A T-cell receptor capable of binding to HPV16 E6 antigen presented by HLA-C*14:02 molecules and its application

Abstract

This invention relates to a T-cell receptor capable of binding to the HPV16 E6 antigen presented by the HLA-C*14:02 molecule. The T-cell receptor comprises: αCDR1 (amino acid sequence as shown in SEQ ID NO:1), αCDR2 (amino acid sequence as shown in SEQ ID NO:3), αCDR3 (amino acid sequence as shown in SEQ ID NO:5), βCDR1 (amino acid sequence as shown in SEQ ID NO:11), βCDR2 (amino acid sequence as shown in SEQ ID NO:13), and βCDR3 (amino acid sequence as shown in SEQ ID NO:15). This invention provides a novel and precise immunotherapy strategy for patients with HLA-C*14:02 genotype HPV-related cervical cancer, significantly expanding the applicable population for TCR-T therapy.

Description

Technical Field

[0001] This invention relates to the field of T-cell receptor technology, and more particularly to a T-cell receptor capable of binding to the HPV16 E6 antigen presented by the HLA-C*14:02 molecule and its application. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors of the female reproductive system. HPV16 is the most prevalent high-risk subtype. The E6 and E7 proteins encoded by the early gene region of HPV16 are two major oncogenic proteins, persistently expressed in virus-infected cells and crucial for maintaining malignant transformation. Therefore, E6 and E7 proteins are not only biomarkers for cervical cancer diagnosis but also ideal targets for developing immunotherapy strategies.

[0003] Currently, treatments for HPV-related cervical cancer primarily include surgery, radiotherapy, and chemotherapy. However, traditional therapies have limited efficacy for recurrent or metastatic cervical cancer and are often accompanied by severe toxic side effects. In recent years, T-cell-based adoptive immunotherapy, particularly T-cell receptor-engineered T-cell (TCR-T) therapy, has opened up new avenues for the treatment of solid tumors. Unlike chimeric antigen receptor (CAR) therapy, TCR-T can recognize antigenic peptides derived from intracellular proteins presented by the major histocompatibility complex (MHC, known as human leukocyte antigen HLA in humans), allowing it to target intracellular oncoproteins such as E6 and E7.

[0004] However, HLA molecules are highly polymorphic, with different individuals expressing different HLA alleles. HLA-C*14:02 is a common but not yet fully explored allele for TCR-T therapy. Currently, there are no reports of T-cell receptors that specifically recognize the HPV16 E6 antigen presented by the HLA-C*14:02 molecule. Therefore, developing high-affinity, high-specificity TCRs targeting the HLA-C*14:02-restricted HPV16 E6 antigen epitope is of great significance for expanding the beneficiary population of TCR-T therapy and meeting the clinical treatment needs of cervical cancer patients with different HLA genotypes. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art, and therefore adopts the following technical solution: The first aspect of the present invention is to provide a T cell receptor, wherein the T cell receptor is capable of binding to the HPV16 E6 antigen presented by the HLA-C*14:02 molecule; the T cell receptor (TCR) includes: a TCR α chain variable domain and a TCR β chain variable domain, wherein the three complementarity-determining regions of the TCR α chain variable domain are: αCDR1 as shown in the amino acid sequence TSGFNG (SEQ ID NO: 1), αCDR2 as shown in the amino acid sequence NVLDGL (SEQ ID NO: 3), and αCDR3 as shown in the amino acid sequence AVREEDSNYQLI (SEQ ID NO: 5), and the three complementarity-determining regions of the TCR β chain variable domain are: βCDR1 as shown in the amino acid sequence LNHNV (SEQ ID NO: 11), βCDR2 as shown in the amino acid sequence YYDKDF (SEQ ID NO: 13), and βCDR3 as shown in the amino acid sequence ATRGESTEAF (SEQ ID NO: 15).

[0006] Chimeric TCRs are prepared by embedding the amino acid sequence of a TCR into any suitable framework structure. As long as the framework structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR region disclosed in the present invention. Therefore, the TCR molecule of the present invention refers to a TCR molecule containing the above-mentioned α chain and / or β chain CDR region sequence and any suitable framework structure.

[0007] The variable domain of the TCRα chain described in this invention is an amino acid sequence that has at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

[0008] Preferably, the amino acid sequence of the variable domain of the TCRα chain is as shown in MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVREEDSNYQLIW (SEQ ID NO: 7).

[0009] The variable domain of the TCRβ chain of the present invention is an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 11.

[0010] Preferably, the amino acid sequence of the variable domain of the TCRβ chain is as shown in MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDTAMYLCATRGESTEAFF (SEQ ID NO: 17).

[0011] Preferably, the constant region of the TCRα chain is mouse-derived.

[0012] Preferably, the amino acid sequence of the TCRα chain is as shown in MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVREEDSNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 9).

[0013] Preferably, the constant region of the TCRβ chain is mouse-derived.

[0014] Preferably, the amino acid sequence of the TCRβ chain is as shown in MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDTAMYLCATRGESTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 19).

[0015] A second aspect of the present invention is to provide a nucleic acid molecule comprising: a nucleic acid sequence encoding a T cell receptor as described above, or a complementary sequence thereof.

[0016] Preferably, the nucleic acid molecule comprises: the nucleotide sequence ACATCTGGGTTCAACGGG (SEQ ID NO: 2) encoding the αCDR1, the nucleotide sequence AATGTTCTGGATGGTTTG (SEQ ID NO: 4) encoding the αCDR2, and the nucleotide sequence GCTGTGAGAGAAGAGGATAGCAACTATCAGTTAATC (SEQ ID NO: 6) encoding the αCDR3.

[0017] Preferably, the nucleic acid molecule comprises: the nucleotide sequence TTGAACCATAACGTC encoding the βCDR1 (SEQ ID NO: 12), the nucleotide sequence TACTATGACAAAGATTTT encoding the βCDR2 (SEQ ID NO: 14), and the nucleotide sequence GCCACCAGGGGAGAAAGCACTGAAGCTTTC encoding the βCDR3 (SEQ ID NO: 16).

[0018] Preferably, the nucleic acid molecule comprises: a nucleotide sequence encoding the variable domain of the TCRα chain, ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACTACAGGACAAAACATTGACCAGCCCACTGAGATGACAGCTACGGAAGGTGCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGGCTGTTCTGGTACCAGCAACATGCTGGCGAAGCACCCACATTTCTGTCTTACAATGTTCTGGATGGTTTGGAGGAGAAAGGTCGTTTTTCTTCATTCCTTAGTCGGTCTAAAGGGTACAGTTACCTCCTTTTGAAGGAGCTCCAGATGAAAGACTCTGCCTCTTACCTTTGCGCTGTGAGAGAAGAGGATAGCAACTATCAGTTAATCTGG (SEQ ID NO: 8).

[0019] Preferably, the nucleic acid molecule comprises: a nucleotide sequence encoding the variable domain of the TCRβ chain: ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTTGACATCCGCTCACCAGGCCTGGGGGACACAGCCATGTACCTCTGTGCCACCAGGGGAGAAAGCACTGAAGCTTTCTTC (SEQ ID NO: 18).

[0020] Preferably, the nucleic acid molecule comprises: the nucleotide sequence ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACTACAGGACAAAACATTGACCAGCCCACTGAGATGACAGCTACGGAAGGTGCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGGCTGTTCTGGTACCAGCAACATGCTGGCGAAGCACCCACATTTCTGTCTTACAATGTTCTGGATGGTTTGGAGGAGAAAGGTCGTTTTTCTTCATTCCTTAGTCGGTCTAAAGGGTACAGTTACCTCCTTTTGAAGGAGCTCCAGATGAAAGACTCTGCCTCTTACCTTTGCGCTGTGAGAGAAGAGGATAGCAACTATCAGTTAATCTGGGGCGCTGGGACCAAGCTAATTATAAAGCCAGACATTCAGAACCCGGATCCTGCCGTTTATCAGTTGCGGGACTCCAAGAGCAGTGATAAATCTGTCTGCCTTTTTACAGACTTTGACAGTCAAACGAATGTGTCCCAGAGTAAGGACAGCGACGTATATATTACAGATAAGACCGTTCTTGACATGAGAAGCATGGACTTCAAAAGCAACTCTGCGGTCGCGTGGAGCAATAAGAGTGATTTCGCCTGTGCGAACGCATTTAACAACAGTATCATCCCCGAAGATACCTTTTTTCCTAGTCCTGAAAGCTCATGTGATGTCAAATTGGTCGAGAAATCCTTTGAAACCGACACCAATCTCAATTTCCAGAACCTGTCCGTTATAGGCTTTAGAATACTTCTTCTCAAGGTGGCCGGATTTAACCTTCTCATGACGCTCCGACTGTGGTCTAGC (SEQ ID NO: 10) encoding the TCR α chain.

[0021] Preferably, the nucleic acid molecule comprises: the nucleotide sequence ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTTGACATCCGCTCACCAGGCCTGGGGGACACAGCCATGTACCTCTGTGCCACCAGGGGAGAAAGCACTGAAGCTTTCTTCGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAAGTATTCCCTCCGGAGGTCGCCGTATTCGAACCGTCAGAAGCTGAAATAAGTCATACTCAAAAGGCGACATTGGTGTGTTTGGCTACAGGATTCTTCCCTGACCACGTAGAACTCAGTTGGTGGGTAAACGGAAAAGAGGTTCATTCCGGTGTTTCTACAGACCCGCAGCCCCTGAAAGAGCAGCCAGCACTGAATGATTCTAGGTATTGTCTCTCATCAAGACTGCGGGTCAGCGCAACCTTCTGGCAGAACCCACGCAATCACTTCAGATGTCAGGTTCAATTCTACGGCCTCTCAGAGAATGACGAATGGACTCAGGATAGGGCCAAGCCGGTAACACAAATCGTGTCTGCTGAGGCGTGGGGACGAGCATGTGGTTTTACCAGTAGCTACCAGCAGGGGGTCCTTTCTGCAACTATTCTTTATGAAATCCTCCTTGGCAAGGCTACATTGTATGCGGTACTCGTATCTGCCCTTGTACTGATGGCTATGGTGAAACGGAAAGACTTC (SEQ ID NO: 20) that encodes the TCR β chain.

[0022] Nucleotide sequences can be codon-optimized; different cells utilize specific codons differently, and codons in the sequence can be changed according to cell type to improve expression levels; codon selection tables for mammalian cells and many other organisms are well known to those skilled in the art.

[0023] The full-length sequence or fragment of the nucleic acid molecule of the present invention can generally be obtained by, but is not limited to, PCR amplification, recombination or artificial synthesis. Currently, the DNA sequence encoding the TCR (or its fragment / or its derivative) of the present invention can be completely obtained by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. The DNA can be a coding strand or a non-coding strand.

[0024] A third aspect of the present invention is to provide a carrier comprising: a nucleic acid molecule as described above.

[0025] Preferably, the vector is a viral vector.

[0026] More preferably, the vector is a retroviral vector or a lentiviral vector.

[0027] A fourth aspect of the present invention is to provide a host cell comprising: a T cell receptor as described above, a nucleic acid molecule as described above, or a vector as described above.

[0028] Preferably, the host cell is a T cell, NK cell, NKT cell, or stem cell.

[0029] More preferably, the host cell is a T cell.

[0030] Most preferably, the host cell is a Jurkat cell.

[0031] A fifth aspect of the present invention is to provide a pharmaceutical composition comprising: a T-cell receptor as described above, and a pharmaceutically acceptable carrier.

[0032] A sixth aspect of the invention is to provide the use of the T-cell receptor, nucleic acid molecule, carrier, or pharmaceutical composition as described above in the preparation of a medicament for treating cervical cancer.

[0033] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention fills the gap in TCR targeting the HLA-C*14:02-restricted HPV16 E6 antigen; the obtained TCR not only has high specificity and recognition activity, but more importantly, after being engineered into T cells, it can effectively kill HPV16-positive cervical cancer cells; this provides a novel and precise immunotherapy strategy for patients with HLA-C*14:02 genotype HPV-related cervical cancer in clinical practice, significantly expanding the applicable population of TCR-T therapy, and has extremely high clinical application value and commercial prospects. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the structure of the T cell receptor in this invention.

[0035] Figure 2 The image shows the flow cytometry results, illustrating the specificity of the TCR-JTR cells of this invention in recognizing target cells. The target cells were HEK-293T cells co-expressing HLA-C*14:02 and HPV16 E6 (experimental group) or HEK-293T cells expressing only HLA-C*14:02 (negative control group).

[0036] Figure 3 The image shows the flow cytometry results, illustrating the ability of JTR cells expressing the TCR of this invention to recognize HLA-C*14:02-positive target cells loaded with the HPV16 E6-derived peptide KFYSKISEY. HLA-C*14:02-positive target cells without the peptide were used as a negative control.

[0037] Figure 4 The figure shows the cytotoxicity experiment results, illustrating the killing efficiency of the TCR-T cells of this invention against target cells. Effector cells were T cells transduced with the TCR of this invention, and target cells were SiHa cells (endogenously expressing HPV16 E6) of the cervical cancer cell line expressing HLA-C*14:02. Untransduced T cells served as a negative control.

[0038] Figure 5 Comparison of ex vivo tumor size between the TCR-T group and the control group in the NDG mouse model bearing SiHa cells (expressing HLA-C*14:02) (day 12 after infusion).

[0039] Figure 6 The bar chart shows the comparison of subcutaneous tumor weight in the right abdomen of NDG mice in the TCR-T group and the control group on day 12 after infusion. Data are expressed as mean ± standard error. *P<0.05. Detailed Implementation

[0040] The specific embodiments of the present invention will be described in detail below.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0042] The word "comprising" or similar terms used in the specification and claims of this patent application mean that the objects preceding "comprising" include the objects listed after "comprising" or their equivalents, and do not exclude other objects.

[0043] Example 1: TCR-JTR Cell Recognition and Screening Experiment 1. TCR Molecular Cloning Based on the amino acid sequence of the T cell receptor described in this invention, a codon-optimized encoding nucleotide sequence was designed and synthesized, cloned into the pHSIB retroviral vector, and the recombinant plasmid pHSIB-TCR was constructed and used after being verified by sequencing.

[0044] 2. Preparation of TCR-JTR cells 2.1 Transfection of 293FT cells to produce virus The day before transfection, 293FT cells in good condition were digested with 2 mL of 1× trypsin at 37°C for 2 minutes. After observing the cells under a microscope and confirming they had become rounded, 2 mL of complete culture medium was added to resuspend the cells, and the cells were collected in a 15 mL centrifuge tube. The tubes were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 2 mL of DMEM complete culture medium containing 10% FBS and without antibiotics was added to resuspend the cells. 5 μL of the cell suspension was mixed thoroughly with 0.4% trypan blue solution at a 1:1 ratio, and the cells were counted using a cell counter. The cell density was adjusted to 1.2 × 10⁻⁶ cells / mL. 6 Add 2 mL of culture medium to each well, seed into a 6-well plate, and incubate overnight at 37°C in a cell culture incubator containing 5% CO2.

[0045] The next day, after the cells have adhered, prepare two 1.5 mL sterile EP tubes: add 125 μL of Opti-MEM and 7.5 μL of Lipofectamine to the first EP tube. TM3000, mix thoroughly and let stand for 5 minutes; add 125 μL Opti-MEM, target gene plasmid pHSIB-TCR, helper plasmid pHIT-60, and VSVG (total 2.5 μg) to the second EP tube, mix thoroughly, then add 5 μL P3000 reagent and mix well. Add the plasmid mixture from the second EP tube to the first EP tube, mix thoroughly, and let stand at room temperature for 15-20 minutes. Slowly add the formed DNA-liposome complex along the well wall to a 293FT cell 6-well plate, gently mix from side to side, and place in a cell culture incubator for further culture.

[0046] Six to eight hours after transfection, replace the original medium in the six-well plate with 2 mL / well of RPMI 1640 complete medium preheated at 37°C containing 10% FBS and 2% P / S antibiotics, and continue culturing in a cell culture incubator. After 36 hours of culture, collect the viral supernatant, centrifuge at 2500 rpm for 5 minutes at 4°C to remove cell debris, and obtain the supernatant containing retroviruses for later use.

[0047] 2.2 Transduction of virus to construct TCR-JTR cells In this invention, "JTR cells" are a type of TCR signaling reporter cell constructed in our laboratory (Wang X, Song X, Li Y, et al. Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data. Journal for ImmunoTherapy of Cancer, 2025, 13(7): e012029). These cells are based on the human acute T-cell leukemia cell line Jurkat. By using gene editing technology, the endogenous T-cell receptor (TCR) and CD4 molecules were knocked out to eliminate the interference of endogenous TCR on the detection results and reduce the recognition background. Simultaneously, a green fluorescent protein (GFP) reporter system regulated by TCR activation signals was introduced into these cells. Specifically, GFP expression is regulated by downstream TCR signaling pathways; only when exogenous TCRs successfully recognize specific antigens and activate downstream signaling pathways will GFP expression be driven, causing the cells to emit green fluorescence.

[0048] Take JTR cells in good growth condition, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in 5 mL of RPMI 1640 complete medium containing 10% FBS and 2% P / S antibiotics. Mix 5 μL of the cell suspension with 0.4% trypan blue solution at a 1:1 ratio and count the cells using a cell counter. Take 5 × 10⁶ cells... 5Add 100 μL of RPMI 1640 complete medium containing 10% FBS and 2% P / S antibiotics to each well, and add 4 µg / mL polybrene and 500 μL of the collected viral supernatant to each well to a final volume of 1 mL. After incubating at room temperature for 10 minutes, centrifuge at 30°C and 2000 rpm for 2 hours, and then place in a cell culture incubator for further culture.

[0049] 2.3 Screening of stable TCR-JTR cells Forty-eight hours after cell transduction, 16 μg / mL of blast fungicide (Beyotime) was added to the culture medium for drug screening. JTR cells that permanently expressed TCRαβ were continuously cultured to obtain the stable transgenic TCR-JTR cell line for subsequent functional testing.

[0050] 3. Preparation of HLA-C*14:02 expressing cells The gene encoding HLA-C*14:02 was cloned into the retroviral vector pHSIB, which is resistant to blast fungicide S, and the virus was packaged using the same method as in step 2.1 above. HEK-293T cells with HLA-A / B / C knockout were then cultured at a rate of 5 × 10⁻⁶ cells / year. 5 Cells were seeded per well in 6-well plates. After the cells adhered and grew well, the above-mentioned viral supernatant was added, along with 4 µg / mL polybrene. After 48 hours, 10 µg / mL blastcin (Beyotime) was added for drug screening. After another 48 hours of culture, HEK-293T cell lines permanently expressing HLA-C*14:02 were obtained.

[0051] 4. Target cells (HLA-C*14:02) + HPV16 E6 + Preparation of ) The gene encoding HPV16 E6 was cloned into the pHSIP retroviral vector containing puromycin resistance, and the virus was packaged using the same method as in step 2.1 above. HEK-293T cells expressing HLA-C*14:02, constructed in step 3 above, were then cultured at a rate of 5 × 10⁻⁶ cells / year. 5 Cells were seeded per well in 6-well plates. After the cells adhered and grew well, the viral supernatant was added, along with 4 µg / mL polybrene. After 48 hours, 0.5 µg / mL puromycin dihydrochloride (Beyotime) was added for drug screening. After another 48 hours of culture, HEK-293T cells that permanently co-expressed HLA-C*14:02 and HPV16 E6 were obtained and used as target cells for subsequent experiments.

[0052] 5. Functional detection of TCR-JTR cells recognizing target cells HEK-293T cells co-expressing HLA-C*14:02 and HPV16 E6, constructed in step 4 above, were used as target cells at a density of 2 × 10⁻⁶. 5 Cells were seeded per well in 24-well plates and allowed to adhere well before use. A negative control group was also set up, consisting of HEK-293T cells (constructed in step 3) that expressed only HLA-C*14:02 and not HPV16 E6.

[0053] The TCR-JTR cells constructed in step 2 were used as effector cells and added to the 24-well plate at an effector-to-target ratio of 2:1 (effector cells: target cells), i.e., 4 × 10⁶ cells were added to each well. 5 TCR-JTR cells were incubated at 37°C for 24 hours. Cells were then collected, stained with a live / dead cell fixative dye and PE-CD8β antibody, and the percentage of GFP-positive cells was determined by flow cytometry to assess TCR. - JTR cell activation status.

[0054] 6. Experimental Results Experimental results are as follows Figure 2 As shown, when the target cells were HEK-293T cells co-expressing HLA-C*14:02 and HPV16 E6, the proportion of GFP-positive cells in TCR-JTR cells was significantly increased, indicating that the TCR was specifically activated. However, when the target cells expressed only HLA-C*14:02 (without HPV16 E6), the proportion of GFP-positive cells in TCR-JTR cells was comparable to the background control, and no significant activation was observed. These results indicate that the T cell receptor provided by this invention can specifically recognize the HPV16 E6 antigen presented by the HLA-C*14:02 molecule, without cross-reactivity to individual HLA molecules.

[0055] Example 2: Identification of HPV16 E6 antigenic epitope 1. Bioinformatics prediction of candidate epitopes To determine the specific antigenic epitopes recognized by the screened positive TCRs, the amino acid sequences of HPV16 E6 and E7 proteins were first analyzed using NetMHCpan-4.1 bioinformatics software to predict candidate antigenic peptides that may bind to the HLA-C*14:02 molecule. The prediction results showed that a total of 9 candidate peptides met the screening criteria, all originating from the HPV16 E6 protein.

[0056] 2. Preliminary verification of candidate epitopes 2.1 Candidate peptide grouping and expression plasmid construction Based on the distribution of the nine candidate peptides on the HPV16 E6 protein, they were divided into three groups: group #1 containing one peptide, group #2 containing five adjacent peptides, and group #3 containing three adjacent peptides. Retroviral expression plasmids capable of expressing the candidate peptides from each group were then constructed.

[0057] 2.2 Preparation of target cells Following the method described in Section 4 of Example 1, plasmids expressing each candidate peptide were transduced into HEK-293T cells that had stably expressed HLA-C*14:02, thereby constructing three target cell lines: C*14:02, HLA-C ... + Antigen peptide #1 + Cells, C*14:02 + Antigen peptide #2 + Cells and C*14:02 + Antigen peptide #3 + cell.

[0058] 2.3 Preliminary Identification and Verification The three types of target cells constructed above were respectively used at 2×10 5 4 × 10⁶ cells / well were seeded into 24-well plates. After cell adhesion, TCR-JTR cells (4 × 10⁶ cells / well) were added at an effector-to-target ratio of 2:1. 5 Cells were incubated at 37°C and 5% CO2 for 24 hours. Cells were then collected, stained with a live / dead cell fixative dye and PE-CD8β antibody, and the percentage of GFP-positive cells was detected by flow cytometry.

[0059] Experimental results showed that only antigen peptide #3 could induce a significant increase in the proportion of GFP-positive cells in TCR-JTR cells, while neither antigen peptide #1 nor antigen peptide #2 showed significant activation. This result indicates that the antigen peptide recognized by the TCR described in this invention is located in the region corresponding to antigen peptide #3.

[0060] 3. Identification of the core antigen peptide 3.1 Synthesis of Antigenic Peptides To further identify the core antigenic epitope recognized by TCR, three candidate peptides contained in antigenic peptide #3 were chemically synthesized. Each peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity greater than 95%. They were dissolved in sterile water and prepared into stock solutions of appropriate concentrations, and stored at -20°C for later use.

[0061] 3.2 Target cell loading experiment The day before the experiment, HEK-293T cells stably expressing HLA-C*14:02 were introduced at a rate of 2×10⁻⁶. 5Cells were seeded per well in 24-well plates and incubated overnight at 37°C with 5% CO2. The next day, after the cells adhered, the synthesized candidate antigen peptide was added to each well to a final concentration of 0.1 µg / mL. The cells were gently mixed and incubated for another 3-4 hours to allow the peptides to bind fully to HLA molecules on the cell surface.

[0062] After incubation, TCR-JTR cells were added to each well as effector cells at an effector-to-target ratio of 2:1, i.e., 4 × 10⁶ cells were added to each well. 5 Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0063] 3.3 Flow cytometry detection After co-incubation for 24 hours, cells from each well were collected and stained with a live / dead cell fixation viability dye and PE-CD8β antibody. The percentage of GFP-positive cells was determined by flow cytometry to assess the activation status of TCR-JTR cells.

[0064] 4. Experimental Results Experimental results are as follows Figure 3 As shown, when sending to HLA-C*14:02 + When HEK-293T cells were loaded with the peptide KFYSKISEY (SEQ ID NO: 21), the proportion of GFP-positive cells in TCR-JTR cells increased significantly, indicating that TCR was specifically activated. However, no significant increase in the proportion of GFP-positive cells was observed in cells loaded with other candidate peptides from this group, or in the negative control group cells without peptide loading.

[0065] These results indicate that the specific antigenic epitope recognized by the T-cell receptor described in this invention is the short peptide KFYSKISEY derived from HPV16 E6 and presented by the HLA-C*14:02 molecule. The identification of this epitope provides a clear molecular basis for further optimizing TCR-T cell therapy strategies and evaluating treatment efficacy.

[0066] Example 3: TCR-T cell killing experiment 1. TCR Molecular Cloning Based on the amino acid sequence of the T cell receptor described in this invention, a codon-optimized encoding nucleotide sequence was designed and synthesized (to avoid mismatch between the TCR and the endogenous TCR, the constant region of the human TCR was replaced with the constant region of the mouse TCR) and cloned into a pMSCV retroviral vector labeled with green fluorescent protein (GFP) to construct the recombinant plasmid pMSCV-TCR. After being verified by sequencing, it was put into use.

[0067] 2. Transfecting 293FT cells to produce viruses Virus packaging was performed according to the method described in Section 2.1 of Example 1.

[0068] 3. Preparation of TCR-T cells 3.1 PBMC cell resuscitation Remove the PBMC cryovials (purchased from Junxing Biotechnology Co., Ltd., rigorously quality-tested to ensure they are free of infectious viruses such as HIV, HBV, HCV, and TP, and negative for bacteria, fungi, and mycoplasma) from liquid nitrogen and quickly place them in a 37°C water bath, gently shaking until completely thawed. Transfer the cell suspension to centrifuge tubes containing pre-warmed T-cell culture medium, centrifuge at 400×g for 8 minutes, and discard the supernatant. Resuspend the cells in an appropriate amount of T-cell culture medium, and mix a small amount of the cell suspension with 0.4% trypan blue solution at a 1:1 ratio, then count the cells using a cell counter. To ensure normal cell growth, add recombinant human IL-2 (2.5 ng / mL), recombinant human IL-7 (5 ng / mL), and recombinant human IL-15 (5 ng / mL) to the T-cell culture medium, adjusting the cell density to an appropriate concentration for later use.

[0069] 3.2 Human T cell activation The T cells in PBMCs are activated using human CD3 / CD28 T cell activation magnetic beads. The specific procedure is as follows (using a 96-well plate as an example): Magnetic bead pretreatment: Thoroughly resuspend the magnetic beads (vortex for at least 30 seconds). Transfer 25 μL of magnetic beads to a sterile flow cytometry tube, add 1 mL of washing buffer, vortex for 5 seconds, place on a magnetic separator, and magnetically separate for 3 minutes. Discard the supernatant. Repeat the washing process once with cell culture medium. Finally, resuspend the magnetic beads in 1 mL of cell culture medium, at which point the magnetic bead concentration is 2.5 × 10⁻⁶. 6 The magnetic beads per mL are sufficient for 10 wells in a 96-well plate.

[0070] T cell activation: Adjust the T cell concentration to 1×10⁶ cells using culture medium. 7 Cells / mL. Add 25 μL of T cell suspension and 75 μL of cell culture medium to each well to make the cell count 2.5 × 10⁶ cells / mL. 5 The well plate has a volume of 100 μL. Add 100 μL of the cleaned magnetic beads (2.5 × 10⁻⁶ beads). 5 (Number of cells), ensuring a magnetic bead:cell ratio of 1:1, with a final well volume of 200 μL. The wells containing the seeded cells and magnetic beads were incubated at 37°C in a 5% CO2 incubator for 48 hours for activation.

[0071] 3.3 Transducing viruses to construct TCR-T cells After T cells were stimulated with CD3 / CD28 magnetic beads for 48 hours, cells and supernatant from each well of a 96-well plate were transferred to a 1.5 mL EP tube. The EP tube was placed on a magnetic rack for 2-3 minutes to separate the magnetic beads from the supernatant, and the supernatant was discarded. The EP tube was removed from the magnetic rack, and the cells were resuspended in 250 μL of T cell culture medium and transferred to a 24-well plate. 4 µg / mL polybrene and 750 μL of the previously collected viral supernatant were added to each well to a final volume of 1 mL. After standing at room temperature for 10 minutes, the cells were centrifuged at 30°C and 2000 rpm for 2 hours for viral infection. After centrifugation, 750 μL of supernatant was discarded, and 750 μL of PBMC culture medium was added. Recombinant human IL-2 (5 ng / mL), recombinant human IL-7 (10 ng / mL), and recombinant human IL-15 (10 ng / mL) were then added. The cells were carefully homogenized and placed in a cell culture incubator for further culture.

[0072] After 48 hours of culture, a portion of the cells were taken and stained with live / dead cell fixation viability dyes and PE-TCRα / β antibodies. The transduction efficiency of TCR was detected by flow cytometry to obtain TCR-T cells.

[0073] 4. Target cell preparation Construction of the SiHa cell line expressing HLA-C*14:02: The gene encoding HLA-C*14:02 was cloned into the retroviral vector pHSIB, which is resistant to blast fungicide S, and the virus was packaged according to the method described in Section 2.1 of Example 1. The HPV16-positive cervical cancer cell line SiHa cells were cultured at 5 × 10⁻⁶ cells / year. 5 Virus supernatant was seeded per well in 6-well plates. After 6 hours of incubation, 4 µg / mL polybrene was added. 48 hours later, 10 µg / mL blastcin (Beyotime) was added for drug screening. Screening continued for one week to obtain a stable SiHa cell line expressing HLA-C*14:02. + SiHa), used for subsequent lethality experiments.

[0074] 5. Experiment on TCR-T cell-targeted killing of SiHa cells 5.1 Experimental Grouping and Co-culture The above-constructed expression HLA-C*14:02 + SiHa cells were used as target cells, with 5 × 10⁻⁶ cells. 3 Cells were seeded per well in 96-well plates and incubated at 37°C in a 5% CO2 incubator until they adhered well and grew well. The following experimental groups were set up: Sample group: target cells and TCR-T cells (effector cells) Control group: Target cells and activated T cells that do not express HPV TCR (control T cells) Total LDH group: target cells and lysis buffer (used to determine maximum LDH release) Add effector cells (or control T cells) at an effector-to-target ratio of 10:1, i.e., add 5 × 10⁶ cells per well. 4 1 effector cell (or control T cell). Incubate at 37°C, 5% CO2 for 12 hours.

[0075] 5.2 Kill Efficiency Test After 12 hours of co-incubation, the killing efficiency of TCR-T cells against target cells was assessed using the Beyotime lactate dehydrogenase cytotoxicity assay kit (WST-8 method, catalog number C0018). The specific procedures were performed according to the kit instructions. After co-culturing, the 96-well plate was centrifuged at 400×g for 5 minutes.

[0076] Carefully aspirate the supernatant from each well and transfer it to a new 96-well plate, 100 μL per well.

[0077] Add 100 μL of WST-8 colorimetric working solution to each well and mix well.

[0078] Incubate in the dark for 10-30 minutes.

[0079] Add 20 μL of stop solution to each well and mix well.

[0080] The absorbance (OD) of each well was measured at 450 nm using a microplate reader. 450 ).

[0081] 5.3 Calculation of lethality The lethality rate was calculated using the following formula, with three replicate wells for each experimental group: Kill rate (%) = (Absorbance of sample group - Absorbance of control group) / (Total absorbance of LDH group - Absorbance of control group) × 100% 6. Experimental Results Experimental results are as follows Figure 4 As shown, the TCR-T cells constructed in this invention exhibit significant targeted killing activity against SiHa cells expressing HLA-C*14:02, with a killing rate far exceeding that of the control T cell group without TCR transduction. This result fully demonstrates that the T cell receptor provided by this invention can endow T cells with potent and specific anti-tumor activity, effectively recognizing and eliminating tumor cells endogenously expressing HPV16 E6 (HLA-C*14:02). + SiHa cells).

[0082] Example 4: In vivo killing experiment of TCR-T cells 1. Experimental Materials and Animals The SPF-grade NDG mice used in this example were purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd., a total of 10 female mice, 6-8 weeks old, weighing 18g-22g. All animal experimental procedures were reviewed and approved by the Laboratory Animal Ethics Committee and strictly adhered to relevant ethical regulations. The preparation method of SiHa target cells expressing HLA-C*14:02 is detailed in Section 4 of Example 3. The preparation method of TCR-T cells is detailed in Section 3 of Example 3.

[0083] 2. Tumor modeling Ten NDG mice were used, and each mouse was subcutaneously inoculated with SiHa cells expressing HLA-C*14:02 in the right abdomen. Before inoculation, the cells were washed twice with PBS and resuspended, and the cell density was adjusted to 1×10⁻⁶. 7 Cells / mL. Take 100 μL of cell suspension (containing 1×10⁶ cells / mL). 6 Mix 100 cells with an equal volume of matrix gel (volume ratio 1:1) to prepare a mixing medium, and keep it on ice for later use. Using a sterile syringe, draw 1 mL of the mixture and slowly inject it subcutaneously into the right abdomen of each mouse. A wheal will form locally after injection. The day of inoculation is defined as day 0.

[0084] 3. TCR-T cell tumor therapy On day 7 post-inoculation, the mouse tumor had grown to approximately 100 mm². 3 The TCR-T cells were infused via tail vein. The cells were washed twice with sterile PBS and the cell density was adjusted to 3 × 10⁻⁶ cells / mL. 7 Cells / mL. 200 μL of cell suspension was reinfused into each mouse via the tail vein, i.e., 6 × 10⁶ cells per mouse. 6 Each TCR-T cell. Control group mice received an equal volume (6 × 10⁶) of untransduced TCR T cells via tail vein infusion. 6 (each / each).

[0085] 4. Intraperitoneal injection of recombinant human IL-2 Starting on day 1 after TCR-T cell infusion via the tail vein, recombinant human IL-2 was administered intraperitoneally every 3 days, with a dose of 100 ng / animal per injection and an injection volume of 50 μL (diluted to 2 μg / mL with sterile PBS). This was repeated for a total of 3 injections.

[0086] 5. Tumor tissue collection and analysis On day 12 after infusion, the experimental animals were euthanized by cervical dislocation. After euthanasia, the subcutaneous tumor on the right side of the mouse's abdomen was completely dissected, washed with pre-cooled PBS to remove surface blood, and blotted dry with filter paper. The gross appearance of the tumor was recorded and photographed (see [link to original text]). Figure 5Then, the weight of each group of tumors was measured using an electronic balance, and the data was recorded.

[0087] 6. Statistical Analysis Experimental data are expressed as mean ± standard error (Mean ± SEM). Independent samples t-tests were used to compare the two groups, and a p-value < 0.05 was considered statistically significant.

[0088] 7. Experimental Results Experimental results are as follows Figure 6 As shown in the figure, compared with the control group, the tumor volume of mice in the TCR-T group was significantly reduced and the tumor weight was significantly decreased, with statistically significant differences (P<0.05). These results indicate that in a tumor model of subcutaneous SiHa cells (expressing HLA-C*14:02) in the right abdomen of NDG mice, TCR-T cell infusion therapy can significantly inhibit tumor growth and effectively reduce tumor weight, confirming that the TCR-T cells described in this invention have potent anti-tumor activity in vivo.

[0089] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A T cell receptor, characterized in that, The T cell receptor is capable of binding to an HPV16E6 antigen presented by an HLA-C*14:02 molecule; the T cell receptor comprises: a TCRa chain variable domain and a TCRP chain variable domain, three complementarity determining regions of the TCRa chain variable domain are: aCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, aCDR2 with an amino acid sequence as shown in SEQ ID NO: 3, and aCDR3 with an amino acid sequence as shown in SEQ ID NO: 5, three complementarity determining regions of the TCRP chain variable domain are: bCDR1 with an amino acid sequence as shown in SEQ ID NO: 11, bCDR2 with an amino acid sequence as shown in SEQ ID NO: 13, and bCDR3 with an amino acid sequence as shown in SEQ ID NO:

15.

2. The T cell receptor of claim 1, wherein, The amino acid sequence of the TCRa chain variable domain is as shown in SEQ ID NO: 7, or / and the amino acid sequence of the TCRP chain variable domain is as shown in SEQ ID NO:

17.

3. The T cell receptor according to claim 1 or 2, characterized in that The amino acid sequence of the TCRa chain is as shown in SEQ ID NO: 9, or / and the amino acid sequence of the TCRP chain is as shown in SEQ ID NO:

19.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises: a nucleic acid sequence encoding the T cell receptor as claimed in any one of claims 1-3, or a complement thereof.

5. The nucleic acid molecule of claim 4, wherein, The nucleic acid molecule comprises: a nucleotide sequence SEQ ID NO: 8 encoding the TCRa chain variable domain, or / and a nucleotide sequence SEQ ID NO: 18 encoding the TCRP chain variable domain.

6. The nucleic acid molecule of claim 4 or 5, wherein, The nucleic acid molecule comprises: a nucleotide sequence SEQ ID NO: 10 encoding the TCRa chain, or / and a nucleotide sequence SEQ ID NO: 20 encoding the TCRP chain.

7. A vector, characterized in that, The vector comprises: the nucleic acid molecule as claimed in any one of claims 4-6.

8. A host cell, characterized in that, The host cell comprises: the T cell receptor as claimed in any one of claims 1-3, the nucleic acid molecule as claimed in any one of claims 4-6, or the vector as claimed in claim 7.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: the T cell receptor as claimed in any one of claims 1-3, and a pharmaceutically acceptable carrier.

10. Use of the T cell receptor as claimed in any one of claims 1-3, the nucleic acid molecule as claimed in any one of claims 4-6, the vector as claimed in claim 7, or the pharmaceutical composition as claimed in claim 9 in the preparation of a medicament for treating cervical cancer.

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