High-affinity fusion protein mutant for recognizing DMF5-TCR and application of high-affinity fusion protein mutant

Abstract

The invention discloses a high-affinity fusion protein mutant for recognizing DMF5-TCR and application of the high-affinity fusion protein mutant, and relates to the technical field of biological medicine. Multiple MHC-peptide fusion protein mutants with significantly improved affinity are obtained through screening, the affinity between DMF5-TCR and MHC-peptide fusion protein can be improved by nearly 250 times at most by using the fusion protein mutants, and the preparation method is relatively simple, significantly reduced in cost and more suitable for large-scale research and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to a high-affinity fusion protein mutant that recognizes DMF5-TCR and its applications. Background Art

[0002] T cell receptor-engineered T cell therapy (TCR-T) is an adoptive T cell therapy. It genetically modifies natural T cells to highly express TCRs on the surface of T cells that can recognize certain specific tumor antigens, precisely recognizes tumor-specific antigens derived from the cell membrane surface or intracellularly, and has high tumor-killing ability, showing unprecedented prospects in the treatment of solid tumors. Melanoma, as one of the most targeted cancer types, is the target of multiple TCR-T clinical trials.

[0003] However, the melanoma antigen (MART-1) recognized by TCR-T cells exists not only in melanoma cells but also in normal melanocytes. Targeting such antigens may cause "on-target, off-tumor" toxicity to normal cells of key organs. DMF5-TCR-T, which is the most widely used in TCR-T cell therapy trials targeting the MART-1 protein, has been reported to have autoimmune toxicity to the eyes, ears, and skin, and there are even patients who became blind after receiving DMF5-TCR-T cell therapy. In addition, in one trial, fatal cardiac toxicity was also observed in 3 patients who received the MART-1 trial.

[0004] To achieve the purpose of effectively eliminating melanoma, DMF5-TCR-T cells must have characteristics such as high efficiency and high permeability. However, when it is very effective, it will have the same killing effect on tissues with low expression levels as on those with high expression levels, resulting in strong autoimmune toxicity. Therefore, a technology is needed to constantly target and label these engineered TCR-Ts, facilitating the rapid and simple detection of antigen-specific T cells for qualitative and quantitative analysis, and quickly causing these TCR-Ts to apoptose after they reach the threshold sufficient to kill tumor cells, reducing the "on-target, off-tumor" toxicity.

[0005] The existing detection technology for targeting and labeling T cells for the purpose of detection is only the MHC-peptide tetramer detection. The specific principle is to use the biotin-streptavidin system to tetramerize the MHC-peptide complex, which can specifically bind to multiple TCRs on the cell surface, improving the affinity and stability of the MHC-peptide complex with TCR. The advantage of this tetramer technology is that it is rapid, direct, sensitive and highly specific. However, its preparation is relatively difficult. Not only is it difficult for the MHC class I heavy chain, β2m, and polypeptide to fold into the MHC-peptide complex (monomer), but also a significant amount of MHC-peptide complex is lost during the process of biotinylating the lysine residue at the C-terminus of the MHC class I heavy chain using BirA enzyme and then performing tetramerization using biotin-avidin binding, resulting in extremely high costs. And because the affinity of the MHC-peptide complex (monomer) is generally low, even when prepared into tetramers, it is difficult to efficiently label a very small number of antigen-specific T cells, causing difficulties in detection and making it difficult to conduct engineering TCR-T research. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a high-affinity fusion protein mutant that recognizes DMF5-TCR, its amino acid sequence and nucleotide sequence, and realizes its use in targeting and labeling DMF5-TCR T cells. Provide an MHC-peptide fusion protein mutant, so that the monomeric mutant has a relatively high binding affinity of MHC-peptide tetramer, and on the basis of not changing its advantages of rapidity, directness, sensitivity and high specificity, significantly reduce the preparation difficulty and detection cost. Better achieve the efficient labeling of a very small number of antigen-specific T cells.

[0007] To achieve the above object, the present invention provides a high-affinity MHC-peptide fusion protein mutant that recognizes DMF5-TCR. The mutant mutates multiple amino acid sites in the MHC-peptide fusion protein with the amino acid sequence shown in SEQ ID NO.1, and the multiple mutated amino acid sites include at least one of R209L, K210R, A213G, A294G and E298D.

[0008] In a preferred embodiment, the present invention provides a high-affinity MHC-peptide fusion protein mutant that recognizes DMF5-TCR, and the mutation method includes any one of the following four:

[0009] K210R; K210R and E298D; K210R, A213G and A294G; R209L, K210R, A213G and A294G.

[0010] Furthermore, the amino acid sequences of the above four mutants are successively shown in SEQ ID NO.2-5.

[0011] The second object of the present invention is to provide polynucleotides encoding the aforementioned high-affinity MHC-peptide fusion protein mutants that recognize DMF5-TCR, so that the encoded monomeric mutants have a higher affinity binding force than the MHC-peptide tetramer, and can better achieve the efficient labeling of a very small number of antigen-specific T cells. To achieve this object, in the basic experimental protocol, the present invention provides polynucleotides encoding the aforementioned mutants, as shown in SEQ ID NO. 6-9.

[0012] The present invention also provides a vector containing the aforementioned polynucleotide and a host cell containing the vector.

[0013] The present invention also provides a method for preparing the aforementioned fusion protein, including the following steps: (1) constructing a coding gene vector expressing the MHC-peptide fusion protein mutant; (2) transfecting the coding gene vector expressed in step (1) into mammalian cells, culturing and then purifying the protein to obtain the fusion protein. Preferably, the vector in step (1) is pcDNA3.1(+); the mammalian cell in step (2) is HEK293F cell.

[0014] The third object of the present invention is to provide the application of the aforementioned high-affinity MHC-peptide fusion protein mutant that recognizes DMF5-TCR or the aforementioned nucleotide in the preparation of a reagent or drug for targeting and binding DMF5-TCR T cells. When applying, the high-affinity MHC-peptide fusion protein mutant is contacted with T cells expressing DMF5-TCR for specific binding, so as to further achieve purposes such as immunostaining and T cell function regulation. Specifically, mainly the high-affinity MHC-peptide fusion protein mutant (monomer) is used to achieve the targeted labeling of DMF5-TCR T cells, which can be detected by flow cytometry (FACS).

[0015] To achieve this object, in the basic experimental protocol, the present invention provides the application of the high-affinity MHC-peptide fusion protein mutant that recognizes DMF5-TCR. The method shown is to construct DMF5-TCR Jurkat T cells, label the cells with the high-affinity MHC-peptide fusion protein mutant (monomer), and detect by flow cytometry (FACS).

[0016] In a preferred embodiment, the present invention provides the application of the high-affinity MHC-peptide fusion protein mutant that recognizes DMF5-TCR, and the proportion of DMF5-TCR Jurkat T cells is 6%-8%.

[0017] Furthermore, the buffer system is 1×PBS system, pH 7.2-7.4.

[0018] Furthermore, the target binding temperature is 4°C and the binding time is 30 min.

[0019] Advantages of the present invention:

[0020] By using the high-affinity MHC-peptide fusion protein mutant that recognizes DMF5-TCR of the present invention, the affinity between DMF5-TCR and the MHC-peptide fusion protein can be increased by nearly 250 times at most. Moreover, the preparation method is relatively simple, the cost is significantly reduced, and it is more suitable for large-scale research applications. Description of the Drawings

[0021] Figure 1 This is the flow cytometry evaluation of the MHC-peptide expression level and positive rate of yeast cells after the second round of magnetic sorting in Example 1 of the present invention.

[0022] Figure 2 This is the sorting result of yeast flow cytometry showing the surface display of MHC-peptide fusion protein in each round in Example 1 of the present invention.

[0023] Figure 3 This is the flow cytometry detection result of yeast monoclonal with significantly improved binding affinity in Example 1 of the present invention.

[0024] Figure 4 This is for use in Example 2 of the present invention The collected peak diagram purified using a purifier and a SuperdexTM 75 Increase 10 / 300GL prepacked column.

[0025] Figure 5 This is the BLI detection result of the refolded and expressed MHC-peptide mutant and DMF5-TCR in Example 2 of the present invention.

[0026] Figure 6 This is the expression of the high-affinity MHC-peptide mutant fusion protein in Example 3 of the present invention. Lanes 1 and 3 are wild-type proteins, and lanes 2 and 4 are mutant fusion proteins with the highest improved affinity.

[0027] Figure 7 This is the verification of the folding conformation of the MHC-peptide fusion protein in Example 3 of the present invention.

[0028] Figure 8 This is the flow cytometry verification that the mutant fusion protein targets and labels a very small number of antigen-specific T cells in Example 3 of the present invention. Detailed Embodiments

[0029] To enable those skilled in the art to better understand the technical solution of the present invention, the following will clearly and completely describe the technical solution in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the scope of protection of the present invention.

[0030] Example 1 Screening of MHC-peptide fusion protein mutants with high affinity

[0031] The MHC-peptide mutants are displayed on the yeast surface using yeast display technology. Since yeast cells are relatively large, they are compatible with magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS), enabling real-time quantitative screening of clones that bind to the target TCR. This screening method offers better visualization and specificity, and can even distinguish MHC-peptides with minor differences in affinity. The MHC-peptide mutant yeast display library used is constructed by linking Aga2p to Agalp anchored on the cell surface via a disulfide bond, thereby displaying the MHC-peptide (amino acid sequence shown in SEQ ID NO.1) on the cell surface. To facilitate subsequent detection of the expression level of the fusion protein, an HA tag is introduced at the carboxyl terminus of the MHC-peptide.

[0032] (1) Take 100 OD of yeast library strains from the -80°C refrigerator and add them to 100 mL of yeast tryptophan auxotrophic (SD-Trp deficient) growth medium. Culture at 30°C and 220 rpm for 3 - 4 h;

[0033] (2) Measure the OD 600 absorbance, centrifuge to remove the deficient growth medium, collect the cell pellet, resuspend the cell pellet in SD-Trp deficient induction medium, adjust the cell concentration to 0.8 OD / mL, and culture at 25°C and 2200 rpm for 24 h; then centrifuge to collect the yeast cells;

[0034] (3) Remove mutants that non-specifically bind to streptavidin magnetic beads through the first round of magnetic sorting

[0035] Resuspend the yeast cells in 40 mL of PBSA (1×PBS + 1% BSA) buffer. After centrifugation and discarding the supernatant, resuspend the yeast cells in 5 mL of PBSA buffer. Add 200 μL of streptavidin magnetic beads and slowly rotate the mixture on a rotary mixer at 4°C for 1 h; transfer the mixture into multiple 1.5 mL centrifuge tubes, place them on a magnetic stand, and wait for 2 - 3 min; collect the yeast cells that do not bind to the magnetic beads, and wash the magnetic beads with 1 mL / tube of selection buffer twice; centrifuge at 2000 rpm for 5 min, discard the supernatant; add 5 mL of PBSA buffer to resuspend.

[0036] (4) Enrich the correctly folded MHC-peptide fusion protein mutants by the second round of magnetic sorting

[0037] Resuspend the yeast with the DMF5-TCR complex at a final concentration of 400 nM TCR, and gently rotate it on a rotary shaker at 4°C for 1 h; centrifuge at 2000 rpm for 5 min, discard the supernatant, and resuspend with 5 mL of PBSA buffer; add 200 μL of magnetic beads, mix well, and gently rotate it on a rotary shaker at 4°C for 30 min; transfer the mixture into multiple 1.5 mL centrifuge tubes, place it on a magnetic stand, and wait for 2 - 3 min; discard the yeast that has not bound to the magnetic beads, and wash the magnetic beads with 1 mL / tube of PBSA buffer twice; then resuspend the magnetic beads with the growth medium and culture at 30°C, 220 rpm for 24 h;

[0038] (5) Wash the magnetic beads with the growth medium, place it on a magnetic stand, and wait for 2 - 3 min; aspirate the supernatant, discard the magnetic beads, and induce with the induction medium for 24 h; evaluate the MHC-peptide expression level and positive rate of yeast cells after the second round of magnetic sorting by flow cytometry. The flow analysis results are as Figure 1 shown. The yeast library display rate increased from 4.02% to 15.56% after magnetic sorting

[0039] (6) Enrich the high-affinity MHC-peptide fusion proteins by multiple rounds of flow cytometry sorting

[0040] The binding proteins used for flow cytometry sorting are the DMF5-TCR tetramer complex and TCR monomers (prepared by the renaturation method in our laboratory, containing the DMF5-TCR α-chain and β-chain, and the C-terminus of the β-chain contains a biotin tag). Streptavidin labeled with PE can specifically bind to the biotin tag on the surface of TCR, and the PE fluorescence intensity is used to characterize the binding degree of pMHC and TCR displayed on the yeast surface; an anti-HA antibody labeled with APC can specifically bind to the HA tag on the surface of yeast cells, and the APC fluorescence intensity is used to characterize the expression of pMHC on the cell surface. Sort the cells that are double positive for PE and APC;

[0041] (7) After two rounds of magnetic sorting, the library capacity decreased to 10 6 Next, take 1 OD of yeast cells in the growth medium and culture at 30°C, 220 rpm for 24 h; centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend the yeast cells with the induction medium, adjust the cell concentration to 0.8 OD / mL, and culture at 25°C, 2200 rpm for 24 h;

[0042] (8) Take 1 OD yeast cells per tube and wash them once with 1x PBSA buffer; prepare 500 nM DMF5-TCR tetramer protein during the flow cytometry process. Mix 13 μg of DMF5-TCR protein with 15 μL of SAPE, supplement PBS to 100 μL, and let it stand for 5 min; incubate the yeast cells with 500 nM DMF5-TCR tetramer for 30 min; then wash the yeast twice with 1x PBSA buffer; resuspend the yeast cells with 1x PBSA and then the flow cytometry experiment can be carried out; sort the cell population that is double positive for PE and APC;

[0043] (9) For the second round of flow cytometry sorting, the concentration of the DMF5-TCR tetramer complex is 200 nM, and other conditions remain unchanged; for the third round of flow cytometry sorting, use 25 μM DMF5-TCR tetramer complex, and other conditions remain unchanged; next, sorting will be carried out using DMF5-TCR monomer. The concentration of DMF5-TCR monomer in the fourth round is 100 nM, and the concentration of DMF5-TCR monomer in the fifth round is 25 nM; strictly sort the cell population that is double positive for PE and APC; The specific sorting results for each round are as Figure 2 shown. It can be found that the double-positive cells are significantly enriched. When sorting with 25 nM TCR monomer subsequently, the double-positive cells still account for more than half of the displayed cells.

[0044] (10) FACS clone identification

[0045] The enriched yeast cells are spread on an SD-Trp growth plate and cultured in an incubator at 30 °C for 2 - 3 days; pick monoclonal colonies and inoculate them in SD-Trp growth medium, and culture them at 30 °C and 220 rpm for 24 h; perform FASC detection according to step (8), where the experimental group uses 25 nM DMF5-TCR monomer, and the concentration of DMF5-TCR monomer in the control group is 0 nM; select the clones with better detection results (as Figure 3 shown) for sequencing to obtain high-affinity MHC-peptide mutants, which are named MHC-peptide mutant fusion proteins RRAAE, RRAAD, RRGGE, and LRGGE respectively. The amino acid sequences are shown in SEQ ID NO.2 - 5 respectively, and the encoding genes are shown in SEQID NO.6 - 9 respectively.

[0046] Example 2 Detection of the binding affinity between high-affinity MHC-peptide mutants and DMF5-TCR

[0047] (1) Renatured expression of high-affinity MHC-peptide mutants

[0048] To accurately detect the binding affinity between the mutant and DMF5-TCR, the MHC-peptide mutant complex was renatured and expressed. The pET-28a-β2m and pET-28a-HLA02 expression plasmids were constructed and transformed into BL21(DE3) Escherichia coli respectively, and β2m (β2-microglobulin) and the extracellular region of HLA-A*0201 (MHC) were expressed in the form of inclusion bodies. IPTG was used to induce the expression of recombinant proteins in E. coli. After disrupting the bacteria, the inclusion bodies were collected by centrifugation, washed multiple times and fully dissolved with 8M urea. To facilitate biotinylation modification after renaturation, the carboxyl terminus of the extracellular region of HLA-A*0201 carried a BirA-specific recognition amino acid sequence.

[0049] (2) Renaturation was carried out according to the following steps (1L reaction system):

[0050] 1. The renaturation buffer (400 mmol / L L-arginine, 100 mmol / L Tris, 2 mmol / L EDTA, pH = 8.0) was pre-cooled at 4°C, 1.54 g of reduced glutathione, 0.31 g of oxidized glutathione and 5 mL of 100 mmol / L PMSF were added, and stirred evenly;

[0051] 2. 0.5 mL of ELAGIGILTV polypeptide (dissolved in DMF, 20 mg / mL) was added and stirred evenly;

[0052] 3. 23.4 mg of β2m was diluted in 5 mL of renaturation buffer and injected into the reaction system in 3 equal portions at intervals of 10 min each;

[0053] 4. After stirring for 30 min, 34 mg of the extracellular region of HLA-A*0201 was diluted in the injection buffer (3 mol / L guanidine hydrochloride, 10 mmol / L sodium acetate, 10 mmol / L EDTA, pH = 4.2) and injected into the reaction system in 3 equal portions at intervals of 10 min each;

[0054] 5. Stir at 4°C and replenish the extracellular region of HLA-A*0201 every 12 h (i.e., repeat step 4), and replenish 2 times in total;

[0055] 6. After 36 h, ultrafiltration concentration was carried out with a 30 kDa molecular weight cut-off ultrafiltration tube and the solution was replaced with PBS;

[0056] 7. Biotinylation modification of the renatured product was carried out with BirA enzyme according to the instructions;

[0057] 8. Purification was carried out using an AKTA purifier and a SuperdexTM 75 Increase 10 / 300GL pre-packed column, and about 12 mL of the peak was collected. The results were as Figure 4As shown, the protein concentration was measured using a Nanodrop ND-1000, aliquoted, and stored frozen at -80 °C for later use.

[0058] (3) The refolded and expressed MHC-peptide mutant and DMF5-TCR were sent for BLI detection, and the detection results are as Figure 5 shown, with the highest binding affinity increased by nearly 250-fold.

[0059] Example 3 Verification of the Targeted Labeling of DMF5-TCR T Cells

[0060] (1) Expression of the MHC-peptide mutant fusion protein

[0061] After transfecting the HEK293F cells with the expression plasmid of the mutant fusion protein RRAAE with the strongest binding affinity for 3 - 4 days, centrifugation was performed at 4000 × g for 20 min to remove the cell supernatant. After filtration through a 0.22 μm filter membrane, purification was carried out using an AKTA protein purifier and a Ni-NTA affinity column.

[0062] The purification steps are as follows:

[0063] 1. Turn on the AKTA protein purifier and the connected control computer. After the instrument is connected to the computer, set the pressure parameter (high pressure 0.5 MPa).

[0064] 2. Place the A and B pump heads in pure water filtered through a 0.45 μm filter membrane, set the flow rate (4 mL / min) and the flushing ratio of the AB pumps (50% B). After the pure water flushes to conductivity equilibrium (about 60 mL), connect the Ni-NTA affinity column (5 mL) to the AKTA purifier and continue to flush with pure water for at least three column volumes.

[0065] 3. Change the flushing ratio of the AB pumps to 0% B, and change the A pump to the binding buffer (50 mM NaH2PO4, 300 mM NaCl, pH 7.4). After conductivity equilibrium, change the A pump to the culture medium supernatant to be purified. After sample loading, change the A pump to the binding buffer until conductivity equilibrium; then change the ratio of the AB pumps to 100% B and change the B pump to the elution buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 7.4) to elute the target protein. The AKTA purifier was flushed to conductivity equilibrium with pure water and the entire system was stored with 20% ethanol (v / v). The harvested fusion protein was ultrafiltered using a 50 KDa pore size ultrafiltration membrane. After replacing the solvent with PBS buffer, the protein concentration was measured using a Nanodrop ND-1000, aliquoted, and stored frozen at -80 °C for later use. SDS-PAGE analysis of the protein was performed as Figure 6 shown.

[0066] (2) Verification of MHC-peptide mutant fusion protein

[0067] Verify the MHC-peptide conformation in the fusion protein by Dot-blot

[0068] 1. Cut an appropriate size of PVDF membrane and activate it in methanol for 1 minute

[0069] 2. Place the activated PVDF membrane in ddH2O and wash it on a shaker for 15 minutes

[0070] 3. Thaw the sample at 4°C

[0071] 4. Take out the activated PVDF membrane and let it air-dry at room temperature until the water film disappears. Spot 2 μl of MHC-peptide mutant fusion protein, blank control (1×PBS), and positive control (wild-type protein) onto the PVDF membrane respectively, and air-dry. Wait for the sample to be absorbed at room temperature, and the color at the spotting area will deepen after absorption

[0072] 5. Blocking: After the sample on the PVDF membrane is completely dry, block it in TBST solution containing 5% skim milk powder, incubate it in a 37°C incubator for 2 h or overnight at 4°C

[0073] 6. Wash the PVDF membrane with TBST 5 times, 5 minutes each time

[0074] 7. Primary antibody incubation: Dilute the primary antibody (Purified anti-human HLA-A,B,C Antibody, purchased from biolegend, catalog number 311402) with TBST solution containing 5% skim milk powder at a dilution ratio of 1:10000, incubate it in a 37°C incubator for 2 h or overnight at 4°C

[0075] 8. Wash the PVDF membrane with TBST 5 times, 5 minutes each time

[0076] 9. Secondary antibody incubation: Dilute the secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG(H+L), purchased from Beyotime, catalog number A0350) with TBST solution containing 5% skim milk powder at a dilution ratio of 1:10000, incubate it in a 37°C incubator for 1.5 h

[0077] 10. Wash the PVDF membrane with TBST 5 times, 5 minutes each time

[0078] 11. Exposure: Blot the TBST solution on the PVDF membrane with filter paper, add ECL hypersensitive color developer (A solution: B solution = 1:1), pre-cool the chemical exposure instrument in advance, perform exposure, observe and record the results. There is an obvious binding between the fusion protein and the W6 / 32 antibody compared with the negative control Figure 7), indicating that the MHC-peptide conformation in the fusion protein is correctly expressed and meets the expected design.

[0079] (3) FACS verification of the targeting label

[0080] 1. Take 2×10 6 Jurkat T cells (the proportion of DMF5-TCR Jurkat T cells is 6%-8%) into a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and then wash three times with 1×PBS buffer.

[0081] 2. Add 200 nM of the MHC-peptide mutant fusion protein RRAAE to the experimental group, add the wild-type fusion protein WT to the control group, and do not add protein to the negative group. Incubate at 4°C for 30 min.

[0082] 3. After incubation, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash three times with 1×PBS buffer.

[0083] 4. Add 100 μL of His tag-FITC fluorescent secondary antibody (diluted 1:500), and incubate at 4°C for 30 min.

[0084] 5. After incubation, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash three times with 1×PBS buffer.

[0085] 6. Finally, resuspend with 300 μL of 1×PBS buffer and perform flow cytometry detection.

[0086] (4) Data analysis is as Figure 8 , and the results show that the provided MHC-peptide fusion protein mutant can target and bind to DMF5-TCR T cells in monomer form. On the basis of not changing its advantages of being rapid, direct, sensitive and highly specific, it significantly reduces the preparation difficulty and detection cost. It better realizes the efficient labeling of a very small number of antigen-specific T cells.

Claims

1. A high-affinity fusion protein mutant that recognizes DMF5-TCR, characterized in that, The fusion protein is an MHC-peptide fusion protein, and the amino acid sequence of the MHC-peptide fusion protein is as shown in SEQ ID NO.

1. The high-affinity fusion protein mutant is obtained by mutating the MHC-peptide fusion protein, and the mutation mode is at least one of the following: (1) R209L; (2) K210R; (3) A213G; (4) A294G; (5) E298D.

2. The high-affinity fusion protein mutant according to claim 1, wherein The mutation mode is any one of the following: (a) K210R; (b) K210R and E298D; (c) K210R, A213G and A294G; (d) R209L, K210R, A213G and A294G.

3. Nucleotides encoding the high-affinity fusion protein mutant according to claim 1 or 2.

4. The nucleotide according to claim 3, wherein The nucleotide sequences encoding the high-affinity fusion protein mutants with mutation modes (a)-(d) are as shown in SEQ ID NOs. 6-9 respectively.

5. A vector containing the nucleotide according to claim 3 or 4.

6. A host cell containing the vector according to claim 5.

7. The method for preparing the high-affinity fusion protein mutant according to claim 1 or 2, characterized in that, It includes the following steps: (1) Construct a coding gene vector for expressing the MHC-peptide fusion protein mutant; (2) Transfect the coding gene vector expressed in step (1) into mammalian cells, and after culturing, perform protein purification to obtain the high-affinity fusion protein mutant.

8. The preparation method according to claim 7, characterized in that, In step (1), the vector is pcDNA3.1(+); in step (2), the mammalian cell is HEK293F cell.

9. Use of the high-affinity fusion protein mutant according to claim 1 or 2, or the nucleotide according to claim 3 or 4 in the preparation of a reagent or drug for specifically binding to DMF5-TCR T cells.

10. The application according to claim 9, characterized in that, During application, the high-affinity MHC-peptide fusion protein mutant is contacted with T cells expressing DMF5-TCR for specific binding.

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