Nano-antibody G11 for enhancing NK cell killing of tumor cells by ADCC effect and its application

Abstract

The present invention relates to a nano antibody G11 and its application for enhancing NK cells to kill tumor cells by using ADCC, wherein the amino acid sequence of the antibody G11 includes CDR1 shown in SEQ ID No. 2, CDR2 shown in SEQ ID No. 3 and CDR3 shown in SEQ ID No. 4. The antibody has high affinity and specificity with CD147 protein, and experiments have shown that the antibody can effectively enhance the killing function of NK cells on cells expressing CD147 protein through antibody-dependent cell-mediated cytotoxicity (ADCC).

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a nano antibody G11 that utilizes ADCC to enhance NK cells from killing tumor cells 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 50kDa-60kDa, belonging to the immunoglobulin superfamily. The CD147 molecule is a single-pass transmembrane glycoprotein, and the gene is located at 19p13.3, consisting of 10 exons spanning approximately 12kb. From the cloned cDNA sequence, it is known that the mRNA length of the CD147 molecule is approximately 1.7kb, and there are about 115 nucleotides before the N-terminal start as a non-coding region, the coding region encodes 269 amino acids, 21 amino acids are signal peptides, 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, also known as basigin and Emmprin, is a transmembrane glycoprotein and a member of the Ig superfamily. It is considered to be an effective inducer of extracellular matrix metalloproteinases (MMPs) and can induce the synthesis of various MMPs. CD147 is located on the plasma membrane and consists of two extracellular immunoglobulin domains (ECI and ECII), a transmembrane domain and an intracellular domain. The monoclonal blocking antibody EIIF4 reacts with ECI and activates the immune activity of the ECI region. The proline 180 and glycine 181 residues in the extracellular domain are essential for CD147-mediated signal transduction and chemotaxis.

[0004] CD147 is expressed on all leukocytes, platelets, and endothelial cells. Through interactions with cyclophilin, monocarboxylate transporter (MCT), Caveolin-1, and integrins, its main biological effects are divided into three areas: cancer, inflammation, and immunity. CD147 regulates cell proliferation, apoptosis, and tumor cell migration, metastasis, and differentiation. It is expressed at high levels in a variety of malignant tumors and plays an important role in the occurrence and development of tumors.

[0005] CD147 is widely expressed in human tumors (such as bone cancer, ovarian cancer, breast cancer, oral cancer, gastric cancer, bladder cancer, lung cancer, prostate cancer, kidney cancer, leukemia, colorectal cancer, glioblastoma, melanoma, esophageal cancer, liver cancer, pancreatic cancer, bile duct cancer, nasopharyngeal carcinoma, etc.), and plays a core role in the progression of many cancers by stimulating the secretion of matrix metalloproteinases (MMP) and cytokines. It can be seen that CD147 is a suitable therapeutic target. Nanobodies are antibodies with naturally missing light chains in the peripheral blood of alpacas. The antibodies only contain one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but they are not as easy to stick to each other as artificially modified single-chain antibody fragments (scFv), and even aggregate into clumps. More importantly, the VHH structure cloned and expressed separately has a structural stability and antigen binding activity comparable to the original heavy chain antibody. 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.).

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

[0007] In order to solve the above technical problems, the present invention provides a nano antibody G11 and its application for enhancing NK cells to kill tumor cells by ADCC. The nano antibody has high affinity and specificity for CD147 protein. Experiments have proved that the antibody can effectively enhance the killing function of NK cells against cells expressing CD147 protein through antibody-dependent cell-mediated cytotoxicity (ADCC).

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

[0009] In a first aspect, the present invention provides a nanobody G11 that utilizes ADCC to enhance NK cell killing of tumor cells, wherein the amino acid sequence of the nanobody G11 comprises CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.3, and CDR3 shown in SEQ ID No.4.

[0010] SEQ ID No. 2: GFTLDYYA.

[0011] SEQ ID No. 3: ISSNGGHTN.

[0012] SEQ ID No. 4: AAVREVRSGYVCYRSVRISYDS.

[0013] In the present invention, CD147 protein is used to immunize alpacas. There is a special antibody lacking light chains, namely heavy chain antibodies, in the blood of camelids and cartilaginous fish. Compared with ordinary antibodies, it only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single domain antibody (also known as nano antibody) consisting only of the heavy chain variable region can be obtained, which has the advantages of small molecular weight, strong tissue penetration, high stability, and low immunogenicity. The prepared G11 antibody has high specificity, and the tumor killing effect of ADCC and CAR-T mediated by it is good.

[0014] The present invention creatively constructs a nano antibody G11 that utilizes ADCC to enhance NK cell killing of tumor cells. The antibody includes three complementary determining regions. Antibody G11 is obtained by amplifying the VHH fragment of the CD147 antibody in alpaca serum. It is a single-domain antibody. The antibody fragment is smaller and easier to penetrate into tissues and cells. It has higher stability. Experiments have shown that it has higher specificity for CD147 protein and can be used for subsequent research on drugs and other aspects.

[0015] Preferably, the amino acid sequence of the Nanobody G11 comprises the sequence shown in SEQ ID No.1.

[0016] SEQ ID No.1:

[0017] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSNGGHTNHADSVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCAAVREVRSGYVCYRSVRISYDSWGQGTQVTVSS.

[0018] In a second aspect, the present invention provides a nucleic acid molecule encoding the nanobody G11 for enhancing NK cell killing of tumor cells using ADCC as described in the first aspect.

[0019] In a third aspect, the present invention provides a recombinant antibody, which comprises a fusion sequence of the nanobody G11 for enhancing NK cell killing of tumor cells by ADCC as described in the first aspect and a human IgG1 Fc fragment.

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

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

[0022] SEQ ID No.5:

[0023] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0024] Preferably, the fused sequence is shown as SEQ ID No.6.

[0025] SEQ ID No.6:

[0026] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSNGGHTNHADSVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCAAVREVRSGYVCYRSVRISYDSWGQGTQVTVSSGGCCACGAAGGCCAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0027] In a fourth aspect, the present invention provides a chimeric antigen receptor molecule, which comprises a human signal peptide, the antibody G11 for enhancing NK cell killing of tumor cells by ADCC as described in the first aspect, a human hinge region, a human transmembrane domain, a human intracellular co-stimulatory domain and a human intracellular signal transduction domain in series.

[0028] Preferably, the human intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, CD3δ, CD3γ or CD3ε.

[0029] Preferably, the human intracellular co-stimulatory domain includes any one or a combination of at least two of CD3ε, CD134, CD3δ, OX40, CD28, OX40L, CD27, CD2, CD30, CD40, CD70, CD3γ, 4-1BB, PD1, ICOS, CDS, ICAM-1, GITR, Dap10, NKG2D, LFA-1 / CD11a / CD18 or CD7.

[0030] Preferably, the human transmembrane domain includes any one or a combination of at least two of CD28, CD3ε, CD8, CD3γ, CD3δ, CD9, TCRβ, CD40, CD152, CD4, CD5, CD3ζ, TCRα, CD16, CD86, CD33, CD37, CD64, CD80, CD134, CD22, CD137, CD154, CD278, CD45, CD279, CD233 or CD314.

[0031] Preferably, the human hinge region includes any one or a combination of at least two of IgG1, CD28, TCRα, CD8, IgG4, CD137 or TCRβ.

[0032] Preferably, the human signal peptide includes any one or a combination of at least two of CD137, GM-CSF, IgE, CD28, CD8, IgG, CD4, TCRα or TCRβ.

[0033] Preferably, the amino acid sequence of the human signal peptide includes the sequence shown in SEQ ID No.7.

[0034] SEQ ID No. 7: MALPVTALLLPLALLLHAARPS.

[0035] Preferably, the amino acid sequence of the human hinge region includes the sequence shown in SEQ ID No.8.

[0036] SEQ ID No.8:

[0037] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0038] Preferably, the amino acid sequence of the human transmembrane domain includes the sequence shown in SEQ ID No.9.

[0039] SEQ ID No. 9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0040] Preferably, the amino acid sequence of the human intracellular co-stimulatory domain includes the sequence shown in SEQ ID No.10.

[0041] SEQ ID No.10:

[0042] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0043] Preferably, the amino acid sequence of the human intracellular signal transduction domain includes the sequence shown in SEQ ID No.11.

[0044] SEQ ID No.11:

[0045] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0046] Preferably, the coding sequence of the chimeric antigen receptor molecule includes that shown in SEQ ID No.12.

[0047] SEQ ID No.12:

[0048] AATMALPVTALLLPLALLLHAARPSAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSNGGHTNHADSVKGRFTISRDNAKSTVYLQMNSLKP EDTAVYYCAAVREVRSGYVCYRSVRISYDSWGQGTQVTVSSAHHSEDPGHEWESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0049] In a fifth aspect, the present invention provides an immune cell, wherein the immune cell comprises the chimeric antigen receptor molecule according to the fourth aspect;

[0050] The immune cells include any one or a combination of at least two of T cells, TIL cells, NK cells, B cells or macrophages.

[0051] In the sixth aspect, a use of the nanobody G11 according to the first aspect for enhancing NK cell killing of tumor cells by ADCC, the nucleic acid molecule according to the second aspect, the recombinant antibody according to the third aspect, the chimeric antigen receptor molecule according to any one of the fourth aspect, or the immune cell according to the fifth aspect in the preparation of a drug for treating a disease associated with high expression of CD147;

[0052] The diseases related to high CD147 expression include solid tumors;

[0053] The solid tumor includes colorectal cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, nasopharyngeal cancer, ovarian cancer or any one thereof or a combination of at least two thereof.

[0054] Compared with the prior art, the present invention has at least the following beneficial effects:

[0055] In the present invention, the nano antibody G11 that utilizes ADCC to enhance NK cell killing of tumor cells includes three complementary determining regions. It has been verified by experiments that the antibody can effectively enhance the killing function of NK cells against cells expressing CD147 protein through antibody-dependent cell-mediated cytotoxicity (ADCC). The antibody fragment is smaller and easier to penetrate into tissues and cells, has higher stability, and has been experimentally proven to have high specificity for CD147 protein. The structure of antibody G11 is simple and easier to transform, and the production cost is low, which is suitable for large-scale production. Alpaca-derived antibodies have a low risk of causing immune response because the alpaca's immune system can produce antibodies with high specificity and affinity for specific antigens, and have weak immunogenicity to humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a flow cytometry assay to detect the specificity of the recombinant single domain antibody.

[0057] Figure 2 This is a flow cytometry assay for detecting the EC50 of a recombinant single domain antibody.

[0058] Figure 3 This is the alpaca antibody concentration-effect curve.

[0059] Figure 4 This is a flow cytometry detection chart of the binding of recombinant single domain antibody to colorectal cancer HCT116 cells.

[0060] Figure 5 This is IncuCyte real-time quantitative live cell imaging analysis of recombinant single-domain antibody-mediated ADCC function, where Figure A is a curve chart of the change of relative fluorescence area over time, and Figure B is a statistical chart of the relative fluorescence area value at the last time point recorded in Figure A.

[0061] Figure 6 It is the expression rate detection of recombinant single-domain antibody CAR-T cells, where Figure A is a schematic diagram of the chimeric antigen receptor structure, and Figure B is the expression rate detection result of the chimeric antigen receptor in CAR-T cells.

[0062] Figure 7 This is IncuCyte real-time quantitative live cell imaging analysis of the killing function of CAR-T cells targeting CD147. Figure A is a curve chart of the change of relative fluorescence area over time, and Figure B is a statistical chart of the relative fluorescence area value at the last time point recorded in Figure A.

[0063] Reagents used in the following examples:

[0064] PBS (bico, CAT# 14190-250).

[0065] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82).

[0066] MonoRab™ Rabbit Anti-Camelid VHH Cocktail [FITC] (GenScript, CAT#A02014).

[0067] Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CAT#L3000001).

[0068] Adjuvant immune adjuvant (GERBU, CAT# 3030).

[0069] HRP-ProteinA (Boster, CAT# BA1080).

[0070] HRP-Streptavidin (Boster, CAT# BA1088).

[0071] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B).

[0072] DNA fragment recovery kit (TakaRa, CAT# 9761).

[0073] Goat anti-Llama IgG (H+L)) Secondary Antibody [HRP] (Novus, AT#NB7242).

[0074] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805).

[0075] Streptavidin PE (eBioscience, CAT# 12-4317-87). DETAILED DESCRIPTION

[0076] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0077] Example 1

[0078] This example is alpaca immunization and serum titer detection

[0079] Three alpacas (No. 73, No. 74 and No. 194) were immunized with CD147 protein, and the immunization experiment was started after recording the ear number of the blank alpaca. Each time, the alpacas were injected on both sides near the cervical lymph nodes, with 2 injections on each side, and about 500 μg of emulsified antigen was injected at each point. After immunization, observe for half an hour to confirm that the alpacas are in good condition and have no discomfort symptoms. Immunization is performed once every 3 weeks, and at least three immunizations are performed. 5 ml of peripheral blood was collected before immunization, two weeks after the second immunization, and the third immunization, respectively. The centrifuge tubes containing blood samples were placed in a centrifuge and centrifuged at 800 g for 10 minutes 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 (PBS solution containing 0.05% Tween 20); add 200 µl / well of blocking solution and block at 37°C for 2 hours; after shaking off the blocking buffer, wash the plate 5 times with PBST; use PBS to graded dilution of the above-collected serum, add 100 µl of graded diluted serum to the 96-well ELISA plate, incubate at room temperature for 1 hour, and use PBS as the control well; shake off the liquid in the well and wash 5 times with PBST; add 100 µl HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour; shake off the liquid in the well and wash the plate 5 times with PBST; add 100 µl / well TMB colorimetric solution; incubate at room temperature in the dark for 10 minutes; add 50 µl / well stop solution; read the OD in the well using a microplate reader 450 value.

[0080] The ratio of the absorbance of the test serum to the blank control was calculated. When the ratio was greater than 2, it was positive, and the positive result with the largest dilution multiple was defined as the titer of the antiserum. The results are shown in Tables 1, 2, and 3. The binding titers of the serum of the three alpacas before immunization with the CD147 protein were 8k, 1k, and 32k, respectively. The binding titers of the serum of the three alpacas after the third immunization with the CD147 protein were all much higher than 64k, indicating that the content of CD147-specific antibodies in the serum of the three alpacas was significantly increased after multiple immunizations, and the immunization was successful, which can be used for the construction of yeast display libraries.

[0081] Table 1

[0082]

[0083] Table 2

[0084]

[0085] Table 3

[0086]

[0087] Example 2

[0088] This example constructs a single domain antibody yeast display library

[0089] (1) VHH antibody fragment cloning

[0090] 100 ml of peripheral blood was collected and PBMCs were separated using lymphocyte separation buffer. RNA was extracted using PrimeScript™II 1 st cDNA was obtained by reverse transcription using the Strand cDNA Synthesis Kit. PCR amplified the alpaca heavy chain antibody sequence using specific primer 1 (upstream primer binds to the signal peptide of the VHH antibody ORF, and downstream primer binds to the CH2 region). Analyze the PCR product by electrophoresis and recover the target fragment. Then, using the first-round PCR product as a template, amplify the heavy chain antibody VHH fragment using specific primer 2 (upstream primer binds to the antibody FR1 region, and downstream primer binds to the anti-Hinge and FR4 regions). Analyze the PCR product by electrophoresis and recover the target fragment. Store the target fragment at -80°C.

[0091] Table 4

[0092]

[0093] (2) Electroporation of library vectors and detection of library capacity and diversity

[0094] The yeast surface display vector pYDisplay and the VHH fragment library obtained above were digested with SfiI endonuclease. The pYDisplay vector fragment was separated by electrophoresis, and the 5000 bp vector fragment was cut out for gel recovery. The yeast competent strain frozen at -80°C was streaked onto a YPD solid medium plate in advance and activated at 30°C for 3 days. A single colony of yeast competent strain was inoculated into 50 ml of YPD medium and shaken at 250 rpm and 30°C for 1 day.

[0095] After mixing the linearized vector fragment and PCR product, electroporate and transform; transfer the yeast competent cells after electroporation to a culture bottle and shake at 220 rpm and 30°C for 1 h. Take 20 μl of the resuspension, dilute it 5000 times with SDCAA medium, take 100 μl, apply it to SDCAA plates, culture for 3 days, calculate the library capacity, and continue to culture the rest of the bacterial solution for 24 hours. Collect the remaining bacterial solution in a 50 ml centrifuge tube, centrifuge at 3000×g for 5 minutes, discard the supernatant, add 10 ml of SDCAA medium to resuspend, and freeze the freezing solution with a volume ratio of 50% glycerol and resuspension solution of 1:1, and freeze at -80°C.

[0096] The results showed that the library capacity of the yeast display library from alpaca No. 73 was 1.6×10 9 The library capacity of the yeast display library from alpaca No. 74 is 1.3×10 9 , the library capacity of the yeast display library from alpaca 194 is 1.1×10 9 After Sanger sequencing, the results showed that the yeast library had good sequence diversity and could be used for subsequent experiments.

[0097] Example 3

[0098] This example performs panning of a yeast display library

[0099] (1) Pretreatment of Streptavidin magnetic beads and yeast cells

[0100] Take 8 ml of yeast library (about 2 × 10 8 Yeast cells) to a 50ml centrifuge tube, centrifuge at 3000×g for 5 minutes, and discard the supernatant; at the same time, dilute sterile 5% PBSA (PBS solution containing 5% BSA) to 0.5% PBSA; resuspend the yeast library with 1ml of 0.5% PBSA, add it to a 1.5ml centrifuge tube, centrifuge at 3000×g for 5 minutes, and discard the supernatant; wash again with 0.5% PBSA. Prepare three 1.5ml centrifuge tubes (two for negative selection of empty magnetic beads and one for positive selection), and add 1ml of 0.5% PBSA to each tube; use a pipette to fully resuspend the aliquoted Streptavidin magnetic beads, and pipette 10μl of magnetic beads into a 1.5ml centrifuge tube; put the tubes in a bag, fix them on a rotating mixer, and rotate and incubate at 4°C for 5 minutes; place the tubes on a magnetic stand for 5 minutes, and remove the supernatant with a pipette; then add 1ml of 0.5% PBSA again, rotate and incubate at 4°C for another 5 minutes, and remove the supernatant.

[0101] (2) Yeast display library screening

[0102] 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, put it in a bag, and rotate and incubate at 4°C for 60 minutes; Place the empty magnetic bead centrifuge tube with yeast on a magnetic rack for 10 minutes, 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 30 minutes. After the incubation, place it on a magnetic rack for 15 minutes, aspirate the yeast solution for later use.

[0103] Biotin-CD147-His magnetic separation: add 100 μl of 50 μg / ml Biotin-CD147-His protein solution (Biotin coupled, diluted with 0.5% PBSA) to a centrifuge tube containing Streptavidin magnetic beads, place the centrifuge tube in a bag, and incubate at 4°C for 60 minutes; then add 1 ml of 0.5% PBSA, let it stand for 5 minutes, keep the centrifuge tube on the magnetic rack, and remove the supernatant with a pipette, then add 1 ml of 0.5% PBSA, remove the centrifuge tube from the magnetic rack, mix it with a pipette, place the centrifuge tube on the magnetic rack again, let it stand for 5 minutes, 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.

[0104] Add the positive selection magnetic beads bound to Biotin-CD147-His to the yeast cells that have completed negative selection, put them into a bag and place them on a rotating mixer, incubate them at 4°C for 60 minutes, and after the incubation, place them on a magnetic rack and let them stand at room temperature for 15 minutes; keep the centrifuge tube on the magnetic rack and use a pipette to absorb the unbound yeast solution; remove the 1.5 ml centrifuge tube from the magnetic rack, and add 1 ml of sterile 0.5% PBSA buffer to the centrifuge tube with a pipette, gently blow the magnetic beads, and then transfer them to a sterile 1.5 ml centrifuge tube, place the centrifuge tube on the magnetic rack, let it stand at room temperature for 5 minutes, and discard the supernatant; repeat the washing twice, and the whole operation is performed in accordance with the sterile operation requirements. After washing, resuspend the magnetic beads and adhered yeast cells with 1 ml of SDCAA medium, pipette 20 μl of the resuspended solution into 180 μl of SDCAA medium and coat two plates, each with a coating volume of 100 μl, then pipette 5 μl of the resuspended solution into 95 μl of SDCAA medium and coat one plate, i.e. a total of 3 plates need to be coated.

[0105] Yeast flow cytometry after panning: The yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into yeast. The yeast that has undergone negative panning with empty magnetic beads and magnetic sorting with Biotin-CD147-His was incubated with CD147 protein containing His tag at 4°C for 60 minutes, then centrifuged to remove the supernatant, added flow antibodies with anti-His tag and anti-V5 tag, incubated at 4°C for 60 minutes, then centrifuged to remove the supernatant, and resuspended the cells in 500 μl of PBS for flow analysis. Before and after sorting, the yeast that can bind to CD147 protein in the yeast antibody display library of alpaca No. 73, alpaca No. 74 and alpaca No. 194 increased by 10%, 41% and 13% respectively, and subsequent experiments can be carried out.

[0106] Example 4

[0107] This example is used for screening of recombinant antibodies

[0108] After sorting, the yeast solution was spread on the SDCAA plate, and a single clone was picked after overnight culture, and the single clone was inoculated into the culture medium to induce expression. After 48 hours, the bacterial solution was incubated with the CD147 protein containing the His tag at 4°C for 60 minutes, and then the supernatant was removed by centrifugation. The precipitate was resuspended with a solution containing a biotin-conjugated anti-His tag flow antibody, and incubated at 4°C for 60 minutes. The supernatant was then removed by centrifugation, and the precipitate was resuspended with a solution containing PE-Streptavidin, and incubated at 4°C for 60 minutes. After the incubation, flow detection was performed. Finally, the monoclonal 74-2-G11 with a high positive rate was selected. 74-2-G11 was lysed with 0.2% SDS (incubated at 95°C for 10 minutes), 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).

[0109] Example 5

[0110] This example is to express the recombinant single domain antibody and detect its binding to the target protein

[0111] A PCR instrument was used to amplify the candidate 74-2-G11 VHH sequence, CMV promoter sequence and IgG1 Fc sequence, 50 μl of the PCR product was taken for electrophoresis analysis and then purified. The nucleic acid sequence of the CMV promoter is shown in SEQ ID No.17.

[0112] SEQ ID No.17:

[0113] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG ACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT.

[0114] The 74-2-G11 VHH sequence, CMV promoter and IgG1 Fc sequence were connected using a PCR instrument, and then 50 μl of the PCR product was taken for electrophoresis analysis and purification. After the obtained PCR product was transiently transfected into HEK293 cells, the harvested cell culture supernatant contained the recombinant single-domain antibody 74-2-G11.

[0115] The binding specificity of 74-2-G11 was tested. The culture supernatant containing 74-2-G11 was mixed with 3×10 5 CHO-S or CHO-S-CD147 cells were incubated at room temperature for 1 h. After centrifugation at 800×g for 5 minutes, the supernatant was discarded and the cells were washed 3 times with PBS. 100 μl of PE-labeled Anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature for 45 minutes in the dark. After centrifugation at 800×g for 5 minutes, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500 μl of PBS for flow cytometry analysis.

[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 had significant binding to CHO-S-CD147 but not to CHO-S cells, indicating that CHO-S-CD147 cells successfully overexpressed CD147 protein and could be used as detection cells for alpaca antibodies. 74-2-G11 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-G11 had good affinity and specificity.

[0117] Example 6

[0118] In this example, the recombinant antibody was purified and the half effective concentration (EC 50 ) Determination

[0119] To determine the EC of 74-2-G11 antibody 50 , transiently transfect the relevant expression plasmid into 293F cells, and shake the flask to culture for antibody expression and purification. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30 ml of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads to the 293F cell shake flask according to the required amount of the sample. Incubate at room temperature for 2 hours in a shaking incubator at 120 rpm.

[0120] Collect the ProteinA beads with a magnetic separation rack and transfer them to a 50 ml centrifuge tube. Wash twice with 30 ml of PBS buffer and deionized water, and resuspend with 1 ml of elution buffer. After incubation at room temperature for 5 minutes, collect the beads with a magnetic separation rack, and transfer the supernatant containing the target antibody to a 15 ml centrifuge tube. After eluting the ProteinA beads twice, combine the eluates, and add neutralization buffer to adjust the pH of the solution. The eluted sample is dialyzed with at least 100 times the volume of the sample with PBS, first dialyzed at 20°C for 2 h, changed the solution once, and then dialyzed at 6°C for 15 h. Finally, determine the protein concentration and filter the sample with a 0.22μm sterile filter membrane and aliquot, and store in a -80°C refrigerator for use.

[0121] Serial dilutions of the target antibody were added to 3×10 5CHO-S-CD147 cells were incubated at room temperature for 1 h. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Add 100 μl of PE-labeled Anti-human IgG antibody (1:500 PBS dilution), mix well, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant, wash the cells three times with PBS, resuspend the cells with 200 μl PBS, and perform flow cytometry analysis.

[0122] The results are as follows Figure 2 and Figure 3 As shown, the EC of 74-2-G11 on CHO-S-CD147 50 The value was 0.25 μg / ml, indicating that 74-2-G11 had a high affinity.

[0123] Example 7

[0124] This example detects the binding of recombinant antibodies to colon cancer cells

[0125] Incubate the colorectal cancer cells HCT116 with 74-2-G11 antibody and positive control antibody at room temperature for 1 h. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant, and wash the cells 3 times with PBS. Add 100μl of APC-labeled Anti-human IgG antibody, mix thoroughly, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant, wash the cells 3 times with PBS, and resuspend the cells with 200μl of 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-G11 significantly binds to HCT116 and can detect 99.6% of HCT116 cells expressing CD147 protein, indicating that 74-2-G11 has a high affinity for CD147 protein.

[0126] Example 8

[0127] This embodiment is used for ADCC function detection

[0128] 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 inoculated into 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-G11 was added at a final concentration of 5 μg / ml.

[0129] 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 5 A and Figure 5 As shown in B, compared with the NK cell group without antibody, 74-2-G11 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-G11 can enhance the killing function of NK cells against SK-OV-3 through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0130] Example 9

[0131] This example is used to construct CAR-T cells based on antibody G11

[0132] (1) Design of CAR molecules

[0133] The target gene structure of the lentiviral vector involved in this example is as follows Figure 6 As shown in A.

[0134] BVH147-010 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CD147 single-domain antibody 74-2-G11 [VHH (74-2-G11) 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).

[0135] (2) Lentivirus preparation

[0136] 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 with lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G using the transfection reagent Lipofectamine 3000. The viral supernatant was collected 48 hours after transfection, centrifuged at 3000rpm for 10-15 minutes at 4°C, filtered through a 0.45 μm pore size filter membrane, and finally ultracentrifuged at 25,000rpm at 4°C for 2-3 hours. The obtained virus concentrate was stored at -80°C and named BVH147-010. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0137] (3) CAR-T cell preparation

[0138] 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 IU / ml for transduction. At the same time, 25 ng / ml anti-CD3 antibody, 25 ng / ml anti-CD28 antibody and 300 IU / ml recombinant hIL-2 were supplemented and cultured in a cell culture incubator (culture temperature was 37°C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 1.5~2×10 6 / ml, and supplemented with 300 IU / ml hIL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for 5 days (culture temperature was 37°C, carbon dioxide concentration was 5%), during which the cell density was maintained at 1 ~ 2×10 6 / ml. The cells were collected on the 10th day after transduction and frozen in liquid nitrogen for future use. The obtained CAR-T cells were named after the corresponding CAR molecules, and the T cells not transduced with lentivirus were named Ctrl T.

[0139] (4) Detection of CAR molecule expression

[0140] The BVH147-010 CAR-T cells to be tested were washed twice with PBS and resuspended in FACS buffer (PBS solution containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, the FITC-labeled anti-VHH antibody was incubated with the CAR-T cells for 1 h. The supernatant was then removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Ctrl T cells as negative control, the CAR molecule expression rate of BVH147-010 cells was detected by flow cytometry. The results are shown in Figure 6 As shown in B, the expression rate of BVH147-010 CAR was 35.6%.

[0141] Example 10

[0142] This example studies the function of CAR-T cells in vivo and in vitro

[0143] In vitro killing experiment of BVH147-010 cells, 1×10 5HCT116-mCherry cells were resuspended at a density of 100 μl / ml and inoculated into a 96-well plate at a volume of 100 μl per well. After being cultured overnight in the IncuCyte 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 software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells. The results are shown in Figure 7 A and Figure 7 As shown in B, compared with Mock-T cells, BVH147-010 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-G11 antibodies have a good killing function on CD147-positive tumor cells.

[0144] In summary, the present invention determines the titer of CD147-specific antibodies in alpaca serum by ELISA, separates alpaca peripheral blood mononuclear cells, extracts RNA and reverse transcribes to obtain cDNA. Using alpaca single-domain antibody-specific primers, amplify the VHH sequence, clone it into a yeast expression plasmid, and 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. Pick a monoclone from the enriched product, and detect the specific binding of the candidate antibody to CD147 by ELISA. After the positive antibody is expressed and purified, its affinity for binding to the CD147 protein is detected. The antibody clone with strong affinity to CD147 is selected to obtain the G11 antibody, and co-cultured with NK cells and SK-OV-3 ovarian cancer cells to detect the antibody-dependent cell-mediated cytotoxicity of each clone. 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 antibody G11 has high specificity and good tumor killing effects of ADCC and CAR-T mediated by it.

[0145] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-CD147 nanobody G11 that utilizes ADCC to enhance NK cell killing of tumor cells, characterized in that: The amino acid sequence of the anti-CD147 nanobody G11 is CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.3 and CDR3 shown in SEQ ID No.

4.

2. The anti-CD147 nanobody G11 for enhancing NK cell killing of tumor cells using ADCC according to claim 1, characterized in that: The amino acid sequence of the anti-CD147 nanobody G11 includes the sequence shown in SEQ ID No.

1.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD147 nanobody G11 according to claim 1 or 2, which utilizes ADCC to enhance NK cell killing of tumor cells.

4. A recombinant antibody, characterized in that: The recombinant antibody comprises the anti-CD147 nano antibody G11 and human IgG1 Fc for enhancing NK cell killing of tumor cells by ADCC as described in claim 1 or 2.

5. The recombinant antibody according to claim 4, characterized in that The sequence of the human IgG1 Fc fragment is shown in SEQ ID No. 5; The sequence of the recombinant antibody is shown as SEQ ID No.

6.

6. A chimeric antigen receptor molecule, characterized in that The chimeric antigen receptor molecule includes a human signal peptide, the anti-CD147 nanoantibody G11 for enhancing NK cell killing of tumor cells using ADCC as described in claim 1 or 2, a human hinge region, a human transmembrane domain, a human intracellular co-stimulatory domain and a human intracellular signal transduction domain.

7. The chimeric antigen receptor molecule according to claim 6, characterized in that The human intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, CD3δ, CD3γ or CD3ε; The human intracellular co-stimulatory domain includes any one or a combination of at least two of CD3ε, CD134, CD3δ, OX40, CD28, OX40L, CD27, CD2, CD30, CD40, CD70, CD3γ, 4-1BB, PD1, ICOS, CDS, ICAM-1, GITR, Dap10, NKG2D, LFA-1 / CD11a / CD18 or CD7; The human transmembrane domain includes any one or a combination of at least two of CD28, CD3ε, CD8, CD3γ, CD3δ, CD9, TCRβ, CD40, CD152, CD4, CD5, CD3ζ, TCRα, CD16, CD86, CD33, CD37, CD64, CD80, CD134, CD22, CD137, CD154, CD278, CD45, CD279, CD233 or CD314; The human hinge region includes any one or a combination of at least two of IgG1, CD28, TCRα, CD8, IgG4, CD137 or TCRβ; The human signal peptide includes any one or a combination of at least two of CD137, GM-CSF, IgE, CD28, CD8, IgG, CD4, TCRα or TCRβ.

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

12.

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

10. Use of the anti-CD147 nanobody G11 according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant antibody according to claim 4 or 5, the chimeric antigen receptor molecule according to any one of claims 6 to 8, or the immune cell according to claim 9 in the preparation of a drug for treating a disease associated with high expression of CD147; The diseases related to high CD147 expression include solid tumors; The solid tumors include colon cancer and / or ovarian cancer.

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