Anti-human GPC3 nano antibody mutants, chimeric antigen receptor and application

Abstract

The invention discloses a group of anti-human GPC3 nano antibody mutants, a chimeric antigen receptor and application, and belongs to the technical field of immunity. The invention provides a group of anti-GPC3 nano antibody mutants which are used for mutating an existing anti-CPG3 nano antibody (NH3), so that the affinity of the antibody is improved. A chimeric antigen receptor is constructed on the basis of the nano antibody mutant, meanwhile, double-stranded DNA of the nano antibody mutant is synthesized to serve as a template for producing mRNA through in-vitro transcription, and then genetic engineering modification is conducted on NK cells through mRNA, so that the CAR-NK cells are obtained. In the CAR-NK cell disclosed by the invention, mRNA can effectively express CAR molecules on the NK cell, and the CAR-NK cell has stronger in-vitro killing ability and shows a better anti-liver cancer effect.

Description

Technical Field

[0001] The present invention belongs to the field of immunotechnology, and specifically relates to a group of anti-human GPC3 nano antibody mutants and chimeric antigen receptors and applications. Background Art

[0002] Glypian-3 (GPC3) is a heparin sulfate proteoglycan (HSPG), a 65kD protein composed of 580 amino acids. The GPC3 protein is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, 14 conserved cysteine ​​residues, and the last 50 residues are modified by heparin sulfate (HS) side chains. This protein is expressed in the liver and kidneys of healthy fetuses, but is hardly expressed in adult 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 still unclear, it has been strongly implicated in the malignant transformation of HCC. Therefore, GPC3 has been identified as a promising target for cancer immunotherapy.

[0003] In 1989, in a study, famous scientists Gross G, Waks T and Eshhar Z gave cytotoxic T cells a gene sequence expressing a specific antibody, which enabled T cells to achieve antigen-specific, non-MHC-restricted activation and enhanced effects. They believed that "giving T cells a chimeric antigen receptor will be an important way for humans to fight tumors in the future." This was the first time in human history that the concept of CAR was proposed. CAR-T therapy, also known as chimeric antigen receptor (CAR) T cell therapy, is the process of modifying human T cells in vitro through genetic engineering and then infusing them back into the patient's body for the treatment of diseases.

[0004] However, with the deepening of research and the increasing number of clinical applications, the drawbacks of CAR-T treatment have gradually been recognized and paid attention to, such as graft-versus-host reaction, cytokine storm and neurotoxicity, tolerance and resistance to solid tumors, process complexity, personalized customization and long production cycle, and expensive treatment costs. Natural killer (NK) is a component of the body's inherent immune system and plays an important role in the body's anti-tumor immune surveillance process. Dario Campana et al. first established the 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 several 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, thereby reducing the possibility of tumor cells escaping killing when targeted by CAR-NK therapy; CAR-NK cells will not undergo immune rejection within days to weeks. Therefore, they have not shown the safety issues of CAR-T in many clinical trials, such as the lack 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 Miaozhuang et al. disclosed an anti-GPC3 nanoantibody in CN 105968209 A, but its affinity is low and its subsequent application is limited. Summary of the invention

[0007] The present invention provides a group of anti-human GPC3 nano antibody mutants and chimeric antigen receptors and applications. The nano antibody mutants have stronger binding ability with targets and are more suitable for the application of CAR-NK technology.

[0008] The present invention provides a group of anti-GPC3 nanobody mutants, wherein the nanobody mutants 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 mutants include 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 to SEQ ID No.4.

[0011] Preferably, the nucleotide sequences of the nanobody mutants are shown in SEQ ID No.5 to SEQ ID No.8.

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

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

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

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

[0016] The hinge region may be derived from human IgG4 or human CD8a;

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

[0018] The sources of the intracellular region include human 41BB or CD3.

[0019] The present invention also provides a linear double-stranded gene obtained by nucleotide amplification of the chimeric antigen receptor, wherein the primers used for amplification include an upstream primer having a nucleotide sequence as shown in SEQ ID No.11 and a downstream primer as shown in SEQ ID No.12.

[0020] The present invention also provides mRNA obtained by in vitro transcription using the linear double-stranded gene as a template.

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

[0022] The present invention 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 drug for treating a subject suffering from a cancer expressing GPC3.

[0023] Beneficial effects: The present invention provides a group of anti-GPC3 nanobody mutants, which mutate the existing anti-CPG3 nanobody (NH3) to improve the affinity of the antibody. In the embodiment, four mutated anti-GPC3 antibodies were compared with NH3 parent antibodies (1-2, 2-2, 3-2, 4-2 and NH3 parent antibodies). The EC values ​​of 1-2, 2-2 and 3-2 were 50 The values ​​were lower than those of the parent antibody NH3, indicating a stronger binding ability.

[0024] The present invention also constructs a chimeric antigen receptor based on the nano antibody mutant, and synthesizes the double-stranded DNA of the nano antibody mutant as a template for in vitro transcription to produce mRNA, and then uses the mRNA to genetically engineer NK cells to obtain CAR-NK cells. In the CAR-NK cells of the present invention, mRNA can effectively express CAR molecules on NK cells, and has a stronger in vitro killing ability, showing a better anti-liver cancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of primer design for preparing mutant antibody library;

[0026] Figure 2 This is a diagram showing the results of small-scale induced expression of anti-GPC3 nanobody mutants;

[0027] Figure 3 This is a graph showing the affinity determination results of anti-GPC3 antibodies detected by ELISA;

[0028] Figure 4 It is the affinity signal diagram of GPC3 protein and antibody NH3;

[0029] Figure 5 It is the affinity signal diagram of GPC3 protein and antibody 1-2;

[0030] Figure 6 It is the affinity signal diagram of GPC3 protein and antibody 2-2;

[0031] Figure 7 It is the affinity signal diagram of GPC3 protein and antibody 3-2;

[0032] Figure 8 It is the affinity signal diagram of GPC3 protein and antibody 4-2;

[0033] Fig. 9 This is a graph showing the expression results of CAR-NK cells prepared based on the 1-2 antibody sequence;

[0034] Fig.10 This is a graph showing the results of the in vitro killing ability test of CAR-NK based on 1-2 antibody;

[0035] Fig.11 It is a structural diagram of the amino acid sequence of the antibody library of the present invention. DETAILED DESCRIPTION

[0036] The present invention provides a group of anti-GPC3 nanobody mutants, wherein the nanobody mutants 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 of the present invention has been disclosed in Chinese patent CN105968209A. The present invention preferably designs a mutation library based on the anti-GPC3 nanobody (parent NH3 antibody) and screens and obtains four mutant antibodies, with clone numbers: 1-2, 2-1, 3-1 and 4-2, and their sequences are shown in Table 1.

[0039] Table 1 Amino acid sequence and nucleotide sequence of antibodies

[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 present invention 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 invention preferably includes 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 includes a human CD8a signal peptide, the amino acid sequence of which is shown in SEQ ID No.79, and the nucleotide sequence of which is shown in SEQ ID No.84; the antibody region preferably includes a mutant sequence or two mutant sequences connected by a flexible connecting peptide; the source of the hinge region preferably includes human IgG4 or human CD8a, wherein the amino acid sequence of the hinge region of IgG4 is shown in SEQ ID No.80, and the nucleotide sequence is shown in SEQ ID No.85, the amino acid sequence of the hinge region of CD8a is shown in SEQ ID No.81, and the nucleotide sequence is shown in SEQ ID No.86; the source of the transmembrane region preferably includes human CD28 or human CD8a, wherein the amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID No.82, and the nucleotide sequence is shown in SEQ ID No.87, the amino acid sequence of the transmembrane region of CD8a is shown in SEQ ID No.83, and the nucleotide sequence is shown in SEQ ID No.88; the source of the intracellular region preferably includes human 41BB and human CD3, wherein the amino acid sequence of 41BB is shown in SEQ ID No.89: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL; the amino acid sequence of human CD3 is shown in SEQ ID No.90: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR; in the embodiment, both are included, the amino acid sequence is shown in SEQ ID No.91, and the nucleotide sequence is shown in SEQ ID No.92.

[0043] In the examples of the present invention, it was verified that mutant 1-2 had the best affinity. Therefore, in the chimeric antigen receptor targeting GPC3 based on mutant 1-2, the antibody fragment can be used once or twice in head-to-tail connection. The two antibody fragments are directly connected with a flexible connecting peptide, and the above structures are sequentially connected in series to obtain the chimeric antigen receptor of the present invention. The amino acid sequences and nucleotide sequences of the various structures of the chimeric antigen receptor targeting GPC3 based on mutant 1-2 in the examples of the present invention are shown in Tables 2 and 3, but they cannot be identified as the entire protection scope of the present invention.

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

[0045]

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

[0047]

[0048]

[0049]

[0050]

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

[0052] The present invention preferably concatenates the sequences in Table 3, and then entrusts Jinweizhi Biotechnology to perform linear double-stranded gene synthesis (minigene) of four structures, and performs PCR amplification using upstream primers and downstream primers; 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 adenylic acid repeats) is generated, 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 Primers (SEQ ID No. 12): ttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt

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

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

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

[0058] The present invention transforms the mRNA into NK cells, and in the embodiment, the transformation is preferably completed by electroporation.

[0059] The present invention 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 drug for treating a subject suffering from a cancer expressing GPC3.

[0060] In the embodiments of the present invention, the mRNAs of the four CAR structural designs can effectively express CAR molecules on NK cells and enhance the killing ability in vitro, and have a strong anti-liver cancer effect.

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

[0062] Example 1

[0063] 1. Primer synthesis and PCR splicing

[0064] According to the NH3 antibody and its nucleotide sequence in Chinese patent CN105968209A, six sets 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. The splicing PCR strategy is as follows: Figure 1 shown.

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

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

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

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

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

[0070] The sixth set of 1 primer (SEQ ID No. 73).

[0071] All primers were commissioned to be synthesized by Genewise Biotechnology Co., Ltd. After synthesis, they were delivered after desalting and purification. They were dissolved in water at a concentration of 10 μM.

[0072] Using a PCR enzyme kit (brand: Toyobo, polymerase KOD MAX), refer to its instructions to mix the components including six sets of primers and amplify the pUCG3 vector to obtain a linearized fragment. In brief, the reaction system includes three steps, the first step is an initial denaturation step at 98°C for 10 seconds; the second step is 35 cycles of amplification steps, each cycle includes denaturation at 98°C for 10 seconds, annealing at 68°C for 45 seconds, and extension at 68°C for 1 minute; and finally a final extension step at 68°C for 1 minute. The nucleic acid was separated by agarose gel electrophoresis, and the cut target sequence was recovered for use.

[0073] The pUCG3 plasmid was obtained by gene synthesis and extracted in small quantities for later use (commissioned to Jinweizhi Biotechnology Co., Ltd.). The full sequence of the pUCG3 plasmid is SEQ ID No.74; after final splicing, the library gene sequence is SEQ ID No.75. The structure diagram of the antibody library amino acid sequence is preferably as follows Fig.11 shown.

[0074] 2. Construction of mutant antibody gene library plasmid

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

[0076] At the same time, referring to the instructions, the homologous recombination kit (Novozyme brand, item number C112) was used to connect the GPC3 mutant library fragment to the vector: pUCG3 linear vector (2000ng), GPC3 mutant library (400ng), 5×CEbuffer, Exnase II enzyme were added to the PCR tube, and the final volume was supplemented with water to 400μl, and the reaction was carried out at 37°C for 15 minutes. The reaction product was recovered using a nucleic acid purification kit (DP214). All nucleic acids were used to electrotransfer 17 TG1 competent cells. In addition to calculating the library size, all bacterial solutions were evenly divided into 17 plates and cultured overnight. The next day, all colonies on the bacterial plate were collected and frozen into 10 cryopreservation tubes with 1ml each;

[0077] 3. Phage display and panning

[0078] Take 60 μL of the frozen bacterial suspension and add it to 30 ml of 2×YT (2xYTAG) medium containing ampicillin and glucose at 200 rpm until the OD 600 =0.75, add helper phage M13K07 (Critical Point brand, item number P006) to a final concentration of 1×10 9 TU / ml, 200 rpm, 37°C, 1h; centrifuge at 3500 rpm to obtain the precipitate, resuspend it in 30 ml 2×YT+Amp+Kan medium (sugar-free), and culture it overnight with shaking and constant temperature;

[0079] The next day, the culture supernatant was collected by centrifugation, and 100 μL of phage supernatant was taken for screening (solid phase panning). First, 900 ul of phosphate buffer containing 5% skimmed milk powder was added to the phage supernatant and incubated for 1 hour. The phage supernatant was further incubated with the GPC3 antigen immobilized on the ELISA plate (each well was coated with 0.5 μg protein, and 8 wells were used each time) for 2 hours, and the unbound phage was removed by 10 phosphate buffer washing steps. Subsequently, the specifically bound phage was eluted with a trypsin solution (0.01%). The eluate was completely added to 10 ml of Escherichia coli TG1 for infection. All bacterial solutions were evenly divided into 3 plates, and after overnight culture, they were divided and frozen into 2 cryopreservation tubes. The above steps were repeated for a total of 3 rounds.

[0080] 4. Sequence analysis and Phage ELISA verification

[0081] Phage ELISA was used to identify the antibody clones after screening. 96 monoclonal clones were randomly selected from the plates cultured overnight after each screening of the GPC3 library, added to each well, supplemented with 1 ml of 2×YTAG liquid medium, and cultured at 37°C and 200 rpm for 5.5 hours. Subsequently, 10 9 The helper phage of TU was cultured for 0.5 hours. Subsequently, the 96-deep-well plate was centrifuged at 3500 rpm for 5 minutes, the supernatant was discarded, and 1 ml of 2×YT liquid culture medium was added to each well and cultured overnight with shaking.

[0082] The next day, take out the ELISA plate pre-coated with GPC3 protein (10ng / well), add 250μL of PBS blocking solution (containing 5% skim milk) to each well, and block for 1 hour. At the same time, centrifuge and take 100μL of the phage supernatant in 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 3 times with TBST, 300μL / well, 1min interval. Add 50μL of phage / well, incubate at room temperature for 1h, and wash again with TBST. Add 50μL of anti-M13-HRP diluted 1:10000 to each well, incubate at room temperature for 1h, and wash 3 times with TBST. Add 50μLTMB colorimetric solution to each well, protect from light for 20min, stop colorimetric development, and read absorbance at 450nm.

[0083] The corresponding monoclonal bacterial solution of the Phage ELISA positive well was sent to Genewise Biotech Co., Ltd. for gene sequencing. The gene sequencing results of the second and third rounds of screening were cross-compared, and the four clones with the highest frequency (1-2, 2-2, 3-2, and 4-2) were selected and cloned into prokaryotic expression plasmids by homologous recombination technology according to the instructions of the kit ((Novozyme brand, item number C112)).

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

[0085] The corresponding expression plasmids of 4 clones (1-2, 2-2, 3-2, 4-2) were taken and transformed into expression competent W3110 (DE3), and coated on culture plates for overnight culture. Subsequently, 2 single clones were selected on the plates of each antibody expression strain for shake culture and turbidity was detected (absorbance at 600 nm wavelength was monitored by ultraviolet spectrophotometry, OD 600 ), until OD 600 When the value was between 0.5 and 0.8, the expression was induced (final concentration 1 mM IPTG). Subsequently, the bacterial solution was cultured overnight at 37°C and 150 rpm, and the supernatant of the overnight culture was collected by centrifugation.

[0086] Refer to the instructions of the filler (Ni Sepharose excel) for affinity purification of the bacterial supernatant. Add benzonase to the expression supernatant and mix well. Balance the filler with PBS. Subsequently, sample the supernatant of each monoclonal clone for use, and mix the remaining supernatant with the balanced filler and incubate for 1 hour. After the incubation is completed, add the filler to the empty column tube of the gravity column, and collect the supernatant after flow-through for subsequent analysis. After the flow-through is completed, use 500mM imidazole to elute the protein. Replace the eluted protein into PBS through an ultrafiltration tube, and detect the protein by SDS-PAGE protein electrophoresis Coomassie Brilliant Blue staining. Wash the stained protein gel and take pictures and record ( Figure 2 ). The purified samples can be used for subsequent applications.

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

[0088] Dissolve 5 μg of GPC3 protein in 5 ml of PBS, mix thoroughly, add 50 μl of GPC3 solution to each well, coat the ELISA plate, and place the microplate on a horizontal shaker for 12 hours at room temperature. Then, discard the supernatant and wash the plate three 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, and the diluent is 3% skim milk. The diluted samples are added to each well and then incubated in a 37°C incubator for 2 hours. The subsequent steps include washing the plate, adding secondary antibodies, washing the plate again, color development and termination, and finally reading the value at a wavelength of 450nm. According to the reading value of each antibody, a curve between concentration and absorbance is drawn, and then the EC is calculated by quartic regression curve fitting. 50 The result is Figure 3 As shown, the EC values ​​of the three antibodies 1-2, 2-2, and 3-2 are 50 The lower the values ​​were than that of the parent antibody NH3, the higher the affinity of the antibody to the target, that is, the stronger the binding ability. However, in the ELISA method, 4-2 did not show a stronger affinity than the parent antibody.

[0090] 7. Kinetic properties of antibodies against antigens

[0091] The affinity of the NH3 antibody mutant to the NH3 parent antibody was determined using a protein interaction instrument (model Octet RH16, manufacturer Sartorius) according to the manufacturer's recommended operating procedures.

[0092] The commercially available recombinant GPC3 protein (10088-H08H Yiqiao Shenzhou) 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, 2-1, 3-2, and 4-2 were all diluted to 153.8 nM, 76.9 nM, 38.5 nM, 19.2 nM, 9.62 nM, and 0 nM using PBST. The instrument-matched probe (model AR2G) pre-wetted in water was activated with NHS / EDC reagent, and then the AR2G probe was used to capture the GPC3 protein diluted with a sodium acetate solution at pH 5.0. After the fixed AR2G probe was blocked with EZ solution, it was respectively reacted with NH3, 1-2, 2-1, 3-2, and 4-2 antibodies diluted with PBST. The solid-phase bound product after full reaction was subjected to dissociation analysis in PBST buffer, and finally the results were analyzed using DataAnalysis12.0 software to obtain the binding rate, dissociation rate, and affinity constant as shown in Table 4 and Figures 4 to 8 shown.

[0093] Table 4 Kinetic properties of antibodies against antigens

[0094] sample Binding force (M) Binding constant (1 / Ms) Dissociation constant (1 / s) <![CDATA[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 2-1 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 sequences

[0097] Four chimeric antigen receptor sequences based on 1-2 antibodies were prepared into plasmids by gene synthesis, and the results and sequences are shown in Tables 2 and 3. After the above four were concatenated, Jinweizhi Bio was commissioned to synthesize the linear double-stranded genes of the four structures, and PCR amplified by 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 T7 promoter and kozac sequence and polyadenylic acid tail (85 adenylic acid repeats) will be generated, 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 (Kaixia T7 RNA Polymerase Kit, catalog number T7P-EE102), and the mRNA was generated by overnight reaction at 37°C in a water bath. The next day, DNase (included in the kit) was added to degrade the template, 1 μL / system, 37°C, 15 minutes; 30 μL of enzyme-free water was added to each reaction system, 50 μL / system of LiCl (Thermo Fisher brand AM9480) was added, mixed, frozen at -80°C for 2 hours to precipitate mRNA, centrifuged at 4°C at maximum speed for 20 minutes, the supernatant was discarded, the precipitate was re-dissolved with enzyme-free water, the concentration was determined, and the aliquots were frozen at -80°C for use.

[0099] The NK cells in the peripheral blood (collected in batch 0126) were isolated using the human NK cell enrichment kit (Stemcell brand number: 19055) according to the instructions. K562 trophoblast cells overexpressing IL-21 and 41BBL were added (the construction process is described in patent CN201811520275). The NK cells were expanded for 12 days according to patent CN201811520275, and the NK density was adjusted to 1*10 8 / ml, use Bio-red electroporator, refer to the instructions, and use 2mm electroporation cup for electrotransfection. Add 30μg mRNA and 200μL / cup of cell suspension to each electroporation cup, set the voltage to 300V, and perform 2 pulses of electrotransfection. Each pulse lasts 800us and the interval is 800ms. After electroporation, transfer the cells to complete culture medium and culture at a density of 2E6 / ml. 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 CAR-NK cell expression rate based on 1-2 antibody sequence

[0101] Take the mRNA samples prepared by the four CAR structures, electrophoretically transfer RNA to cells 12 hours later, about 1E6 / sample, 100μL system, biotin-labeled target protein GPC31μg / sample (KACTUS GPC-HM431B-100μg), incubate at room temperature for 30min, 1ml PBS400g, wash once for 3min, PE Streptavidin 1:400, 100μL / sample, incubate at room temperature in the dark for 15-20min, 1ml PBS 400g, wash once for 3min, resuspend in 300μL PBS, and detect on the machine.

[0102] The results are as follows Fig. 9 As shown, the mRNAs of the four CAR structure designs can effectively express CAR molecules on NK cells. GPC3 protein can indicate the expression of CAR molecules with binding ability. After the mRNA of structure 1 was transfected into NK cells, about 91% of the cells could detect CAR expression, which was more significant than structures 2, 3 and 4 (48.8%, 47.4% and 37.7%, respectively).

[0103] 3. CAR-NK in vitro specific killing based on 1-2 antibody sequence

[0104] Common liver cancer target cells were used to determine the killing ability of CAR-NK prepared by mRNA. 2×10 4 Target cells (HepG2-luc, purchased from Nanjing Kebai Biotechnology) were suspended in 50 μL of culture medium. Subsequently, 50 μL of 4×10 4The cell suspension of CAR-NK was prepared in the same process, and the CAR-NK (labeled as 10HL) constructed by mRNA targeting an irrelevant antigen (targeting CD33) was used as a control. Incubate for 4 hours at a temperature of 37°C. During this period, CAR-NK cells interact with target cells to evaluate the killing effect of CAR-NK cells on target cells. After the experiment, 100 μL of the prepared fluorescein substrate (Novozyme brand DD1203, prepared according to the instructions) was added to each well, the intensity of chemiluminescence was detected by an enzyme reader, and the data of each well in the experimental group was recorded. In addition, 2×10 4 Add 100 μL of the prepared luciferin substrate to each well of the target cell and record the maximum luminescence intensity. Draw a histogram with the ratio of the luminescence intensity of each group to the maximum luminescence intensity as the cell killing percentage. The larger the ratio, the stronger the killing effect.

[0105] The results are as follows Fig.10 As shown, the four structures based on 1-2 antibodies all have the effect of killing target cells HepG2, which is stronger than the killing effect of NK cells modified with irrelevant antibody CAR (the control group is marked as 10HL). Among them, the killing effect of structure 1 and structure 2 is more obvious than that of structure 3 and structure 4, but structure 1 has the best CAR-NK killing effect and has a better anti-liver cancer effect in in vitro experiments.

[0106] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, 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 mutant has a higher affinity for GPC3 than the anti-GPC3 nanobody; The amino acid sequence of the anti-GPC3 nanobody is shown in SEQ ID No.

9.

2. The nanobody mutant according to claim 1, characterized in that The nanobody mutants include any 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 to SEQ ID No.

4.

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

8.

4. A chimeric antigen receptor targeting GPC3 based on the Nanobody mutant according to any one of claims 1 to 3.

5. The chimeric antigen receptor according to claim 4, 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 which are connected in sequence.

6. The chimeric antigen receptor according to claim 5, characterized in that The source of the signal peptide includes human CD8a signal peptide; The antibody region includes one mutant sequence or two mutant sequences connected by a flexible connecting peptide; The hinge region may be derived from human IgG4 or human CD8a; The source of the transmembrane region includes human CD28 or human CD8a; The sources of the intracellular region include human 41BB or CD3.

7. A linear double-stranded gene obtained by nucleotide amplification of the chimeric antigen receptor according to any one of claims 4 to 6, characterized in that: The primers used 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.

8. mRNA obtained by in vitro transcription using the linear double-stranded gene according to claim 7 as a template.

9. A genetically engineered CAR-NK cell, characterized in that: The CAR-NK cells are obtained by transforming NK cells with the mRNA according to claim 8, and express the nanobody mutant according to any one of claims 1 to 3.

10. Use of the Nanobody mutant according to any one of claims 1 to 3, the Chimeric Antigen Receptor according to any one of claims 4 to 6, or the CAR-NK cell according to claim 9 in the preparation of a medicament for treating a subject suffering from a cancer expressing GPC3.

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