Mini-protein inhibitor of CTLA-4 protein with high affinity

Abstract

The invention discloses a mini-protein inhibitor of CTLA-4 protein with high affinity, belongs to the technical field of biological medicine, and the mini-protein inhibitor of the CTLA-4 protein with the high affinity has the ability of being combined with the CTLA-4 protein on the surface of a T cell during use, and can be used for inhibiting the CTLA-4 protein on the surface of the T cell. The mini-protein inhibitor has certain T cell proliferation inhibition and melanoma cell apoptosis ability, ensures accurate targeting immune checkpoints, shows specific recognition and binding ability to CTLA-4 protein, has relatively high affinity, can be widely applied to treatment of various diseases related to CTLA-4 receptors, is compatible with existing cancer immunotherapy schemes, and can be widely applied to treatment of various diseases related to CTLA-4 receptors. And a new possibility is provided for combined treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a mini-protein inhibitor of CTLA-4 protein with high affinity. Background Art

[0002] CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that plays an important role in the immune system. It is a co-stimulatory molecule of T lymphocytes that regulates immune responses. CTLA-4 inhibits T cell activation and proliferation by competitively binding to B7 molecules (CD80 and CD86). CTLA-4 is believed to play a role in maintaining immune tolerance and preventing autoimmunity, but it can also be used by cancer cells to evade immune surveillance.

[0003] In recent years, immune checkpoint inhibitors targeting CTLA-4 have shown great potential in the treatment of various cancers. However, the CTLA-4 antibodies currently developed are large in size and relatively expensive. Therefore, the present invention provides a mini protein inhibitor with low cost, high expression level and high affinity for new drug development. Summary of the invention

[0004] 1. Technical problems to be solved: In view of the problems existing in the prior art, the purpose of the present invention is to provide a mini-protein inhibitor of CTLA-4 protein with high affinity, which has the ability to bind to the CTLA-4 protein on the surface of T cells. The mini-protein inhibitor exhibits specific recognition and binding ability to the CTLA-4 protein, and its affinity is also high, and can be widely used in the treatment of various diseases related to the CTLA-4 receptor.

[0005] 2. Technical solution: To solve the above problems, the present invention adopts the following technical solutions.

[0006] A mini protein inhibitor of CTLA-4 protein with high affinity, including protein sequence design and analysis steps at the computer level: S1. Use molecular docking technology to simulate the interaction between antibodies and CTLA-4 to predict possible binding modes; S2. Use the ProteinMPNN algorithm to design the amino acid sequence of the antibody based on the three-dimensional structure information of the protein to ensure that it has specific biological functions; S3. The designed antibody sequence is evaluated for binding affinity using the Prodigy platform to ensure that it has high affinity for human CTLA-4 protein.

[0007] A further improvement is that the prediction analysis software in step S1 uses AlphaFold software and PyMOL protein visualization software to perform structure prediction analysis.

[0008] A further improvement is that the main structure of the novel CTLA-4 antibody is mainly composed of three helices and four key amino acid residues (serine 1, tyrosine 6, threonine 34, glutamic acid 33).

[0009] Further improvements include: a method for preparing the protein: S4, constructing and culturing the expression strain of the tagged recombinant protein; S5, purification steps for protein cleavage purification, Ni-NTA affinity chromatography and gel filtration chromatography; S6. In vitro binding assay for proteins; S7. T cell toxicity assay - T cell proliferation assay.

[0010] A further improvement is that the strain culture method in step S4 comprises the following steps: S41, using NcoI and XhoI restriction enzymes to perform restriction ligation to ensure that the sequence is correctly inserted into the pET-28a(+) expression vector; S42, transforming the synthesized expression plasmid into BL21 (DE3) competent cells, placing them on ice for 30 minutes, then subjecting them to a heat shock treatment at 42°C for 50 seconds, and then rapidly cooling them to reduce the effect of heat shock on the cells; S43, adding 100 μL LB liquid medium at 37°C for 45 minutes to resuscitate the cells, and spreading the resuscitated cells on LB solid medium containing kanamycin resistance, and culturing in a 37°C incubator for 12 hours to screen for successfully transformed cells; S44, select a single colony and transfer it to 30 mL of LB liquid medium containing kanamycin, culture it on a shaker at 37°C and 200 rpm for 10 hours, then transfer it to 1 L of medium with the same conditions and continue to culture until OD600 reaches 0.8; S45, add 0.5 mM IPTG to induce expression for 6 hours at 30°C and 200 rpm on a shaker to express the target mini-protein inhibitor.

[0011] A further improvement is that the protein purification method in step S5 includes the following steps: S51. The cultured strain containing the recombinant CTLA-4 mini-protein inhibitor was centrifuged at 5000 rpm for 10 minutes to collect the bacteria; the bacteria were resuspended in 30 mL of PBS buffer, and the cells were disrupted by ultrasonic disruption, and then centrifuged at 13000 rpm for 30 minutes at 4°C to collect the supernatant containing the recombinant protein; S52, adding the supernatant to a pre-cooled Ni-NTA affinity chromatography column, and incubating at 4°C for 25 minutes to promote the affinity binding of the recombinant protein to the Ni-NTA resin on the nickel column through its His tag; after the unbound impurities flow out with the supernatant, the chromatography column is washed with a washing buffer for 5-6 column volumes to remove the impurities; S53, adding 15 mL of elution buffer and incubating at 4°C for 5 minutes to dissolve and elute the recombinant protein bound to the column; S54. The purified protein was analyzed by SDS-PAGE electrophoresis.

[0012] A further improvement is that the in vitro protein binding experimental method in step S6 includes the following steps: S61, CTLA-4 was selected as the target protein, and CTLA-4 was incubated with Cy5 dye in the dark at 4°C for 30 minutes; S62, free dye was effectively removed by gel filtration chromatography to obtain stained CTLA-4 at a concentration of 200 nM; S63, the novel mini-protein inhibitor was diluted stepwise in 16 steps, with the highest concentration being 1.85 mM, and stained CTLA-4 was added sequentially at a final concentration of 100 nM; S64, after mixing, incubate at 4°C for 30 minutes, centrifuge at 13000 rpm for 10 minutes, take the supernatant for affinity measurement, and the measured affinity is 2.7e-04 M; S65. Use MST technology to accurately measure the affinity between mini protein inhibitors and CTLA-4.

[0013] A further improvement is that the method for T cell toxicity determination-T cell proliferation experiment in step S7 comprises the following steps: S71. Collect T cells from the culture system and count the cells using a hemacytometer or an automated cell counter to determine the initial number of cells; S72, placing the collected T cells in a centrifuge tube, and centrifuging at 300 g for 5 minutes to collect the cell precipitate; S73, discard the supernatant without disturbing the cell pellet; S74. Resuspend the cell pellet with 10 mL of CFSE staining solution and incubate in a cell culture incubator at 37°C for 20 minutes to allow CFSE to fully bind to the cells. S75. After staining, add 40 mL of fresh culture medium to the cells and continue incubation at 37°C for 5 minutes to dilute and remove the dye that is not bound to the cells; S76. Centrifuge again at 300 g for 5 min, discard the supernatant, and resuspend the cells in an appropriate amount of fresh culture medium; S77. Cultivate CFSE-labeled T cells according to the experimental design, either alone or in co-culture with antigen presenting cells, or add the novel CTLA-4 mini-protein inhibitor for intervention; S78. After the culture period, the T cells were analyzed using flow cytometry; S79. Evaluate the proliferation of T cells by measuring the CFSE fluorescence intensity of the cells.

[0014] A further improvement is that the culture medium and reagents used in the protein preparation process include LB liquid culture medium, LB solid culture medium, Ni-NTA affinity chromatography buffer, wherein; LB liquid medium included 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride; LB solid medium includes 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar; The wash buffer consisted of 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 30 mM imidazole; The elution buffer consisted of 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 250 mM imidazole.

[0015] 3. Beneficial effects: Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: The present invention is reasonably designed, has high specificity and affinity (272 μM), can bind to CTLA-4 protein, has certain T cell proliferation inhibition and melanoma cell apoptosis capabilities, and ensures precise targeting of immune checkpoints.

[0016] In the present invention, the mini-protein inhibitor of the present application is compatible with existing cancer immunotherapy regimens, providing new possibilities for combined therapy.

[0017] In the present invention, the designed sequences are different, mainly because the targets are different, and the designed small protein sequences are also different. By adding a T cell proliferation experiment, it is observed whether the designed small protein will affect T cell proliferation.

[0018] It should be noted that the structures not introduced in the present invention are the same as the prior art or can be implemented by using the prior art, and are not described in detail here because they do not involve the design points and improvement directions of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a three-dimensional structural analysis diagram of the docking of the CTLA-4 protein of the present invention and its mini-protein inhibitor molecule; Figure 2 is an SDS-PAGE diagram of the expression of the miniprotein inhibitor of the present invention; Figure 3 Schematic diagram of in vitro binding analysis of CTLA-4 protein and its mini-protein inhibitor of the present invention; Figure 4 Schematic diagram of the T cell proliferation experiment of the present invention. DETAILED DESCRIPTION

[0020] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0021] In the description of the present invention, those skilled in the art can easily gain insight into other aspects and advantages of the present disclosure from the detailed description below. Only exemplary embodiments of the present disclosure are shown and described in the detailed description below. As will be appreciated by those skilled in the art, the content of the present disclosure enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the descriptions in the drawings and specification of the present application are merely exemplary and not restrictive.

[0022] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0023] In the present invention, a mini protein inhibitor of CTLA-4 protein was designed by using the method of artificial intelligence deep learning, using software such as AlphaFold, docking, and kinetic simulation. The target protein gene was fully synthesized at Sangon Biotech (Shanghai) Co., Ltd. and connected to the pET-28a (+) expression vector, with Nco1 and Xho1 as restriction sites.

[0024] The Escherichia coli BL21 (DE3) competent cells used in the experiment were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0025] The media and reagents used included: LB liquid medium: including 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, the volume was adjusted to 1 L, and then sterilized under high pressure at 121°C for 20 minutes.

[0026] LB solid culture medium: including 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar. The volume was adjusted to 1 L, and then sterilized under high pressure at 121°C for 20 minutes. After cooling slightly to about 60°C, it was poured into the clean bench to prepare the plate.

[0027] Ni-NTA affinity chromatography buffer: Wash buffer: 2.7 mM KCl, 137 mM NaCl, 10 mM NaHPO4, 1.8 mM KH2PO4, 30 mM Imidazole.

[0028] Elution buffer: 2.7 mM KCl, 137 mM NaCl, 10 mM NaHPO4, 1.8 mM KH2PO4, 250 mM Imidazole.

[0029] To promote its growth, 50 μg / mL kanamycin was added as the antibiotic concentration. For vector cloning and protein expression purification steps, please refer to the Molecular Cloning Experiment Guide.

[0030] 1. Computer-based protein sequence design and analysis The present invention relates to a new type of artificial antibody developed using a computer-aided design method, which specifically binds to human CTLA-4 protein and is expected to be used in the immunotherapy of autoimmune diseases and cancer. During the design process, the interaction between the antibody and CTLA-4 is first simulated by molecular docking technology to predict possible binding modes. Then, the ProteinMPNN algorithm is used to design the amino acid sequence of the antibody based on the three-dimensional structural information of the protein to ensure that it has specific biological functions. Finally, the designed antibody sequence is evaluated for binding strength using the Prodigy platform to ensure that it has high affinity with the human CTLA-4 protein.

[0031] To further verify the structure and function of the new antibody, the present invention uses AlphaFold software for structural prediction, and uses protein visualization software such as PyMOL to analyze the structure of the designed new antibody in detail. The analysis results show that the main structure of the new antibody for CTLA-4 is mainly composed of three helices. Through structural analysis, we identified four key amino acid residues (serine 1, tyrosine 6, threonine 34, glutamic acid 33), such as Figure 1 , these residues play a crucial role in the binding of antibodies to CTLA-4.

[0032] The antibody is designed to effectively block the binding between CTLA-4 and its ligand B7 molecules (CD80 and CD86), thereby regulating the activation and proliferation of T cells. Through this mechanism, the antibody of the present invention is expected to enhance the immune system's ability to kill tumor cells and show potential in the treatment of autoimmune diseases.

[0033] The novel CTLA-4 antibody design method of the present invention not only improves the accuracy and efficiency of the design, but also provides a solid theoretical basis for subsequent experimental verification and drug development by accurately identifying key amino acids. Through this method, we are able to design mini protein inhibitors with high affinity and specificity, which are expected to play an important role in immune regulation and the treatment of related diseases.

[0034] 2. Protein Preparation 1) Construct and culture the expression strain of the tagged recombinant protein: The present invention provides a method for efficiently expressing a novel human CTLA-4 mini-protein inhibitor. The method first involves fully synthesizing a plasmid containing a nucleic acid sequence of a human CTLA-4 mini-protein inhibitor at Sangon Biotech (Shanghai) Co., Ltd., and performing restriction digestion and ligation using NcoI and XhoI restriction enzymes to ensure that the sequence is correctly inserted into a pET-28a(+) expression vector; subsequently, the synthesized expression plasmid is transformed into BL21(DE3) competent cells, and after standing on ice for 30 minutes, a heat shock treatment at 42°C is performed for 50 seconds, and then the cells are rapidly cooled to reduce the effect of heat shock on the cells; next, 100 μL of The cells were revived in LB liquid medium for 45 minutes, and the revived cells were spread on LB solid medium containing kanamycin resistance and cultured in a 37°C incubator for 12 hours to screen successfully transformed cells; then, single colonies were selected and transferred to 30 mL of LB liquid medium containing kanamycin, cultured on a shaker at 37°C and 200 rpm for 10 hours, and then transferred to 1 L of culture medium under the same conditions and continued to be cultured until OD600 reached 0.8; finally, 0.5 mM IPTG was added to induce expression for 6 hours on a shaker at 30°C and 200 rpm, thereby efficiently expressing the target mini-protein inhibitor.

[0035] 2) Protein purification In the present invention, we describe in detail a method for efficiently purifying a novel mini-protein inhibitor of human CTLA-4, which includes three main steps: cleavage purification, Ni-NTA affinity chromatography and gel filtration chromatography.

[0036] First, the cultured strain containing the recombinant CTLA-4 mini-protein inhibitor was centrifuged at 5000 rpm for 10 minutes to collect the cells. The cells were resuspended in 30 mL of PBS buffer, and then ultrasonically disrupted, and then centrifuged at 13000 rpm for 30 minutes at 4°C to collect the supernatant containing the recombinant protein.

[0037] Next, the supernatant was added to the pre-cooled Ni-NTA affinity chromatography column and incubated at 4°C for 25 minutes to promote the affinity binding of the recombinant protein to the Ni-NTA resin on the nickel column through its His tag. After the unbound impurities flowed out with the supernatant, the chromatography column was rinsed with 5-6 column volumes of washing buffer to remove the impurities. Finally, 15 mL of elution buffer was added and incubated at 4°C for 5 minutes to dissolve and elute the recombinant protein bound to the column. The purified protein was analyzed by SDS-PAGE electrophoresis, as shown in Figure 2. Figure 2 shown.

[0038] 3. In vitro binding assay In order to verify the affinity between the new mini-protein inhibitor and CTLA-4 protein, this study used microthermophoresis (MST) technology for measurement. Microthermophoresis is an optical method for characterizing the properties of biomolecules. It obtains information such as the mode and kinetic constants of molecular interactions by detecting the changes in the electrophoretic mobility of biomolecules in a temperature gradient.

[0039] In the experiment, we selected CTLA-4 as the target protein and incubated CTLA-4 with Cy5 dye in the dark for 30 minutes at 4°C. Cy5 is a commonly used fluorescent dye with a high molar extinction coefficient that can produce bright fluorescent signals and is suitable for fluorescent labeling of biological molecules. The free dye was effectively removed by gel filtration chromatography, and finally the stained CTLA-4 with a concentration of 200 nM was obtained. Gel filtration chromatography is a separation technology based on molecular size that can effectively separate dye-bound proteins from unbound dye.

[0040] Subsequently, the new mini-protein inhibitor was diluted stepwise in 16 gradients, with the highest concentration being 1.85 mM, and stained CTLA-4 was added in sequence with a final concentration of 100 nM. After mixing, it was incubated at 4°C for 30 minutes, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was taken for affinity measurement. The measured affinity was 2.7e-04 M. Through MST technology, we can accurately determine the affinity between mini-protein inhibitors and CTLA-4, providing important data for evaluating its effectiveness as a potential therapeutic agent.

[0041] 4. T cell toxicity assay-T cell proliferation assay In the present invention, in order to evaluate the effect of the novel CTLA-4 mini-protein inhibitor on T cell proliferation, we conducted a detailed T cell proliferation experiment using CFSE (Carboxy fluorescein succinimidylester), a cell dye. The following is a detailed description of the experimental steps: First, collect T cells from the culture system and count the cells using a hemocytometer or an automated cell counter to determine the initial number of cells. Then, place the collected T cells in a centrifuge tube and centrifuge at 300 g for 5 minutes to collect the cell pellet. Carefully discard the supernatant, taking care not to disturb the cell pellet. Resuspend the cell pellet with 10 mL of CFSE staining solution, which is a fluorescent dye that can covalently bind to cell surface proteins and is used to track cell proliferation. Incubate in a cell culture incubator at 37°C for 20 minutes to allow CFSE to fully bind to the cells.

[0042] After staining, add 40 mL of fresh culture medium to the cells and continue incubation at 37°C for 5 minutes to dilute and remove the dye that is not bound to the cells. Centrifuge again at 300 g for 5 minutes, discard the supernatant, and resuspend the cells with an appropriate amount of fresh culture medium to prepare for subsequent experiments. CFSE-labeled T cells can be cultured according to the experimental design, either alone or co-cultured with antigen-presenting cells, or the new CTLA-4 mini-protein inhibitor can be added for intervention.

[0043] After the culture period, the T cells are analyzed using a flow cytometer. By measuring the CFSE fluorescence intensity of the cells, the proliferation of the T cells can be assessed. Proliferating T cells undergo cell division, which leads to the dilution of CFSE. Therefore, by analyzing the distribution of fluorescence intensity, the proliferation rate of T cells can be determined. Figure 4 As shown, the treatment group had no significant effect on T cell proliferation compared to the control group. In summary, the present application provides a mini protein inhibitor of CTLA-4 protein, and successfully induced expression in the E. coli system to achieve rapid production of antibodies. The technical scope of this invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

[0044] The protein nucleotide sequence of the present invention is: ATGGGCCATCATCATCATCATAGCGGCGGCGCGCGCTATACCTATGAAGTGGCGGTGCGCGCGGTGCGCTATCTGCATGCGCTGGGCGATCCGGAAGCGGCGCGCAAAGTGGTGGAAGAAACCGCGGCGATGCTGGAAAGCGCGGGCGATCCGGCGCTGGCGGCGGAACTGCGCGCGCTGTGGGAAGAACTG The amino acid sequence of the protein is: MGHHHHHHSGGARYTYEVAVRAVRYLHALGDPEAARKVVEETAAMLESAGDPALAAELRALWEEL.

[0045] In summary, the present application provides a mini-protein inhibitor of CTLA-4 protein, and successfully induces expression in the E. coli system to achieve rapid production of antibodies.

[0046] The above-described embodiments only express a certain implementation mode of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A mini-protein inhibitor of CTLA-4 protein with high affinity, characterized in that Including computer-level protein sequence design and analysis steps: S1. Use molecular docking technology to simulate the interaction between antibodies and CTLA-4 to predict possible binding modes; S2. Use the ProteinMPNN algorithm to design the amino acid sequence of the antibody based on the three-dimensional structure information of the protein to ensure that it has specific biological functions; S3. The designed antibody sequence is evaluated for binding affinity using the Prodigy platform to ensure that it has high affinity for human CTLA-4 protein.

2. A mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 1, characterized in that: The prediction analysis software in step S1 uses AlphaFold software and PyMOL protein visualization software to perform structure prediction analysis.

3. A mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 1, characterized in that: The main structure of the novel CTLA-4 antibody is mainly composed of three helices and four key amino acid residues (serine 1, tyrosine 6, threonine 34, and glutamate 33).

4. A mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 1, characterized in that: The invention also comprises a method for preparing the protein: S4, constructing and culturing the expression strain of the tagged recombinant protein; S5, purification steps for protein cleavage purification, Ni-NTA affinity chromatography and gel filtration chromatography; S6. In vitro binding assay for proteins; S7. T cell toxicity assay - T cell proliferation assay.

5. A mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 4, characterized in that: The strain culture method in step S4 comprises the following steps: S41, using NcoI and XhoI restriction enzymes to perform restriction ligation to ensure that the sequence is correctly inserted into the pET-28a(+) expression vector; S42, transforming the synthesized expression plasmid into BL21 (DE3) competent cells, placing them on ice for 30 minutes, then subjecting them to a heat shock treatment at 42°C for 50 seconds, and then rapidly cooling them to reduce the effect of heat shock on the cells; S43, adding 100 μL LB liquid medium at 37°C for 45 minutes to resuscitate the cells, and spreading the resuscitated cells on LB solid medium containing kanamycin resistance, and culturing in a 37°C incubator for 12 hours to screen for successfully transformed cells; S44, select a single colony and transfer it to 30 mL of LB liquid medium containing kanamycin, culture it on a shaker at 37°C and 200 rpm for 10 hours, then transfer it to 1 L of medium with the same conditions and continue to culture until OD600 reaches 0.8; S45, add 0.5 mM IPTG to induce expression for 6 h at 30°C, 200 rpm on a shaker to express the target mini-protein inhibitor.

6. A mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 4, characterized in that: The protein purification method in step S5 comprises the following steps: S51. The cultured strain containing the recombinant CTLA-4 mini-protein inhibitor was centrifuged at 5000 rpm for 10 minutes to collect the bacteria; the bacteria were resuspended in 30 mL of PBS buffer, and the cells were disrupted by ultrasonic disruption, and then centrifuged at 13000 rpm for 30 minutes at 4°C to collect the supernatant containing the recombinant protein; S52, adding the supernatant to a pre-cooled Ni-NTA affinity chromatography column, and incubating at 4°C for 25 minutes to promote the affinity binding of the recombinant protein to the Ni-NTA resin on the nickel column through its His tag; after the unbound impurities flow out with the supernatant, the chromatography column is washed with a washing buffer for 5-6 column volumes to remove the impurities; S53, adding 15 mL of elution buffer and incubating at 4°C for 5 minutes to dissolve and elute the recombinant protein bound to the column; S54. The purified protein was analyzed by SDS-PAGE electrophoresis.

7. A mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 4, characterized in that: The in vitro binding experimental method for the protein in step S6 comprises the following steps: S61, CTLA-4 was selected as the target protein, and CTLA-4 was incubated with Cy5 dye in the dark at 4°C for 30 minutes; S62, free dye was effectively removed by gel filtration chromatography to obtain stained CTLA-4 at a concentration of 200 nM; S63, the novel mini-protein inhibitor was diluted stepwise in 16 steps, with the highest concentration being 1.85 mM, and stained CTLA-4 was added sequentially at a final concentration of 100 nM; S64, after mixing, incubate at 4°C for 30 minutes, centrifuge at 13000 rpm for 10 minutes, take the supernatant for affinity measurement, and the measured affinity is 2.7e-04 M; S65. Use MST technology to accurately measure the affinity between mini protein inhibitors and CTLA-4.

8. The mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 4, characterized in that: The method for T cell toxicity assay-T cell proliferation assay in step S7 comprises the following steps: S71. Collect T cells from the culture system and count the cells using a hemacytometer or an automated cell counter to determine the initial number of cells; S72, placing the collected T cells in a centrifuge tube, and centrifuging at 300 g for 5 minutes to collect the cell precipitate; S73, discard the supernatant without disturbing the cell pellet; S74. Resuspend the cell pellet with 10 mL of CFSE staining solution and incubate in a cell culture incubator at 37°C for 20 minutes to allow CFSE to fully bind to the cells. S75. After staining, add 40 mL of fresh culture medium to the cells and continue incubation at 37°C for 5 minutes to dilute and remove the dye that is not bound to the cells; S76. Centrifuge again at 300 g for 5 min, discard the supernatant, and resuspend the cells in an appropriate amount of fresh culture medium; S77. Cultivate CFSE-labeled T cells according to the experimental design, either alone or in co-culture with antigen presenting cells, or add the novel CTLA-4 mini-protein inhibitor for intervention; S78. After the culture period, the T cells were analyzed using flow cytometry; S79. Evaluate the proliferation of T cells by measuring the CFSE fluorescence intensity of the cells.

9. The mini-protein inhibitor of CTLA-4 protein with high affinity according to claim 4, characterized in that: The culture medium and reagents used in the protein preparation process include LB liquid culture medium, LB solid culture medium, Ni-NTA affinity chromatography buffer, wherein; LB liquid medium included 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride; LB solid medium includes 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar; The wash buffer consisted of 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 30 mM imidazole; The elution buffer consisted of 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 250 mM imidazole.

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