A pan-tumor antigen antibody A11 and its application

Abstract

The present invention discloses a pan-tumor antigen antibody A11 and its application. The amino acid sequence of CDR1 of the pan-tumor antigen antibody A11 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 of the pan-tumor antigen antibody A11 is shown in SEQ ID NO.2; the amino acid sequence of CDR3 of the pan-tumor antigen antibody A11 is shown in SEQ ID NO.3. The pan-tumor antigen antibody A11 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. At the same time, the antibody can enhance the ability of NK cells to kill tumor cells through ADCC.

Description

Technical Field

[0001] The present invention belongs to the field of antibody biotechnology and relates to a pan-tumor antigen antibody A11 and an application thereof. Background Art

[0002] The immunoglobulin superfamily (IgSF) consists of at least one protein with an Ig 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 50 kDa-60 kDa and belongs to the immunoglobulin superfamily. The CD147 molecule is a single-pass transmembrane glycoprotein. The gene is located at 19p13.3 and consists 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. 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, the middle 185 amino acids constitute the extracellular domain, and a total of 24 amino acids from 206-229 are transmembrane regions. CD147 is expressed in almost all tumor cells.

[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, 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 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 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] Therefore, there is an urgent need to provide an alpaca antibody with CD147 as a therapeutic target for the preparation of antibody drugs or cell drugs. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a pan-tumor antigen antibody A11 and its application, which can penetrate into tissues and cells, especially the blood-brain barrier, has strong stability, can maintain stability under extreme temperature and pH conditions, can still maintain biological activity at high temperatures up to 90 degrees, has a simple structure, can be modified, and has high specificity.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a pan-tumor antigen antibody A11, the amino acid sequence of CDR1 of the pan-tumor antigen antibody A11 is shown as SEQ ID NO.1; the amino acid sequence of CDR2 of the pan-tumor antigen antibody A11 is shown as SEQ ID NO.2; the amino acid sequence of CDR3 of the pan-tumor antigen antibody A11 is shown as SEQ ID NO.3.

[0009] SEQ ID NO.1: GRSFNNYA.

[0010] SEQ ID NO. 2: INWSGIAT.

[0011] SEQ ID NO. 3: AAHSGAKWLRPPSYDY.

[0012] The pan-tumor antigen antibody A11 of the present invention is small in size, can penetrate into tissues and cells, especially can penetrate the blood-brain barrier, has strong stability, can maintain stability under extreme temperature and pH conditions, can still maintain biological activity at high temperatures up to 90 degrees, has a simple structure, can be modified, and has high specificity.

[0013] 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 nanobody) 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.

[0014] The present invention uses CD147 protein to immunize alpacas. After determining the titer of CD147-specific antibodies in alpacas serum by ELISA, alpacas peripheral blood mononuclear cells (PBMCs) are separated, RNA is extracted and reverse transcribed to obtain cDNA. Alpaca single-domain antibody-specific primers are used to amplify the VHH sequence and clone it into a yeast expression plasmid to construct a yeast display library. Then, the yeast display library is positively and negatively screened with biotin-CD147 protein-streptavidin magnetic beads or streptavidin magnetic beads 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. Antibody clones with strong affinity to CD147 are selected, co-cultured with NK cells and SK-OV-3 ovarian cancer cells, and the antibody-dependent cell-mediated cytotoxicity (ADCC) of each clone is detected. At the same time, the CD147 single domain antibody sequence was cloned into the CAR expression vector to construct CAR-T cells with CD147 single domain antibody as the antigen binding domain, and its ability to kill CD147-positive HCT116 cells in vitro was tested. The results showed that alpaca antibodies have high specificity and good tumor killing effects of ADCC and CAR-T mediated by them.

[0015] Preferably, the amino acid sequence of the pan-tumor antigen antibody A11 is as shown in SEQ ID NO.4.

[0016] SEQ ID NO.4:

[0017] QVQLVESGGGLVQPGGSLRLSCAADGRSFNNYAMAWFRQAPGKEREFVAAINWSGIATTYGDSVKGRFTISRDDTKKTLYLQMNNLKPEDAAVYYCAAHSGAKWLRPPSYDYWGQGTQVTVSS.

[0018] In a second aspect, the present invention provides a heavy chain antibody, which comprises the variable region sequence of the pan-tumor antigen antibody A11 described in the first aspect and the full-length or partial amino acid sequence of the crystallizable segment of human immunoglobulin Ig Fc.

[0019] Preferably, the Ig Fc comprises the full-length or partial sequence of the Fc fragment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

[0020] Preferably, the Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.5.

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

[0022] SEQ ID NO.5:

[0023] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0024] SEQ ID NO.6:

[0025] QVQLVESGGGLVQPGGSLRLSCAADGRSFNNYAMAWFRQAPGKEREFVAAINWSGIATTYGDSVKGRFTISRDDTKKTLYLQMNNLKPEDAAVYYCAAHSGAKWLRPPSYDYWGQGTQVTVSSGGCCACGAAGGCCAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0026] In a third aspect, the present invention provides a nucleic acid molecule encoding the pan-tumor antigen antibody A11 described in the first aspect.

[0027] 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 region, a signal transduction domain and a constant region; the antigen binding domain comprises the pan-tumor antigen antibody A11 described in the first aspect.

[0028] Preferably, the signal peptide comprises any one or a combination of at least two of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ.

[0029] Preferably, the hinge region comprises any one of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ, or a combination of at least two thereof.

[0030] Preferably, the transmembrane region includes any one 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, or a combination of at least two of them.

[0031] Preferably, the signal transduction domain comprises a co-stimulatory domain and a human CD3 intracellular signal transduction domain.

[0032] Preferably, the 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] Preferably, the human CD3 intracellular signal transduction domain includes any one of CD3ζ, CD3ε, CD3γ or CD3δ, or a combination of at least two of them.

[0034] Preferably, the signal peptide comprises a human CD8 signal peptide having an amino acid sequence as shown in SEQ ID NO.7.

[0035] Preferably, the hinge region comprises the human IgG4 hinge region as shown in SEQ ID NO.8.

[0036] Preferably, the transmembrane region comprises the human CD8 transmembrane region as shown in SEQ ID NO.9.

[0037] Preferably, the signal transduction domain includes the human 4-1BB intracellular region as shown in SEQ ID NO.10 and / or the human CD3ζ intracellular region as shown in SEQ ID NO.11.

[0038] SEQ ID NO.7: MALPVTALLLPLALLLHAARPS.

[0039] SEQ ID NO.8:

[0040] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0041] SEQ ID NO.9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0042] SEQ ID NO.10:

[0043] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0044] SEQ ID NO.11:

[0045] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0046] Preferably, the chimeric antigen receptor comprises the human CD8 signal peptide shown in SEQ ID NO.7, the first aspect, the human IgG4 hinge region shown in SEQ ID NO.8, the human CD8 transmembrane region shown in SEQ ID NO.9, the human 4-1BB intracellular region shown in SEQ ID NO.10 and the human CD3ζ intracellular region shown in SEQ ID NO.11.

[0047] In a fifth aspect, the present invention provides an expression vector, comprising a gene encoding the chimeric antigen receptor according to the fourth aspect.

[0048] 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.

[0049] In a sixth aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the fifth aspect.

[0050] 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.

[0051] 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.

[0052] Preferably, the chimeric antigen receptor immune cell comprises any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages.

[0053] In the eighth aspect, the present invention provides the use of the pan-tumor antigen antibody A11 described in the first aspect, the nucleic acid molecule 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, or the chimeric antigen receptor immune cell described in the seventh aspect in the preparation of a product that enhances NK cell killing of tumor cells.

[0054] In the ninth aspect, the present invention provides the use of the pan-tumor antigen antibody A11 described in the first aspect, the nucleic acid molecule 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, or the chimeric antigen receptor immune cell described in the seventh aspect in the preparation of a drug or preparation that can prevent and / or treat cancer or tumors; the cancers include colorectal cancer and ovarian cancer.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The pan-tumor antigen antibody A11 of the present invention is small in size, one-tenth the size of ordinary antibodies, and can penetrate into tissues and cells, especially the blood-brain barrier;

[0057] (2) The pan-tumor antigen antibody A11 of the present invention has strong stability and can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90 degrees;

[0058] (3) The pan-tumor antigen antibody A11 of the present invention has a simple structure, and nano-antibodies are easier to transform, such as humanization and multivalent construction, and have low production costs and are suitable for large-scale production;

[0059] (4) The pan-tumor antigen antibody A11 of the present invention is highly specific. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens, and has weak immunogenicity to humans. The risk of nano-antibodies causing immune responses is low. The alpaca antibodies prepared by the present invention are highly specific, and the tumor killing effects of ADCC and CAR-T mediated by them are good. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The specificity diagram of the recombinant single domain antibody is detected by flow cytometry;

[0061] Figure 2 Detection of EC of recombinant single domain antibodies by flow cytometry 50 picture;

[0062] Figure 3 This is a graph of the concentration-effect curve of alpaca antibodies tested on CHO-S-CD147 cells;

[0063] Figure 4 This is a flow cytometry analysis of the binding of recombinant single domain antibodies to colorectal cancer HCT116 cells;

[0064] Figure 5A This is a graph showing the change in relative fluorescence area over time in the IncuCyte real-time quantitative live cell imaging analysis of the ADCC function mediated by recombinant single domain antibodies;

[0065] Figure 5B for Fig. 7A Statistical graph of relative fluorescence area values ​​at the last time point recorded;

[0066] Fig. 6A The structure of the chimeric antigen receptor and the expression of the chimeric antigen receptor in CAR-T cells are shown in the figure.

[0067] Figure 6B This is a graph showing the expression rate of chimeric antigen receptors in CAR-T cells;

[0068] Fig. 7A This is a graph showing the change in relative fluorescence area over time in the results of IncuCyte real-time quantitative live cell imaging analysis of the killing function of CAR-T cells targeting CD147;

[0069] Figure 7B for Fig. 7A Statistical graph of relative fluorescence area values ​​recorded at the last time point. DETAILED DESCRIPTION

[0070] 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.

[0071] 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.

[0072] Example 1

[0073] Related reagents:

[0074] Adjuvant immune adjuvant (GERBU, CAT# 3030);

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

[0076] Alpaca immunization and antibody titer determination.

[0077] Three alpacas (73#, 74# and 194#) were immunized with CD147 recombinant protein. Each time, the alpacas were injected on both sides near the cervical lymph nodes, with 2 injections on each side, and about 500 μg of emulsified antigen was injected at each point. After immunization, the alpacas were observed for half an hour to confirm that they were in good condition. Immunization was performed once every 3 weeks, and 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 collected blood samples were placed in a centrifuge and centrifuged at 800×g for 10 min, and the upper serum was collected. Use sterile PBS to dilute CD147-His recombinant protein to a final concentration of 1 µg / mL, take a 96-well ELISA plate, add 1 µg / mL CD147-His recombinant protein at 100 µL / well, and coat overnight at 4°C; then discard 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 h; after discarding the blocking buffer, wash the plate 5 times with PBST; use PBS to graded dilution of the above serum, add 100 µL of graded diluted serum to the 96-well plate, incubate at 25°C for 1 h, and use PBS as the control well; discard the liquid in the well and wash 5 times with PBST; add 100 µL HRPanti-Llama IgG (H+L) antibody (1:50000 dilution), incubate at 25°C for 1 h; shake off the liquid in the well, wash the plate 5 times with PBST; add 100 µL / well TMB colorimetric solution; incubate at 25°C in the dark for 15 min; add 50 µL / well stop solution; read the OD in the well using a microplate reader 450 value.

[0078] The binding titers of the sera of the three alpacas after three immunizations to CD147 protein were significantly higher than those of the sera before immunization, indicating that the content of CD147-specific antibodies in the sera of the three alpacas was significantly increased after multiple rounds of immunization and could be used for the construction of yeast display libraries.

[0079] Example 2

[0080] Related reagents:

[0081] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B);

[0082] DNA fragment recovery kit (TakaRa, CAT# 9761).

[0083] Construction of a single domain antibody yeast display library.

[0084] (1) VHH antibody fragment cloning

[0085] 100 mL of peripheral blood was collected, PBMCs were separated using lymphocyte separation buffer, and RNA was extracted. st Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. Using PBMC cDNA as a template, specific primers (upstream primer binds to the signal peptide of VHH antibody ORF, primer sequence is SEQ ID NO.12, downstream primer binds to CH2 region, primer sequence is SEQ ID NO.13) were used to PCR amplify the alpaca heavy chain antibody sequence.

[0086] SEQ ID NO. 12: GTCCTGGCTGCTCTTCTACAAGG.

[0087] SEQ ID NO. 13: GGTACGTGCTGTTGAACTGTTCC.

[0088] The PCR products were analyzed by agarose gel electrophoresis, 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 using specific primers (the upstream primer binds to the antibody FR1 region, and the 5' end contains the SfiI restriction site GGCCCAGCCGGCC, and the primer sequence is SEQ ID NO.14; the downstream primer binds to the anti-Hinge and FR4 regions, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC, and the primer sequence is SEQ ID NO.15).

[0089] SEQ ID NO.14:

[0090] ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTGG.

[0091] SEQ ID NO.15:

[0092] GATCACTAGTGGGGTCTTC GCTGTGGTGCG.

[0093] Analyze the PCR products by agarose gel electrophoresis, and recover and separate the target fragment with a molecular weight of about 450 bp. Store the target fragment at -80°C (the storage solution contains 1 / 10 volume of 3 M sodium acetate, 1 μg / μL glycogen, and 80% anhydrous ethanol).

[0094] (2) Electroporation of library vectors and detection of library capacity and diversity

[0095] The yeast surface display vector plasmid pYDisplay and the VHH fragment library obtained above were digested with SfiI endonuclease. The pYDisplay vector fragment was separated by agarose gel and recovered. The yeast competent strain frozen at -80°C was streaked onto a YPD solid medium plate in advance and activated at 30°C for 5 days. A single colony of yeast competent strain was inoculated into 50 mL YPD medium and shaken at 250 rpm and 30°C for 2 days. The linearized vector fragment and PCR product were mixed and added to the electroporation cup for electroporation transformation; the yeast competent strain after electroporation was transferred to a culture bottle and shaken at 220 rpm and 30°C for 1 h. Take 20 μL of the resuspension, dilute 5000 times with SDCAA, take 100 μL, and culture on the SDCAA plate for 3 days to calculate the storage capacity, and the remaining bacterial solution was cultured for 24 h. The remaining bacterial solution was collected in a 50 mL centrifuge tube, centrifuged at 3000×g for 5 min, and the supernatant was discarded. Add 10 mL SDCAA to resuspend, mix with 50% glycerol: resuspension solution = 1:1, and freeze at -80°C. The results showed that the library capacity of the single domain antibody yeast display library obtained from PBMC of 73# alpaca, 74# alpaca and 194# alpaca was 1.6×10 9 , 1.3×10 9 and 1.1×10 9 After Sanger sequencing, the results showed that the yeast library had large sequence differences, no repeated sequences, and good diversity.

[0096] Example 3

[0097] Related reagents:

[0098] PBS (bico, CAT# 14190-250);

[0099] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805);

[0100] Streptavidin PE (eBioscience, CAT# 12-4317-87).

[0101] Panning of yeast display libraries.

[0102] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0103] Take 10 mL of yeast library (about 2×10 8Yeast cells) to a 50 mL centrifuge tube, centrifuge at 3000×g for 5 min, and remove the supernatant; at the same time, dilute sterile 5% PBSA (PBS+5% BSA) to 0.5% PBSA; resuspend the yeast library with 1 mL 0.5% PBSA, centrifuge at 3000×g for 5 min, and wash twice. Prepare 3 1.5 mL centrifuge tubes (2 centrifuge tubes for negative selection of empty magnetic beads, 1 centrifuge tube for positive selection), add 1 mL 0.5% PBSA to each centrifuge tube; fully resuspend the aliquoted streptavidin magnetic beads, pipette 10 μL of magnetic beads into the 1.5 mL centrifuge tube, fix it on the rotating mixer, and rotate and incubate at 4°C for 5 min; place the centrifuge tube on the magnetic stand for 5 min, and discard the supernatant; add 1 mL 0.5% PBSA again, rotate and incubate at 4°C for another 5 min, and discard the supernatant.

[0104] (2) Yeast display library screening

[0105] Negative selection with empty magnetic beads: Place the centrifuge tube on a magnetic rack, add the washed yeast solution to the centrifuge tube containing empty magnetic beads, and rotate and incubate at 4°C for 60 min; place the empty magnetic bead centrifuge tube containing yeast on a magnetic rack for 10 min, pipette the yeast solution into a new empty magnetic bead centrifuge tube. After rotating and incubating at 4°C for 30 min, place it on a magnetic rack for 15 min, pipette the yeast solution into a centrifuge tube, and set aside.

[0106] 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, and incubate at 4°C for 60 min; then add 1 mL of 0.5% PBSA, let stand for 5 min, keep the centrifuge tube fixed on the magnetic rack, and discard the supernatant, then add 1 mL of 0.5% PBSA, blow and mix, place the centrifuge tube on the magnetic rack again, let stand for 5 min, keep the centrifuge tube fixed on the magnetic rack, and discard the supernatant; repeat the above washing steps once again to obtain the 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 place them on a rotating mixer. After 60 min of rotation incubation at 4°C, place on a magnetic stand and stand at 25°C for 15 min; keep the centrifuge tube on the magnetic stand and use a pipette to absorb the unbound yeast solution; remove the 1.5 mL centrifuge tube from the magnetic stand and add 1 mL of sterile 0.5% PBSA buffer to the centrifuge tube with a pipette, gently blow the magnetic beads, then transfer them to a sterile 1.5 mL centrifuge tube, place the centrifuge tube on a magnetic stand, stand at 25°C for 5 min, and discard the supernatant; repeat the wash twice, and perform the whole operation 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 apply two plates, each with a volume of 100 μL, and then pipette 5 μL of the resuspension into 95 μL SDCAA medium to apply one plate, that is, a total of 3 plates are required.

[0107] (3) Flow cytometry detection of yeast after selection

[0108] The yeast expression vector contains a V5 tag, which indicates whether the expression vector has been successfully transferred into yeast. The yeast that has undergone empty magnetic bead negative panning and Biotin-CD147-His magnetic sorting was incubated with CD147 protein containing His tag at 4°C for 60 min, and then the supernatant was removed by centrifugation. Flow cytometry antibodies with anti-His tag and anti-V5 tag were added, incubated at 4°C for 60 min, and then the supernatant was removed by centrifugation, and the cells were resuspended in 500 μL PBS for flow analysis. Before and after sorting, the number of yeast that can bind to CD147 protein in the yeast antibody display library of 73# alpaca, 74# alpaca and 194# alpaca increased by 10%, 41% and 13% respectively, and subsequent experiments can be carried out.

[0109] After sorting, the yeast solution was spread on SDCAA plates, and single clones were picked after overnight culture, and the single clones were 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, then centrifuged and discarded the supernatant, and the precipitate was resuspended with a solution containing biotin-conjugated anti-His tag flow antibody, incubated at 4°C for 60 min, then centrifuged and discarded the supernatant, and the precipitate was resuspended with a solution containing PE-Streptavidin, incubated at 4°C for 60 min, and flow detection was performed after incubation. Finally, a single clone 74-2-A11 with a high positive rate was selected. 74-2-A11 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 sequencing (the remaining bacterial solution was stored at -20°C).

[0110] Example 4

[0111] Related reagents:

[0112] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82).

[0113] Expression of recombinant single domain antibodies and detection of binding to target proteins.

[0114] PCR amplified the 74-2-A11 VHH sequence, CMV promoter sequence and IgG1 Fc sequence, and electrophoresed and purified the PCR product (the band size of CMV is 750 bp, the band size of Fc is 1400 bp, and the band size of VHH is 450 bp). Then, the 74-2-A11 VHH sequence, CMV promoter and IgG1 Fc sequence were connected, and the PCR product was electrophoresed and purified. After the obtained PCR product was transfected into HEK293 cells, the harvested cell culture supernatant contained the recombinant single-domain antibody 74-2-A11.

[0115] To detect the binding specificity of 74-2-A11, the culture supernatant containing 74-2-A11 was incubated with CHO-S or CHO-S-CD147 cells for 1 h, and the cells were washed 3 times with PBS. PE-labeled Anti-human IgG antibody (1:500 dilution) was added, incubated for 45 min, and the cells were washed 3 times with PBS before flow cytometry analysis.

[0116] The results are as follows Figure 1As shown, the expression supernatant of the negative control group had no significant binding to CHO-S or CHO-S-CD147; the positive control antibody only had significant binding to CHO-S-CD147, indicating that CHO-S cells did not express background CD147 protein, and CHO-S-CD147 cells could be used as detection cells for alpaca antibodies. 74-2-A11 did not bind to CHO-S cells, but had significant binding to CHO-S-CD147, and could detect most CHO-S-CD147 cells expressing CD147 protein, indicating that 74-2-A11 had good affinity and specificity.

[0117] Example 5

[0118] Related reagents:

[0119] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82).

[0120] Purification and half effective concentration (EC) of recombinant antibodies 50 ) determination.

[0121] To determine the EC of 74-2-A11 antibody 50 , transfect the relevant expression plasmid into 293F cells for antibody expression and purification. The target recombinant antibody contains human IgG fragments and can be affinity purified using Protein A magnetic beads. Wash the Protein A magnetic beads twice with 30 mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20 mg IgG / mL Protein A magnetic beads) to the 293F cell shaker according to the required amount of the sample. Incubate at 120 rpm and 25°C for 4 h in a shaking incubator. Collect the Protein A magnetic beads with a magnetic separation rack, wash twice with 30 mL PBS buffer and deionized water, and resuspend with 1 mL elution buffer. After incubation at 25°C 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 25°C for 2 h, then dialyzed at 8°C for 16 h after changing the solution once. Finally, the sample was filtered with a 0.22 μm sterile filter membrane and stored in a -80°C refrigerator for use.

[0122] Target antibodies were diluted in series and incubated with CHO-S-CD147 cells at 25℃ for 1 h. Centrifuge at 800×g for 5 min at 25℃, discard the supernatant, and wash the cells 3 times with PBS. Add 100 μL PE-labeled Anti-human IgG antibody (1:500 dilution), mix thoroughly, and incubate at 25℃ in the dark for 45 min. Centrifuge at 800×g for 5 min at 25℃, discard the supernatant, wash the cells 3 times with PBS, and resuspend the cells with 200 μL PBS for flow cytometry analysis.

[0123] The results are as follows Figure 2 and Figure 3 As shown, the EC of 74-2-A11 on CHO-S-CD147 50 The value was 0.4241 μg / mL, indicating that 74-2-A11 had a high affinity.

[0124] Example 6

[0125] Binding detection of recombinant antibodies to colorectal cancer cells.

[0126] Incubate the colorectal cancer cells HCT116 with 74-2-A11 antibody and positive control antibody at 25℃ for 1 h. Centrifuge at 800×g at 25℃ for 5 min, discard the supernatant, and wash the cells three times with PBS. Add 100 μL of APC-labeled Anti-human IgG antibody, mix thoroughly, and incubate at 25℃ in the dark for 45 min. Centrifuge at 800×g at 25℃ for 5 min, discard the supernatant, wash the cells three times with PBS, and resuspend the cells with 200 μL PBS for flow cytometric analysis. Figure 4 As shown, the positive control antibody significantly binds to HCT116, indicating that HCT116 expresses CD147 protein and can be used as a detection cell for alpaca antibodies; 74-2-A11 significantly binds to HCT116 and can detect 98.1% of HCT116 cells expressing CD147 protein, indicating that 74-2-A11 has a high affinity for the CD147 protein of HCT116.

[0127] Example 7

[0128] ADCC functional detection.

[0129] 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 with 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-A11 was added at a final concentration of 5 μg / mL.

[0130] The real-time quantitative live cell imaging and analysis platform IncuCyte was used for detection. The changes in the fluorescence signals of target cells in each group were calculated using the IncuCyte software. The lower the signal value, the fewer cells in the group, and the better the NK cell killing effect. Figure 5A and Figure 5B As shown, compared with the NK cell group without antibody, 74-2-A11 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-A11 can enhance the killing function of NK cells on tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0131] Example 8

[0132] Related reagents:

[0133] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014);

[0134] Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CAT#L3000001).

[0135] Construction of CAR-T cells based on target antibodies.

[0136] (1) Design of CAR molecules

[0137] The target gene structure of the lentiviral vector involved in this example is as follows Fig. 6A shown.

[0138] BVH147-007 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CD147 single-domain antibody 74-2-A11 [VHH (74-2-A11) 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).

[0139] (2) Lentivirus preparation

[0140] The above CAR molecule expression sequence was fully synthesized and cloned into the lentiviral vector pCDH-EF1α-MCS plasmid. The above lentiviral vector expression plasmids were co-transfected with lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G in 293T cells. The viral supernatant was collected 48 h after transfection, centrifuged at 3000 rpm for 15 min at 4°C, filtered through a 0.45 μm pore size filter membrane, and finally ultracentrifuged at 25,000 rpm for 3 h at 4°C. The obtained virus concentrate was stored at -80°C and named BVH147-007.

[0141] (3) CAR-T cell preparation

[0142] 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 for 24 h (culture temperature was 37°C, carbon dioxide concentration was 5%). Lentivirus was added at an MOI of 5 TU / mL for transduction, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were supplemented and cultured in a cell culture incubator (culture temperature was 37°C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 2×10 6 / mL, and supplemented with 300 IU / mL of hIL-2. The cells were collected on the 10th day after transduction and frozen in liquid nitrogen for later use. The obtained CAR-T cells continued to use the naming of the corresponding CAR molecules, and the T cells not transduced with lentivirus were named Ctrl T.

[0143] (4) Detection of CAR molecule expression

[0144] The BVH147-007 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). FITC-labeled anti-VHH antibody was incubated with CAR-T cells for 1 h. The supernatant was then removed by centrifugation and washed twice with FACS buffer. Ctrl T cells were used as negative controls, and the CAR molecule expression rate of BVH147-007 cells was detected by flow cytometry. The results are shown in Figure 2. Figure 6B As shown, the expression rate of BVH147-007 CAR was 36.1%.

[0145] Example 9

[0146] In vitro killing assay of BVH147-007 cells.

[0147] Use culture medium at 1×10 5HCT116-mCherry cells were resuspended at a density of 100 μL / mL and inoculated in a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte live cell imaging analysis system, CAR-T cells were added for co-culture at an effect-target ratio of 3:1, and the killing effect of CAR-T cells on tumor cells was recorded in real time. The changes in the mCherry fluorescence signal of target cells in each group were calculated using the IncuCyte software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells. The results are shown in Fig. 7A and Figure 7B As shown in the figure, compared with Mock-T cells, BVH147-007 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-A11 antibody have a good killing function on CD147-positive tumor cells.

[0148] In summary, the pan-tumor antigen antibody A11 of the present invention is small in size, can penetrate into tissues and cells, especially can penetrate the blood-brain barrier, has strong stability, can maintain stability under extreme temperature and pH conditions, can still maintain biological activity at high temperatures up to 90 degrees, has a simple structure, can be modified, and has high specificity.

[0149] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

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

3.

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

4.

3. A heavy chain antibody, characterized in that The heavy chain antibody comprises the single domain antibody targeting CD147 according to claim 1 or 2 and the full length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin; The Ig Fc comprises the full length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

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

5. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, a signal transduction domain and a constant region; The antigen binding domain comprises the single domain antibody targeting CD147 according to claim 1 or 2; 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 region 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 signal transduction domain includes a co-stimulatory domain and a human CD3 intracellular signal transduction domain; The 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 human CD3 intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, CD3ε, CD3γ or CD3δ; The signal peptide includes a human CD8 signal peptide whose amino acid sequence is shown in SEQ ID NO.7; The hinge region includes the human IgG4 hinge region as shown in SEQ ID NO.8; The transmembrane region includes the human CD8 transmembrane region shown in SEQ ID NO.9; The signal transduction domain includes the human 4-1BB intracellular region as shown in SEQ ID NO.10 and / or the human CD3ζ intracellular region as shown in SEQ ID NO.11; The chimeric antigen receptor includes the human CD8 signal peptide shown in SEQ ID NO.7, the single-domain antibody targeting CD147 according to claim 1 or 2, the human IgG4 hinge region shown in SEQ ID NO.8, the human CD8 transmembrane region shown in SEQ ID NO.9, the human 4-1BB intracellular region shown in SEQ ID NO.10 and the human CD3ζ intracellular region shown in SEQ ID NO.

11.

6. An expression vector, characterized in that: The expression vector comprises the gene encoding the chimeric antigen receptor according to claim 5; 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 according to claim 5.

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

8. A chimeric antigen receptor immune cell, characterized in that The chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 5; The chimeric antigen receptor immune cell comprises the expression vector of claim 6 and / or the recombinant lentivirus of claim 7; The chimeric antigen receptor immune cells include any one of T cells, B cells, NK cells, mast cells or macrophages, or a combination of at least two of them.

9. Use of the single-domain antibody targeting CD147 according to claim 1 or 2, the nucleic acid molecule according to claim 4, the chimeric antigen receptor according to claim 5, the expression vector according to claim 6, the recombinant lentivirus according to claim 7 or the chimeric antigen receptor immune cell according to claim 8 in the preparation of a product that enhances NK cell killing of ovarian cancer cells.

10. Use of the single-domain antibody targeting CD147 according to claim 1 or 2, the nucleic acid molecule according to claim 4, the chimeric antigen receptor according to claim 5, the expression vector according to claim 6, the recombinant lentivirus according to claim 7 or the chimeric antigen receptor immune cell according to claim 8 in the preparation of a drug or preparation for preventing and / or treating colorectal cancer and ovarian cancer.

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