Method for screening human-mouse MR1 cross-restrictive high-affinity TCR by using phage display technology

Abstract

The invention relates to a method for screening a human-mouse cross MR1 restriction high-affinity TCR (T cell receptor) by using a phage display technology, and relates to the technical field of T cell receptors. According to the invention, a phage display technology is utilized to prepare an MAIT cell single-chain T cell receptor (scTCR) phage library, the scTCR phage library is subjected to biological elutriation through an MR1 / 5-OP-RU tetramer, and Phage-ELISA identifies that TCR6 clone (TCR6) has specific binding with human and mouse MR1 / 5-OP-RU and has higher affinity compared with other clone.

Description

Technical Field

[0001] The present invention relates to the technical field of T cell receptors, and more specifically, to a method for screening human-mouse cross-reactive MR1-restricted high-affinity TCRs using phage display technology. Background Art

[0002] MAIT (Mucosal-associated invariant T) cells are a type of innate-like immune cells with a conserved TCR (T cell antigen receptor) repertoire, usually composed of a constant TCRα chain (Va7.2-Ja33) paired with a limited TCRβ chain repertoire, showing structural conservation. MAIT cell TCRs can recognize microbial metabolites, such as the riboflavin metabolite 5-OP-RU presented to MHC-class-I-related protein 1 (MR1). MAIT cells are enriched in peripheral blood, liver, lung, and mucosal tissues and are one of the main immune cells of the mucosal barrier; they have the functional characteristic of rapid response and can play an important role in anti-infection immunity, inflammation regulation, and tumorigenesis and development by secreting IFN-γ, TNF-α, IL-17, and granzyme B without clonal expansion. In the future, the functional regulation of MAIT cells may provide new ideas for the treatment of infectious diseases, inflammatory diseases, and tumors, as well as the development of new immunotherapies targeting MAIT cells.

[0003] Phage display technology is an in vitro selection platform that studies the interaction between polypeptides or proteins and target molecules by displaying them on the surface of phages. It can use the coat protein on the surface of phages to fuse and express foreign proteins (such as TCRs) on the surface of phages while keeping the phage DNA internally carrying the gene encoding the foreign protein. By this method, a direct association between phenotype and genotype can be achieved, thus quickly discovering target molecules by screening phage particles with specific binding abilities. The success of using phage display of short peptides and antibodies has led to the development of this technology. Due to the wide application of TCRs in immunotherapy, it has become an important binding motif developed by phage display. Although phage display has been widely used in antibody presentation, TCRs are relatively large and complex molecules, making their presentation on the surface of phages challenging. Summary of the Invention

[0004] The present invention prepares a phage display library of single-chain T cell receptors (scTCRs) of MAIT cells by using phage display technology, biopans the scTCR phage library with MR1 / 5-OP-RU tetramers, and Phage-ELISA identifies that the 6th clone of TCR (TCR6) has specific binding to both human and murine MR1 / 5-OP-RU and has a higher affinity than other clones.

[0005] According to the object of the present invention, a method for screening human-mouse cross-reactive MR1-restricted high-affinity TCRs by using phage display technology is provided, including the following steps:

[0006] (1) Sort MAIT cells from peripheral blood mononuclear cells, extract the RNA of the MAIT cells, and obtain cDNA by RT-PCR amplification;

[0007] In the first round of PCR, using the cDNA as a template, TRAF-1 as the forward primer and TRAR-1 as the reverse primer, amplify the Vα chain fragment; then, using TRBF1-1, TRBF2-1, TRBF3-1, and TRBF4-1 as the forward primers and TRBR-1 as the reverse primer respectively, amplify 4 types of Vβ chain fragments;

[0008] In the second round of PCR, using the Vα as a template, TRAF-2 as the forward primer and TRAR-2 as the reverse primer, amplify the Vα-2 chain fragment, and using TRBF1-2, TRBF2-2, TRBF3-2, and TRBF4-2 as the forward primers and TRBR-2 as the reverse primer respectively, amplify 4 types of Vβ-2 chain fragments;

[0009] The sequence of TRAF-1 is shown as SEQ ID NO:1, the sequence of TRAR-1 is shown as SEQ ID NO:2, the sequence of TRBF1-1 is shown as SEQ ID NO:3, the sequence of TRBF2-1 is shown as SEQ ID NO:4, the sequence of TRBF3-1 is shown as SEQ ID NO:5, the sequence of TRBF4-1 is shown as SEQ ID NO:6, the sequence of TRBR-1 is shown as SEQ ID NO:7, the sequence of TRAF-2 is shown as SEQ ID NO:8, the sequence of TRAR-2 is shown as SEQ ID NO:9, the sequence of TRBF1-2 is shown as SEQ ID NO:10, the sequence of TRBF2-2 is shown as SEQ IDNO:11, the sequence of TRBF3-2 is shown as SEQ ID NO:12, the sequence of TRBF4-2 is shown as SEQ ID NO:13, and the sequence of TRBR-2 is shown as SEQ ID NO:14;

[0010] The Vα-2 chain fragments are respectively assembled with each of the Vβ-2 chain fragments into complete scTCR fragments by using a linker peptide;

[0011] The linearized phagemid vector pComb3X is respectively ligated with each of the scTCR fragments by homologous recombination to construct a recombinant phagemid vector pComb3X-scTCR, and the ligation product is transformed into Escherichia coli; then, superinfection is carried out by using the helper phage M13K07 to obtain recombinant phages, namely, an scTCR library is obtained;

[0012] (2) A tetramer formed by protein MR1 and the riboflavin metabolite 5-OP-RU is coated on a solid-phase carrier; then, the recombinant phages obtained in step (1) are incubated with the solid-phase carrier, the unbound target molecule solution is discarded, and then the specifically bound phages are eluted with an eluent to obtain a specific eluate; the specific eluate is cultured, single colonies with clear edges are selected, shake culture is carried out, plasmid digestion identification is carried out, and then sequencing is carried out;

[0013] (3) The identified positive monoclonal is prepared into a phage solution, and monoclonal Phage-ELISA identification is carried out on the phage scTCR; tetramers of human and murine protein MR1 and the riboflavin metabolite 5-OP-RU are respectively coated, the tested single-clone phage solution is added, and the helper phage M13K07 with the same titer is used as a negative control, and a polyclonal mixed phage solution is used as a positive control. Horseradish peroxidase-labeled Anti-M13 antibody is added to carry out Phage-ELISA, and the absorbance value at a wavelength of 450 nm is detected by an enzyme-linked immunosorbent assay instrument; a TCR with an OD450 value significantly higher than that of the positive control is used as a high-affinity TCR, and the significance is p<0.05. The clone shown in SEQ ID NO:15 has a significant difference, that is, the TCR shown in SEQ ID NO:15 is a human-mouse MR1-restricted high-affinity TCR.

[0014] Preferably, in step (1), the sorting method is magnetic bead sorting.

[0015] Preferably, in step (2), the solid-phase carrier is a sulfate latex microsphere.

[0016] Preferably, in step (2), the tetramer is a tetramer of human protein MR1 and the riboflavin metabolite 5-OP-RU.

[0017] Preferably, in step (1), each of the Vα-2 chain fragments is assembled with each of the Vβ-2 chain fragments into respective complete scTCR fragments by the overlap extension PCR method.

[0018] Preferably, in step (1), the transformation is electrotransformation.

[0019] Generally speaking, compared with the prior art by the above technical solutions conceived by the present invention, the following technical advantages are mainly presented:

[0020] (1) The present invention uses phage display technology to prepare a single-chain T cell receptor (scTCR) phage library of MAIT cells. The scTCR phage library is biopanned by MR1 / 5-OP-RU tetramer. Phage-ELISA identifies that the 6th clone of TCR (TCR6) has specific binding to both human and murine MR1 / 5-OP-RU and has a higher affinity than other clones.

[0021] (2) The TCR displayed in the phage library of the present invention is a single-chain TCR, which can greatly improve the display efficiency. The phage library is constructed by inserting DNA fragments encoding TCR into a phage expression vector. In the present invention, the full-length TCR α-chain and β-chain are designed into a single-chain TCR (scTCR), and the variable regions of the α-chain and β-chain of TCR (Vα and Vβ) are connected by a flexible linker peptide (a 15-mer rich in glycine and serine) to form a single-chain molecule. This format has a relatively small molecular weight (about 32 kDa), which helps to improve the display efficiency.

[0022] (3) The present invention can use phage display technology to construct a unique scTCR phage library of MAIT cells, screen the single-chain TCR of MAIT cells through multiple rounds of biopanning, and after the screening is completed, use phage enzyme-linked immunosorbent assay (Phage-ELISA) to verify the specificity and affinity of TCR with human and murine MR1-5-OP / RU tetramers.

[0023] (4) The present invention coats the MR1 / 5-OP-RU tetramer on sulfuric acid latex microspheres as a solid-phase carrier, making the operation of biopanning simpler and easier.

[0024] (5) The 6th phage clone of TCR has specificity and high affinity for both human and murine MR1 / 5-OP-RU: After verification by monoclonal Phage-ELISA experiment, among the 19 clones after biopanning, the 6th clone of TCR has significant specificity and high affinity for both human and murine MR1 tetramers.

[0025] (6) The present invention has scalability: The MAIT cell TCR screened by this system can be identified by both human MR1 tetramer and murine MR1 tetramer. Therefore, the 6th clone of TCR that can recognize both human and murine MR1 screened out can provide a basis for the establishment of MAIT cell TCR-T cell lines, MAIT cell TCR-T cell therapy, and MAIT cell TCR transgenic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flow chart for screening human and murine MR1 - reactive and high - affinity TCRs by phage display technology.

[0027] Figure 2 : A. Electrophoresis results of Vα amplification. A visible band was observed between 250 - 500 bp in lane 1 of the product, which was consistent with the theoretical value of the fragment; B. Electrophoresis results of Vβ amplification. Visible bands were observed between 250 - 500 bp in the products of lanes 1, 2, 3, and 4, which were consistent with the theoretical value of the fragment; C. Electrophoresis results of scTCR amplification. Visible bands were observed between 750 - 1000 bp in the products of lanes 1, 2, 3, and 4, which were consistent with the theoretical value of the fragment.

[0028] Figure 3 : Enzyme digestion verification showed that the recombinant phagemid pCcomb3x - scTCR was successfully constructed. Control was the plasmid without enzyme digestion. Visible bands were observed between 250 - 500 bp in the enzyme digestion products of lanes 1 - 10, which were consistent with the theoretical value of the scTCR fragment.

[0029] Figure 4 : Flow cytometry verification showed that MR1 / 5 - OP - RU tetramers could be effectively adsorbed on Beads.

[0030] Figure 5 : A. Phage - ELISA detected and identified that clone TCR6 was reactive with human MR1 / 5 - OP - RU and had a higher affinity compared to other clones; B. Phage - ELISA detected and identified that clone TCR6 was reactive with murine MR1 / 5 - OP - RU and had a higher affinity compared to other clones. In the above figures, the vertical axis represents the OD450nm value, and the horizontal axis represents different TCR clone numbers and colony numbers. The bar graph shows the detection results of each sample, and the black dots represent the specific data points of the repeated experiments. The bars of different colors represent samples of different groups. The significance level of statistical analysis is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0031] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] The method for screening human and murine MR1 - restricted high - affinity TCRs using phage display technology according to the present invention includes the following steps:

[0033] 1. Construction of the single-chain TCR phage library of MAIT cells.

[0034] 1.1 RNA extraction from MAIT cells derived from healthy donors and synthesis of cDNA;

[0035] 1.2 Design and synthesis of single-chain TCR primers;

[0036] 1.3 Synthesis and purification of Vα and Vβ;

[0037] 1.4 SOE-PCR splicing of the scTCR gene;

[0038] 1.5 Construction of the recombinant phagemid vector pComb3X-scTCR and electrotransformation.

[0039] 2. Enrichment and screening of the scTCR phage library of MAIT cells.

[0040] 2.1 Coating of MR1 / 5-OP-RU tetramers on the solid-phase carrier sulfate latex microspheres (Beads);

[0041] 2.2 Nonspecific elution of phages in each round;

[0042] 2.3 Amplification and purification of the eluate in each round;

[0043] 2.4 Preliminary identification of the screening results of the phage scTCR library.

[0044] 3. Screening and identification of high-affinity positive phage clones.

[0045] 3.1 Preparation of monoclonal positive phages;

[0046] 3.2 Screening of positive phage clones by Phage-ELISA;

[0047] 3.3 Sequencing and analysis of the gene sequence of TCR6.

[0048] Specifically, the method for screening human-mouse cross-reactive MR1-restricted high-affinity TCRs using phage display technology of the present invention is as Figure 1 shown, and includes the following steps:

[0049] 1. Construction of the single-chain TCR phage library of MAIT cells.

[0050] 1.1 RNA extraction from MAIT cells derived from healthy donors and synthesis of cDNA;

[0051] Obtain peripheral blood mononuclear cells (PBMCs) from healthy donors, isolate MAIT cells using magnetic beads, extract RNA, and amplify the full-length cDNA of the gene by RT-PCR.

[0052] 1.2 Design and synthesis of single-chain TCR primers

[0053] Download the constant alpha-chain V-region gene TRAV1-2, C-region gene TRAC of human MAIT cells, the most common V-region genotypes of the MAIT cell beta-chain, the TRBV6 family (including TRBV6-1, TRBV6-5, TRBV6-2), TRAV6-4, TRBV20-1, TRBV4-2, and the TRBC2*01 sequence of the C-region gene from the IMGT database, and design specific primers for amplifying the variable regions (Vα) of the MAIT cell alpha-chain and the variable region (Vβ) of the beta-chain. The downstream primer of the alpha-chain and the upstream primer of the beta-chain contain a partial oligonucleotide chain (linker peptide, Linker) composed of 15 amino acids (Gly 4 Ser) 3 composition.

[0054] (1) Design of the first-round PCR primers:

[0055] TRAF-1: Complementary to the 5' end of TRAV1-2;

[0056] TRBF1-1: Complementary to the 5' end of TRBV20-1;

[0057] TRBF2-1: Complementary to the 5' end of TRBV4-1;

[0058] TRBF3-1: Complementary to the 5' end of the TRBV6 family;

[0059] TRBF4-1: Complementary to the 5' end of TRBV6-5;

[0060] TRAR-1 / TRBR-1: Designed to anneal to the 5' gene segment of TRAC / TRBC2*01.

[0061] (2) Design of the second-round PCR primers:

[0062] TRBF1-2: Complementary to the 5' end of TRBV20-1, and introducing the Va-linker homologous sequence and the second half of the linker; TRBF2-2: Complementary to the 5' end of TRBV4-1, and introducing the Va-linker homologous sequence and the second half of the linker; TRBF3-2: Complementary to the starting end of the TRBV6 family, and introducing the Va-linker homologous sequence and the second half of the linker; TRBV4-2: Complementary to the 5' end of TRBV6-4, and introducing the Va-linker homologous sequence and the second half of the linker;

[0063] TRBR-2: It is related to the 5' gene segment of TRBC2*01 and introduces plasmid homologous sequences;

[0064] TRAR-2: It is complementary to the 5' end of TRAC and introduces the first half of the linker.

[0065] The primer sequences are as follows:

[0066]

[0067] 1.3 Synthesis and purification of Vα and Vβ;

[0068] Specifically, the results of Vα and Vβ obtained by PCR amplification using MAIT cell cDNA in the present invention are as Figure 2 shown and include the following steps:

[0069] In the first round of PCR, cDNA is used as the template, TRAF-1 is used as the forward primer, and TRAR-1 is used as the reverse primer to amplify Vα. Using TRBF1-1 / TRBF2-1 / TRBF3-1 / TRBF4-1 as the forward primer and TRBR-1 as the reverse primer, 4 types of Vβ chain fragments are amplified respectively, and the fragments are recovered and purified.

[0070] In the second round of PCR, Vα is used as the template, TRAF-2 is used as the forward primer, and TRAR-2 is used as the reverse primer to amplify Vα-2. Using TRBF1-2 / TRBF2-2 / TRBF3-2 / TRBF4-2 as the forward primer and TRBR-2 as the reverse primer, 4 types of Vβ-2 chain fragments are amplified respectively, and the fragments are recovered and purified.

[0071] Since TRBF3-1 is a common universal primer for TRBV6-1, TRBV6-5, and TRBV6-2, and because there is a CDR3 region (Complementarity Determining Region 3) in the V region of the β chain, which has extremely high diversity, the amplified Vβ-2 chain fragments will involve multiple fragments.

[0072] 1.4 SOE-PCR splicing of the scTCR gene; Using the linker peptide Linker, the purified products of the above-mentioned second-round PCR products Vα-2 and Vβ-2 genes are assembled into a complete scTCR gene by overlap extension PCR (SOE-PCR).

[0073] 1.5 Construction and electrotransformation of the recombinant phagemid vector pComb3X-scTCR;

[0074] Specifically, the results of constructing the recombinant phagemid pComb3X-scTCR in the present invention are as Figure 3 shown and include the following steps:

[0075] The linearized phagemid vector pComb3X and the scTCR fragment were ligated by homologous recombination to construct the recombinant phagemid vector pComb3X-scTCR, and the above ligation product was electrotransformed into Escherichia coli TG1. With the reinfection of the helper phage M13K07, a recombinant phage scTCR library was obtained.

[0076] 2. Enrichment screening of the MAIT cell scTCR phage library.

[0077] 2.1 The MR1 / 5-OP-RU tetramer was coated on the solid-phase carrier sulfate latex microspheres (Beads).

[0078] Specifically, the results of using the tetramer to coat Beads in the present invention are as Figure 4 shown, including the following steps:

[0079] Take about 100 μL of sulfate latex microspheres into a sterile tube, wash with PBS, resuspend with 100 μL of PBS and add the MR1 / 5-OP-RU tetramer, vortex and mix well at 4 °C, incubate for 24 hours, wash and then block overnight with BSA.

[0080] 2.2 Nonspecific elution of phages in each round;

[0081] Adjust the phage library titer to 10 10 pfu and incubate with 5×10 7 beads / mL to allow sufficient binding. After centrifugation, wash with TBST (0.1% Tween-20), centrifuge the beads to the bottom of the tube, discard the unbound target molecule solution, and repeat the washing 5 times. Subsequently, elute the specifically bound phages with the eluent, and neutralize the eluent with the neutralizing solution. Take a part for titer determination, and use the remaining for amplification. The solution obtained after this round of panning is called the first-round specific eluate.

[0082] The above steps are the first-round screening process. The phages screened out are used for the next round of panning after being expanded in culture. A total of three rounds of panning are carried out. The concentrations of the MR1 tetramer-coated beads used in the second and third rounds of panning are reduced to 1×10 7 beads / mL and 5×10 6 beads / mL respectively; the eluents used in the second and third rounds of panning are changed to TBST containing 0.25% and 0.5% Tween-20; the number of washing times after phage incubation in the second and third rounds of panning is increased to 10 times and 15 times.

[0083] 2.3 Amplification and purification of each round of eluate;

[0084] The eluted phages were added to the TG1 bacterial solution in the early logarithmic growth phase, and helper phages were added for infection. Antibiotics were supplemented, and after shaking and amplification culture, the supernatant after centrifugation was precipitated overnight with PEG / NaCl solution. After centrifugation to discard the supernatant, it was resuspended with TBS buffer, centrifuged again, and precipitated with 1 / 5 of the supernatant volume of PEG / NaCl. After centrifugation, the supernatant was discarded, and it was resuspended with TBS buffer to obtain the phage solution after screening and amplification.

[0085] 2.4 Preliminary identification of the screening results of the phage scTCR library;

[0086] Randomly select well-grown and clearly defined single colonies from the culture dishes after the third round of enrichment screening, perform shaking culture, plasmid digestion identification, and NGS sequencing.

[0087] 3. Screening of high-affinity positive phage clones.

[0088] 3.1 Preparation of monoclonal positive phages;

[0089] The identified positive monoclonal phages were prepared into phage solutions, and each positive phage clone was diluted to the same titer (1×10 10 PFU), and monoclonal Phage-ELISA identification was performed on phage scTCR with the same titer.

[0090] 3.2 Screening of positive phage clones by Phage-ELISA;

[0091] Specifically, the results of Phage-ELISA using human and murine MR1 / 5-OP-RU are as Figure 5 shown, including the following steps:

[0092] The MR1 / 5-OP-RU tetramers of human and murine were respectively coated on the plates, the single-clone phage solution to be tested was added, and the helper phage M13K07 with the same titer was used as the negative control, and the polyclonal mixed phage solution was used as the positive control. Horseradish peroxidase-labeled Anti-M13 antibody was added to perform the Phage-ELISA experiment, and the absorbance value at a wavelength of 450 nm was detected in the microplate reader. The method for determining high affinity is: the OD450 value is significantly higher than that of the positive control, and the inter-group difference is tested by independent sample t-test. The significance markers are: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0093] 3.3 Sequencing and analyzing the gene sequence of TCR6;

[0094] The TCR clone No. 6 was subjected to NGS sequencing, and the clone with the sequence shown in SEQ ID NO: 15 had significant differences, that is, the TCR shown in SEQ ID NO: 15 was a human-mouse MR1-restricted high-affinity TCR.

[0095] According to the analysis of the IMGT database, its type is as follows:

[0096]

[0097] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening human-mouse crossover MR1-restricted high-affinity TCR using phage display technology, characterized in that: The following steps are involved: (1) sorting MAIT cells from peripheral blood mononuclear cells, extracting RNA from the MAIT cells, and obtaining cDNA by RT-PCR amplification; In the first round of PCR, the cDNA was used as a template, TRAF-1 was used as a forward primer, and TRAR-1 was used as a backward primer to amplify the Vα chain fragment; then TRBF1-1, TRBF2-1, TRBF3-1, and TRBF4-1 were used as forward primers, and TRBR-1 was used as a backward primer to amplify four types of Vβ chain fragments respectively; The second round of PCR uses the Vα as a template, TRAF-2 as a forward primer, TRAR-2 as a backward primer, to amplify the Vα-2 chain fragment, and TRBF1-2, TRBF2-2, TRBF3-2, TRBF4-2 as forward primers, TRBR-2 as a backward primer, to amplify four types of Vβ-2 chain fragments respectively; The sequence of TRAF-1 is shown in SEQ ID NO:1, the sequence of TRAR-1 is shown in SEQ ID NO:2, the sequence of TRBF1-1 is shown in SEQ ID NO:3, the sequence of TRBF2-1 is shown in SEQ ID NO:4, the sequence of TRBF3-1 is shown in SEQ ID NO:5, the sequence of TRBF4-1 is shown in SEQ ID NO:6, the sequence of TRBR-1 is shown in SEQ ID NO:7, the sequence of TRAF-2 is shown in SEQ ID NO:8, the sequence of TRAR-2 is shown in SEQ ID NO:9, the sequence of TRBF1-2 is shown in SEQ ID NO:10, the sequence of TRBF2-2 is shown in SEQ ID NO:11, the sequence of TRBF3-2 is shown in SEQ ID NO:12, the sequence of TRBF4-2 is shown in SEQ ID NO:13, and the sequence of TRBR-2 is shown in SEQ ID NO:14; Using a connecting peptide, the Vα-2 chain fragment is assembled with each of the Vβ-2 chain fragments into a complete scTCR fragment; The linearized phagemid vector pComb3X is connected to each of the scTCR fragments by homologous recombination to construct a recombinant phagemid vector pComb3X-scTCR, and the connection product is transformed into Escherichia coli; then the helper phage M13K07 is used for reinfection to obtain a recombinant phage, that is, a scTCR library; (2) coating the tetramer formed by protein MR1 and riboflavin metabolic intermediate 5-OP-RU on a solid phase carrier; then incubating the recombinant phage obtained in step (1) with the solid phase carrier, discarding the unbound target molecule solution, and then eluting the specifically bound phage with an eluent to obtain a specific eluate; culturing the specific eluate, selecting single colonies with clear edges, shaking the bacteria, extracting the plasmid, digesting and identifying the plasmid, and then sequencing; (3) The identified positive monoclonal clones are prepared into phage solutions, and the phage scTCR is identified by monoclonal Phage-ELISA; the human and mouse protein MR1 and the riboflavin metabolic intermediate 5-OP-RU tetramer are coated respectively, and the single clone phage solution to be tested is added, and the same titer auxiliary phage M13K07 is used as a negative control, and the polyclonal mixed phage solution is used as a positive control, and horseradish peroxidase-labeled Anti-M13 antibody is added to perform Phage-ELISA, and the absorbance value at a wavelength of 450nm is detected in an ELISA instrument; the OD450 value significantly higher than the positive control is regarded as a high-affinity TCR, and the significance is p<0.05, and the clone with the sequence shown in SEQ ID NO: 15 has a significant difference, that is, the TCR with the sequence shown in SEQ ID NO: 15 is a human-mouse MR1-restricted high-affinity TCR.

2. The method for screening human-mouse crossover MR1-restricted high-affinity TCR using phage display technology as claimed in claim 1, characterized in that: In step (1), the sorting method is magnetic bead sorting.

3. The method for screening human-mouse crossover MR1-restricted high-affinity TCR using phage display technology as claimed in claim 1, characterized in that: In step (2), the solid phase carrier is sulfate latex microspheres.

4. The method for screening human-mouse crossover MR1-restricted high-affinity TCR using phage display technology as claimed in claim 1, characterized in that: In step (2), the tetramer is a tetramer of human protein MR1 and riboflavin metabolic intermediate 5-OP-RU.

5. The method for screening human-mouse crossover MR1-restricted high-affinity TCR using phage display technology as claimed in claim 1, characterized in that: In step (1), the Vα-2 chain fragment is assembled with each of the Vβ-2 chain fragments into each complete scTCR fragment by overlapping extension PCR.

6. The method for screening human-mouse crossover MR1-restricted high-affinity TCR using phage display technology as claimed in claim 1, characterized in that: In step (1), the transformation is electroporation transformation.

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