A pan-tumor antigen antibody E1 and its application

Abstract

The present invention discloses a pan-tumor antigen antibody E1 and its application. The amino acid sequence of CDR1 of the pan-tumor antigen antibody E1 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 of the pan-tumor antigen antibody E1 is shown in SEQ ID NO.2; the amino acid sequence of CDR3 of the pan-tumor antigen antibody E1 is shown in SEQ ID NO.3. The pan-tumor antigen antibody E1 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 invention belongs to the field of antibody biotechnology and relates to a pan-tumor antigen antibody E1 and an application thereof. Background Art

[0002] Composition of the immunoglobulin superfamily Due to the progress of cell surface markers, monoclonal antibodies and genetic engineering research, nearly 100 members of the IGSF have been found in recent years, mainly including: 1. T, B cell antigen recognition receptors and signal transduction molecules. 2. MHC and related molecules. 3. Ig receptors. 4. Certain cytokine receptors. 5. Molecules related to nervous system function. 6. Some leukocyte differentiation antigens (CD). Characteristics of the immunoglobulin superfamily. 1. Structural characteristics of IGSF Members of the IGSF contain 1-7 diverse functional regions. The secondary structure of the functional region is composed of 3-5 parallel β-folds, each forming a plane with two parallel β sheets. Most functional regions have a disulfide bond; the two β sheets are vertically connected to form a spherical structure. This folding method of the peptide chain is called the immunoglobulin fold (Ig gold). The function of the IGSF is based on recognition, so it is also called the cognoglobuin superfamily. The basic ways of ISGF recognition are as follows (1) IGSF and IGSF recognize each other, such as Fc receptor recognition of IgG Fc segment. (2) Mutual recognition between IGSF and integrin, such as ICAM-2 and IFA-1. (3) Mutual recognition between IGSF and other molecules, such as TCR recognition of MHC class I or II molecules and Ag complexes.

[0003] CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a transmembrane glycoprotein containing 269 amino acids and belongs to the immunoglobulin superfamily (IgSF). Studies have confirmed that the high expression of CD147 molecules in tumor cells is related to the degree of differentiation, infiltration and metastasis of 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.). CD147 degrades the extracellular matrix of tumor cells by binding to matrix metalloproteinases (MMPs), thereby promoting tumor infiltration and spread. While CD147 interacts with MMPs, it can also produce VEGF, stimulate tumor vascular endothelial cells, promote tumor vascular reconstruction, and enhance the proliferation and invasion of tumor cells. Both 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] 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

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a pan-tumor antigen antibody E1 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.

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

[0007] In the first aspect, the present invention provides a pan-tumor antigen antibody E1, the amino acid sequence of CDR1 of the pan-tumor antigen antibody E1 is shown as SEQ ID NO.1; the amino acid sequence of CDR2 of the pan-tumor antigen antibody E1 is shown as SEQ ID NO.2; the amino acid sequence of CDR3 of the pan-tumor antigen antibody E1 is shown as SEQ ID NO.3.

[0008] SEQ ID NO.1: GFTLDYYA.

[0009] SEQ ID NO. 2: ISASDGRP.

[0010] SEQ ID NO.3:ATACPYSGSYYYTEGLDY.

[0011] The pan-tumor antigen antibody E1 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.

[0012] There is a special antibody lacking light chains in the blood of camelids and chondrichthyes, 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.

[0013] 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.

[0014] Preferably, the amino acid sequence of the pan-tumor antigen antibody E1 is as shown in SEQ ID NO.4.

[0015] SEQ ID NO.4:

[0016] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISASDGRPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACPYSGSYYYTEGLDYWGQGTQVTVSS.

[0017] In a second aspect, the present invention provides a heavy chain antibody, which comprises the variable region sequence of the pan-tumor antigen antibody E1 described in the first aspect and the full-length or partial amino acid sequence of the crystallizable segment of human immunoglobulin Ig Fc.

[0018] 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.

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

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

[0021] SEQ ID NO.5:

[0022] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] SEQ ID NO.6:

[0024] Question VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0025] In a third aspect, the present invention provides a nucleic acid molecule encoding the pan-tumor antigen antibody E1 described in the first aspect.

[0026] 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 E1 described in the first aspect.

[0027] 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.

[0028] Preferably, the hinge region comprises any one of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ, or a combination of at least two thereof.

[0029] 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.

[0030] Preferably, the signal transduction domain comprises a co-stimulatory domain and a human CD3 intracellular signal transduction domain.

[0031] 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.

[0032] 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.

[0033] Preferably, the signal peptide comprises a human CD8 signal peptide having an amino acid sequence as shown in SEQ ID NO.7.

[0034] Preferably, the hinge region comprises the human IgG4 hinge region as shown in SEQ ID NO.8.

[0035] Preferably, the transmembrane region comprises the human CD8 transmembrane region as shown in SEQ ID NO.9.

[0036] 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.

[0037] SEQ ID NO.7: MALPVTALLLPLALLLHAARPS.

[0038] SEQ ID NO.8: ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0039] SEQ ID NO.9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0040] SEQ ID NO.10:

[0041] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0042] SEQ ID NO.11:

[0043] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0044] 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.

[0045] In a fifth aspect, the present invention provides an expression vector, comprising a gene encoding the chimeric antigen receptor according to the fourth aspect.

[0046] 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.

[0047] In a sixth aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the fifth aspect.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In an eighth aspect, the present invention provides a pharmaceutical composition, comprising the chimeric antigen receptor immune cell described in the seventh aspect and / or the pan-tumor antigen antibody E1 described in the first aspect.

[0052] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0053] Preferably, the auxiliary materials include any one or a combination of at least two of a carrier, a surfactant, a disintegrant, a coating material, an excipient, a solubilizer, a diluent, a pH regulator, a binder, a wetting agent, a colorant, an emulsifier, an antibacterial agent, a solubilizer, an osmotic pressure regulator, a filler, an antioxidant or a buffer.

[0054] In the ninth aspect, the present invention provides the use of the pan-tumor antigen antibody E1 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.

[0055] In the tenth aspect, the present invention provides the use of the pan-tumor antigen antibody E1 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.

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

[0057] (1) The pan-tumor antigen antibody E1 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;

[0058] (2) The pan-tumor antigen antibody E1 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;

[0059] (3) The pan-tumor antigen antibody E1 of the present invention has a simple structure and is easier to transform than ordinary antibodies, such as humanization and multivalent construction, and has low production costs and is suitable for large-scale production;

[0060] (4) The pan-tumor antigen antibody E1 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

[0061] Figure 1 This is the amplification diagram of the VHH gene of the alpaca antibody;

[0062] Figure 2 The enrichment diagram of yeast library before and after sorting was detected by flow cytometry;

[0063] Figure 3 The specificity diagram of the recombinant single domain antibody is detected by flow cytometry;

[0064] Figure 4 Detection of EC of recombinant single domain antibodies by flow cytometry 50 picture;

[0065] Figure 5 This is a graph of the concentration-effect curve of alpaca antibodies tested on CHO-S-CD147 cells;

[0066] Figure 6 This is a flow cytometry analysis of the binding of recombinant single domain antibodies to colorectal cancer HCT116 cells;

[0067] Fig. 7A 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;

[0068] Figure 7B for Fig. 7A Statistical graph of relative fluorescence area values ​​at the last time point recorded;

[0069] Fig. 8A The structure of the chimeric antigen receptor and the expression of the chimeric antigen receptor in CAR-T cells are shown in the figure.

[0070] Figure 8B This is a graph showing the expression rate of chimeric antigen receptors in CAR-T cells;

[0071] Fig. 9A This is a curve of the change of relative fluorescence area over time in the result diagram of the killing function of CAR-T cells targeting CD147 analyzed by IncuCyte real-time quantitative living cell imaging;

[0072] Fig. 9B for Fig. 9A Statistical graph of relative fluorescence area values ​​recorded at the last time point. DETAILED DESCRIPTION

[0073] 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.

[0074] 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.

[0075] Example 1

[0076] Related reagents:

[0077] Adjuvant immune adjuvant (GERBU, CAT# 3030);

[0078] Goat anti-Llama IgG (H+L)) Secondary Antibody [HRP] (Novus, AT#NB7242).

[0079] Alpaca immunization and antibody titer determination.

[0080] Three alpacas (73#, 74# and 194#) were immunized with CD147 protein, and the immunization experiment was started after recording the ear number of the blank alpaca. Each time, the alpacas were injected on both sides near the cervical lymph nodes, with 2 injections on each side, and 800 μg of emulsified antigen was injected at each point. After immunization, the alpacas were observed for half an hour to confirm that they were in good condition and had no discomfort symptoms. Immunization was performed once every 3 weeks, and 3 immunizations were performed. 15 mL of peripheral blood was collected before immunization, two weeks after the second immunization, and the third immunization, respectively. The centrifuge tubes containing blood samples were placed in a centrifuge and centrifuged at 1800×g for 5 min to collect the upper serum. Dilute CD147-His recombinant protein with sterile PBS to a final concentration of 1 µg / mL, take a new 96-well ELISA plate, add 1 µg / mL CD147-His recombinant protein to 100 µL / well, and coat overnight at 4°C; then shake off the antigen coating solution, wash 5 times with PBST (containing 0.08% Tween 20); add 200 µL / well of blocking solution and block at 37°C for 4 h; after shaking off the blocking buffer, wash the plate 5 times with PBST; use PBS to graded dilution of the above-collected serum, add 100 µL of graded diluted serum to the 96-well ELISA plate, incubate at 25°C for 3 h, and use PBS as the control well; shake off the liquid in the well and wash 7 times with PBST; add 100 µL HRP anti-Llama IgG (H+L) antibody (1:50000 dilution), incubate at 25°C for 3 h; shake off the liquid in the well, wash the plate 3 times with PBST; add 100 µL / well TMB colorimetric solution; incubate at 25°C in the dark for 25 min; add 50 µL / well stop solution; read the OD in the well using a microplate reader 450 value.

[0081] The ratio of the absorbance of the test serum to the blank control was calculated. When the ratio was greater than 2, it was positive, and the positive result with the largest dilution multiple was defined as the titer of the antiserum. The results are shown in Tables 1, 2, and 3. The binding titers of the serum of the three alpacas before immunization with CD147 protein were 8 k, 1 k, and 32 k, respectively. The binding titers of the serum of the three alpacas after the third immunization with CD147 protein were much higher than 64 k, 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.

[0082] Table 1

[0083]

[0084] Table 2

[0085]

[0086] Table 3

[0087]

[0088] Example 2

[0089] Related reagents:

[0090] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B);

[0091] DNA fragment recovery kit (TakaRa, CAT# 9761).

[0092] Construction of a single domain antibody yeast display library.

[0093] (1) VHH antibody fragment cloning

[0094] After confirming that the three alpacas’ sera contained CD147-specific antibodies, 150 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.

[0095] SEQ ID NO. 12: GTCCTGGCTGCTCTTCTACAAGG.

[0096] SEQ ID NO. 13: GGTACGTGCTGTTGAACTGTTCC.

[0097] The results are as follows Figure 1 As shown, 1% agarose was used for electrophoresis analysis of PCR products, and the target fragment with a molecular weight of about 750 bp was recovered and separated. The first-round PCR product was then used as a template to amplify the heavy chain antibody VHH fragment using specific primers (the upstream primer was combined with the antibody FR1 region, and the 5' end contained the SfiI restriction site GGCCCAGCCGGCC, and the primer sequence was SEQ ID NO.14; the downstream primer was combined with the anti-Hinge and FR4 regions, and the 5' end contained the SfiI restriction site GGCCACGAAGGCC, and the primer sequence was SEQ ID NO.15).

[0098] SEQ ID NO.14:

[0099] ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTGG.

[0100] SEQ ID NO.15:

[0101] GATCACTAGTGGGGTCTTC GCTGTGGTGCG.

[0102] 1% agarose gel electrophoresis was used to analyze the PCR products, and the target fragment with a molecular weight of about 450 bp was recovered and separated ( Figure 1 The target fragment was stored at -80°C (the storage solution contained 1 / 10 volume of 3 M sodium acetate, 1 μg / μL glycogen, and 80% anhydrous ethanol).

[0103] (2) Electroporation of library vectors and detection of library capacity and diversity

[0104] The yeast surface display vector pYDisplay and the VHH fragment library obtained above were digested with SfiI endonuclease. The pYDisplay vector fragment was separated using 1% agarose gel, and a 5000 bp vector fragment was cut for gel recovery. The yeast competent strain frozen at -80°C was streaked onto a YPD solid medium plate in advance and activated at 31°C for 4 days. A single colony of yeast competent strain was inoculated into 50 mL YPD medium and cultured at 300 rpm and 31°C for 3 days. The linearized vector fragment and PCR product were mixed and added to the electroporation cup for electroporation; the yeast competent strain after electroporation was transferred to a culture bottle and cultured at 300 rpm and 31°C for 3 h. Take 20 μL of the resuspension, dilute 5000 times with SDCAA, take 100 μL, apply it to the SDCAA plate, culture for 3 days, calculate the reservoir capacity, and continue to culture the rest of the bacterial solution for 24 h. The remaining bacterial liquid was collected into a 50 mL centrifuge tube, centrifuged at 5000×g for 15 min, the supernatant was discarded, 10 mL SDCAA was added for resuspending, 50% glycerol: resuspending liquid was mixed at a ratio of 1:1, and frozen at -80°C. The results showed that the single domain antibody yeast display library obtained from 73# alpaca PBMC had a library capacity of 1.6×10 9 The single domain antibody yeast display library obtained from 74# alpaca PBMC has a capacity of 1.3×10 9 The single domain antibody yeast display library obtained from 194# alpaca PBMC has a capacity of 1.1×10 9 After Sanger sequencing, the results showed that the yeast library had large sequence differences, no repeated sequences, good diversity, and could be used for subsequent experiments.

[0105] Example 3

[0106] Related reagents:

[0107] PBS (bico, CAT# 14190-250);

[0108] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805);

[0109] Streptavidin PE (eBioscience, CAT# 12-4317-87).

[0110] Panning of yeast display libraries.

[0111] (1) Pretreatment of streptavidin magnetic beads and yeast cells

[0112] Take 10 mL of yeast library (about 2×10 8 Yeast cells) to a 50 mL centrifuge tube, centrifuge at 5000×g for 15 min, and discard the supernatant; at the same time, dilute sterile 5% PBSA (PBS+5% BSA) to 0.5% PBSA; resuspend the yeast library with 2 mL0.6% PBSA, add it to a 1.5 mL centrifuge tube, centrifuge at 5000×g for 8 min, and discard the supernatant; wash again with 0.5% PBSA. Prepare three 1.5 mL centrifuge tubes (two for negative panning with empty magnetic beads and one for positive panning), and add 1 mL of 0.5% PBSA to each tube; use a pipette to fully resuspend the aliquoted streptavidin magnetic beads, and pipette 10 μL of magnetic beads into a 1.5 mL centrifuge tube; put the tubes in a bag, fix them on a rotating mixer, and rotate and incubate at 4°C for 15 min; place the tubes on a magnetic stand for 15 min, and remove the supernatant with a pipette; then add 1 mL of 0.5% PBSA again, rotate and incubate at 4°C for another 15 min, and remove the supernatant.

[0113] (2) Yeast display library screening

[0114] Negative selection with empty magnetic beads: Place the centrifuge tube on the magnetic rack, add the washed yeast solution to the centrifuge tube containing the empty magnetic beads, put it in a bag, and rotate and incubate at 4°C for 80 min; Place the empty magnetic bead centrifuge tube incubated with yeast on the magnetic rack for 20 min, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube, being careful not to aspirate the old magnetic beads, and rotate and incubate at 4°C for 20 min. After the incubation, place it on the magnetic rack for 25 min, aspirate the yeast solution and add it to a new empty magnetic bead centrifuge tube.

[0115] Biotin-CD147-His magnetic separation: add 100 μL of 50 μg / mL Biotin-CD147-His protein solution (biotin-coupled, diluted with 0.6% PBSA) to a centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and rotate and incubate at 4°C for 80 min; then add 1 mL of 0.6% PBSA, let it stand for 15 min, keep the centrifuge tube on the magnetic rack, and remove the supernatant with a pipette, then add 1 mL of 0.6% PBSA, remove the centrifuge tube from the magnetic rack, mix it with a pipette, place the centrifuge tube on the magnetic rack again, let it stand for 15 min, keep the centrifuge tube on the magnetic rack, and remove the supernatant with a pipette; repeat the above washing steps once more to obtain positive selection magnetic beads coated with Biotin-CD147-His. Add the positive selection magnetic beads bound to Biotin-CD147-His to the yeast cells that have completed negative selection, and put them into a bag and place them on a rotating mixer. Incubate them at 4°C for 80 min. After the incubation, place them on a magnetic rack and let them stand at 25°C for 25 min. Keep the centrifuge tube on the magnetic rack and use a pipette to absorb the unbound yeast solution. Remove the 1.5 mL centrifuge tube from the magnetic rack and add 1 mL of sterile 0.6% PBSA buffer to the centrifuge tube with a pipette. Gently blow the magnetic beads and then transfer them to a sterile 1.5 mL centrifuge tube. Place the centrifuge tube on a magnetic rack and let it stand at 25°C for 15 min. Discard the supernatant. Repeat the washing twice. The entire operation is performed in accordance with the aseptic operation requirements. After washing, resuspend the magnetic beads and adhered yeast cells with 1 mL SDCAA medium, pipette 20 μL of the resuspension into 180 μL SDCAA medium and coat two plates, each with a volume of 100 μL, then pipette 5 μL of the resuspension into 95 μL SDCAA medium and coat one plate, i.e. a total of 3 plates need to be coated.

[0116] 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 the His tag at 4°C for 80 min, then centrifuged to remove the supernatant, added flow cytometry antibodies with anti-His tag and anti-V5 tag, incubated at 4°C for 80 min, then centrifuged to remove the supernatant, resuspended the cells in 500 μL PBS, and analyzed by flow cytometry. The results are shown in Figure 2 As shown, before and after sorting, the yeast capable of binding to CD147 protein in the yeast antibody display libraries of 73# alpaca, 74# alpaca and 194# alpaca increased by 10%, 41% and 13% respectively, which can be used for subsequent experiments.

[0117] After sorting, the yeast solution was spread on the SDCAA plate, and a single clone was picked after overnight culture, and the single clone was inoculated into the culture medium to induce expression. After 48 hours, the bacterial solution was incubated with CD147 protein containing His tag at 4°C for 60 min, and then the supernatant was removed by centrifugation. The precipitate was resuspended with a solution containing biotin-conjugated anti-His tag flow antibody, and incubated at 4°C for 80 min. The supernatant was then removed by centrifugation, and the precipitate was resuspended with a solution containing PE-Streptavidin, and incubated at 4°C for 80 min. After incubation, flow detection was performed. Finally, the monoclonal clone 194-1-E1 with a high positive rate was selected. 194-1-E1 was lysed with 0.2% SDS (incubated at 95°C for 20 min), centrifuged, and 0.5 μL of the bacterial supernatant was taken as a template for PCR amplification and sent for testing (the remaining bacterial solution was stored at -20°C).

[0118] Example 4

[0119] Related reagents:

[0120] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82).

[0121] Expression of recombinant single domain antibodies and detection of binding to target proteins.

[0122] The candidate 194-1-E1 VHH sequence, CMV promoter sequence and IgG1 Fc sequence were amplified using a PCR instrument. 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The band size of CMV was 750 bp, the band size of Fc was 1400 bp, and the band size of VHH was 450 bp. The target band was cut from the gel and the PCR product was purified. The 194-1-E1 VHH sequence, CMV promoter and IgG1 Fc sequence were connected using a PCR instrument, and then 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The target band was cut from the gel and the PCR product was purified. After the obtained PCR product was transiently transfected into HEK293 cells, the harvested cell culture supernatant contained the recombinant single domain antibody 194-1-E1.

[0123] The binding specificity of 194-1-E1 was tested. The culture supernatant containing 194-1-E1 was mixed with 3×10 5CHO-S or CHO-S-CD147 cells were incubated at 25℃ for 2 h. After centrifugation at 1000×g at 25℃ for 15 min, the supernatant was discarded and the cells were washed 4 times with PBS. 100 μL PE-labeled Anti-human IgG antibody (1:500 dilution) was added and incubated at 25℃ in the dark for 60 min. After centrifugation at 1000×g at 25℃ for 15 min, the supernatant was discarded and the cells were washed 5 times with PBS. The cells were resuspended in 500 μL PBS for flow cytometry analysis.

[0124] The results are as follows Figure 3 As shown, the expression supernatant of the negative control group had no significant binding to CHO-S or CHO-S-CD147; the positive control antibody had significant binding to CHO-S-CD147 but not to CHO-S cells, indicating that CHO-S cells did not express CD147 protein, and CHO-S-CD147 cells successfully overexpressed CD147 protein and could be used as detection cells for alpaca antibodies. 194-1-E1 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 194-1-E1 had good affinity and specificity.

[0125] Example 5

[0126] Related reagents:

[0127] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82).

[0128] Purification and half effective concentration (EC) of recombinant antibodies 50 ) determination.

[0129] To determine the EC of 194-1-E1 antibody 50, transiently transfect the relevant expression plasmid into 293F cells, and shake the flask to culture for antibody expression and purification. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 50 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 shake flask according to the required amount of the sample. Incubate at 220 rpm and 25°C in a shaking incubator for 2 h. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50 mL centrifuge tube. After washing twice with 30 mL PBS buffer and deionized water, resuspend them with 1 mL elution buffer. After incubation at 25°C for 15 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, the eluates were combined and the neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with PBS at least 100 times the volume of the sample, first at 25°C for 4 h, then the solution was changed once and then dialyzed at 8°C for 20 h. Finally, the protein concentration was determined and the sample was filtered with a 0.22 μm sterile filter membrane and aliquoted, and stored in a -80°C refrigerator for use.

[0130] Serial dilutions of the target antibody were added to 3×10 5 CHO-S-CD147 cells were incubated at 25℃ for 1 h. Centrifuged at 800×g for 5 min at 25℃, discarded the supernatant, and washed the cells 3 times with PBS. Added 100 μL PE-labeled Anti-human IgG antibody (1:500 dilution), mixed thoroughly, and incubated at 25℃ in the dark for 45 min. Centrifuged at 800×g for 5 min at 25℃, discarded the supernatant, washed the cells 3 times with PBS, and resuspended the cells with 200 μL PBS for flow cytometry analysis.

[0131] The results are as follows Figure 4 and Figure 5 As shown, the EC of 194-1-E1 on CHO-S-CD147 50 The value was 0.1253 μg / mL, indicating that 194-1-E1 had a high affinity.

[0132] Example 6

[0133] Binding detection of recombinant antibodies to colorectal cancer cells.

[0134] Incubate the colorectal cancer cells HCT116 with 194-1-E1 antibody and positive control antibody at 25℃ for 1 h. Centrifuge at 900×g at 25℃ for 15 min, discard the supernatant, and wash the cells 5 times with PBS. Add 100 μL APC-labeled Anti-human IgG antibody, mix thoroughly, and incubate at 25℃ in the dark for 80 min. Centrifuge at 900×g at 25℃ for 15 min, discard the supernatant, wash the cells 3 times with PBS, resuspend the cells with 200 μL PBS, and perform flow cytometric analysis. Figure 6 As shown, the positive control antibody significantly binds to HCT116, indicating that HCT116 expresses CD147 protein and can be used as a detection cell for alpaca antibodies; 194-1-E1 significantly binds to HCT116 and can detect 99.6% of HCT116 cells expressing CD147 protein, indicating that 194-1-E1 has a high affinity for the CD147 protein of HCT116.

[0135] Example 7

[0136] ADCC functional 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 194-1-E1 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. Fig. 7A and Figure 7B As shown, compared with the NK cell group without antibody, 194-1-E1 antibody can significantly enhance the killing effect of NK cells on tumor cells (the relative fluorescence area at the last time point was statistically significant P<0.05), indicating that 194-1-E1 can enhance the killing function of NK cells on tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0139] Example 8

[0140] Related reagents:

[0141] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014);

[0142] Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CAT#L3000001).

[0143] Construction of CAR-T cells based on target antibodies.

[0144] (1) Design of CAR molecules

[0145] The target gene structure of the lentiviral vector involved in this example is as follows Fig. 8A shown.

[0146] BVH147-002 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CD147 single-domain antibody 194-1-E1 [VHH (194-1-E1) 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).

[0147] (2) Lentivirus preparation

[0148] The above CAR molecule expression sequence was fully synthesized and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning, so that it was expressed under the regulation of the human EF-1α promoter and Kozak sequence. The above lentiviral vector expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G using the transfection reagent Lipofectamine 3000 according to the relevant instructions. The viral supernatant was collected 60 h after transfection, centrifuged at 4000 rpm for 10 min at 4°C, filtered through a 0.45 μm pore size filter membrane, and finally ultracentrifuged at 25,000 rpm at 4°C for 2h. The obtained virus concentrate was stored at -80°C and named BVH147-002. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0149] (3) CAR-T cell preparation

[0150] 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 5 TU / mL for transduction. At the same time, 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, and 300 IU / mL recombinant hIL-2 were supplemented and cultured in a cell culture incubator (culture temperature was 37°C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 2×10 6 / mL, and supplemented with 300 IU / mL of hIL-2. On the 5th 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 7 days (culture temperature was 37°C, carbon dioxide concentration was 5%), during which the cell density was maintained at 2×10 6 / mL. On the 12th day after transduction, the cells were collected and frozen in liquid nitrogen for future use. The obtained CAR-T cells were named after the corresponding CAR molecules, and the T cells not transduced with lentivirus were named Ctrl T.

[0151] (4) Detection of CAR molecule expression

[0152] The BVH147-002 CAR-T cells to be tested were washed twice with PBS and resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, the FITC-labeled anti-VHH antibody was incubated with the CAR-T cells for 2 h. The supernatant was then removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Ctrl T cells as a negative control, the CAR molecule expression rate of BVH147-002 cells was detected by flow cytometry. The results are shown in Figure 8B As shown, the expression rate of BVH147-002 CAR was 88.5%.

[0153] Example 9

[0154] In vitro killing assay of BVH147-002 cells.

[0155] 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 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 Fig. 9A and Fig. 9B As shown in the figure, compared with Mock-T cells, BVH147-002 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 194-1-E1 antibody have a good killing function on CD147-positive tumor cells.

[0156] In summary, the pan-tumor antigen antibody E1 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.

[0157] 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 amino acid sequence of CDR1 of the single-domain antibody targeting CD147 is shown as SEQ ID NO.1; the amino acid sequence of CDR2 of the single-domain antibody targeting CD147 is shown as SEQ ID NO.2 and the amino acid sequence of CDR3 of the single-domain antibody targeting CD147 is shown as 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 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 as described in claim 1 or 2, the nucleic acid molecule as described in claim 4, the chimeric antigen receptor as described in claim 5, the expression vector as described in claim 6, the recombinant lentivirus as described in claim 7, and the chimeric antigen receptor immune cell as described in claim 8 in the preparation of a product that enhances NK cell killing of cancer cells; the cancers include colorectal cancer and ovarian cancer.

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