A group of anti-human gpc3 nanobody mutants and chimeric antigen receptors and applications

Abstract

The application discloses a group of anti-human GPC3 nanobody mutants and chimeric antigen receptors and application, and belongs to the technical field of immunity. The application provides a group of anti-GPC3 nanobody mutants, and the existing anti-CPG3 nanobody (NH3) is subjected to mutation, so that the affinity of the antibody is improved. The application also constructs a chimeric antigen receptor based on the nanobody mutant, simultaneously synthesizes double-stranded DNA of the nanobody mutant as a template for producing mRNA through in-vitro transcription, and then utilizes the mRNA to genetically engineer NK cells, so as to obtain CAR-NK cells. In the CAR-NK cells, the mRNA can effectively express CAR molecules on the NK cells, and has a stronger in-vitro killing ability, and shows a better anti-liver cancer effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunology, and particularly relates to a group of anti-human GPC3 nanobody mutants and chimeric antigen receptors and applications. BACKGROUND

[0002] Glypian-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) consisting of a 580-amino acid 65-kD protein. GPC3 protein is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, 14 conserved cysteine residues, and the last 50 residues are modified by heparan sulfate (HS) side chains. This protein is expressed in the liver and kidney of healthy fetuses, but in adults it is almost not expressed in tissues other than placental tissue. Studies have shown that GPC3 is specifically expressed in hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, lung squamous cell carcinoma, hepatoblastoma, Wilms tumor, yolk sac tumor and some pediatric cancers. Although the exact function of GPC3 is not clear, it has been strongly suggested that it is related to the malignant transformation of HCC. Therefore, GPC3 has been identified as a promising target for cancer immunotherapy.

[0003] In 1989, famous scientists Gross G, Waks T and Eshhar Z in a study, the gene sequence expressing a specific antibody was given to cytotoxic T cells, which enabled T cells to achieve antigen-specific, non-MHC restricted activation and enhancement of its effects. They believe: "Giving T cells a chimeric antigen receptor will be an important way for humans to fight tumors in the future." This is the first time in human history that the concept of CAR has been proposed. CAR-T therapy, also known as chimeric antigen receptor (CAR) T cell therapy, is to modify human T cells in vitro by genetic engineering, and then return them to the patient's body for the treatment of diseases.

[0004] However, as research deepens and clinical applications continue to increase, the drawbacks produced during CAR-T therapy have gradually been recognized and focused on, such as graft-versus-host reaction, cytokine storm and neurotoxicity, resistance and tolerance to solid tumors, process complexity, personalized customization and long production cycle, and expensive treatment costs, etc. Natural killer (NK) is an integral part of the body's innate immune system and plays an important role in the body's antitumor immune surveillance process. Dario Campana et al. first established a PB-NK cell platform in 2005 and successfully introduced the CAR structure into primary NK cells for the first time.

[0005] CAR-NK cells have multiple advantages over CAR-T cells: unlike CAR-T cells, CAR-NK cells retain the intrinsic ability to recognize and target tumor cells through their natural receptors, so that the possibility of tumor cells escaping killing is reduced when CAR-NK cells are targeted for treatment; CAR-NK cells do not cause immune rejection within several days to several weeks. Therefore, they do not exhibit the safety problems of CAR-T in many clinical trials, such as the distress of cytokine release syndrome; NK cells do not require strict HLA matching and have no potential to cause graft-versus-host disease, which is an important risk of CAR-T cell immunotherapy.

[0006] He Miao Zhuang et al. disclosed an anti-GPC3 nanobody in CN 105968209A, but its affinity is low, and its subsequent application is limited. SUMMARY

[0007] The present application provides a group of anti-human GPC3 nanobody mutants and chimeric antigen receptors and applications, which have stronger binding capacity to the target and are more suitable for the application of CAR-NK technology.

[0008] The present application provides a group of anti-GPC3 nanobody mutants, which have higher GPC3 affinity than the anti-GPC3 nanobody.

[0009] The amino acid sequence of the anti-GPC3 nanobody is shown in SEQ ID No. 9.

[0010] Preferably, the nanobody mutant includes any one of the following: mutant 1-2, mutant 2-2, mutant 3-2 and mutant 4-2, whose amino acid sequences are shown in SEQ ID No. 1-SEQ ID No. 4 in turn.

[0011] Preferably, the nucleotide sequence of the nanobody mutant is shown in SEQ ID No. 5-SEQ ID No. 8 in turn.

[0012] The present application also provides a chimeric antigen receptor for targeting GPC3 based on the above-mentioned nanobody mutant.

[0013] Preferably, the structure of the chimeric antigen receptor includes a signal peptide, an antibody region, a hinge region, a transmembrane region and an intracellular region connected in turn.

[0014] Preferably, the source of the signal peptide includes a human CD8a signal peptide.

[0015] The antibody region includes one mutant sequence or two mutant sequences connected by a flexible connecting peptide.

[0016] The source of the hinge region comprises human IgG4 or human CD8a;

[0017] The source of the transmembrane region comprises human CD28 or human CD8a;

[0018] The source of the intracellular region comprises human 41BB or CD3.

[0019] The application also provides a linear double-stranded gene amplified by nucleotides of the above-mentioned chimeric antigen receptor, wherein the primers for amplification comprise an upstream primer with a nucleotide sequence as shown in SEQ ID No. 11 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 12.

[0020] The application also provides an mRNA obtained by in vitro transcription using the above-mentioned linear double-stranded gene as a template.

[0021] The application also provides a genetically engineered CAR-NK cell, which is obtained by transforming an NK cell with the above-mentioned mRNA and expressing the above-mentioned nanobody mutant.

[0022] The application also provides the use of the above-mentioned nanobody mutant or the above-mentioned chimeric antigen receptor or the above-mentioned CAR-NK cell in the preparation of a medicament for treating a subject suffering from a cancer expressing GPC3.

[0023] Beneficial effects: the application provides a group of anti-GPC3 nanobody mutants, which are obtained by mutating an existing anti-CPG3 nanobody (NH3), so as to improve the affinity of the antibody. In the examples, the anti-GPC3 antibodies after mutation (1-2, 2-2, 3-2 and 4-2) are compared with the parent antibody NH3 (1-2, 2-2, 3-2, 4-2 and NH3 parent antibody), and the EC 50 values of the three antibodies 1-2, 2-2 and 3-2 are lower than that of the parent antibody NH3, indicating that they have stronger binding capacity.

[0024] The application also constructs a chimeric antigen receptor based on the nanobody mutant, synthesizes double-stranded DNA of the nanobody mutant as a template for producing mRNA by in vitro transcription, and genetically engineers an NK cell with the mRNA, so as to obtain a CAR-NK cell. In the CAR-NK cell of the application, the mRNA can effectively express a CAR molecule on the NK cell, and has stronger in vitro killing capacity and better anti-liver cancer effect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A schematic diagram of primer design for preparing a mutant antibody library;

[0026] Figure 2 A small-scale induced expression result diagram of the anti-GPC3 nanobody mutant;

[0027] Figure 3 Figure for affinity determination results of ELISA detection of anti-GPC3 antibody;

[0028] Figure 4 Figure for GPC3 protein and antibody NH3 affinity signal;

[0029] Figure 5 Figure for GPC3 protein and antibody 1-2 affinity signal;

[0030] Figure 6 Figure for GPC3 protein and antibody 2-2 affinity signal;

[0031] Figure 7 Figure for GPC3 protein and antibody 3-2 affinity signal;

[0032] Figure 8 Figure for GPC3 protein and antibody 4-2 affinity signal;

[0033] Figure 9 Figure for CAR-NK cell expression results prepared based on 1-2 antibody sequence;

[0034] Figure 10 Figure for CAR-NK in vitro killing capacity detection results based on 1-2 antibody;

[0035] Figure 11 Figure for structure of amino acid sequence of antibody library of the application. DETAILED DESCRIPTION

[0036] The application provides a group of anti-GPC3 nanobody mutants, which have higher GPC3 affinity than the anti-GPC3 nanobody.

[0037] The amino acid sequence of the anti-GPC3 nanobody is shown in SEQ ID No. 9.

[0038] The anti-GPC3 nanobody has been disclosed in Chinese patent CN105968209A, and the application preferably designs a mutant library based on the anti-GPC3 nanobody (parent NH3 antibody) and screens four mutant antibodies, with clone numbers being 1-2, 2-2, 3-2 and 4-2, and sequences being shown in Table 1.

[0039] Table 1: Amino acid sequence and nucleotide sequence of antibody

[0040] Antibody code Amino acid sequence Gene sequence NH3 SEQ ID No. 9 SEQ ID No. 10 1-2 SEQ ID No. 1 SEQ ID No. 5 2-2 SEQ ID No. 2 SEQ ID No. 6 3-2 SEQ ID No. 3 SEQ ID No. 7 4-2 SEQ ID No. 4 SEQ ID No. 8

[0041] The application also provides a chimeric antigen receptor targeting GPC3 based on the above-mentioned nanobody mutant.

[0042] The structure of the chimeric antigen receptor of the present application preferably comprises a signal peptide, an antibody region, a hinge region, a transmembrane region and an intracellular region connected in sequence; wherein the source of the signal peptide preferably comprises a human CD8a signal peptide, the amino acid sequence is shown as SEQ ID No. 79, and the nucleotide sequence is shown as SEQ ID No. 84; the antibody region preferably comprises a mutant sequence or two mutant sequences connected by a flexible linker peptide; the source of the hinge region preferably comprises human IgG4 or human CD8a, wherein the amino acid sequence of the hinge region of IgG4 is shown as SEQ ID No. 80, the nucleotide sequence is shown as SEQ ID No. 85, the amino acid sequence of the hinge region of CD8a is shown as SEQ ID No. 81, and the nucleotide sequence is shown as SEQ ID No. 86; the source of the transmembrane region preferably comprises human CD28 or human CD8a, wherein the amino acid sequence of the transmembrane region of CD28 is shown as SEQ ID No. 82, the nucleotide sequence is shown as SEQ ID No. 87, the amino acid sequence of the transmembrane region of CD8a is shown as SEQ ID No. 83, and the nucleotide sequence is shown as SEQ ID No. 88; the source of the intracellular region preferably comprises human 41BB and human CD3, wherein the amino acid sequence of 41BB is shown as SEQ ID No. 89: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL; the amino acid sequence of human CD3 is shown as SEQ ID No. 90: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR; both of which are included in the embodiment, the amino acid sequence is shown as SEQ ID No. 91, and the nucleotide sequence is shown as SEQ ID No. 92.

[0043] It is found that mutant 1-2 has the best affinity through verification in the embodiment of the present application, so the chimeric antigen receptor targeting GPC3 based on mutant 1-2 is obtained, wherein the antibody fragment can be used once or twice in head-to-tail, the two antibody fragments are directly connected by a flexible linker peptide, and the above structure is sequentially connected in sequence, thereby obtaining the chimeric antigen receptor of the present application. The amino acid sequence and the nucleotide sequence of each structure of the chimeric antigen receptor targeting GPC3 based on mutant 1-2 in the embodiment of the present application are shown in Table 2 and Table 3, but it cannot be simply regarded as the entire protection scope of the present application.

[0044] Table 2 Structure sequence of CAR-NK (amino acid)

[0045]

[0046] Table 3 Structure sequence of CAR-NK (nucleotide)

[0047]

[0048]

[0049]

[0050]

[0051] The present application also provides a linear double-stranded gene amplified by the nucleotide of the above-mentioned chimeric antigen receptor, and the primers for amplification include an upstream primer with a nucleotide sequence as shown in SEQ ID No. 11 and a downstream primer as shown in SEQ ID No. 12.

[0052] The present application preferably concatenates the sequences in Table 3, and entrusts Jinweizhi Biological to synthesize linear double-stranded genes (minigenes) of four structures, and then performs PCR amplification by using the upstream primer and the downstream primer; after PCR amplification, double-stranded DNA containing a T7 promoter (SEQ ID No. 93: TAATACGACTCACTATAA) and a kozac sequence (gccacc) and a polyadenylic acid tail (85 adenosine repeats) is produced, which can be used as a template for further in vitro transcription to produce mRNA.

[0053] Upstream primer (SEQ ID No. 11): taatacgactcactataagccaccATGGCCCTGCCCGTGACC;

[0054] Downstream primer (SEQ ID No. 12): tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttCCTTGGAGGCAGGGCCTGCATG.

[0055] The present application also provides mRNA obtained by in vitro transcription using the above-mentioned linear double-stranded gene as a template.

[0056] The present application preferably uses an in vitro transcription kit to reverse transcribe the above-mentioned linear double-stranded gene into mRNA.

[0057] The present application also provides a genetically engineered CAR-NK cell, which is obtained by transforming an NK cell by the above-mentioned mRNA and expressing the above-mentioned nanobody mutant.

[0058] The mRNA of the application transforms NK cells, and in the examples, the transformation is preferably accomplished using the method of electroporation.

[0059] The application also provides the use of the above-mentioned nanobody mutant or the above-mentioned chimeric antigen receptor or the above-mentioned CAR-NK cell in the preparation of a medicament for treating a subject suffering from a cancer expressing GPC3.

[0060] In the examples of the application, the mRNA of the four CAR structure designs can effectively express CAR molecules on NK cells and enhance the in vitro killing ability, and have a strong anti-liver cancer effect.

[0061] In order to further illustrate the application, a group of anti-human GPC3 nanobody mutants and chimeric antigen receptors and applications provided by the application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the application.

[0062] Example 1

[0063] 1. Synthesis of primers and PCR splicing

[0064] According to the NH3 antibody and its nucleotide sequence in Chinese patent CN105968209A, six groups of degenerate primers were designed and mixed (10 pM of each primer per reaction), so as to splice the full-length sequence based on the bridge PCR reaction, and the splicing PCR strategy is as shown in Figure 1 .

[0065] The first group has 1 primer (SEQ ID No. 13);

[0066] The second group has 28 primers (SEQ ID No. 14-SEQ ID No. 41);

[0067] The third group has 15 primers (SEQ ID No. 41-SEQ ID No. 56);

[0068] The fourth group has 1 primer (SEQ ID No. 57);

[0069] The fifth group has 15 primers (SEQ ID No. 58-SEQ ID No. 72);

[0070] The sixth group has 1 primer (SEQ ID No. 73).

[0071] All primers were entrusted to be synthesized by Jinweizhi Biotechnology Co., Ltd., and after synthesis, they were purified by desalting to the specified size and delivered, and were dissolved in water at a concentration of 10 μM when delivered.

[0072] The PCR enzyme kit (brand: Toyobo, polymerase KOD MAX) was used to mix the components including six groups of primers according to the instructions and amplify the pUCG3 vector to obtain a linearized fragment. Briefly, the reaction system contains three steps, the first step is the initial denaturation step of 98℃ for 10 seconds; the second step is the amplification step of 35 cycles, each cycle includes denaturation of 98℃ for 10 seconds, annealing of 68℃ for 45 seconds, and extension of 68℃ for 1 minute; and finally, a final extension step of 68℃ for 1 minute is performed. The nucleic acid was separated by agarose gel electrophoresis, and the cut target sequence was recovered for standby.

[0073] The pUCG3 plasmid was obtained by gene synthesis method, and a small amount of extraction was prepared (commissioned by Jinweizhi Biotechnology Co., Ltd.). The full sequence of pUCG3 plasmid is SEQ ID No. 74; after final splicing, the library gene sequence is SEQ ID No. 75, and the structure diagram of the amino acid sequence of the antibody library is preferably as shown in Figure 11

[0074] 2. Mutant antibody gene library plasmid construction

[0075] The pUCG3 vector-F (SEQ ID No. 76: GGTGGCAGCGATTACAAGGATGACGATGACAAG) primer and the pUCG3 vector-R (SEQ ID No. 77: GGCCATCGCCGGCTGGGCCGCGAG) primer were used to amplify the pUCG3 vector to obtain a linearized fragment according to the instructions of the PCR enzyme kit (Toyobo KOD MAX). The amplification program was set as follows: 98℃ pre-denaturation for 10 seconds; 35 amplification cycles, each cycle including 98℃ denaturation for 10 seconds, 68℃ annealing for 45 seconds, and 68℃ extension for 1 minute; and 68℃ re-extension for 1 minute. The obtained fragment sequence is SEQ ID No. 78.

[0076] At the same time, according to the instructions, the GPC3 mutant library fragment was connected with the vector by using the homologous recombination kit (Novagen brand, item number C112): The pUCG3 linear vector (2000 ng), GPC3 mutant library (400 ng), 5×CE buffer, and Exnase II enzyme were added to the PCR tube, and water was added to make the final volume 400 μl, and the reaction was carried out at 37℃ for 15 minutes. The reaction product was recovered using the nucleic acid purification kit (DP214). All nucleic acids were used for electrotransformation of 17 TG1 competent cells, and all bacterial solutions were evenly divided into 17 plates except for calculating the library size, and cultured overnight. The next day, all colonies on the plates were collected, and 1 ml of each was frozen as 10 cryotubes;

[0077] 3. Phage display and panning

[0078] ​Recover one vial of frozen bacteria, take 60 μl and add to 30 ml of 2xYT (2xYTAG) medium containing ampicillin and glucose, 200 rpm, until OD 600 = 0.75, add helper phage M13K07 (Stratagene, cat. P006) to a final concentration of 1 x 10 9 TU / ml, 200 rpm, 37°C, 1 h; centrifuge at 3500 rpm to pellet, resuspend in 30 ml 2xYT + Amp + Kan medium (without sugar), shake and incubate overnight at 37°C;

[0079] The next day, centrifuge to collect the culture supernatant, take 100 μl of the phage supernatant and use it for screening (solid phase panning). First, add 900 ul of phosphate buffer containing 5% skimmed milk to the phage supernatant and incubate for 1 hour. Further incubate the phage supernatant with the GPC3 antigen immobilized on an enzyme-linked plate (0.5 μg of protein per well, use 8 wells per time) for 2 hours, unbound phage is removed by 10 washing steps with phosphate buffer. Then, elute the specifically bound phage with a trypsin solution (0.01%). Add the eluate to 10 ml of E. coli TG1 for infection. Divide all bacteria equally on 3 plates and incubate overnight. Then, freeze the bacteria as 2 vials. Repeat the above steps for 3 rounds.

[0080] 4. Sequence analysis and Phage ELISA verification

[0081] Use Phage ELISA to identify the selected antibody clones. Randomly pick 96 single clones from the GPC3 library after each round of screening from the overnight culture plates, add each clone to a well, complete with 1 ml of 2xYTAG liquid medium, incubate at 37°C, 200 rpm for 5.5 hours. Then, add 10 9 TU of helper phage to each well, continue incubation for 0.5 hours. Then, centrifuge the 96 deep well plate at 3500 rpm for 5 minutes, discard the supernatant, then add 1 ml of 2xYT liquid medium to each well, shake and incubate overnight.

[0082] Next day, take out the pre-coated GPC3 protein ELISA plate (10 ng / well), add 250 μL PBS blocking solution (containing 5% skim milk) to each well, and block for 1 hour. At the same time, centrifuge 100 μL of the phage supernatant from the 96-deep well plate rescued yesterday, transfer it to a new 96-well plate, and add an equal amount of blocking solution to each well for 1 hour. Wash the ELISA plate with TBST 3 times, 300 μL / well, at 1 min intervals. Add 50 μL phage / well, incubate at room temperature for 1 h, and wash again with TBST. Add 50 μL anti-M13-HRP diluted 1:10000 / well, incubate at room temperature for 1 h, and wash with TBST 3 times. Add 50 μL TMB color developing solution / well, avoid light for 20 min, stop color development, and read the absorbance at 450 nm.

[0083] Take the corresponding monoclonal phage from the positive wells of the Phage ELISA, and send it to the Goldengene Biotechnology Co., Ltd. for gene sequencing. Based on the gene sequencing results of the second and third rounds of screening, cross-comparison was performed, and the four clones with the highest frequency of occurrence (1-2, 2-2, 3-2, and 4-2) were selected. According to the instructions of the test kit (Novagen brand, item number C112), they were cloned into prokaryotic expression plasmids by homologous recombination technology.

[0084] 5. Small-scale induction expression and purification of anti-GPC3 antibodies

[0085] Take the corresponding expression plasmids of the four clones (1-2, 2-2, 3-2, and 4-2), and transform them into expression competent W3110 (DE3). Then, inoculate the culture plates and incubate them overnight. Subsequently, pick two single colonies from each antibody-expressing strain plate, shake the culture, and detect the turbidity (monitor the absorbance at 600 nm wavelength by ultraviolet spectrophotometry, OD 600 ) until the OD 600 value is between 0.5 and 0.8, and then induce expression (final concentration of 1 mM IPTG). Subsequently, incubate the bacterial solution at 37°C and 150 rpm overnight, and centrifuge to collect the supernatant of the overnight culture.

[0086] Refer to the instructions of the filler (Ni Sepharose excel) for affinity purification of the supernatant of the bacterial solution. Add benzonase to the expression supernatant and mix well. Equilibrate the filler with PBS. Then, take a sample of the supernatant of each single colony for standby, and mix the remaining supernatant with the equilibrated filler and incubate for 1 hour. After incubation, add the filler to the gravity column empty column tube, and collect the supernatant after flow-through for subsequent analysis. After flow-through is completed, elute the protein with 500 mM imidazole. Replace the eluted protein into PBS through an ultrafiltration tube, and detect the protein by SDS-PAGE protein electrophoresis and Coomassie blue staining. Wash the stained protein gel and take a photo recordFigure 2 ). The purified sample can be used for subsequent application.

[0087] 6. Using common indirect ELISA method to determine the binding between antibody mutants and GPC3 protein

[0088] Dissolve 5 μg of GPC3 protein in 5 ml of PBS, mix well, and add 50 μl of GPC3 solution to each well to coat the ELISA plate. Place the microplate on a horizontal shaker and incubate at room temperature for 12 hours. Then, discard the supernatant and wash the plate 3 times with 200 μl of PBST to remove unbound GPC3. Add 3% skim milk and incubate in a 37°C incubator for 30 minutes to block the plate.

[0089] Select 1-2, 2-2, 3-2, 4-2 and NH3 parent antibodies for gradient dilution in 3% skim milk. Add the diluted samples to each well, and then incubate in a 37°C incubator for 2 hours. The subsequent steps include washing the plate, adding secondary antibody, washing the plate again, developing color, and stopping, and finally reading at a wavelength of 450 nm. According to the readings of each antibody, plot the concentration versus absorbance curve, and then calculate the EC 50 value by four-parameter regression curve fitting. The results are shown in Figure 3 The EC 50 values of 1-2, 2-2, and 3-2 antibodies are lower than that of the parent antibody NH3, indicating that the antibodies have higher affinity to the target and stronger binding ability. However, 4-2 does not show stronger affinity than the parent antibody in the ELISA method.

[0090] 7. Kinetic performance of antibodies to antigens

[0091] Use the protein interaction instrument (model Octet RH16, manufacturer Sartorius) to determine the affinity of NH3 antibody mutants and NH3 parent antibodies, and refer to the recommended operation method of the manufacturer.

[0092] The commercially available recombinant GPC3 protein (10088-H08H Yi Qiao God) was diluted to a concentration of 5 μg / mL using a sodium acetate solution at pH 5.0. In addition, the antibodies NH3, 1-2, 2-2, 3-2, 4-2 were all diluted to concentrations of 153.8 nM, 76.9 nM, 38.5 nM, 19.2 nM, 9.62 nM and 0 nM using PBST. The water pre-wetted instrument matching probe (model AR2G) was activated with NHS / EDC reagent, and then the GPC3 protein diluted with sodium acetate solution at pH 5.0 was captured using the AR2G probe. After the immobilized AR2G probe was blocked with EZ solution, it was reacted with the PBST-diluted NH3, 1-2, 2-2, 3-2, 4-2 antibodies, respectively. The solid-phase conjugate after sufficient reaction was dissociated in PBST buffer, and finally the results were analyzed using DataAnalysis 12.0 software to obtain the binding rate, dissociation rate and affinity constant as shown in Table 4 and Figures 4-8

[0093] Table 4 Kinetic performance of antibodies to antigens

[0094] Sample Binding force (M) Binding constant (1 / Ms) Dissociation constant (1 / s) Precision R 2 ]] NH3 3.11E-08 3.71E+05 1.15E-02 0.9901 1-2 2.38E-08 3.58E+05 8.53E-03 0.9868 2-2 3.05E-08 3.35E+05 1.02E-02 0.9897 3-2 2.72E-08 3.49E+05 9.50E-03 0.9866 4-2 2.95E-08 2.81E+05 8.28E-03 0.9831

[0095] Example 2

[0096] 1. Construction of CAR-NK cells based on 1-2 antibody mutant sequence

[0097] Four kinds of chimeric antigen receptor sequences based on 1-2 antibody were prepared into plasmids by gene synthesis, and the results and sequences are shown in Tables 2 and 3. The above four kinds of sequences were connected in series, and the linear double-stranded gene synthesis of the four structures was entrusted to Goldengene Biological Technology Co., Ltd. PCR amplification was performed using the upstream primer shown in SEQ ID No. 11 and the downstream primer shown in SEQ ID No. 12. After PCR amplification, double-stranded DNA containing a T7 promoter, kozac sequence and poly A tail (85 adenosine repeats) was produced, which can be used as a template for further in vitro transcription to produce mRNA.

[0098] The system was configured according to the instructions of the in vitro transcription kit (Qiagen T7 RNA Polymerase Kit, product number T7P-EE102), and the mRNA was generated by overnight reaction in a water bath at 37°C. The next day, the template was degraded by adding DNase (included in the kit), 1 μL per system, 37°C, 15 minutes; each reaction system was supplemented with 30 μL of enzyme-free water, 50 μL of LiCl (Sigma-Aldrich brand AM9480) was added per system, mixed well, and the mRNA was precipitated at -80°C for 2 hours, centrifuged at 4°C for 20 minutes at maximum speed, the supernatant was discarded, the precipitate was resuspended with enzyme-free water, the concentration was determined, and the aliquots were frozen at -80°C for later use.

[0099] ​NK cells in peripheral blood (batch 0126) were isolated using the NK cell enrichment kit (Stemcell brand number: 19055) according to the instructions, and K562 feeder cells overexpressing IL-21 and 41BBL (the construction process is described in patent CN201811520275) were added, and the NK cells were expanded for 12 days according to patent CN201811520275, and the NK density was adjusted to 1*10 8 6E6 / ml, and the cells were transferred into complete medium after the electric shock. After 12 hours, the expression of CAR molecules can be detected, or functional experiments such as target cell killing can be performed.

[0100] 2. Detection of expression rate of CAR-NK cells prepared based on the antibody sequences of 1-2

[0101] Take four mRNA samples prepared from CAR structures, and after 12 hours of electric conversion of the cells, about 1E6 / sample, 100 μL system, biotin-labeled target protein GPC3 μg / sample (KACTUS GPC-HM431B-100 μg), incubate at room temperature for 30 min, 1 ml PBS 400g, 3 min wash once, PE Streptavidin 1:400 dilution, 100 μL / sample, incubate at room temperature for 15-20 min, 1 ml PBS 400g, 3 min wash once, 300 μL PBS resuspension, and detect on the machine.

[0102] The results are shown in Figure 9 Table 1. The mRNA of the four CAR structures can effectively express CAR molecules on NK cells. GPC3 protein can indicate the expression amount of CAR molecules with binding ability. About 91% of cells transfected with mRNA of structure 1 can detect CAR expression, which is more significant than structures 2, 3 and 4 (48.8%, 47.4% and 37.7%, respectively).

[0103] 3. Specific killing of CAR-NK prepared based on the antibody sequences of 1-2 in vitro

[0104] Common hepatocellular carcinoma target cells were used to determine the killing ability of mRNA-prepared CAR-NK. 2*10 4 4 ​CAR-NK cell suspensions were prepared using a CAR-NK cell suspension targeting an irrelevant antigen (CD33) constructed from mRNA prepared in the same process (labeled 10HL). Both suspensions were incubated for 4 hours at 37°C. During this period, CAR-NK cells interacted with target cells, and the cytotoxic effect of CAR-NK cells on target cells was evaluated. After the experiment, 100 μL of prepared luciferin substrate (Novizan brand DD1203, prepared according to the instructions) was added to each well, and the intensity of chemiluminescence was measured using a microplate reader. Data for each well in the experimental group were recorded. An additional 2 × 10⁶ cells were added separately. 4 100 μL of the prepared luciferin substrate was added to each well of the target cells, and the maximum luminescence intensity was recorded. The ratio of the luminescence intensity to the maximum luminescence intensity of each group was used as the killing percentage to plot a histogram. The larger the ratio, the stronger the killing effect.

[0105] The results are as follows Figure 10 As shown, all four structures based on antibodies 1-2 exhibited cytotoxic activity against HepG2 target cells, demonstrating stronger cytotoxicity than NK cells modified with an unrelated antibody CAR (labeled as 10HL in the control group). Structures 1 and 2 showed more pronounced cytotoxic activity than structures 3 and 4, but structure 1 exhibited the best CAR-NK cytotoxic activity and demonstrated better anti-hepatocellular carcinoma activity in in vitro experiments.

[0106] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A group of anti-GPC3 nanobody mutants, characterized in that, The nanobody mutants are selected from any of the following: mutants 1-2, 2-2, 3-2 and 4-2, whose amino acid sequences are as shown in SEQ ID No. 1 to SEQ ID No. 4 in sequence.

2. The nanobody mutant according to claim 1, characterized in that, The nucleotide sequences of the nanobody mutants are shown in SEQ ID No. 5 to SEQ ID No.

8.

3. A chimeric antigen receptor targeting GPC3 based on the nanobody mutant of claim 1 or 2.

4. The chimeric antigen receptor according to claim 3, characterized in that, The structure of the chimeric antigen receptor includes a signal peptide, an antibody region, a hinge region, a transmembrane region, and an intracellular region connected in sequence.

5. The chimeric antigen receptor according to claim 4, characterized in that, The source of the signal peptide includes human CD8a signal peptide; The antibody region includes a single anti-GPC3 nanobody mutant sequence or two anti-GPC3 nanobody mutant sequences linked by a flexible linker peptide. The hinge region may be derived from human IgG4 or human CD8a; The transmembrane region is derived from human CD28 or human CD8a; The intracellular region is derived from human 41BB or CD3.

6. A linear double-stranded gene obtained by nucleotide amplification of the chimeric antigen receptor according to any one of claims 3 to 5, characterized in that, The primers used for amplification include an upstream primer with nucleotide sequences as shown in SEQ ID No. 11 and a downstream primer as shown in SEQ ID No.

12.

7. mRNA obtained by in vitro transcription using the linear double-stranded gene of claim 6 as a template.

8. A genetically engineered CAR-NK cell, characterized in that, The CAR-NK cells are obtained by transforming NK cells with the mRNA described in claim 7 and expressing the nanobody mutant described in claim 1 or 2.

9. The use of the nanobody mutant of claim 1 or 2, or the chimeric antigen receptor of any one of claims 3 to 5, or the CAR-NK cell of claim 8 in the preparation of a medicament for treating a subject with liver cancer.

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