A specific recognition of hla-a 01:01 presents qvdyyglyy specific force enhanced t cell receptor and its application

Abstract

The application provides a specific recognition HLA-A 01:01 presented QVDYYGLYY specific stress enhanced T cell receptor and application thereof. The T cell receptor comprises a TCR alpha chain and a TCR beta chain paired with each other, the two paired chains respectively comprise sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and under the single bond condition of constant force single molecule determination, the stress enhanced bond presents a bond life prolonged with the increase of acting force. The application also provides a coding nucleic acid, an expression construct, an engineered immune cell, a pharmaceutical composition, a cell in vitro preparation method and a binding and function evaluation method; and a pharmaceutical use thereof is directed to HPV16 related diseases which are positive to HLA-A 01:01 and present the peptide-HLA complex on diseased cells.

Description

Technical Field

[0001] This application relates to antigen-specific receptors and engineered cell technology, providing a T-cell receptor that recognizes the HLA-A 01:01-QVDYYGLYY complex, corresponding gene expression materials, and immune cell products, and also relates to methods for single-molecule biomechanical evaluation and cellular function evaluation of this receptor. The recognized QVDYYGLYY is a nonapeptide derived from the human papillomavirus type 16 (HPV16) E2 protein. Background Technology

[0002] For TCR-engineered immune cells, the recognition target is the antigen complex composed of peptides on the cell surface and major histocompatibility complex molecules. Therefore, to develop recognition tools targeting specific lesions, it is necessary to simultaneously identify the epitope peptide, the HLA molecule that presents the peptide, and the α / β pairing chain that constitutes the receptor, rather than relying on only one of these pieces of information.

[0003] QVDYYGLYY and HLA-A 01:01 constitute the antigen recognition target of interest in this application. When designing receptors for HPV16-related lesions, even if the E2-derived epitope and its HLA restriction have been identified, it is still necessary to obtain receptor sequences with a defined pairing relationship and examine target binding, antigen-dependent cellular responses, and non-target reactions.

[0004] The binding state of receptors at the cell interface is also affected by mechanical loading. For receptor-ligand pairs exhibiting trapping bond behavior, an increase in the interaction force within a certain non-zero force range can be accompanied by an increase in average bond lifetime; further increases in the interaction force may shorten the bond lifetime. The force-dependent information provided by this process is different from the static affinity information and cannot be directly inferred from other TCR results.

[0005] Therefore, this application focuses on the identified QVDYYGLYY specific paired receptor, organizing its molecular identity, expression conformation, engineered cells, and detection methods, and incorporating changes in single-bond lifetime with interaction forces into the evaluation. Actual identification of diseased cells also requires consideration of the target complex's presentation state, with peptide loading assays and recognition of naturally processed antigens evaluated separately. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art by providing an α / β T cell receptor capable of recognizing HLA-A 01:01-QVDYYGLYY, defining its molecular identity by two defined pairing strand sequences, and describing its mechanical properties by force-enhanced binding; at the same time, it provides the corresponding coding material, cell preparation method, and in vitro evaluation pathway for this receptor.

[0007] In a first aspect, the present invention provides a T-cell receptor comprising a paired TCRα chain and a TCRβ chain, wherein the TCRα chain contains the amino acid sequence shown in SEQ ID NO:2, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO:4; the T-cell receptor specifically binds to a complex composed of HLA-A 01:01 and a peptide with the amino acid sequence shown in SEQ ID NO:5, and exhibits force-enhanced binding under single-bond conditions measured by constant force single-molecule assay; force-enhanced binding refers to a situation in which, within at least a non-zero force range, the average lifetime of the single-molecule bond formed between the receptor and the complex is significantly prolonged with increasing force.

[0008] In one embodiment, the TCRα chain and TCRβ chain form a membrane-bound heterodimer, and the receptor can be provided as a separate or recombinant α / β receptor, which can form a membrane-bound heterodimer in cellular expression.

[0009] In one embodiment, under the same measurement conditions of temperature, molecular orientation, surface density, contact time, preload, detection platform, and statistical method, there are three non-zero constant forces F1, F2, and F3, satisfying F1 < F2 < F3, and τ(F2) is significantly greater than τ(F1) and significantly greater than τ(F3), to present a capture-slip transition; wherein, τ(F) is the average single-molecule bond lifetime under the force F, and the significance is determined according to a predetermined statistical method.

[0010] In the second aspect, it contains a first nucleic acid sequence encoding a TCRα chain and a second nucleic acid sequence encoding a TCRβ chain; the first nucleic acid sequence and the second nucleic acid sequence are disposed in the same nucleic acid molecule, or are disposed in different nucleic acid molecules respectively.

[0011] In one embodiment, the first nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO:1, and the second nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO:3. SEQ ID NO:1 and SEQ ID NO:3 respectively provide the nucleotide sequences of the two strands; different nucleic acid sequences resulting from codon degeneracy or codon optimization may also be used while keeping the corresponding amino acid sequences unchanged.

[0012] Thirdly, the expression construct can be the expression cassette itself or a vector containing the expression cassette; the two chains can be expressed separately by promoters, or they can be expressed under the drive of a common promoter via the 2A peptide coding region or the internal ribosome entry site. The 2A peptide coding sequence and the internal ribosome entry site are different co-expression arrangements, and the latter is not interpreted as a linker peptide coding sequence.

[0013] In one embodiment, the vector is a lentiviral vector, and the expression cassette uses CMV as a promoter and arranges the TCRβ chain coding region, the 2A peptide coding region, and the TCRα chain coding region in a 5′ to 3′ direction; the TCRβ chain coding region located upstream of the 2A peptide coding region does not contain a stop codon, so that translation can continue to the 2A peptide coding region and the TCRα chain coding region.

[0014] Fourthly, engineered immune cells carry nucleic acid molecules or combinations of nucleic acid molecules, or nucleic acid expression constructs, and express T cell receptors on the cell surface.

[0015] In one embodiment, the cell forms that may be used include CD8+ T cells, CD4+ T cells, CD4 / CD8 double-positive engineered T cells, natural killer T cells, γδ T cells, and T lineage cells derived from stem cells; wherein, CD8+ T cells are the specific embodiment.

[0016] To address the mispairing issue between the endogenous TCR strand and the introduced strand, the expression of the endogenous TCR can be reduced, or control can be achieved through gene knockout. In the CD8+ T cell implementation, the endogenous TRAC gene can be knocked out or its expression reduced.

[0017] In a fifth aspect, a pharmaceutical composition contains engineered immune cells and a pharmaceutically acceptable carrier, diluent, or excipient.

[0018] In the sixth aspect, the use of engineered immune cells in the preparation of medicaments for treating HPV16-related diseases in subjects who are HLA-A 01:01 positive and whose diseased cells present the HLA-A 01:01-QVDYYGLYY complex. The disease name itself does not substitute for the HLA and the presentation conditions of the target complex.

[0019] In one embodiment, the HPV16-related disease is persistent HPV16 infection, HPV16-related precancerous lesions, or HPV16-related malignancies; the malignant tumor is selected from cervical cancer, head and neck squamous cell carcinoma, anal cancer, and penile cancer. This use limits the target lesion cells to having the target antigen complex, rather than using E2 expression alone as a sufficient condition of applicability.

[0020] In a seventh aspect, a method for preparing engineered immune cells in vitro involves introducing nucleic acid molecules or combinations of nucleic acid molecules or nucleic acid expression constructs into immune cells; culturing the introduced immune cells; detecting the expression of a target T cell receptor on the cell surface and collecting cells expressing the receptor; and, in preparing engineered immune cells, using CD8+ T cells as the starting cells and knocking out or reducing the expression of the endogenous TRAC gene before, simultaneously with, or after the introduction.

[0021] In the eighth aspect, a method for in vitro evaluation of the force-binding properties of T cell receptors includes: adjusting the surface density of the T cell receptor and the HLA-A 01:01-QVDYYGLYY complex; confirming single-bond conditions using Poisson statistics or equivalent methods; applying different non-zero constant forces to the formed single-molecule bonds using atomic force microscopy to obtain bond survival times at each force level; calculating the average bond lifetime and confidence interval according to a predetermined data analysis method; determining that force-enhanced binding properties exist when F1 < F2 and τ(F2) is significantly greater than τ(F1); and determining that capture-slip switching properties exist when F3 > F2 and τ(F3) is significantly less than τ(F2); wherein, parallel settings are provided for HLA-A 01:01-irrelevant peptide complex, HLA mismatched complex, blocking conditions, or a reference TCR control to identify nonspecific adhesion.

[0022] In a ninth aspect, a method for in vitro quality evaluation of engineered immune cells includes detecting the binding specificity of the engineered immune cells to the HLA-A 01:01-QVDYYGLYY complex; co-culturing the engineered immune cells with HLA-A01:01 positive target cells and comparing antigen responses under QVDYYGLYY loading, irrelevant peptide loading, and unloaded peptide conditions; and determining the mechanodependent binding characteristics of the expressed T cell receptor according to the method for in vitro evaluation of the mechanosensitive binding properties of T cell receptors.

[0023] Beneficial effects: The identified α / β pairing sequence provides a consistent molecular benchmark for receptor construction and identity verification, allowing descriptions of coding nucleic acids, expression materials, and engineered cells to be mapped to the same receptor.

[0024] Force-enhanced binding adds a single-molecule dynamics dimension to the evaluation of target recognition. By comparing bond lifetimes under different constant forces, it is possible to distinguish between the lifetime-increasing range and the lifetime-decreasing range after further force application, avoiding the use of static binding information to describe the mechanical response of the receptor.

[0025] By incorporating receptor binding, antigen-dependent cellular response, and mechanical assays into the evaluation, a common detection framework for comparing constructs and engineered cells can be established. Comparisons with other TCRs are based on data from controls under the same conditions, without pre-setting killing advantage or statistical significance in the absence of corresponding data. Attached Figure Description

[0026] Figure 1 A schematic diagram showing the structure and core sequence location of the TCRα and TCRβ chains; Figure 2 This is a schematic diagram of the TCRα and TCRβ expression vectors, in which the TCRα and TCRβ chains are tandemly linked by the P2A sequence. Figure 3The binding of engineered T cells to the HLA-A 01:01-QVDYYGLYY multimer and the results of control T cells; Figure 4 The average bond lifetime-force curve and trap-slip transition of the TCR-HLA-A 01:01-QVDYYGLYY monomer complex are shown. Figure 5 The results show the killing effect of engineered T cells and control T cells on target cells expressing endogenous QVDYYGLYY antigen. Detailed Implementation

[0027] The technical solutions in this embodiment will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0029] I. Terminology for In Vitro Evaluation and Judgment In this specification, “QVD-TCR” refers to the receptor defined by the paired strand sequences listed in SEQ ID NO:2 and SEQ ID NO:4.

[0030] The “target complex” refers to HLA-A 01:01-QVDYYGLYY.

[0031] In the context of single-molecule force testing in this application, "force-enhanced bonding" is also referred to as "capture bond," both referring to the same type of force-dependent bond lifetime increase phenomenon.

[0032] "Specific binding" is evaluated by comparing target and non-target conditions. Non-target conditions may include complexes formed by HLA-A 01:01 and irrelevant peptides, combinations of QVDYYGLYY and non-restricted HLA, or HLA conditions without target peptide loading. Detection can be performed using multimer staining, cell binding, surface plasmon resonance, reporter gene, or cell function readout. Positive thresholds, number of replicates, and statistical methods are determined prior to analysis.

[0033] The "single-bond condition" describes the detection state where a single contact-recorded effective binding event is primarily contributed by a single TCR-pMHC bond. The binding probability is controlled by adjusting the surface molecular density and contact conditions, and verified using Poisson statistics or equivalent methods. No single adhesion event is directly equated to a single-bond event.

[0034] The average bond lifetime is denoted by τ(F), where F is the constant force. τ(F) can be obtained by combining the mean of the event duration, maximum likelihood estimation, or representative lifetime parameters from a pre-selected kinetic model. Lifetime estimation methods should be consistent within the same comparison, and the corresponding confidence intervals should be reported.

[0035] The determination of force-enhanced bonding requires the existence of a non-zero force level F1 < F2, causing τ(F2) to increase significantly relative to τ(F1); the capture-slip transition also requires the existence of F3 > F2, causing τ(F3) to decrease significantly relative to τ(F2). During the comparison, temperature, buffer system, molecular orientation, geometry, surface density, contact time, preload, loading conditions, and detection platform are controlled, and pre-defined statistical methods are employed.

[0036] In vitro quality assessment yields information in three dimensions: receptor binding specificity to the target complex, the response of cells expressing the receptor under antigenic conditions, and the force-dependent kinetics of the receptor-complex single bond. These three dimensions correspond to different detection endpoints and are not interchangeable.

[0037] In antigen recognition assays, exogenous QVDYYGLYY loading is used to evaluate peptide-dependent sensitivity. Whether target cells can present the target complex through their own antigen processing needs to be recorded separately from peptide loading conditions and cannot be determined solely by a positive exogenous peptide loading result.

[0038] II. Sequence and Location Information Nucleotide and amino acid sequences are numbered SEQ ID NO:1 to SEQ ID NO:11. The lengths in Table 1 below are counted character by character according to the sequence appendix of this document; the length of nucleic acids includes the terminal stop codon, while the length of proteins does not include the stop codon.

[0039] Table 1 Serial Number Corresponding content Check length SEQ ID NO:1 TCRα chain encodes nucleic acid 816 nt, including the stop codon SEQ ID NO:2 TCRα chain amino acid sequence 271 aa SEQ ID NO:3 TCRβ chain encodes nucleic acid 930 nt, including the stop codon SEQ ID NO:4 TCRβ chain amino acid sequence 309 aa SEQ ID NO:5 QVDYYGLYY epitope peptide 9 aa SEQ ID NO:6, 7, 8 α chain CDR1, CDR2, CDR3 6 aa, 7 aa, 12 aa SEQ ID NO:9, 10, 11 β chain CDR1, CDR2, CDR3 5 aa, 6 aa, 14 aa The CDR fragments of the two chains are identified by the sequences listed in the table below, with positions counted consecutively starting from the first amino acid of SEQ ID NO:2 or SEQ ID NO:4. In Table 2, CDR3 uses the original sequences containing boundary residues, namely SEQ ID NO:8 and SEQ ID NO:11; SEQ ID NO:7 and SEQ ID NO:10 are CDR2 of the two chains, respectively.

[0040] Table 2 Excerpt Serial Number amino acid sequence length Full chain position αCDR1 SEQID NO:6 DSAIYN 6 aa 46–51 αCDR2 SEQID NO:7 IQSSQRE 7 aa 69–75 αCDR3 SEQID NO:8 CAVDTGGFKTIF 12 aa 109–120 βCDR1 SEQID NO:9 MNHEY 5 aa 46–50 βCDR2 SEQ ID NO:10 SMNVEV 6 aa 68–73 βCDR3 SEQ ID NO:11 CASSSGDGNYGYTF 14 aa 110–123 The above-described locations are used to verify the position of the listed CDR fragments within the intact chain, and do not additionally presuppose the cleavage site of the signal peptide or the precise boundary between the variable and constant regions. The receptor structure in this application is based on the listed complete paired sequences, and the structural diagram is not intended as a precise labeling of residue boundaries.

[0041] Example 1: Acquisition and sequence alignment of paired receptors Starting with tumor-infiltrating T lymphocytes from HPV-positive tumor tissues, α / β chain pairing information and corresponding transcriptomic characteristics were obtained through single-cell TCR sequencing and RNA sequencing. Candidate analysis was conducted by combining single-cell transcriptomic and clonogenic characteristics, followed by cloning and sequencing of candidate T cells.

[0042] The obtained paired nucleotide sequences are listed in SEQ ID NO:1 and SEQ ID NO:3, and the corresponding protein sequences are listed in SEQ ID NO:2 and SEQ ID NO:4. SEQ ID NO:1 is 816 nt, and translation terminates after forming 271 aa; SEQ ID NO:3 is 930 nt, and translation terminates after forming 309 aa. The correspondence between the two sets of nucleic acids and the listed proteins was checked according to the complete reading frame.

[0043] Sample source, pairing criteria, and receptor identity are recorded separately. Sequence verification is used to confirm the correspondence between nucleic acids and proteins, and does not replace the detection of QVDYYGLYY-HLA-A 01:01 binding or stress phenotype.

[0044] Example 2: Double-strand expression and preparation of engineered T cells The coding regions of both strands were configured in a construct suitable for expression in mammalian cells. The co-expression arrangement of TCRβ-2A-TCRα was driven by the CMV promoter, and codon optimization was performed without altering the amino acid sequence of the target strand.

[0045] When assembling the co-expression cassette, verify the reading frame at the junction of TCRβ and 2A, and ensure that the upstream TCRβ coding region does not contain a stop codon. SEQ ID NO:1 and SEQ ID NO:3 serve as verification criteria for each single-strand coding sequence; the complete co-expression cassette also requires verification of the 2A sequence, the junction region, and the processing status of the final expression product.

[0046] The construct was introduced into peripheral blood T cells already stimulated against CD3 / CD28 using lentivirus, and the expression of the target receptor on the cell surface was detected after culture. In an embodiment that reduces endogenous pairing interference, TRAC was knocked out; an embodiment that reduces its expression can also be used for endogenous TCR control.

[0047] Cell collection can detect cell viability, transduction rate, target TCR surface expression, CD4 / CD8 composition, sterility, mycoplasma, endotoxin, vector copy number, and replicating virus, with supplementary evaluation using target pMHC-related potency assays. The decision thresholds for each item should be determined according to the actual detection protocol used.

[0048] Example 3: Evaluation of the binding of the target complex to HLA and peptide dependence HLA-A 01:01-QVDYYGLYY monomers and fluorescently labeled multimers were prepared. Engineered T cells were contacted with the target multimer, HLA-A 01:01-irrelevant peptide multimer, and HLA-mismatched multimer, respectively, and the binding signals were compared using flow cytometry.

[0049] In addition, HLA-A 01:01 positive target cells were used to establish functional comparisons, with QVDYYGLYY loading, irrelevant peptide loading, and no peptide loading conditions. The target cell killing mediated by engineered cells under each condition was compared to evaluate the dependence of cellular response on the target peptide.

[0050] The above analysis employed pre-selected positive thresholds, control distributions, independent replicate counts, and statistical methods. Detection results should be recorded in accordance with specific HLA conditions, peptide treatment methods, and receptor expression status to avoid merging binding signals and functional responses into the same endpoint.

[0051] Example 4: Determination of force bonding and capture-slip transition Using an atomic force microscope with constant force control, QVD-TCR or cells expressing this receptor were placed on one side of the detection system, while the HLA-A 01:01-QVDYYGLYY complex was placed on the other side. Single-bond detection states were established by controlling the molecular density and contact conditions on both sides, and the results were verified using Poisson statistics or equivalent methods.

[0052] After fixing the contact time and preload, the survival time of each bonding event was recorded under multiple non-zero constant force levels F1, F2 to Fn. For each force level, consistent survival analysis, maximum likelihood fitting, or other predefined methods were used to calculate τ(F) and its confidence interval.

[0053] First, examine whether the average bond lifetime at higher force levels significantly increases relative to lower force levels to determine force-enhanced bonding. Then, examine whether further increasing the force significantly reduces the average bond lifetime to determine capture-slip transition. Specific force levels, confidence levels, and significance analyses are performed according to the pre-experimental protocol; schematic curves are not used to replace primal event analysis.

[0054] Simultaneously set up irrelevant peptide-HLA-A 01:01 complexes, HLA mismatch complexes, blocking conditions, or reference TCRs. Identify nonspecific adhesion using each control, and report the number of effective events, censored events, original survival time distribution, fitted model, replicate experiments, and significance analysis for each control at different strength levels.

[0055] Example 5: Engineered Cell Response and Overall Quality Evaluation Engineered T cells expressing QVD-TCR were co-cultured with HLA-A 01:01-positive target cells expressing QVDYYGLYY antigen. Response sensitivity was evaluated using a targeted TCR-T killing assay and compared with corresponding controls. Multiple effector-target ratios and time points were included in the experiment, but the specific values ​​were recorded according to the actual implementation protocol.

[0056] Evaluation endpoints can include early activation markers, cytokines, degranulation, real-time impedance or imaging killing, residual target cell count, and T cell proliferation. Each endpoint reflects a different aspect of the antigen-dependent response and should not be presented as a validated effect without corresponding readings.

[0057] For the same engineered cell product, a quality evaluation was performed by combining the specific information from Example 3, the force dynamics information from Example 4, and the cell response information from this example.

[0058] Sequence Appendix

[0059] TCRα chain SEQ ID NO:1 (TCRα chain encodes nucleic acid; length: 816 nt, including stop codon) ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACACAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTCTGTGCTGTGGATACTGGAGGCTTCAAAACTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA。

[0060] SEQ ID NO: 2 (TCR α chain amino acid sequence; length: 271 aa) METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVDTGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS.

[0061] TCR β chain SEQ ID NO: 3 (TCR β chain encoding nucleic acid; Length: 930 nt, including stop codon) ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTCGGGGGACGGGAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA.

[0062] SEQ ID NO:4 (TCRβ chain amino acid sequence; length: 309 aa) MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSSGDGNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF.

[0063] SEQ ID NO:5 (HPV16 E2-derived epitope peptide; length: 9 aa) QVDYYGLYY.

[0064] SEQ ID NO:6 (TCRα CDR1; Length: 6 aa) DSAIYN.

[0065] SEQ ID NO:7 (TCRα CDR2; Length: 7 aa) IQSSQRE.

[0066] SEQ ID NO:8 (TCRα CDR3; Length: 12 aa) CAVDTGGFKTIF.

[0067] SEQ ID NO:9 (TCRβ CDR1; Length: 5 aa) MNHEY.

[0068] SEQ ID NO:10 (TCRβ CDR2; Length: 6 aa) SMNVEV.

[0069] SEQ ID NO:11 (TCRβ CDR3; Length: 14 aa) CASSSGDGNYGYTF.

Claims

1. A T-cell receptor, characterized in that, The device comprises paired TCRα and TCRβ chains, wherein the TCRα chain contains the amino acid sequence shown in SEQ ID NO:2, and the TCRβ chain contains the amino acid sequence shown in SEQ ID NO:4; the T cell receptor specifically binds to a complex consisting of HLA-A 01:01 and a peptide with the amino acid sequence shown in SEQ ID NO:5, and exhibits force-enhanced binding under single-bond conditions measured by constant force single-molecule assay; force-enhanced binding refers to a significant increase in the average lifetime of the single-molecule bond formed between the receptor and the complex as the force increases within at least a non-zero force range.

2. The T cell receptor according to claim 1, characterized in that, The TCRα chain and TCRβ chain form a membrane-bound heterodimer.

3. The T-cell receptor according to claim 1, characterized in that, Under the same measurement conditions of temperature, molecular orientation, surface density, contact time, preload, detection platform and statistical method, there are three non-zero constant forces F1, F2 and F3, which satisfy F1 < F2 < F3, and τ(F2) is significantly greater than τ(F1) and significantly greater than τ(F3), so as to present a capture-slip transition; where τ(F) is the average single bond lifetime under the force F, and the significance is determined according to a predetermined statistical method.

4. A nucleic acid molecule or combination of nucleic acid molecules for encoding the T cell receptor of claim 1, characterized in that, It contains a first nucleic acid sequence encoding the TCRα chain and a second nucleic acid sequence encoding the TCRβ chain; the first nucleic acid sequence and the second nucleic acid sequence are disposed in the same nucleic acid molecule, or are disposed in different nucleic acid molecules.

5. The nucleic acid molecule or combination of nucleic acid molecules according to claim 4, characterized in that, The first nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO:1, and the second nucleic acid sequence is the nucleotide sequence shown in SEQ ID NO:

3.

6. A nucleic acid expression construct, characterized in that, The expression cassette contains the nucleic acid molecule or combination of nucleic acid molecules as described in claim 4, or a vector loading the expression cassette; the first nucleic acid sequence and the second nucleic acid sequence are expressed through their respective promoters, or co-expressed under the same promoter through a 2A peptide coding sequence or an internal ribosome entry site.

7. The nucleic acid expression construct according to claim 6, characterized in that, The vector is a lentiviral vector, and the expression cassette uses CMV as a promoter and arranges the TCRβ chain coding region, 2A peptide coding region and TCRα chain coding region in a 5′ to 3′ direction; The TCRβ chain coding region, located upstream of the 2A peptide coding region, does not contain a stop codon, so that translation can continue to the 2A peptide coding region and the TCRα chain coding region.

8. An engineered immune cell, characterized in that, The cell carries the nucleic acid molecule or combination of nucleic acid molecules as described in claim 4, or the nucleic acid expression construct as described in claim 6, and expresses the T cell receptor as described in claim 1 on the cell surface.

9. The engineered immune cells according to claim 8, characterized in that, The cells were CD8+ T cells, and their endogenous TRAC gene was knocked out or its expression was reduced.

10. A pharmaceutical composition, characterized in that, It contains engineered immune cells as described in claim 8 or 9, and contains a pharmaceutically acceptable carrier, diluent, or excipient.

11. Use of the engineered immune cells as described in claim 8 or 9 in the preparation of a medicament for treating HPV16-related diseases in subjects who are HLA-A 01:01 positive and whose lesion cells present with the HLA-A 01:01-QVDYYGLYY complex.

12. The use according to claim 11, characterized in that, The HPV16-related diseases are persistent HPV16 infection, HPV16-related precancerous lesions, or HPV16-related malignant tumors; the malignant tumors are selected from cervical cancer, head and neck squamous cell carcinoma, anal cancer, and penile cancer.

13. A method for preparing the engineered immune cells of claim 8 or 9 in vitro, characterized in that, The nucleic acid molecule or combination of nucleic acid molecules as described in claim 4 or the nucleic acid expression construct as described in claim 6 is introduced into immune cells; the introduced immune cells are cultured; the expression of the target T cell receptor on the cell surface is detected and cells expressing the receptor are collected; In preparing the engineered immune cells according to claim 9, CD8+ T cells are used as the starting cells, and the endogenous TRAC gene is knocked out or its expression is reduced before, during or after introduction.

14. A method for in vitro evaluation of the force-binding properties of the T cell receptor according to claim 1, characterized in that, include: The surface density of the T cell receptor and the HLA-A 01:01-QVDYYGLYY complex was adjusted, and the single bond conditions were confirmed by combining Poisson statistics or equivalent methods. Atomic force microscopy was used to apply different non-zero constant forces to the formed monomolecular bonds, and the bond survival time at each force level was obtained. The average bond lifetime and confidence interval were calculated according to a predetermined data analysis method. When F1 < F2 and τ(F2) is significantly greater than τ(F1), the bond is determined to have force-enhanced binding properties. When F3 > F2 and τ(F3) is significantly less than τ(F2), the bond is determined to have capture-slip transition properties. Parallel HLA-A 01:01-irrelevant peptide complex, HLA mismatch complex, blocking condition or reference TCR control were set to identify nonspecific adhesion.

15. A method for in vitro quality evaluation of the engineered immune cells according to claim 8 or 9, characterized in that, The binding specificity of the engineered immune cells to the HLA-A 01:01-QVDYYGLYY complex was detected; the engineered immune cells were co-cultured with HLA-A 01:01 positive target cells, and the antigen response was compared under conditions of QVDYYGLYY loading, irrelevant peptide loading, and no peptide loading; and the mechanodependent binding characteristics of the expressed T cell receptor were determined according to the method of claim 14.

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