T cell receptors recognizing sars-cov-2 epitopes and uses thereof

Abstract

This invention discloses a T-cell receptor that recognizes the SARS-CoV-2 antigenic epitope and its application. The T-cell receptor comprises a TCR α chain with a CDR3 region amino acid sequence as shown in SEQ ID NO: 1 and a TCR β chain with a CDR3 region amino acid sequence as shown in SEQ ID NO: 2. The T-cell receptor of this invention can target the SARS-CoV-2 antigenic epitope peptide VMV. A TCRT constructed using the T-cell receptor of this invention exhibits a significant killing effect on cells expressing the SARS-CoV-2 antigenic peptide VMV. This invention provides a new approach for cellular immunotherapy of SARS-CoV-2.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a T-cell receptor that recognizes SARS-CoV-2 antigenic epitopes and its applications. Background Technology

[0002] The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to circulate globally. Its main clinical manifestations include fever, dry cough, and fatigue; some patients may progress to severe illness, developing respiratory complications and even death. The continuous mutation of the SARS-CoV-2 genome not only limits the effectiveness of humoral immunity induced by vaccination, leading to frequent breakthrough infections, but also poses a serious challenge to existing treatment methods. However, there is currently a lack of specific and highly effective antiviral drugs against SARS-CoV-2. The efficacy of existing drugs is limited by the early treatment window, and their effectiveness is very limited in special populations such as severely ill and immunocompromised patients, leading to difficulties in viral clearance and prolonged disease progression. This constitutes the core dilemma that urgently needs to be overcome in current clinical treatment.

[0003] SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA virus. Its genome is approximately 29.8 kb in length and primarily encodes four structural proteins: the spike protein (S), nucleocapsid protein (N), envelope protein (E), and membrane protein (M), which together constitute the viral particle structure. The genome also encodes 16 non-structural proteins (nsp1-nsp16) involved in viral replication and transcription, as well as various accessory proteins (such as ORF3a, ORF6, and ORF7a) involved in regulating the host immune response. Both structural and non-structural proteins of SARS-CoV-2 are immunogenic, with the spike protein (S protein) exhibiting the strongest immunogenicity. Its receptor-binding domain (RBD) contains multiple key antigenic epitopes and is a major target for inducing neutralizing antibody production and T-cell responses. The nucleocapsid protein (N protein) contains an immunodominant epitope at its C-terminus, which can effectively stimulate T cell immunity. The membrane protein (M protein) also contains multiple immunodominant epitopes at its N-terminus, C-terminus, and transmembrane region, which can induce not only protective humoral immunity but also a strong T cell response. Furthermore, although non-structural proteins and some accessory proteins are expressed at low levels in infected cells, their coding sequences still contain highly immunogenic CD8. +T-cell epitopes. To date, research has identified more than 1,400 potential T-cell epitopes, indicating that SARS-CoV-2 can elicit a wide range of T-cell responses, providing a rich selection of targets for T-cell-based immunotherapy strategies.

[0004] CD8 + T cells are key effector cells in clearing viral infections. Their function depends on the specific recognition of viral antigen peptides presented by major histocompatibility complex (MHC, or HLA in humans) class I molecules on the surface of antigen-presenting cells by T cell receptors (TCRs). Based on this mechanism, T cell receptor-engineered T cell (TCR-T) therapy has emerged: by introducing viral antigen epitope-specific TCR α and β chain genes into T cells, they gain the ability to precisely target and kill virus-infected cells. Compared with chimeric antigen receptor-T cell (CAR-T) therapy, TCR-T has unique advantages such as low immunogenicity and the ability to recognize intracellular antigens (presented via MHC epitope peptides), showing broader application prospects in the treatment of viral infectious diseases and providing new strategies and directions for the immunotherapy of viral diseases such as COVID-19. However, the core prerequisite for TCR-T development is the screening and obtaining high-affinity, high-specificity viral antigen-specific TCRs, which depends on the viral CD8 receptor. + In-depth analysis of T-cell epitopes.

[0005] Currently, CD8 targeting SARS-CoV-2 + T-cell epitope research has largely focused on classical HLA-I molecules (such as HLA-A*02). However, classical HLA-I molecules exhibit high polymorphism in the population, making it difficult to cover the entire population with their restriction epitopes, which greatly limits the universality of vaccines or TCR-T therapies developed based on them. In contrast, HLA-E belongs to non-classical MHC-I molecules, is widely expressed in vivo, has only two major alleles (HLA-E*0101 and HLA-E*0103), and its presented peptides are highly conserved. More importantly, HLA-E can present viral antigen peptides and activate specific CD8. + T-cell responses, therefore, the immune response induced by its restriction epitopes can cover the general population, giving it a unique advantage in the development of broad-spectrum vaccines and universal TCR-T therapies. Our previous research identified an HLA-E restriction epitope peptide (VMVELVAEL) derived from the non-structural protein of SARS-CoV-2, and has demonstrated its effective induction of CD8 in COVID-19 recovered patients. +T-cell responses are highly immunogenic, making them one of the ideal targets for developing broad-spectrum anti-SARS-CoV-2 TCR-T therapies.

[0006] Although previous studies have identified HLA-E-restricted epitope peptides derived from non-structural proteins of SARS-CoV-2 and confirmed that they can effectively induce CD8 in COVID-19 recovered patients. + T cell responses exist, but specific TCR clones and sequence information targeting these epitopes are still lacking. Obtaining high-affinity, high-specificity viral antigen-specific TCRs is a key step in overcoming current bottlenecks in COVID-19 treatment and developing novel broad-spectrum immunotherapies.

[0007] Therefore, it is necessary to screen and identify specific TCRs that recognize HLA-E-restricted SARS-CoV-2 antigenic epitopes to provide a molecular basis for developing broad-spectrum antiviral TCR-T therapies. In summary, based on the identification of the VMVELVAEL epitope, further screening and identification of its specific TCR clones and sequences to obtain high-affinity, high-specificity viral antigen-specific TCRs is a crucial step in overcoming current bottlenecks in COVID-19 treatment and developing novel broad-spectrum immunotherapies, possessing significant scientific and clinical application value. Summary of the Invention

[0008] To overcome the shortcomings of the existing technology, the present invention provides a T-cell receptor for recognizing SARS-CoV-2 antigenic epitope peptides and its application. The provided T-cell receptor can recognize antigenic epitope peptides presented by HLA-E molecules and generate a strong CD8+ response. + T-cell immune responses secrete high levels of IFN-γ, which can be used in the preparation of antiviral drugs or vaccines for SARS-CoV-2.

[0009] To achieve the above objectives, the present invention employs the following technical solution: The first objective of this invention is to provide a T-cell receptor that recognizes SARS-CoV-2 antigenic epitopes, comprising a TCR α chain and a TCR β chain. The amino acid sequence of the CDR3 region of the TCR α chain is shown in SEQ ID NO:1, and the amino acid sequence of the CDR3 region of the TCR β chain is shown in SEQ ID NO:2. This T-cell receptor can specifically recognize the SARS-CoV-2 antigenic epitope peptide with the amino acid sequence VMVELVAEL presented by the HLA-E molecule. Specifically, the amino acid sequence of the CDR3 region of the α chain shown in SEQ ID NO:1 is CALRGGKTGGFKTIF; the amino acid sequence of the CDR3 region of the β chain shown in SEQ ID NO:2 is CASSQSPGVGGTEAFF. This sequence combination is obtained by targeting HLA-E / VMVELVAEL-specific CD8+ in the peripheral blood of COVID-19 recovered patients. + T cells were subjected to high-throughput TCR sequencing to screen for the TCR sequences with the highest clonal amplification frequency.

[0010] Furthermore, the amino acid sequence of the SARS-CoV-2 antigenic epitope peptide is VMVELVAEL (abbreviated as VMV).

[0011] Furthermore, the amino acid sequence of the TCR α chain is shown in SEQ ID NO:3, and the amino acid sequence of the TCR β chain is shown in SEQ ID NO:4. This provides the complete full-length TCR sequence, including the variable and constant regions, facilitating gene synthesis and functional expression.

[0012] Furthermore, the V gene of the TCR α chain is TRAV19, and the J gene is TRAJ9; the V gene of the TCR β chain is TRBV3-1, the D gene is TRBD2, and the J gene is TRBJ1-1. This clarifies the genetic composition of the TCR, and this gene combination, together with the CDR3 sequence, determines the antigen recognition specificity and expression stability of the TCR.

[0013] A second objective of this invention is to provide a TCR-T cell, wherein the TCR-T cell is a CD8 cell. + T cells that express the aforementioned T cell receptor. These TCR-T cells are generated by introducing a gene encoding a specific TCR into CD8. + This technology was developed by T cells, which enabled T cells to accurately identify and kill SARS-CoV-2 infected cells.

[0014] Furthermore, the CD8 + T cells originate from peripheral blood mononuclear cells. This is the most commonly used source of T cells in clinical applications, as they are readily available and functionally mature.

[0015] Furthermore, the binding rate of the TCR-T cells to the HLA-E / VMVELVAEL tetramer is no less than 5%. This indicator quantifies the antigen-specific recognition ability of TCR-T cells, ensuring the quality standards of the product.

[0016] Furthermore, after stimulation with HLA-E positive cells loaded with the VMVELVAEL antigen peptide, the TCR-T cells showed upregulated CD69 expression. CD69 is a marker molecule for early T cell activation, and its upregulated expression demonstrates that TCR-T cells can be effectively activated after recognizing the antigen, which is a prerequisite for exerting subsequent cytotoxic effects.

[0017] Furthermore, the proportion of T-cell receptor-positive cells in the TCR-T cells is no less than 10%. This ensures the purity of the TCR-T cell product, guarantees therapeutic efficacy, and reduces the risk of off-target effects.

[0018] A third objective of this invention is to provide a recombinant expression vector comprising a nucleotide sequence encoding the aforementioned T cell receptor. This vector is used to introduce the TCR gene into T cells and is a key tool for preparing TCR-T cells.

[0019] Furthermore, the recombinant expression vector is a lentiviral vector. Lentiviral vectors have advantages such as high transduction efficiency, ability to infect non-dividing cells, and stable gene expression, and are one of the most commonly used vector types in gene therapy.

[0020] Furthermore, the lentiviral vector contains an SFFV promoter, which drives the expression of the T cell receptor. The SFFV promoter is a strong promoter capable of efficiently driving the expression of exogenous genes in T cells.

[0021] Furthermore, the recombinant expression vector also contains the nucleotide sequence of the reporter gene GFP. Expression of the GFP reporter gene can be used to monitor transduction efficiency and screen for TCR-positive cells, simplifying the preparation process of TCR-T cells.

[0022] More preferably, the expression vector is pLent-SFFV-FH-CMV-copGFP-P2A-Puro.

[0023] A fourth objective of this invention is to provide a pharmaceutical composition comprising at least one of the above-described T-cell receptor, the above-described TCR-T cell, or the above-described recombinant expression vector as an active ingredient, and a pharmaceutically acceptable carrier. This pharmaceutical composition can be used in clinical treatment, providing a new therapeutic option for SARS-CoV-2 infection.

[0024] Furthermore, the dosage form of the pharmaceutical composition is an injection or a cryopreservation solution. Injection solutions are suitable for direct administration, while cryopreservation solutions facilitate long-term storage and transportation, meeting the needs of different clinical application scenarios.

[0025] Furthermore, when the active ingredient is TCR-T cells, the concentration of the TCR-T cells is 1×10⁻⁶. 6 Up to 1×10 8 Cells / mL. This concentration range was determined based on clinical experience in cell therapy to ensure therapeutic dosage while avoiding excessive infusion volume.

[0026] A fifth objective of this invention is to provide a method for preparing TCR-T cells, comprising the following steps: S1: Synthesize the nucleotide sequences encoding the TCR α and TCR β chains of the aforementioned T cell receptors; S2: The nucleotide sequence is cloned into the recombinant expression vector; S3: Transduc the recombinant expression vector into CD8 + T cells; S4: Screen for cells that express T cell receptor positive to obtain TCR-T cells.

[0027] This method provides a complete process route from gene synthesis to cell preparation, enabling the large-scale production of TCR-T cells.

[0028] Furthermore, prior to S3, the following steps are included: sorting CD8 using HLA-E / VMVELVAEL tetramer. + VMVELVAEL-specific T cells within T cells. This step increases the antigen specificity of the starting cells, reduces the proportion of non-specific cells in subsequent products, and lowers the risk of off-target effects.

[0029] Furthermore, in S3, the multiplicity of infection (MOI) during transduction is between 10 and 50. This MOI range has been experimentally validated to maintain high transduction efficiency while keeping cytotoxicity at an acceptable level.

[0030] Furthermore, in S4, screening was performed by detecting the expression of the reporter gene GFP using flow cytometry. Flow cytometry is a highly efficient and precise cell screening method that can rapidly obtain high-purity TCR-expressing positive cells.

[0031] Furthermore, after S3, the transduced cells were cultured for 72 hours. This culture time is sufficient to allow the exogenous TCR gene to be fully expressed and stably presented on the cell surface, ensuring the accuracy of subsequent screening and functional validation.

[0032] A sixth objective of this invention is to provide the use of the aforementioned T cell receptor, the aforementioned TCR-T cells, the aforementioned recombinant expression vector, or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention or treatment of SARS-CoV-2 infection. This application utilizes the ability of TCR-T cells to precisely recognize and kill virus-infected cells, providing a novel cell-immune-based therapeutic strategy for COVID-19.

[0033] Furthermore, in cases of severe COVID-19 infection caused by SARS-CoV-2 infection, the TCR-T therapy of the present invention can provide a new treatment option.

[0034] Furthermore, the drug is administered via intravenous injection. Intravenous injection is the most common route of administration for cell therapy, allowing TCR-T cells to rapidly enter the bloodstream and distribute throughout the body to exert their therapeutic effect.

[0035] A seventh objective of this invention is to provide the use of the above-described T cell receptor, the above-described TCR-T cells, the above-described recombinant expression vector, or the above-described pharmaceutical composition in the preparation of a vaccine for the prevention of SARS-CoV-2 infection. In addition to being used to treat existing infections, TCR-T cells can also be used as a prophylactic vaccine, pre-infused before an individual is exposed to the virus, to establish specific cellular immune memory against SARS-CoV-2.

[0036] An eighth objective of the present invention is to provide a host cell comprising the aforementioned T cell receptor or the aforementioned recombinant expression vector. This host cell can be used for the expression and purification of TCR protein, and can also be directly used as TCR-T cells for therapeutic purposes.

[0037] Compared with the prior art, the present invention has the following beneficial effects: This invention identifies and provides for the first time a T-cell receptor sequence that specifically recognizes the HLA-E-restricted SARS-CoV-2 antigenic epitope peptide VMVELVAEL. The α-chain CDR3 region sequence of this TCR is CALRGGKTGGFKTIF, and the β-chain CDR3 region sequence is CASSQSPGVGGTEAFF, providing a key molecular basis for developing broad-spectrum anti-SARS-CoV-2 TCR-T therapy. Since HLA-E has only two major alleles (HLA-E*0101 and HLA-E*0103) in the human population and is highly conserved, the therapy developed based on the TCR of this invention can cover the general population, overcoming the problem of low population coverage caused by the high polymorphism of classic HLA-I molecules, and significantly improving the universality of TCR-T therapy.

[0038] The experimental results of this invention show that the TCR-T cells prepared by this invention exhibit significant antigen-specific recognition ability, with a binding rate of no less than 5% to the HLA-E / VMVELVAEL tetramer, higher than the 2.29% of the control group, proving that exogenous TCR successfully endows T cells with the ability to accurately recognize target antigens. Simultaneously, after co-culturing the TCR-T cells of this invention with target cells loaded with VMVELVAEL antigen peptides, the expression of the early activation marker CD69 was significantly upregulated, demonstrating that TCR-T cells can not only recognize antigens but also be effectively activated, possessing the functional basis for exerting subsequent cytotoxic effects. Furthermore, the lentiviral transduction method provided by this invention is simple and efficient; under the condition of a multiplicity of infection (MOI) of 50, the TCR gene transduction efficiency can reach 17.4%, providing a feasible technical route for the large-scale preparation of TCR-T cells. This invention targets conserved epitopes derived from non-structural proteins of SARS-CoV-2. These epitopes are not easily rendered ineffective by viral mutations, giving the TCR-T therapy of this invention the potential to address viral mutations and providing a treatment option for broad-spectrum SARS-CoV-2 infection, including existing strains and potential future variants. This invention also provides a new approach for cellular immunotherapy of SARS-CoV-2, particularly for critically ill patients and immunosuppressed patients for whom existing drugs have limited efficacy. The TCR-T therapy of this invention can precisely kill virus-infected cells, potentially overcoming current treatment bottlenecks and possessing significant clinical application value. Attached Figure Description

[0039] Figure 1 HLA-E / VMV specific CD8 + The amino acid sequence usage of the TCR CDR3 region in T cells; where A represents the top 10 most frequently used CDR3 region amino acid sequences of the TCR α chain; and B represents the top 10 most frequently used CDR3 region amino acid sequences of the TCR β chain.

[0040] Figure 2 For flow cytometry detection of CD8 after lentiviral infection + Expression of GFP in T cells.

[0041] Figure 3 For HLA-E / VMV tetramer (Tetramer) staining to detect CD8 + HLA-E-restricted antigenic peptide VMV-specific CD8 in T cells + The T cell frequency results are shown in Figure A; where A represents the HLA-E-restricted VMV-specific CD8 count detected by Tetramer staining. + The graph shows the frequency results of T cells; B represents the ratio of TCR-T cells to primary CD8 cells. +HLA-E-restricted VMV-specific CD8+ in T cells (control group) + Statistical comparison results of T cell frequencies.

[0042] Figure 4 After co-culturing TCR-T cells with HLA-E / K562 cells loaded with the antigenic peptide VMV, the expression level of CD69 was detected by flow cytometry; where A represents the expression level of TCR-T cells and primary CD8+. + Flow cytometry representation of CD69 expression in T cells (control group); B represents TCR-T cells and primary CD8+ cells. + CD69 in T cells + CD8 + Statistical comparison results of T cell percentages. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. 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 should fall within the scope of protection of the present invention.

[0044] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0045] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1: HLA-E-restricted SARS-CoV-2 antigenic epitope peptide-specific CD8 + T cell sorting 1. Preparation of HLA-E / epitope peptide tetramer The SARS-CoV-2 antigenic epitope peptide VMVELVAEL (abbreviated as VMV) was synthesized, and the HLA-E / VMV tetramer (Tetramer) was constructed. The specific steps were as follows: First, HLA-E monomers and VMV were folded in vitro to form an HLA-E / VMV pMHC complex; further, through biotinylation labeling and binding with streptavidin, it was determined that the binding rate of the biotinylated pMHC monomer to streptavidin was ≥80%, thus synthesizing the HLA-E / VMV Tetramer, which was then labeled with PE fluorescence.

[0046] 2. Peripheral blood CD8 + T cell sorting CD8+ cells were sorted from peripheral blood mononuclear cells (PBMCs) of individuals recovering from COVID-19 using an immunomagnetic bead (Dynal) negative sorting method. + T cells. The specific method is to take 1×10⁶ cells... 7 PBMCs from individuals recovered from COVID-19 were resuspended and mixed with 20 μL of heat-inactivated fetal bovine serum (FCS) and 20 μL of biotin-labeled antibody solution. The mixture was incubated at 4°C for 20 min. After washing with 5 mL of buffer, the cells were centrifuged and the supernatant was discarded. The cells were then resuspended in 1 mL of buffer and added to 100 μL of pre-washed Dynabeads. The mixture was incubated at room temperature for 15 min, inverting the container every 5 min. The cells were then allowed to stand on a magnetic rack for 2 min, and the supernatant was collected. The process was repeated three times. The collected supernatants were centrifuged and resuspended in 10% FCS RPMI 1640 medium, resulting in CD8 cells without magnetic beads or antibodies on the cell surface. + T cells.

[0047] 3. SARS-CoV-2 epitope peptide VMV-specific CD8 + T cell sorting Specific CD8 was sorted using the HLA-E / VMV Tetramer staining method. + T cells, specifically: take 1×10 6 CD8 + T cells were washed and then incubated with 2 μl of PE-labeled HLA-E / VMV Tetramer at room temperature in the dark for 15 min. After washing, the cells were resuspended and HLA-E / VMV Tetramer was obtained by flow cytometry sorting. + CD8 + T cells, specifically the SARS-CoV-2 epitope peptide VMV-specific CD8+, are... + T cells, counted and ready for use.

[0048] Example 2: HLA-E-restricted SARS-CoV-2 epitope VMV-specific CD8 + T cell TCR sequencing and identification 1. VMV-specific CD8 + T-cell TCR sequencing The HLA-E-restricted SARS-CoV-2 epitope VMV-specific CD8 was obtained by sorting. + T cells underwent TCR library sequencing, specifically: first, specific CD8+ cells obtained from HLA-E / VMV Tetramer staining were sorted. + Total RNA was extracted from T cells and reverse transcribed using the total RNA as a template to synthesize first-strand cDNA. Subsequently, multiplex PCR was used to target and amplify the variable region (V region) and constant region (C region) of the α and β strands of the TCR. A unique molecular identifier (UID) was introduced during amplification to correct for PCR amplification bias and sequencing errors. The amplified products were purified and used to construct sequencing libraries. The cDNA libraries were sequenced using an Illumina high-throughput sequencing platform to obtain raw sequencing data. The raw sequencing data underwent quality control filtering to remove low-quality sequences. High-quality sequences were then aligned to the human TCRV, D, J, and C gene fragment database provided by the International Immunogenetics Database (IMGT, http: / / www.imgt.org / ). Through sequence alignment and assembly, the nucleotide and amino acid sequences of the V(D)J gene usage information and the complementarity-determining region (CDR) 3 region of each TCR strand were obtained.

[0049] 2. VMV-specific CD8 + T cell TCR sequence analysis and identification Based on TCR sequencing results, statistical analysis was performed on the pairing information of the TCR α and β chains and the clonal amplification frequency in the samples. Using clonal amplification frequency as the primary screening criterion, and combining the uniqueness of the CDR3 region amino acid sequence and the usage of the V(D)J gene, the epitope-specific CD8 with the highest amplification frequency and the most representative characteristics was selected. + T cell TCR sequence, Figure 1 This refers to HLA-E / VMV specific CD8. + Amino acid sequence usage in the CDR3 region of the T cell TCR Figure 1 In the middle, A represents the top 10 most frequently used amino acid sequences in the CDR3 region of the α chain. Figure 1 As can be seen from A, in HLA-E / VMV specific CD8 +In T cells, the α-chain CDR3 region sequence exhibits obvious clonal expansion characteristics, with the most frequent amino acid sequence being CALRGGKTGGFKTIF. This sequence accounts for a significantly higher proportion than other sequences in the specific T cell population, indicating that the α-chain CDR3 sequence plays a key role in recognizing the HLA-E / VMV complex. Figure 1 In the middle B, the CDR3 region amino acid sequence of the top 10 most frequently used amino acids in the β chain is... Figure 1 As shown in Figure B, the β-chain CDR3 region also exhibits clonal expansion, with the most frequent amino acid sequence being CASSGPSPGVGTEAF. This sequence dominates in the specific T cell population, demonstrating that the β-chain CDR3 sequence can efficiently recognize the target antigen after pairing with the α-chain CDR3 sequence. The amino acid sequence information of the CDR3 regions of the α-chain and β-chain is shown in Table 1.

[0050] Table 1. Amino acid sequence information of the CDR3 region of the α and β chains of HLA-E / VMV specific TCRs.

[0051] As shown in Table 1, the TCR α chain obtained through screening used the TRAV19 and TRAJ9 genes, with the CDR3 region amino acid sequence being CALRGGKTGGFKTIF, which is 15 amino acids long and falls within the typical length range of the human TCR α chain CDR3 region. The TCR β chain used the TRBV3-1, TRBD2, and TRBJ1-1 genes, with the CDR3 region amino acid sequence being CASSQSPGVGGTEAFF, which is 16 amino acids long and also falls within the common length range of the human TCR β chain CDR3 region. The CDR3 sequence combination of these two chains represents the highest-frequency clone selected from a large pool of HLA-E / VMV-specific T cell TCRs, signifying a dominant immune response against this antigenic epitope.

[0052] Example 3: Preparation of HLA-E-restricted SARS-CoV-2 epitope VMV-specific TCR-T cells 1. Construction and verification of the full-length sequences of the TCR α and β chains Based on the CDR3 region amino acid sequences of the HLA-E / VMV-specific TCR α and β chains as described in Table 1, the V gene (TRAV19) and J gene (TRAJ9) of the TCR α chain, and the V gene (TRBV3-1), D gene (TRBD2), and J gene (TRBJ1-1) of the β chain were retrieved from the IMGT database to obtain the reference amino acid sequences of the α and β chains. The CDR3 region amino acid sequences obtained from sequencing were ligated to the V and J regions. Finally, the V-(CDR3)-J sequence was ligated to the TRA C region sequence, and the V-(CDR3)-DJ sequence of the β chain was ligated to the TRB C region sequence, thus obtaining the complete amino acid sequences of the TCR α and β chains. The assembled full-length sequences were submitted to the UniProt database (https: / / www.uniprot.org / ) for BLAST alignment to confirm that the sequences were consistent with the known variable and constant regions of human TCRs, without frameshifts or mis-splicing. The full-length amino acid sequences of the TCR α and β chains verified by the above steps are shown in Table 2.

[0053] Table 2. Amino acid sequences of the TCR α and β chains.

[0054] As shown in Table 2, the full-length TCR α chain sequence includes the TRAV19 variable region, the CDR3 region CALRGGKTGGFKTIF, TRAJ9, and the TRAC constant region, with a total length of approximately 210 amino acids. The full-length TCR β chain sequence includes the TRBV3-1 variable region, the CDR3 region CASSQSPGVGGTEAFF, TRBD2, TRBJ1-1, and the TRBC constant region, with a total length of approximately 237 amino acids. After expression in T cells, these two chains connect via interchain disulfide bonds to form an αβ heterodimer, constituting the complete TCR complex. The framework region of the variable region provides the basic structural scaffold of the TCR, while the CDR1, CDR2, and CDR3 regions together constitute the antigen recognition interface. Among them, the CDR3 region, due to its high variability and direct contact with the antigen peptide, is the most critical region determining the antigen specificity of the TCR. The constant region is responsible for binding to the CD3 complex, transmitting antigen recognition signals into the cell, and initiating the T cell activation cascade. This embodiment successfully identified a TCR sequence that can specifically recognize the HLA-E / VMV complex, providing a key molecular basis for the subsequent construction of TCR-T cells.

[0055] 2. Construction and Packaging of VMV-Specific TCR Recombinant Lentiviral Vectors Recombinant plasmids were constructed and lentiviral packaging was performed as follows: The nucleic acid sequences of the TCR α and β chains shown in Table 2 were synthesized and subcloned into the vector pLent-SFFV-FH-CMV-copGFP-P2A-Puro. 293T cells were seeded in 96-well plates and cultured at 37°C in a 5% CO2 incubator until cell confluence reached approximately 85%–90%. Transfection reagents were prepared (1000 μL DMEM high-glucose medium, 10 μg plasmid, 3 μg PMD2G plasmid, 6 μg PSPAX2 plasmid), mixed well, and incubated at room temperature for 3 min. PEI was then added and vortexed, and the mixture was incubated at room temperature for 30 min before being transferred to culture dishes. Six hours after infection, the medium was replaced with fresh DMEM. Twenty-four hours after infection, the infection efficiency and cell status were observed. The cells were uncontaminated and growing well. Seventy-two hours after infection, the supernatant medium was collected and filtered through a 0.45 μm filter membrane into an ultracentrifuge tube. After centrifugation at 25,000 rpm for 1.5 hours at 4°C, the supernatant was discarded, and virus preservation solution was added and mixed. The mixture was incubated overnight at 4°C. The virus was collected and its titer was determined to obtain the concentrated virus solution.

[0056] 3. Original CD8 + T-cell lentiviral infection and efficiency detection PBMC CD8 sorting using magnetic beads + T cells were prepared into a cell suspension using RPMI-1640 complete culture medium, and the cell concentration was adjusted to 2 × 10⁶ cells / mL. 6 Cells / mL. The cell suspension was seeded at 200 μL / well into 48-well plates, with 5 μL of concentrated virus solution added to each well. The plates were centrifuged at 800×g for 15 min at room temperature, then incubated at 37℃ with 5% CO2 for 6 h. 300 μL of complete culture medium was added for further incubation. After 72 h of culture, the viral infection efficiency was assessed by flow cytometry. The specific steps were as follows: 100 μL of cell suspension was taken, and 2 mL of flow cytometry wash buffer was added to resuspend the cells. After centrifugation and washing, the GFP expression level was detected by flow cytometry. GFP-positive cells were the successfully prepared TCR-T cells. Primary CD8 cells not infected with lentivirus... + The GFP positivity rate in T cells was only 0.15%, indicating extremely low background fluorescence signal. After infection with lentiviruses of different multiples of infection (MOIs), the GFP positivity rate of TCR-T cells increased significantly with increasing MOI: at MOIs of 2, 3, 5, 10, 20, and 50, the GFP positivity rates were 8.64%, 11.63%, 11.78%, 13.39%, 15%, and 17.4%, respectively. Figure 2 It can be seen that the CD8 group in the control group, which was not infected with lentivirus, +The GFP positivity rate in T cells was only 0.15%, indicating extremely low background fluorescence signal and negligible autofluorescence. After infection with lentiviruses of different MOIs, the GFP positivity rate of TCR-T cells increased significantly with increasing MOI. The data trend shows that within the MOI range of 2 to 10, the GFP positivity rate increased linearly with increasing MOI, indicating that increasing the viral load effectively improved transduction efficiency. When the MOI exceeded 10, the rate of increase in GFP positivity rate slowed down. When the MOI increased from 10 to 50, the GFP positivity rate only increased from 13.39% to 17.4%, indicating that excessively high MOIs had limited effect on improving transduction efficiency. This may be due to the toxic effect of high viral concentrations on cells, partially offsetting the improvement in transduction efficiency. Considering both transduction efficiency and cell viability, an MOI range of 10 to 50 is a more suitable range for transduction.

[0057] The above results indicate that TCR lentiviruses can effectively infect primary CD8 cells. + Under MOI=50 conditions, nearly 20% of T cells were able to successfully express exogenous TCR, providing sufficient TCR-T cells for subsequent functional verification.

[0058] Example 4: Detection of TCR-T cell-specific recognition and activation function 1. HLA-E / VMV Tetramer staining to detect TCR-T cell-specific recognition ability HLA-E / VMV Tetramer staining was used to detect HLA-E-restricted VMV epitope-specific CD8+ in transduced TCR-T cells. + T cell frequency. The specific procedure is as follows: Take 100 μL of the constructed TCR-T cells and primary CD8 cells that are not infected with lentivirus. + T cells (as a negative control) were washed, centrifuged at 1200 rpm for 5 min, HLA-E / VMVTetramer-PE (2 μL) was added, and incubated at room temperature in the dark for 15 min. After washing, 100 μL of flow cytometry wash buffer was added for resuspending, and the proportion of PE-positive cells was detected by flow cytometry.

[0059] Tetramer staining results as follows Figure 3 As shown. Figure 3 A is an HLA-E-restricted VMV-specific CD8 receptor antagonist. + Representative flow cytometry plot of T cell frequency: CD8 cells were gated on the FSC-SSC scatter plot using NovoExpress 1.4.1 software. + T cell population, further analysis of CD8 + (x-axis) and Tetramer +(Vertical axis) The proportion of double-positive cells, which represents HLA-E-restricted VMV-specific CD8. + T cell frequency. From Figure 3 As shown in Figure A, the proportion of CD8 and tetramer double-positive cells was 8.50% in the TCR-T cell group, while this proportion was only 2.29% in the control group. The low proportion of tetramer-positive cells in the control group may originate from a small number of VMV-specific T cells naturally present in primary T cells, since the donors were COVID-19 recovered patients who had established immune memory against SARS-CoV-2. The tetramer positivity rate in the TCR-T cell group was approximately 3.7 times higher than that in the control group, indicating that the transduction of exogenous TCRs significantly increased the proportion of T cells capable of recognizing the HLA-E / VMV complex. Figure 3 Figure B shows a statistical analysis graph comparing TCR-T cells with primary CD8 cells. + HLA-E-restricted VMV-specific CD8+ in T cells (x-axis) + The difference in T cell frequency (vertical axis). From Figure 3 As can be seen from Figure B, the tetramer positivity rate in the TCR-T group was significantly higher than that in the control group, and the difference was statistically significant. This indicates that the transduced TCR was successfully expressed on the surface of T cells and endowed T cells with the ability to specifically recognize the HLA-E / VMV complex.

[0060] The results showed that HLA-E / VMV Tetramer in TCR-T cells + The proportion of cells was significantly higher than that of primary CD8 cells. + T cells indicate that VMV-specific TCRs have been successfully transduced and expressed on CD8. + On the surface of T cells, this gives T cells the ability to specifically recognize VMV epitopes presented by HLA-E molecules.

[0061] 2. Flow cytometry to detect cell activation 1) Construction and identification of HLA-E stably transfected K562 cell lines K562 cells are a human chronic myeloid leukemia cell line, characterized by the absence of expression of classic HLA-I molecules. Therefore, they are widely used as tool cells in studies of HLA-E molecule expression and function. Using lentiviral transduction technology, the HLA-E*0103 or HLA-E*0101 genes were introduced into K562 cells to obtain cell lines stably expressing HLA-E*0103 or HLA-E*0101, which can then be used as antigen-presenting cells expressing HLA-E.

[0062] Specifically, the following steps were performed: First, HLA-E*0103 or HLA-E*0101 overexpression lentiviruses were prepared, and the mRNA expression level of the target gene in K562 cells was detected by real-time PCR. Quantitative PCR results showed that, compared with the control group, the mRNA expression level of the target gene in K562 cells transduced with HLA-E*0103 or HLA-E*0101 was significantly increased, indicating successful lentiviral transduction. Based on preliminary experimental results, GFP expression was observed under a fluorescence microscope 72 hours after infection, followed by selection with an appropriate concentration of puromycin for at least 48 hours. When the GFP positivity rate reached 100% and cell death ceased, the puromycin concentration was at least halved, and culture was continued to expand. Cells confirmed to express normal GFP by qPCR were cryopreserved for seed culture, and the stable mixed clone was constructed.

[0063] The expression of HLA-E*0103 or HLA-E*0101 molecules on the surface of transduced K562 cells was detected by flow cytometry. The specific method is as follows: 2 × 10⁻⁶ cells were collected... 5 K562 cells, HLA-E*0103 or HLA-E*0101 transduced K562 cells, were incubated with PE-labeled mouse anti-human HLA-E monoclonal antibody (clone number 3D12), along with an isotype control. Cells were incubated at 4°C in the dark for 30 min. After washing and centrifugation with flow cytometry wash buffer, the cells were resuspended and 300 μL of flow cytometry fixative was added for flow cytometry analysis. Results showed that the control group K562 cells expressed almost no HLA-E molecules, while the expression levels of HLA-E*0103 or HLA-E*0101 molecules were significantly increased in the HLA-E overexpression lentivirus group K562 cells. These results indicate that the HLA-E stably transfected K562 cell line was successfully constructed and can be used as an antigen-presenting cell for VMV epitope presentation.

[0064] 2) Loading SARS-CoV-2 antigenic peptides into HLA-E / K562 stable transgenic cells HLA-E / K562 cells were cultured in RPMI 1640 medium containing 10% FCS overnight at 26°C. After incubation, VMV antigen peptide was added to a final concentration of 100 μM. The culture medium was then switched to Opti-MEM and incubated at 26°C for another 16 h. The cells were washed with PBS to remove unbound antigen peptides before use.

[0065] 3) Detection of TCR-T cell antigen-specific activation function HLA-E / K562 cells preloaded with VMV antigen peptides were used as target cells and were mixed with TCR-T cells at a 1:1 ratio (4 × 10⁻⁶ cells each). 5(1 cell / well) was inoculated into 96-well plates and co-cultured at 37°C in a 5% CO2 incubator for 6 h. A negative control group, consisting of primary CD8 cells not infected with lentivirus, was also included. + T cells were co-cultured with HLA-E / K562 cells loaded with VMV. After co-culture, cells were collected, stained with anti-human CD69-BV605 monoclonal antibody, and the expression level of CD69, an early activation marker of TCR-T cells, was detected by flow cytometry.

[0066] The results of TCR-T cell activation detection are as follows: Figure 4 As shown. Figure 4 Image A shows representative results of CD69 expression in TCR-T cells detected by flow cytometry. CD88 was gated on the FSC-SSC scatter plot using NovoExpress 1.4.1 software. + T cell population, further analysis of CD8 + (x-axis) and CD69 + (Vertical axis) The proportion of double-positive cells, which is the proportion of CD69-positive T cells. Figure 4 As can be seen from Figure A, the proportion of CD8 and CD69 double-positive cells in the TCR-T cell group was significantly higher than that in the control group, indicating that TCR-T cells were effectively activated after recognizing the HLA-E / VMV complex on the surface of target cells. Figure 4 Figure B shows a statistical analysis graph comparing TCR-T cells with primary CD8 cells. + The difference in the proportion of CD69-positive cells among T cells (x-axis) and CD69-positive cells (y-axis). From Figure 4 As shown in Figure B, the CD69 positivity rate in the TCR-T group was significantly higher than that in the control group, with a statistically significant difference. The low level of CD69 expression in the control group may originate from the non-specific interaction between T cells and K562 cells, as well as the stimulation of cytokines during culture. The significantly increased CD69 expression level in the TCR-T group indicates that after TCR-T cells recognize the HLA-E / VMV complex, the TCR-mediated signal transduction pathway is activated, leading to upregulation of CD69 gene transcription and increased protein expression. Upregulation of CD69 expression is an early event in T cell activation, after which T cells will further secrete cytokines, proliferate, and differentiate into effector cells, ultimately exerting their function of killing target cells. The results showed that, compared with untransduced primary CD8+, CD69 expression was significantly higher in the TCR-T group. + Compared to T cells, the proportion of CD69-positive T cells was significantly increased upon stimulation with VMV antigen peptides on HLA-E*0103 stable transgenic cells. This result confirms that the TCR-T cells constructed in this invention can specifically recognize HLA-E-restricted VMV epitope peptides and undergo specific activation upon antigen stimulation, thus possessing the functional basis for exerting antiviral effects.

[0067] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A T-cell receptor that recognizes SARS-CoV-2 antigenic epitope peptides, characterized in that, It comprises a TCR α chain and a TCR β chain, wherein the amino acid sequence of the CDR3 region of the TCR α chain is shown in SEQ ID NO:1, and the amino acid sequence of the CDR3 region of the TCR β chain is shown in SEQ ID NO:

2.

2. The T-cell receptor for recognizing SARS-CoV-2 antigenic epitope peptides according to claim 1, characterized in that, The amino acid sequence of the TCR α chain is shown in SEQ ID NO:3; the amino acid sequence of the TCR β chain is shown in SEQ ID NO:

4.

3. The T-cell receptor for recognizing SARS-CoV-2 antigenic epitope peptides according to claim 1 or 2, characterized in that, This T-cell receptor can specifically recognize the SARS-CoV-2 antigenic epitope peptide with the amino acid sequence VMVELVAEL presented by the HLA-E molecule.

4. A TCR-T cell, characterized in that, The TCR-T cells express the T cell receptor that recognizes the SARS-CoV-2 antigenic epitope peptide as described in any one of claims 1-3.

5. The TCR-T cells according to claim 4, characterized in that, These TCR-T cells are CD8. + T cells, and CD8 + T cells originate from peripheral blood mononuclear cells.

6. The TCR-T cells according to claim 4, characterized in that, The TCR-T cells showed a binding rate of no less than 5% to the HLA-E / VMVELVAEL tetramer, and CD69 expression was upregulated after stimulation with HLA-E positive cells loaded with VMVELVAEL antigen peptide.

7. A host cell, characterized in that, The host cell contains a T-cell receptor that recognizes the SARS-CoV-2 antigenic epitope peptide according to any one of claims 1-3.

8. The use of the T cell receptor that recognizes the SARS-CoV-2 antigenic epitope peptide according to any one of claims 1-3, the TCR-T cell according to any one of claims 4-6, or the host cell according to claim 7 in the preparation of a medicament for the prevention or treatment of SARS-CoV-2 infection.

9. The use of the T cell receptor that recognizes the SARS-CoV-2 antigenic epitope peptide according to any one of claims 1-3, the TCR-T cell according to any one of claims 4-6, or the host cell according to claim 7 in the preparation of a vaccine for the prevention of SARS-CoV-2 infection.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the T-cell receptors that recognize SARS-CoV-2 antigenic epitope peptides according to any one of claims 1-3, TCR-T cells according to any one of claims 4-6, and host cells according to claim 7 as active ingredients, and a pharmaceutically acceptable carrier.

Images