High affinity t cell receptors recognizing smith antigen peptides and uses thereof

Abstract

The application relates to the field of biological medicine, and specifically discloses a high-affinity T cell receptor for recognizing Smith antigen peptide and application thereof. The T cell receptor comprises a TCR alpha chain and a beta chain variable domain with specific sequences, the variable domain contains specific CDR3 sequences for targeting a Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB115:01 complex, and a specific site cysteine mutation is introduced into the constant region of the alpha chain and the beta chain, so that the KD value of the target complex can reach the nanomolar level. A bispecific complex and a pharmaceutical composition based on the T cell receptor can be used for preparing a drug for treating systemic lupus erythematosus or lupus nephritis, and have the advantages of high target recognition specificity, high binding affinity and good structural stability; the matching preparation method steps are controllable and reproducible, can be adapted to large-scale production, and provide a new molecular tool for precise treatment of related autoimmune diseases.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to a high-affinity T-cell receptor that recognizes Smith antigen peptides and its applications. Background Technology

[0002] T-cell receptors are glycoproteins on the surface of T-cell membranes, usually existing as αβ heterodimers. They specifically recognize pMHC complexes formed by antigenic peptides presented on the master histocompatibility complex (MHC), thereby mediating T-cell activation and immune responses. They are core molecules in the immune system's recognition of abnormal cells and important targets for immunotherapy. Smith antigens are key autoantigens in autoimmune diseases such as systemic lupus erythematosus (SLE). Their derived peptides can bind to specific molecules to form pMHC complexes, which can trigger abnormal immune responses and cause tissue and organ damage. Lupus nephritis is a common and serious complication of these diseases.

[0003] Current immunotherapies targeting Smith antigens are limited. Naturally derived T-cell receptors exhibit weak binding affinity to Smith antigen peptide-associated pMHC complexes, failing to meet the requirements for precise recognition and targeting, thus hindering the effective implementation of precise immunotherapy targeting Smith antigens. This application aims to provide a high-affinity T-cell receptor with a specific structure. Through optimized design of the variable and constant regions, it enhances the recognition affinity and specificity for Smith antigen peptide complexes, providing a feasible molecular basis for targeted therapy of Smith antigen-related autoimmune diseases. Summary of the Invention

[0004] To address the problem of the lack of high affinity and high specificity T cell receptors for the Smith antigen peptide-pMHC complex in existing technologies, this application provides a high affinity T cell receptor for recognizing the Smith antigen peptide and its application.

[0005] In a first aspect, this application provides a high-affinity T-cell receptor for recognizing Smith antigen peptides, employing the following technical solution:

[0006] A high-affinity T-cell receptor that recognizes the Smith antigen peptide, the T-cell receptor comprising a TCRα chain variable domain and a TCRβ chain variable domain, and capable of specifically binding to the Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB1 The 15:01 complex; the TCRα chain variable domain contains a CDR3α sequence, the amino acid sequence of which is shown in SEQ ID NO:2; the TCRβ chain variable domain contains a CDR3β sequence, the amino acid sequence of which is shown in SEQ ID NO:4; the TCRα chain also contains an artificially introduced C constant region, the C constant region containing Thr48 to Cys mutations; the TCRβ chain also contains an artificially introduced C constant region, the C constant region containing Ser57 to Cys mutations; by surface plasmon resonance detection, the T cell receptor is associated with Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB1 The dissociation equilibrium constant KD of the complex binding at 15:01 is 1.0 × 10⁻⁶. -8 M to 2.0×10 -8 M.

[0007] By employing the above technical solution, the CDR3 sequence is the core functional region for TCR-specific recognition of the pMHC complex, and specific CDR3α and CDR3β sequences can precisely match the Smith antigen peptide-HLA-DRB1. The spatial conformation of the 15:01 complex enables specific recognition of the target antigen and avoids non-specific binding. The site-directed mutation of cysteine ​​introduced into the constant region can form an additional artificial interchain disulfide bond between the extracellular constant regions of the TCR α and β chains, stabilizing the natural spatial structure of the TCR αβ heterodimer, reducing the probability of mismatch between the two chains, improving the protein's correct folding efficiency and in vitro and in vivo structural stability, and ultimately achieving nanomolar-level high affinity binding to the target complex, significantly improving the TCR's recognition efficiency and binding specificity for the target antigen.

[0008] Preferably, the amino acid sequence of the TCRα chain variable domain is encoded by the nucleotide sequence shown in SEQ ID NO:1, which aligns to TRAV9-2 at genomic positions 329474 to 329752. 01 gene and TRAJ47 from 880953 to 881004 The 02 gene, wherein the coding nucleotide sequence of the TCRα chain variable domain is 320 bp to 350 bp in length; the amino acid sequence of the TCRβ chain variable domain is encoded by the nucleotide sequence shown in SEQ ID NO:3, which is aligned to TRBV11-2 at genomic positions 202760 to 203045. 01 gene, TRBD2 02 gene and TRBJ2-7 from 566110 to 566157 The 01 gene, wherein the nucleotide sequence encoding the variable domain of the TCRβ chain is 330 bp to 360 bp in length.

[0009] By adopting the above technical solution, the variable domain nucleotide sequence is derived from a functionally rearranged TCR gene obtained by single-cell sequencing of patient samples. It has no frameshift mutations or premature stop codons and can complete correct transcription and translation in host cells to form a variable domain structure with complete antigen recognition function. The sequence alignment to the identified TRA and TRB genomic functional gene sites clarifies the human gene origin of the variable domain, which can reduce the risk of immunogenicity caused by heterologous sequences and meet the application requirements of subsequent in vivo drug delivery and engineered T cell modification.

[0010] Preferably, the amino acid sequence of the C constant region of the TCRα chain is encoded by the nucleotide sequence shown in SEQ ID NO:5, and the C constant region is derived from TRAC. 01 Exon 1; The amino acid sequence of the C constant region of the TCRβ chain is encoded by the nucleotide sequence shown in SEQ ID NO:6, and the C constant region is derived from TRBC1. 01 or TRBC2 01 exon 1.

[0011] By adopting the above technical solution, using the exon 1 sequences of human TRAC and TRBC genes as the constant region backbone, the core structural domains of TCR structure stability and interstrand pairing are preserved. At the same time, it adapts to the codon bias of human expression system and improves the expression efficiency of full-length protein. The constant region sequence and the upstream variable domain sequence form a complete open reading frame, which can realize the continuous translation of full-length TCR protein, providing a complete structural basis for the correct folding of TCR and the realization of antigen recognition function.

[0012] Preferably, the T cell receptor is associated with the Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB1. The binding rate ka of the 15:01 complex is 3.0 × 10⁻⁶. 4 M -1 ・s -1 Up to 5.0×10 4 M -1 ・s -1 The dissociation rate kd is 5.0 × 10⁻⁶. -4 s -1 Up to 7.0×10 -4 s -1 .

[0013] By adopting the above technical solution, the range of binding kinetic parameters is reasonably summarized based on the measured values ​​of surface plasmon resonance experiments, ensuring a suitable dynamic balance between binding and dissociation between TCR and the target pMHC complex. This enables TCR to rapidly recognize and stably bind to the target complex, ensuring target recognition efficiency, while avoiding non-specific protein retention caused by excessive binding. It adapts to the targeted recognition and action process under in vivo physiological conditions, providing a stable molecular binding basis for subsequent in vivo therapeutic applications.

[0014] Preferably, the complete amino acid sequence of the TCRα chain is shown in SEQ ID NO:9, and the complete amino acid sequence of the TCRβ chain is shown in SEQ ID NO:10.

[0015] By adopting the above technical solution, the full-length amino acid sequence is the complete primary structure of the TCR α chain and β chain, which fully covers the antigen recognition region of the variable domain and the structural stability region of the constant region. It can autonomously fold to form a functional TCR molecule with a complete spatial conformation. The sequence has no redundant amino acid residues and can be directly used for gene synthesis and protein expression without additional sequence optimization and modification, which simplifies the subsequent experimental operation and industrial scale-up process.

[0016] Preferably, the T cell receptor further comprises a flexible linker and an immunomodulatory fragment, forming an ImmTAAI bispecific complex; the amino acid sequence of the flexible linker is shown in SEQ ID NO:7, and the number of repeats is 1 to 5; the immunomodulatory fragment is a PD-L1 functional fragment, and its amino acid sequence is shown in SEQ ID NO:8.

[0017] By adopting the above technical solution, the TCR and PD-L1 functional fragments are fused through a flexible connector. The flexible connector can form a suitable spatial interval between the two functional domains, eliminating the influence of spatial steric hindrance between the two functional domains, and ensuring that the antigen recognition function of the TCR and the immunomodulatory function of the PD-L1 functional fragment can function independently and normally. The formed bispecific complex can accurately deliver the immunomodulatory functional fragment to the lesion area enriched with Smith antigen through the targeted recognition of the TCR, realizing local targeted immunomodulation, reducing the risk of side effects from systemic administration, and expanding the clinical application scenarios of TCR.

[0018] Preferably, the ImmTAAI bispecific complex is formed by co-expressing the coding sequence of the TCRβ chain and the coding sequence of the linker-PD-L1 chain via overlapping PCR.

[0019] By employing the above technical solution, the TCRβ chain coding sequence and the linker-PD-L1 coding sequence are seamlessly linked using overlapping PCR technology, ensuring the integrity of the open reading frame of the fusion gene without base insertions or deletions, thus guaranteeing the continuous and correct translation of the fusion protein. Furthermore, co-expression with the TCRα chain coding sequence allows the two chains to naturally pair and fold correctly in the expression system, forming a structurally complete and functionally normal bispecific complex, effectively improving the preparation efficiency and correct folding rate of the fusion protein.

[0020] Secondly, this application provides a method for preparing a high-affinity T-cell receptor that recognizes Smith antigen peptides, using the following technical solution:

[0021] A method for preparing a high-affinity T-cell receptor that recognizes Smith antigen peptides, comprising the following steps:

[0022] S1. The coding sequences of the TCRα and TCRβ chains synthesized by the gene, wherein the coding sequence of the TCRα chain includes a variable-domain coding sequence and a constant-region coding sequence, and the coding sequence of the TCRβ chain includes a variable-domain coding sequence and a constant-region coding sequence;

[0023] S2. Design primers containing NcoI and NotI restriction sites, amplify TCRα and TCRβ chain fragments by PCR, and construct Vα-Linker-Vβ sequence for prokaryotic expression vector by overlapping PCR ligation.

[0024] S3. The amplification product and pET-28a vector were double-digested with NcoI-HF and NotI-HF, respectively. After the digestion products were inactivated, they were ligated with T4 DNA ligase to construct the recombinant vector.

[0025] S4. Transform the recombinant vector into Escherichia coli BL21DE3 and inoculate it into LB medium containing kanamycin and culture until OD. 600 The value was 0.5 to 0.9. After adding IPTG to induce expression, the bacterial cells were collected by centrifugation.

[0026] S5. Collect inclusion bodies from lysed bacterial cells, denature them, add renaturation buffer, and renaturate overnight at 2°C to 6°C with stirring.

[0027] S6. After filtration, the refolded solution was purified by anion exchange chromatography and gel filtration chromatography to obtain TCR protein.

[0028] By adopting the above technical solution, through gene synthesis, high-fidelity PCR amplification, and specific double enzyme digestion, the TCR recombinant expression vector can be constructed accurately and rapidly, ensuring that the target gene is inserted into the preset site of the expression vector in the correct direction and reading frame. The TCR protein is efficiently induced and expressed using the E. coli prokaryotic expression system. Then, through inclusion body denaturation, gradient renaturation, and two-step chromatography purification, impurities, endotoxins, and misfolded proteins can be effectively removed to obtain high-purity, high-activity TCR protein. The entire process is controllable, reproducible, and adaptable to different needs of small-scale laboratory preparation and large-scale production.

[0029] Thirdly, this application provides a pharmaceutical composition, which adopts the following technical solution:

[0030] A pharmaceutical composition for use with a high-affinity T-cell receptor for recognizing the Smith antigen peptide, and a pharmaceutically acceptable carrier; said pharmaceutically acceptable carrier is selected from one or more of physiological saline, phosphate-buffered saline, Tris buffer, and HEPES buffer; said pharmaceutical composition is formulated as an injection or lyophilized powder for administration via intravenous or subcutaneous injection.

[0031] By adopting the above technical solutions and using physiological saline and various physiological buffer solutions as pharmaceutically acceptable carriers, the pH value and osmotic pressure of the system can be maintained close to the human physiological environment, avoiding protein denaturation and inactivation during storage and administration, and stabilizing the biological activity of TCR. The formulation can be made into injections or lyophilized powder injections, which are suitable for clinical intravenous or subcutaneous injection. The lyophilized powder injection can further improve the long-term storage stability of the product, extend its shelf life, and meet the needs of different clinical drug use scenarios.

[0032] Thirdly, this application provides an application of a high-affinity T-cell receptor for recognizing Smith antigen peptides, employing the following technical solution:

[0033] Application of a high-affinity T-cell receptor for recognizing Smith antigen peptides, for use in the preparation of a drug for treating Smith antigen-related diseases, namely systemic lupus erythematosus or lupus nephritis.

[0034] By employing the above-mentioned technical solution, the high-affinity TCR, its derived bispecific complex, or related pharmaceutical compositions can be used as active ingredients to prepare drugs for treating Smith antigen-related diseases. Through the specific recognition of the Smith antigen peptide-HLA complex by the TCR, abnormal autoimmune response sites related to the disease can be precisely targeted, achieving targeted intervention for systemic lupus erythematosus and lupus nephritis. This provides a novel molecular tool and pathway for the treatment of such autoimmune diseases, reducing the risk of systemic side effects from nonspecific immune intervention.

[0035] In summary, this application has the following beneficial effects:

[0036] 1. This application employs a technique that combines the variable domains of the TCRα and β chains containing a specific CDR3 sequence with the artificial introduction of cysteine ​​mutations at specific sites in the constant region, thereby obtaining a peptide that can specifically recognize the Smith antigen peptide -RVLGLVLLRGENLVS-HLA-DRB1. The 15:01 complex has a high affinity for T-cell receptors, with a dissociation equilibrium constant KD reaching the nanomolar level, exhibiting extremely strong target binding specificity and affinity.

[0037] 2. In this application, the preferred technical solution is to connect the TCR to the PD-L1 functional fragment via a flexible linker to form an ImmTAAI bispecific complex. By combining the TCR's targeting recognition capability with the PD-L1's immunomodulatory function, a fusion product with both precise target localization and immunomodulatory function is obtained, effectively expanding the functional application boundaries of the TCR.

[0038] 3. The preparation method of this application, through a complete process of gene synthesis, PCR amplification, double enzyme digestion vector construction, prokaryotic induction expression and multi-step chromatography purification, realizes the standardized preparation of high-affinity TCR protein, can stably obtain high-purity active TCR protein, has good process reproducibility, and can be adapted to the needs of large-scale protein production.

[0039] 4. In this application, a preferred technical solution is to use a high-affinity TCR or its bispecific complex in combination with a pharmaceutically acceptable carrier to prepare a drug composition in the form of an injection or lyophilized powder for injection. This composition can be administered via intravenous or subcutaneous injection, fully meeting the actual needs of clinical administration and providing a stable formulation basis for drug development.

[0040] 5. This application uses high-affinity TCRs, their bispecific complexes, or related pharmaceutical compositions as active ingredients to prepare drugs for treating Smith antigen-related diseases. These drugs can precisely target the core pathogenic targets of systemic lupus erythematosus, especially lupus nephritis, providing a novel technical tool for the precision treatment of this type of autoimmune disease. Attached Figure Description

[0041] Figure 1 This is the SPR binding curve of the Smith-TCR protein and the Smith antigen-pMHC complex provided in this application; the detection results of the SA chip are shown on the horizontal axis as time (s) and the vertical axis as response value (RU). Different concentrations of TCR protein specifically bind to the Smith antigen-pMHC complex, proving that the TCR can specifically recognize the target antigen.

[0042] Figure 2 This application provides a schematic diagram of the alignment between the TCRα chain and the TRA genome; it shows the alignment results of a 10X single-cell sequencing sequence with the TRA genome, indicating that the TCRα chain variable domain sequence aligned to TRA genome positions 329474-329752 (TRAV9-2). 01)+AGC+880953--881004 (TRAJ47 02), sequence matching degree 100%;

[0043] Figure 3 This application provides a schematic diagram of the alignment between the TCRβ chain and the TRB genome; it shows the alignment results of a 10X single-cell sequencing sequence with the TRB genome, indicating that the TCRβ chain variable domain sequence is aligned to TRB genome position 202760-203045 (TRBV11-2). 01) +CGCTTAGCGGGA (TRBD2) 02) +566110--566157 (TRBJ2-7 01), sequence matching degree 100%. Detailed Implementation

[0044] The present application will be further described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0045] Technical Concept: Systemic lupus erythematosus (SLE) is a chronic inflammatory disease characterized by autoimmune disorders. Lupus nephritis is its most common and serious complication. Smith antigen is a key autoantigen in the disease progression, and its derived peptides are related to HLA-DRB1. The pMHC complex formed at 15:01 is the core target driving abnormal autoimmune responses. Currently, immunotherapies targeting this target are scarce. The core problem is that the affinity of natural TCRs for this target pMHC complex is generally at the micromolar level, which cannot meet the needs of clinical targeted therapy. At the same time, natural TCRs have defects such as high interchain mismatch rate and poor in vivo structural stability. Existing TCR engineering technology has not yet completed specific sequence screening and structural optimization for this specific target, making it impossible to obtain a targeting molecule with high specificity, high affinity and structural stability. As a result, precision immunotherapy against Smith antigen is difficult to implement effectively.

[0046] This technical approach first uses single-cell sequencing and comparison with the IMGT database to screen and obtain the peptide that specifically recognizes the Smith antigen: RVLGLVLLRGENLVS-HLA-DRB1. The TCR sequence of the 15:01 complex clearly identifies the core CDR3α and CDR3β sequences that determine target recognition specificity, fundamentally solving the problem of accurate target recognition. Furthermore, by introducing cysteine ​​mutations at specific sites in the constant regions of the TCR α and β chains, artificial interchain disulfide bonds are formed, significantly reducing the probability of interchain mismatches and improving protein structural stability. This ultimately achieves nanomolar-level high-affinity binding to the target complex. Simultaneously, a bispecific complex was constructed based on this core TCR, establishing a standardized preparation method and a drug composition system suitable for clinical administration. This comprehensively solves the core problem of the lack of effective target molecules in existing Smith antigen-targeted therapy, providing a novel technical tool for the precision treatment of related autoimmune diseases.

[0047] I. Experimental Materials

[0048] Unless otherwise specified, the experimental materials used in the following embodiments of the present invention, including bacterial strains, cells, vectors, enzyme reagents, and culture media, are all commercially available conventional biological reagents; and the specific experimental procedures, unless otherwise specified, are all conventional molecular biology, protein expression and purification, and cell engineering procedures in the art. A detailed list of core experimental materials is as follows:

[0049] Strains and cells: Escherichia coli DH5α competent cells, Escherichia coli BL21DE3 competent cells; TCR gene knockout PD1 / NFAT-Luc / JurkatT cell line, catalog number CBP74018, purchased from Nanjing Baike Biotechnology Co., Ltd.; Expi293F suspension expression cells.

[0050] Vectors and plasmids: prokaryotic expression vector pET-28a; lentiviral backbone vector; lentiviral packaging helper plasmids pMD2.G, pRSV.rev, pMDL / pRRL.

[0051] Enzymes and reagents: PrimeSTAR®Max high-fidelity DNA polymerase, NcoI-HF restriction endonuclease, NotI-HF restriction endonuclease, T4 DNA ligase; BugbusterMasterMix protein lysis buffer, IPTG inducer, DTT dithiothreitol; BCA protein concentration assay kit; HiTrapQHP anion exchange chromatography column, Superdex75 gel filtration chromatography column; SA biotin affinity chip, brand Cytiva, catalog number BR100531.

[0052] Culture media: LB bacterial medium, RPMI 1640 cell medium, and Expi 293F serum-free suspension expression medium.

[0053] Example 1: Synthesis and sequence verification of the TCR coding sequence. The purpose of this example is to determine and verify the full-length coding sequence of the high-affinity TCR of the present invention, ensuring that the sequence has no frameshift, no premature stop codons, and has complete protein expression capability.

[0054] Based on 10X single-cell sequencing results from peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE), sequence alignment and rearrangement analysis were performed using the IMGT international immunogenetic database. This ultimately determined the full-length coding sequence of the TCRα chain to be 615 bp and the full-length coding sequence of the TCRβ chain to be 735 bp. The two independent single-cell sequencing results showed only one synonymous base difference that did not cause an amino acid change; therefore, the second sequencing sequence was used as the baseline for subsequent experiments.

[0055] Artificially synthesized full-length gene fragments encoding the TCRα and TCRβ chains with NcoI / NotI restriction sites at both ends, wherein:

[0056] The TCRα chain variable domain coding sequence is 1-334bp, corresponding to the sequence number SEQ ID NO:1. According to the IMGT database, this sequence is derived from the functional rearrangement of the TRAV9-201 gene and the TRAJ4702 gene in the TRA genome. The rearrangement sites are TRA genome 329474--329752TRAV9-201+AGC+880953--881004TRAJ4702.

[0057] The TCRβ chain variable domain coding sequence is 1-346 bp, corresponding to the sequence number SEQ ID NO:3. According to the IMGT database, this sequence is derived from the functional rearrangement of the TRBV11-201, TRBD202 and TRBJ2-701 genes in the TRB genome. The rearrangement sites are TRB genome 202760--203045TRBV11-201+CGCTTAGCGGGATRBD202+566110--566157TRBJ2-701.

[0058] The synthesized gene fragment was validated by Sanger sequencing. The results showed that the sequence was completely consistent with the design, with no base deletions, missense mutations, or frameshift mutations, and the reading frame was intact, making it suitable for subsequent vector construction and protein expression experiments.

[0059] Example 2: Construction of TCR recombinant expression vector. In this example, a prokaryotic recombinant expression vector for in vitro protein preparation and a lentiviral recombinant vector for stable expression in eukaryotic cells were constructed, providing a foundation for the subsequent preparation and functional verification of TCR.

[0060] 1. PCR amplification of the target fragment

[0061] Using the TCR-encoding gene verified in Example 1 as a template, specific primers were designed for PCR amplification, wherein:

[0062] The full-length TCRα chain fragment was amplified using primer pair P01-α-NcoⅠ-F / P02-α-R;

[0063] The full-length TCR β chain fragment was amplified using primer pair PO4-β-F / PO5-β-R;

[0064] For the construction of prokaryotic expression vectors, primer pair P01-α-F / P05-β-R was used to obtain the Vα-Linker-Vβ single-stranded fragment by overlapping PCR amplification.

[0065] The nucleotide sequences of the primers used are as follows:

[0066] P01-α-NcoⅠ-F:ATGCTCCCATGGCTCAGAAGGTAACTCAAGCG

[0067] P02-α-R: GCTACCGCCACCTGGTTTTACTGTCAGTCTGGTCCCTGCTCC

[0068] P03-Linker: GTAAAACCAGGTGGCGGTAGCGAGGGCGGTGGCAGCGAAGGTGGCGGTAGCGAGGGCGGTGGCAGCGAAGGTGGCACCGGTAATGCTGGT

[0069] P04-β-F:GGCACCGGTAATGCTGGTGTCACTCAGACCCCAAAATTC

[0070] P05-β-R:TTCTGCGGCCGCTGTGACCGTGAGCCTGG

[0071] The PCR reaction system consisted of: 25 μL of 2×PrimeSTAR® MaxDNA Polymerase, 2 μL of template DNA, 2 μL of upstream primer, 2 μL of downstream primer, and enzyme-free water to a final volume of 50 μL.

[0072] The PCR reaction program was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 55℃ annealing for 5s, 72℃ extension for 15s, for a total of 35 cycles; 72℃ final extension for 2min.

[0073] The amplified product was verified by 1% agarose gel electrophoresis, and the band size was exactly as expected. The purified target fragment was obtained by gel extraction and recovery.

[0074] 2. Construction of prokaryotic recombinant expression vectors

[0075] The purified PCR amplification product and the pET-28a empty vector were subjected to double digestion with NcoI-HF and NotI-HF, respectively. The digestion system consisted of 10 μL of 10×rCutSmart™ Buffer, 2 μL of NcoI-HF, 2 μL of NotI-HF, 1 μg of target DNA, and enzyme-free water to a final volume of 100 μL. The digestion reaction was terminated by incubation at 37°C for 1 h and then by water incubation at 80°C for 20 min. The digestion products were recovered by agarose gel electrophoresis to obtain the linearized vector backbone and the target gene fragment.

[0076] The ligation system was prepared according to a 10:1 molar ratio of target fragment to vector: 2 μL T4 DNA ligase, 2 μL 10×T4 DNA ligase buffer, target fragment, linearized vector, and enzyme-free water to a final volume of 20 μL. After ligation at 16°C for 2 h, the ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing kanamycin, and incubated overnight at 37°C. Single colonies were picked for colony PCR identification and Sanger sequencing verification to obtain the fully correct TCR-pET-28a recombinant prokaryotic expression vector.

[0077] 3. Construction of lentiviral recombinant vectors

[0078] The coding sequences of the TCRα and β chains were cloned into a lentiviral backbone vector to construct a recombinant lentiviral vector containing the TCRβ-P2A-TCRα-T2A-eGFP expression cassette. All expression elements were regulated by the EF1α constitutive promoter. The eGFP reporter gene was used for subsequent sorting and expression verification of positive cells.

[0079] The recombinant lentiviral vector, verified by sequencing, was mixed with packaging helper plasmids pMD2.G, pRSV.rev, and pMDL / pRRL in a molar ratio of 1:2:1:4, and transfected into Expi293F cells using liposome transfection. Cell culture supernatants were collected at 48 h and 72 h post-transfection, and the virus was concentrated by ultracentrifugation at 19000 rpm for 2 h at 4 °C to obtain high-titer TCR recombinant lentiviral vectors. These vectors were then aliquoted and stored at -80 °C for later use.

[0080] Example 3: Prokaryotic induction expression and purification of TCR protein. In this example, TCR protein was efficiently prepared using a prokaryotic expression system. High-purity and high-activity TCR protein was obtained through inclusion body refolding and multi-step chromatography purification.

[0081] The TCR-pET-28a recombinant vector, verified in Example 2, was transformed into Escherichia coli BL21DE3 competent cells. Single colonies were picked and inoculated into LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm on a shaker until the bacterial culture reached OD. 600 When the value reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 0.5 mM, and continue inducing expression at 37℃ for 4 h. After induction, collect the bacterial pellet by centrifugation at 8000 rpm for 10 min at 4℃.

[0082] Add BugbusterMasterMix lysis buffer to the bacterial cell pellet, resuspend the cells, and lyse on ice for 30 min. Centrifuge at 12,000 rpm for 20 min at 4 °C, and collect the pellet as TCR protein inclusion bodies. Dissolve the inclusion body pellet in 8 M urea solution, add DTT to a final concentration of 10 mM, and incubate at 37 °C for 30 min to fully denature and depolymerize the protein. Slowly add the denatured protein solution dropwise to pre-cooled renaturation buffer, which consists of 0.4 M arginine, 100 mM Tris (pH 8.1), 2 mM EDTA, 6.5 mM β-mercaptoethylamine, and 1.87 mM cystamine. Renaturate overnight at 4 °C with low-speed magnetic stirring.

[0083] After refolding, the refolding solution was clarified and filtered through a 0.45 μm filter membrane, and then loaded onto a pre-equilibrated HiTrapQHP anion exchange chromatography column. Elution was performed using a linear gradient of 0-1 M NaCl, with the elution curve monitored by a UV detector. The target protein elution peak was collected. The collected protein peak solution was concentrated and then loaded onto a Superdex 75 gel filtration chromatography column for further purification. A single, symmetrical protein elution peak was collected, which is the purified TCR protein.

[0084] The purified protein was subjected to SDS-PAGE electrophoresis, and the results showed that the protein band was single and the purity was >90%. The protein concentration was determined using the BCA protein quantification kit, and the protein concentration was adjusted to 1 mg / mL. After aliquoting, the protein was stored at -80℃ for later use in subsequent affinity testing and formulation preparation.

[0085] Example 4: Detection of the affinity and specificity of the TCR for the target pMHC complex. This example uses surface plasmon resonance (SPR) technology to verify the affinity of the TCR of the present invention for the target Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB1. The binding affinity and specificity of the 15:01 complex were determined, and its core binding kinetic parameters were clarified.

[0086] 1. Preparation of the target Smith antigen-pMHC complex

[0087] cDNA encoding the extracellular domains of the HLA-DR15 α and β chains were cloned into pHLsec secretory expression vectors, respectively. A fos / junleucine zipper structure cleavable by enterokinase was introduced at the C-terminus of the construct to promote proper MHC dimerization. A BirA site for biotinylation and a 6×His tag for purification were simultaneously introduced at the C-terminus of the HLA-DR15 β chain. The Smith antigen peptide RVLGLVLLRGENLVS was covalently linked to the N-terminus of the HLA-DR15 β chain via a linker cleavable by coagulation factor Xa, and a Strep-II purification tag was introduced at its anterior end.

[0088] The constructed recombinant vector was transiently transfected into Expi293FGnTI- / - cells. Six days after transfection, the cell culture supernatant was collected and purified in multiple steps by dialysis, immobilized metal affinity chromatography, and size exclusion chromatography to obtain the correctly folded biotinylated Smith antigen peptide-pMHC complex. The complex was then aliquoted and stored at -80°C.

[0089] 2. SPR combined experiment

[0090] The Biacore T200 molecular interaction instrument was used for detection. The biotinylated Smith antigen-pMHC complex was immobilized on the surface of the SA chip at a concentration of 5 μg / mL, a flow rate of 10 μL / min, and the immobilized response value was controlled between 1000-2000 RU.

[0091] The TCR protein purified in Example 3 was diluted with Running Buffer, which consisted of 10 mM M HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween-20 at pH 7.4. A gradient dilution of 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM was performed. The buffer was flowed through the chip surface at a flow rate of 30 μL / min, with a binding time of 90 s and a dissociation time of 300 s. After each cycle, the chip was rinsed with 3 M magnesium chloride solution as a regeneration buffer to remove bound protein and restore the chip baseline.

[0092] 3. Data Analysis

[0093] The sensor images were fitted using BIAcoreEvaluation software, and the binding kinetic parameters of the TCR-target pMHC complex were calculated: dissociation equilibrium constant KD = 1.56 × 10⁻⁶. -8 M, binding rate ka = 3.71 × 10 4 M -1 ・s -1 The dissociation rate kd = 5.77 × 10 -4 s -1 The results show that the TCR of the present invention has extremely high binding affinity and specificity to the target Smith antigen-pMHC complex, enabling precise target identification.

[0094] Example 5: Preparation of eukaryotic host cells stably expressing TCR. In this example, eukaryotic host cells that can stably express the TCR of the present invention were constructed by lentiviral transfection, providing a foundation for subsequent cellular functional verification and cell therapy development.

[0095] First, the recombinant lentivirus prepared in Example 2 was titered on the TCR gene knockout PD1 / NFAT-Luc / JurkatT cell line, and the viral infection titer was calculated in TU / mL. PD1 / NFAT-Luc / JurkatT cells were seeded into 24-well plates at a seeding density of 1 × 10⁶ cells per well. 6 Cells were incubated with TCR recombinant lentivirus at an MOI ratio of 10 and placed in a 37°C, 5% CO2 incubator.

[0096] 48-72 hours after transfection, cells were sorted using a BDFACSAria flow cytometer, and viable cells that were GFP-positive and PI-negative were collected, which were the preliminary TCR-positive cells. The sorted positive cells were seeded into RPMI 1640 complete medium and continuously passaged in a 37°C, 5% CO2 incubator for amplification. After amplification, the cell surface expression level of TCR was detected by flow cytometry. The results showed that TCR was stably expressed on the cell surface with a positive rate >95%, successfully obtaining an engineered T cell line stably expressing the high-affinity TCR of this invention.

[0097] Example 6: Preparation of ImmTAAI bispecific complex. This example is based on the TCR of the present invention to construct an ImmTAAI bispecific complex targeting Smith antigen, thereby integrating target recognition and immune regulation functions.

[0098] The ImmTAAI bispecific complex is composed of the TCR, a flexible linker, and a PD-L1 functional fragment fused together according to the present invention. The amino acid sequence of the flexible linker is SEQ ID NO:7, and the amino acid sequence of the PD-L1 functional fragment is SEQ ID NO:8. Using overlap PCR technology, the coding sequence of the TCRβ chain is seamlessly ligated to the linker-PD-L1 coding sequence to obtain the TCRβ-linker-PD-L1 fusion gene fragment.

[0099] The fusion gene fragment and the TCRα chain coding sequence were co-cloned into a eukaryotic expression vector and co-transfected into Expi293F cells for transient expression. Six days post-transfection, the cell culture supernatant was collected and purified by affinity chromatography and gel filtration chromatography to obtain the correctly folded ImmTAAI bispecific complex. SPR assay showed that this complex retained high affinity binding activity to the target Smith antigen-pMHC complex, and can be used for subsequent targeted immunomodulatory studies.

[0100] Example 7: Preparation of a pharmaceutical composition containing a TCR / ImmTAAI bispecific complex. This example is based on the TCR and bispecific complex of the present invention to prepare a pharmaceutical composition that can be used for clinical administration, and clarifies its dosage form and preparation process.

[0101] 1. Preparation of Injectables

[0102] Take the TCR protein purified in Example 3 or the ImmTAAI bispecific complex prepared in Example 6, dilute it with sterile physiological saline to a final protein concentration of 0.5 mg / mL, add pharmaceutical grade glycerol to a final concentration of 5% as a co-solvent, stir thoroughly, filter through a 0.22 μm sterile filter membrane for sterilization, dispense 1 mL into sterile injection bottles under a Class A clean environment, stopper and cap to obtain TCR injection, and store at 2-8℃.

[0103] 2. Preparation of lyophilized powder injections

[0104] Take the TCR protein purified in Example 3 or the ImmTAAI bispecific complex prepared in Example 6, dilute it with sterile pH 7.4 phosphate buffer to a final protein concentration of 1 mg / mL, add pharmaceutical-grade mannitol to a final concentration of 10% as a lyophilization protectant, mix thoroughly, filter through a 0.22 μm sterile filter membrane for sterilization, and aliquot into sterile vials under a Class A clean environment. Place the aliquoted samples in a freeze dryer and pre-freeze at -40℃ for 4 hours, then turn on the vacuum system for gradient temperature drying for 24 hours. Seal under vacuum, and after capping, obtain the TCR lyophilized powder for injection. Store at 2-8℃ protected from light. Reconstitute with sterile physiological saline before use.

[0105] III. Sequence Information

[0106] The following are the complete sequence information of SEQ ID NO:1 to SEQ ID NO:10 involved in this invention. All sequences have been verified in the above embodiments and can be directly used for the implementation and reproduction of this invention.

[0107] SEQ ID NO:1: GGAAATTCAGTGACCCAGATGGAAGGGCCAGTGACTCTCTCAGAAGAGGCCTTCCTGACTATAAACTGCACGTACACAGCCACAGGATACCCTTCCCTTTTCTGGTATGTCCAATATCCTGGAAGGTCTACAGCTCCTCCTGAAAGCCACGAAGGCTGATGA CAAGGGAAGCAACAAAGGTTTTGAAGCCACATACCGTAAAGAAACCACTTCTTTCCACTTGGAGAAGGGCTCAGTTCAAGTGTCAGACTCAGCGGTGTACTTCTGTGCTCTGAGTAGCTATGGAAACAAGCTGGTCTTTGGCGCAGGAACCATTCTGAGAGTCAAGTCCT

[0108] SEQ ID NO:2:CALSSYGNKLVF

[0109] SEQ ID NO:3:GAAGCTGGAGTTGCCCAGTCTCCCAGATATAAGATTATAGAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCATGCTACCCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTTCTGATTCAGTTTCAGAATAACGGTGTAGTGGATGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACCCCACTCTCAAGATCCAGCCTGCAATTTGAGGACTCGGCCGTGTATCTCTGTGCCAGCAGCTCGCTTAGCGGGAGCTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAG

[0110] SEQ ID NO:4:CASSSLSGSSYEQYF

[0111] SEQ ID NO:5:ACCAAGTCGGACGTGACCCGAGCCACAAAGTTTACCCCGGAGATCGTGTCGTGCCCAAGACCTACACCTGGTTCGCCAGCGGCATCGACGCCAGCTGGAAGAGCAACGGCGCCACCGAGTGCAAGGTGTCCAACACCTACATGCCCAGCGCCCTGCAGAGCGACCTGACCTACAAGCTGAGCCTGTCCACCGAGTACTGCGAGGCCACCCACAAGACCCGGGTGGACAAGAGCGTGGACTGCGCCATCAAG

[0112] SEQ ID NO:6:AAGCTGGAGTTCGCCCCCAAGGTGGCCGTCTTCGAGCCCAGCGAGGCCGAGATCTCCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGCGTCAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGCCTGCGGGTGTCCGCCACCTTCTGGCAGAACCCCCGCAACCACTTCAGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTCACCCAGATCGTGAGCGCCGAGGCCTGGGGCCGGGCCGAC

[0113] SEQ ID NO:7:GGGGS

[0114] SEQ ID NO:8:FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY

[0115] SEQ ID NO:9:

[0116] GNSVTQMERPVTSLREEAFLTINCTYTATGYPSFLWYVQYLEKVYSSSKSATKADDKGSNKGFEATYRKETTSFHLEEGSVQVSDSSVYFCLSSYGNKLVFAGTILRVKS

[0117] TKSDVTRATKFTPDIVSVPKTYTWFASGIDASWKSNGATECKVSNTYMPALQSDLTYKLSLSTEYCEATHKTRVDKSVDCAIK

[0118] SEQ ID NO:10:

[0119] EAGVAQSPDRIRIEKRQRVAFWCAIPYSGHATFYWYQQILGQPGKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDPTLKIQPAIQELGAVYLCSASSLSGSSYEQYFPGTRLTVT

[0120] KLVEFPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD

[0121] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A high-affinity T-cell receptor for recognizing Smith antigen peptides, characterized in that: The T cell receptor contains a TCRα chain variable domain and a TCRβ chain variable domain, and can specifically bind to the Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB1. The 15:01 complex; the TCRα chain variable domain contains a CDR3α sequence, the amino acid sequence of which is shown in SEQ ID NO:2; the TCRβ chain variable domain contains a CDR3β sequence, the amino acid sequence of which is shown in SEQ ID NO:4; the TCRα chain also contains an artificially introduced C constant region, the C constant region containing Thr48 to Cys mutations; the TCRβ chain also contains an artificially introduced C constant region, the C constant region containing Ser57 to Cys mutations; by surface plasmon resonance detection, the T cell receptor is associated with Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB1 The dissociation equilibrium constant KD of the complex binding at 15:01 is 1.0 × 10⁻⁶. -8 M to 2.0×10 -8 M.

2. The high-affinity T-cell receptor for recognizing Smith antigen peptides according to claim 1, characterized in that: The amino acid sequence of the TCRα chain variable domain is encoded by the nucleotide sequence shown in SEQ ID NO:1, which aligns to TRAV9-2 at genomic positions 329474 to 329752. 01 gene and TRAJ47 from 880953 to 881004 The 02 gene, wherein the coding nucleotide sequence of the TCRα chain variable domain is 320 bp to 350 bp in length; the amino acid sequence of the TCRβ chain variable domain is encoded by the nucleotide sequence shown in SEQ ID NO:3, which is aligned to TRBV11-2 at genomic positions 202760 to 203045. 01 gene, TRBD2 02 gene and TRBJ2-7 from 566110 to 566157 The 01 gene, wherein the nucleotide sequence encoding the variable domain of the TCRβ chain is 330 bp to 360 bp in length.

3. The high-affinity T-cell receptor for recognizing Smith antigen peptides according to claim 1, characterized in that: The amino acid sequence of the C constant region of the TCRα chain is encoded by the nucleotide sequence shown in SEQ ID NO:5, and the C constant region is derived from TRAC. 01 Exon 1; The amino acid sequence of the C constant region of the TCRβ chain is encoded by the nucleotide sequence shown in SEQ ID NO:6, and the C constant region is derived from TRBC1. 01 or TRBC2 01 exon 1.

4. The high-affinity T-cell receptor for recognizing Smith antigen peptides according to claim 1, characterized in that: The T cell receptor targets Smith antigen peptide-RVLGLVLLRGENLVS-HLA-DRB1 The binding rate ka of the 15:01 complex is 3.0 × 10⁻⁶. 4 M -1 ・s -1 Up to 5.0×10 4 M -1 ・s -1 The dissociation rate kd is 5.0 × 10⁻⁶. -4 s -1 Up to 7.0×10 -4 s -1 .

5. A high-affinity T-cell receptor for recognizing Smith antigen peptides according to claim 1, characterized in that: The complete amino acid sequence of the TCRα chain is shown in SEQ ID NO:9, and the complete amino acid sequence of the TCRβ chain is shown in SEQ ID NO:

10.

6. A high-affinity T-cell receptor for recognizing Smith antigen peptides according to claim 1, characterized in that: The T-cell receptor further comprises a flexible linker and an immunomodulatory fragment, forming an ImmTAAI bispecific complex; the amino acid sequence of the flexible linker is shown in SEQ ID NO:7, and the number of repeats is 1 to 5; the immunomodulatory fragment is a PD-L1 functional fragment, and its amino acid sequence is shown in SEQ ID NO:

8.

7. A high-affinity T-cell receptor for recognizing Smith antigen peptides according to claim 6, characterized in that: The ImmTAAI bispecific complex is formed by co-expressing the coding sequence of the TCRβ chain and the coding sequence of the linker-PD-L1 chain via overlapping PCR.

8. A method for preparing a high-affinity T-cell receptor that recognizes Smith antigen peptides, characterized in that, A high-affinity T-cell receptor for recognizing Smith antigen peptides as described in any one of claims 1-7, comprising the following steps: S1. The coding sequences of the TCRα and TCRβ chains synthesized by the gene, wherein the coding sequence of the TCRα chain includes a variable-domain coding sequence and a constant-region coding sequence, and the coding sequence of the TCRβ chain includes a variable-domain coding sequence and a constant-region coding sequence; S2. Design primers containing NcoI and NotI restriction sites, amplify TCRα and TCRβ chain fragments by PCR, and construct Vα-Linker-Vβ sequence for prokaryotic expression vector by overlapping PCR ligation. S3. The amplification product and pET-28a vector were double-digested with NcoI-HF and NotI-HF, respectively. After the digestion products were inactivated, they were ligated with T4 DNA ligase to construct the recombinant vector. S4. Transform the recombinant vector into Escherichia coli BL21DE3 and inoculate it into LB medium containing kanamycin and culture until OD. 600 The value was 0.5 to 0.

9. After adding IPTG to induce expression, the bacterial cells were collected by centrifugation. S5. Collect inclusion bodies from lysed bacterial cells, denature them, add renaturation buffer, and renaturate overnight at 2°C to 6°C with stirring. S6. After filtration, the refolded solution was purified by anion exchange chromatography and gel filtration chromatography to obtain TCR protein.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a high-affinity T-cell receptor for recognizing Smith antigen peptides as described in any one of claims 1 to 7, and a pharmaceutically acceptable carrier; said pharmaceutically acceptable carrier is selected from one or more of physiological saline, phosphate buffer, Tris buffer, and HEPES buffer; said pharmaceutical composition is formulated as an injection or lyophilized powder for injection and administered by intravenous or subcutaneous injection.

10. An application of a high-affinity T-cell receptor that recognizes Smith antigen peptides, characterized in that, A high-affinity T-cell receptor for recognizing Smith antigen peptides as described in any one of claims 1-7, wherein the high-affinity T-cell receptor is used to prepare a medicament for treating Smith antigen-related diseases, wherein the Smith antigen-related diseases are systemic lupus erythematosus or lupus nephritis.

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