CHO-S-CD147, and could detect that most CHO-S-CD147 cells expressed CD147 protein, indicating that 74-2-G9 had good affinity and specificity.

[0126] Example 7

[0127] This example is used for purification of recombinant antibodies and determination of half effective concentration (EC50)

[0128] To determine the EC50 of the 74-2-G9 antibody, the relevant expression plasmid was transiently transfected into 293F cells and cultured in a shaking flask to express and purify 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 of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads to the 293F cell shaking flask according to the required amount of the sample (calculated as 1 mL of magnetic beads per 20 mg of IgG). Incubate at room temperature for 2 hours in a shaking incubator at 120 rpm.

[0129] Collect the Protein A beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. Wash twice with 30mL of PBS buffer and deionized water, and resuspend with 1mL of elution buffer. After incubation at room temperature for 5min, collect the beads with a magnetic separation rack, and transfer the supernatant containing the target antibody to a 15mL centrifuge tube. After eluting the Protein A beads twice, combine the eluates, and add neutralization buffer to adjust the pH of the solution. The eluted sample is dialyzed with PBS, first at 20℃ for 2h, then dialyzed at 4℃ for 14~16h after changing the solution once. Finally, determine the protein concentration and filter the sample with a 0.22μm sterile filter membrane and aliquot it, and store it in a -80°C refrigerator for use.

[0130] Serial dilutions of the target antibody were added to 3×10 5 Incubate CHO-S-CD147 cells at room temperature for 1 hour. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Add 100μL of PE-labeled Anti-human IgG antibody (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant, wash the cells three times with PBS, resuspend the cells with 200μL of PBS, and perform flow cytometry analysis.

[0131] The results are as follows Figure 2 and Figure 3 As shown, the EC50 value of 74-2-G9 for CHO-S-CD147 was 0.234 μg / mL, indicating that 74-2-G9 had a high affinity.

[0132] Example 8

[0133] This example is used to detect the binding effect of recombinant antibodies on colorectal cancer cells

[0134] Incubate HCT116 colon cancer cells with 74-2-G9 antibody and positive control antibody at room temperature for 1 hour, and wash the cells three times with PBS. Add 100 μL of APC-labeled Anti-human IgG antibody and incubate for 45 minutes. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant, wash the cells three times with PBS, and resuspend the cells with 200 μL of PBS for flow cytometric analysis. Figure 4 As shown, the positive control antibody has significant binding to HCT116, indicating that HCT116 expresses CD147 protein and can be used as a positive control cell for the detection of alpaca antibodies; 74-2-G9 significantly binds to HCT116, and can detect 99.2% of HCT116 cells expressing CD147 protein, indicating that 74-2-G9 has a high affinity for the CD147 protein of HCT116.

[0135] Example 9

[0136] This test case is used for ADCC function detection

[0137] SK-OV-3 cells (ovarian cancer cells overexpressing red fluorescent protein) 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 1:1 / well and cultured overnight. NK cells were added at an effector-target ratio of 8:1, and the target antibody 74-2-G9 was added at a final concentration of 5 μg / mL.

[0138] The real-time quantitative live cell imaging and analysis platform IncuCyte was used for detection. The changes in the fluorescence signals of target cells in each group were calculated using the IncuCyte Sx5 software. The lower the signal value, the fewer cells in the group and the better the NK cell killing effect. Figure 5 Middle A and Figure 5 As shown in B, compared with the NK cell group without antibody addition, 74-2-G9 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 74-2-G9 can enhance the killing function of NK cells against SK-OV-3 through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0139] Example 10

[0140] This example is used to construct CAR-T cells based on the target antibody

[0141] (1) Design of CAR molecules

[0142] The target gene structure of the lentiviral vector involved in this example is as follows Figure 6 As shown in A.

[0143] The CAR molecule BVH147-009 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CD147 single-domain antibody 74-2-G9 [VHH (74-2-G9) 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).

[0144] (2) Lentivirus preparation

[0145] The above CAR molecule expression sequence was cloned into the lentiviral vector pCDH-EF1α-MCS plasmid, and 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. The viral supernatant was collected 48 hours after transfection, centrifuged at 3000rpm for 10-15min at 4°C, filtered through a 0.45μm pore size filter membrane, and finally ultracentrifuged at 25,000rpm at 4°C for 2-3h. The obtained virus concentrate was stored at -80°C and named BVH147-009. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0146] (3) CAR-T cell preparation

[0147] PBMCs from healthy donors were revived in AIM V medium, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were added, and cultured in a cell culture incubator for 24 h (culture temperature was 37 ° C, carbon dioxide concentration was 5%). The obtained T cells were washed, and lentivirus was added at an MOI of 5TU / 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 (1.5~2)×10 6 / mL, and supplemented with 300IU / mL 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 1~2×10 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.

[0148] (4) Detection of CAR molecule expression

[0149] The BVH147-009 CAR-T cells to be tested were washed twice with PBS and resuspended in FACS buffer (PBS solution containing 0.1% sodium azide and 0.4% BSA). FITC-labeled anti-VHH antibody was incubated with CAR-T cells for 1 hour. The supernatant was then removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Ctrl T cells were used as negative controls, and the CAR molecule expression rate of BVH147-009 cells was detected by flow cytometry. The results are shown in Figure 2. Figure 6 As shown in B, the expression rate of BVH147-009 CAR was 9.3%.

[0150] Embodiment 11

[0151] This example is used to study the in vitro function of CAR-T cells

[0152] In vitro killing experiments of BVH147-009 cells were performed. 5 HCT116-mCherry cells were resuspended at a density of 100 μL / mL and inoculated into a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte Sx5 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 Sx5 software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells. The results are shown in Figure 7 Middle A and Figure 7 As shown in Figure B, compared with Mock-T cells, BVH147-009 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 74-2-G9 antibodies have a good killing function on CD147-positive tumor cells.

[0153] In summary, the 74-2-G9 antibody prepared by the present invention has high affinity and specificity for CD147 protein, and experiments have shown that the antibody can effectively enhance the killing function of NK cells against cells expressing CD147 protein through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0154] 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, characterized in that: The amino acid sequence of the single-domain antibody includes CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.3 and CDR3 shown in SEQ ID No.

4.

2. The single domain antibody according to claim 1, characterized in that The amino acid sequence of the single-domain antibody includes the sequence shown in SEQ ID No.

1.

3. A heavy chain antibody, characterized in that The heavy chain antibody comprises the variable region sequence of the anti-CD147 single domain antibody according to claim 1 or 2 and the sequence of the crystallizable segment Ig Fc of human immunoglobulin.

4. The heavy chain antibody according to claim 3, characterized in that The sequence of the human immunoglobulin crystallizable segment Ig Fc includes the full-length or partial amino acid sequence of Ig Fc; The Ig Fc comprises any one or a combination of at least two of IgG1, IgG2, IgG3 or IgG4; The sequence of the IgG1 Fc fragment is shown in SEQ ID No. 5; The amino acid sequence of the heavy chain antibody includes the sequence shown in SEQ ID No.

6.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD147 single domain antibody according to claim 1 or 2 or the heavy chain antibody according to claim 3 or 4.

6. A chimeric antigen receptor molecule, characterized in that The sequence of the chimeric antigen receptor molecule includes a signal peptide, the anti-CD147 single domain antibody according to claim 1 or 2, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signal transduction domain; The signal peptide includes any one or a combination of at least two of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ; The hinge region includes any one or a combination of at least two of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ; The transmembrane domain includes any one or a combination of at least two 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; The intracellular co-stimulatory domain includes any one or a combination of at least two 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; The intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, CD3ε, CD3γ or CD3δ.

7. The chimeric antigen receptor molecule according to claim 6, characterized in that The amino acid sequence of the signal peptide includes the sequence shown in SEQ ID No.7; The amino acid sequence of the hinge region includes the sequence shown in SEQ ID No.8; The amino acid sequence of the transmembrane domain includes the sequence shown in SEQ ID No.9; The amino acid sequence of the intracellular co-stimulatory domain includes the sequence shown in SEQ ID No.10; The amino acid sequence of the intracellular signal transduction domain includes the sequence shown in SEQ ID No.11; The coding sequence of the chimeric antigen receptor molecule includes the sequence shown in SEQ ID No.

12.

8. A recombinant lentivirus, characterized in that: The recombinant lentivirus comprises the coding sequence of the chimeric antigen receptor molecule of claim 7.

9. An immune cell, characterized in that The immune cell comprises a chimeric antigen receptor molecule expressing claim 6 or 7; The type of immune cells includes any one or a combination of at least two of T cells, TIL cells, B cells, NK cells or macrophages.

10. Use of the anti-CD147 single domain antibody according to claim 1 or 2, the heavy chain antibody according to claim 3 or 4, the nucleic acid molecule according to claim 5, the chimeric antigen receptor molecule according to claim 6 or 7, the recombinant lentivirus according to claim 8 or the immune cell according to claim 9 in the preparation of a drug for treating a disease associated with high expression of CD147; The diseases related to high CD147 expression include solid tumors; The solid tumor includes any one or a combination of at least two of liver cancer, bile duct cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, lung cancer or nasopharyngeal cancer.

Images