T cell receptors and uses thereof

Abstract

The application provides a T cell receptor and application thereof, and particularly relates to a T cell receptor (TCR) capable of specifically recognizing a glioblastoma-associated antigen and application thereof, wherein the glioblastoma-associated antigen can form a complex with HLA-A*0201 and be presented to the surface of a glioblastoma cell. The T cell of a patient is reformed into a cytotoxic T cell (TCR-T) specific to the GB-associated antigen. When the gene-edited TCR-T cell is input into the patient, the TCR-T cell specific to the GB-associated antigen is activated by specific recognition when encountering the antigen complex on the tumor cell, thereby expanding in the patient and achieving the effect of treating the tumor by killing the tumor cell. The application provides a new way for treating glioblastoma.

Description

Technical Field

[0001] This invention belongs to the technical field of T-cell receptors (TCRs) for glioblastoma and their applications. Specifically, it relates to T-cell receptors (TCRs) capable of recognizing glioblastoma, glioblastoma-specific T cells obtained from the aforementioned TCRs, methods for their preparation, and their applications in the prevention, treatment, and diagnosis of glioblastoma-related tumors. Background Technology

[0002] Glioblastoma (GB) is the most common and aggressive primary brain tumor, and one of the most challenging malignant tumors of all. As the disease progresses, patients diagnosed with this tumor often have a poor prognosis and a low quality of life. In recent years, the standard treatment for newly diagnosed malignant gliomas has included surgery, temozolomide combined with radiotherapy, chemotherapy, and alternating electric field therapy. However, if recurrence occurs, there are no standard treatment interventions, and patient survival is less than 9 months or even shorter. Furthermore, with the continuous improvement and development of immunotherapy methods, the application of immunotherapy in GB should not be underestimated, and promising cellular immunotherapy is gradually developing in GB treatment. Immunotherapy for GB is a promising alternative to conventional treatments, with the potential for long-term benefits in generating a sustainable anti-tumor response.

[0003] Currently, tumor immunotherapy strategies targeting glioblastoma-associated antigens (GAAs) mainly include specific tumor vaccines and adoptive T-cell therapy. Tumor DC vaccines developed around GAAs have entered early clinical trials in glioblastoma patients, with preliminary verification of their safety and efficacy; however, their long-term clinical efficacy requires further validation. Adoptive T-cell therapy targeting cytomegalovirus (CMV) mainly includes tumor-infiltrating lymphocytes (TILs) and in vitro-induced specific TIL reinfusion. However, the isolation and culture of TILs are demanding, requiring a long time to reach the required cell count for clinical treatment. More importantly, the number of tumor tissues from which TILs can be successfully isolated is still very limited, thus restricting the clinical application of TILs. Due to the long production cycle, significant individual variability, and poor tolerability of these traditional T-cell therapies, their clinical application has certain limitations. The combined use of glioblastoma-associated antigen vaccines with other anti-tumor therapies has shown promising results in preclinical trials, but its clinical efficacy remains exploratory. Clinical studies on the application of TCR-T in solid tumors are increasing annually. As one of the important driving factors for the occurrence, development, invasion, metastasis, drug resistance and recurrence of glioblastoma, endogenous antigenic peptides of GB have a very broad application prospect in GB. The development of TCR-T cell immunotherapy with glioblastoma-associated antigens with stronger effect and better tolerance is very promising.

[0004] By using a non-viral gene transduction method based on CRISPR-Cas9, TCRs capable of recognizing GB-related antigens are transferred into human primary T cells, transforming the patient's T cells into GB-related antigen-specific cytotoxic T cells (TCR-T). When these gene-edited TCR-T cells are introduced into the patient, these GB-related antigen-specific TCR-T cells are activated upon encountering the HLA-GB antigen complex on tumor cells, thus proliferating in the patient's body and killing tumor cells to achieve a therapeutic effect. Therefore, to achieve specific T-cell immunotherapy for GB-related malignant tumors, it is still necessary to focus on screening for GB antigen-specific cytotoxic T cells. By transducing human T cells with the obtained TCRs to obtain GB antigen-specific TCR-T cells, these cells can kill tumor cells and play a role in cell immunotherapy for GB-related malignant brain tumors, enhancing the patient's immune capacity, improving prognosis, and preventing tumor recurrence to some extent.

[0005] Therefore, developing a TCR-T cell immunotherapy regimen that can kill glioblastoma cells is of great clinical significance and is expected to improve the survival prognosis of glioblastoma patients. Summary of the Invention

[0006] Traditional TCR screening requires repeated stimulation with antigen peptides to obtain specific T cells, which is difficult and tedious, involving numerous repetitive experiments and extensive screening. This invention directly targets TCRs on the tumor microenvironment's tumor intraepithelial neoplasia (TIL). Through single-cell transcriptome sequencing combined with immunomics analysis, and by comparing and screening a large number of sequencing results, a group of TCRs specifically binding to glioblastoma was identified, and their specific function was verified.

[0007] The primary objective of this invention is to provide a T-cell receptor (TCR) capable of recognizing glioblastoma (GB)-associated tumors.

[0008] A T-cell receptor capable of recognizing and binding GB antigens, wherein the CDR3β sequence of the TCRβ chain variable region is SEQ ID NO: 6, or an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 6.

[0009] The T cell receptor, the CDR3α sequence of the TCRα chain variable region is SEQ ID NO: 3, or an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 3.

[0010] The T cell receptor, the variable region of the TCRα chain further includes CDR1α and CDR2α, wherein the CDR1α sequence is SEQ ID NO: 1 or has an amino acid sequence with at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 1, and the CDR2α sequence is SEQ ID NO: 2 or has an amino acid sequence with at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 2; the variable region of the TCRβ chain further includes CDR1β and CDR2β, wherein the CDR1β sequence is SEQ ID NO: 4 or has an amino acid sequence with at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 4, and the CDR2β sequence is SEQ ID NO: 5 or has an amino acid sequence with at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 5.

[0011] The T-cell receptor, wherein the T-cell receptor is an αβ heterodimer, further comprises a constant region TRAC of the TCR α chain and a constant region TRBC1 and / or TRBC2 of the TCR β chain; preferably, the α-chain amino acid sequence of the TCR is SEQ ID NO: 9 or an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 9, and the β-chain amino acid sequence of the TCR is SEQ ID NO: 13 or an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 13.

[0012] The T cell receptor, wherein one or two amino acid residues of one or more CDRs can be replaced by another amino acid residue, including the following groups: G, A; S, A, T; F, Y, W; D, E; N, Q and I, L, V. Two or three residues in each group can be substituted for each other, for example, G can be replaced by A, and S can be replaced by A or T.

[0013] The TCR of this invention includes a variable region (V) of the TCRα chain and a variable region of the TCRβ chain; the variable region of the TCRα chain includes three complementary determining regions (CDRs), with CDR1α sequence SEQ ID NO: 1, CDR2α sequence SEQ ID NO: 2, and CDR3α sequence SEQ ID NO: 3; the variable region of the TCRβ chain includes three complementary determining regions, with CDR1β sequence SEQ ID NO: 4, CDR2β sequence SEQ ID NO: 5, and CDR3β sequence SEQ ID NO: 6.

[0014] The T-cell receptor is able to recognize the GB antigen complex that binds to HLA-A*0201.

[0015] A second objective of this invention is to provide a nucleic acid molecule encoding a T-cell receptor, comprising a nucleic acid sequence encoding the T-cell receptor or its complementary sequence, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the TCR.

[0016] A third object of the present invention is to provide a vector containing the aforementioned nucleic acid molecules; preferably, the vector is a CRISPR / Cas9 editing vector. This vector can assist in the expression of the aforementioned TCR receptor.

[0017] A fourth object of the present invention is to provide an isolated host cell containing the CRISPR / Cas9 editing vector or the nucleic acid molecule encoding the T cell receptor.

[0018] A fifth object of the present invention is to provide a cell obtained by electroporation of the nucleic acid molecule encoding the T cell receptor or the carrier; preferably, the cell is a T cell or a stem cell; more preferably, the cell is a T cell or a stem cell derived from a GB patient.

[0019] A sixth object of the present invention is to provide a pharmaceutical composition comprising the cells described above.

[0020] A seventh object of the present invention is to provide the use of the above-described T cell receptor, the above-described nucleic acid molecule encoding the T cell receptor, the above-described vector, the above-described isolated host cell, or the above-described cell in the preparation of any of the following products:

[0021] i) Application in the preparation of TCR-T products;

[0022] ii) Use in the preparation of pharmaceutical compositions for the treatment of glioblastoma;

[0023] iii) Application in the preparation of diagnostic agents for glioblastoma;

[0024] iiii) Application in the preparation of glioblastoma antigen detection products.

[0025] The present invention further provides a method for detecting glioblastoma. The method for detecting glioblastoma of the present invention includes contacting a sample containing glioblastoma cells with any of the present invention's TCRs (including their functional portions and functional variants), peptides, proteins, nucleic acids, recombinant expression vectors, host cells, or antibodies or their antigen-binding portions, thereby forming a complex, and detecting the complex, wherein the detection of the complex indicates the presence of glioblastoma.

[0026] This invention uses single-cell transcriptome sequencing combined with immunomics analysis to compare and screen a large number of sequencing results, and found a group of TCRs that can specifically bind to glioblastoma.

[0027] In this invention, "GB-specific TCR (GB-TCR)" refers to a T cell receptor that can specifically recognize and bind to the GB-HLA-A*0201 complex; "GB-specific T cell (GB-TCR-T)" refers to a T cell that has been transduced with a nucleic acid or vector encoding the "GB-specific TCR" to express the "GB-specific TCR".

[0028] The three complementary determinants of the α-chain variable region of the TCR described in this invention are:

[0029] CDR1α-TYDTSDPSYGLF (SEQ ID NO: 1);

[0030] CDR2α-IYQGSYDQQNATEGRYSLNFQKARKSAN (SEQ ID NO: 2);

[0031] CDR3α-CAMRDPPMFSGGYNKLIF (SEQ ID NO: 3);

[0032] The three complementary determinants of the variable region of the TCRβ chain are:

[0033] CDR1β-EQHLGHNAMY (SEQ ID NO: 4);

[0034] CDR2β-FVYSLEERVENNSVPSRFSPECPNSSHLF (SEQ ID NO: 5);

[0035] CDR3β-CASSPGTGSYEQYFGPGTRLTVT (SEQ ID NO: 6).

[0036] One or two amino acid residues in one or more CDRs can be replaced by another amino acid residue. Typically, in these variants, some amino acids are replaced by conserved amino acids. These conserved amino acids include the following groups: G, A; S, A, T; F, Y, W; D, E; N, Q and I, L, V.

[0037] Chimeric TCRs can be prepared by embedding the CDR region amino acid sequence of the present invention into a 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 and / or β chain CDR region sequence and a suitable framework structure.

[0038] The variable region of the TCRα chain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 7; the variable region of the TCRβ chain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 11.

[0039] The TCR molecule of the present invention is a heterodimer composed of α and β chains. Specifically, the α chain of the heterodimeric TCR molecule includes a variable region and a constant region, and the amino acid sequence of the variable region of the α chain includes CDR1α (SEQ ID NO: 1), CDR2α (SEQ ID NO: 2), and CDR3α (SEQ ID NO: 3) of the aforementioned α chain. Preferably, the TCR molecule includes the amino acid sequence of the α chain variable region, SEQ ID NO: 7. The nucleotide sequence of the α chain variable region of the TCR molecule is SEQ ID NO: 8.

[0040] SEQ ID NO: 7: TCRα chain variable region amino acid sequence:

[0041] ITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRDPPMFSGGYNKLIFGAGTRLAVH

[0042] SEQ ID NO: 8: TCRα chain variable region nucleotide sequence:

[0043] ATAACTCAAA CCCAACCAGG AATGTTCGTG CAGGAAAAGG AGGCTGTGAC TCTGGACTGCACATATGACA CCAGTGATCC AAGTTATGGT CTATTCTGGT ACAAGCAGCC CAGCAGTGGG GAAATGATTTTTCTTATTTA TCAGGGGTCT TATGACCAGC AAAATGCAAC AGAAGGTCGC TACTCATTGA ATTTCCAGAAGGCAAGAAAA TCCGCCAACC TTGTCATCTC CGCTTCACAA CTGGGGGACT CAGCAATGTA CTTCTGTGCAATGAGAGACC CACCCATGTT TTCTGGTGGC TACAATAAGC TGATTTTTGG AGCAGGGACC AGGCTGGCTGTACAC

[0044] On the other hand, the β-chain of the heterodimeric TCR molecule includes a variable region and a constant region, and the amino acid sequence of the variable region of the β-chain includes CDR1β (SEQ ID NO: 4), CDR2β (SEQ ID NO: 5), and CDR3β (SEQ ID NO: 6) of the aforementioned β-chain. Preferably, the TCR molecule includes the amino acid sequence of the variable region of the β-chain, SEQ ID NO: 11. More preferably, the nucleotide of the variable region of the β-chain of the TCR molecule is SEQ ID NO: 12.

[0045] SEQ ID NO:11 Sequence:

[0046] VTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSPGTGSYEQYFGPGTRLTVT

[0047] SEQ ID NO:12 Sequence:

[0048] GTTACGCAGA CACCAAGACA CCTGGTCATG GGAATGACAA ATAAGAAGTC TTTGAAATGTGAACAACATC TGGGTCATAA CGCTATGTAT TGGTACAAGC AAAGTGCTAA GAAGCCACTG GAGCTCATGTTTGTCTACAG TCTTGAAGAA CGGGTTGAAA ACAACAGTGT GCCAAGTCGC TTCTCACCTG AATGCCCCAACAGCTCTCAC TTATTCCTTC ACCTACACAC CCTGCAGCCA GAAGACTCGG CCCTGTATCT CTGCGCCAGCAGCCCCGGGA CAGGGTCTTA CGAGCAGTAC TTCGGGCCGG GCACCAGGCT CACGGTCACA

[0049] In a preferred embodiment of the present invention, the constant region of the TCR molecule is the human constant region. Those skilled in the art know or can obtain the amino acid sequence of the human constant region by consulting relevant books or the publicly available database of IMGT (International Immunogenetic Information System). The constant region sequence contained in the α chain of the TCR molecule of the present invention can be "TRAC", and the constant region sequence contained in the β chain of the TCR molecule can be "TRBC1" or "TRBC2". Preferably, the amino acid sequence of the α chain of the TCR molecule of the present invention is SEQ ID NO: 9, and the nucleotide sequence of the α chain is SEQ ID NO: 10; the amino acid sequence of the β chain is SEQ ID NO: 13, and the nucleotide sequence of the β chain is SEQ ID NO: 14.

[0050] SEQ ID NO:9 Sequence:

[0051] ITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRDPPMFSGGYNKLIFGAGTRLAVHPYIQNPDPA

[0052] SEQ ID NO:10 Sequence:

[0053] ATAACTCAAA CCCAACCAGG AATGTTCGTG CAGGAAAAGG AGGCTGTGAC TCTGGACTGCACATATGACA CCAGTGATCC AAGTTATGGT CTATTCTGGT ACAAGCAGCC CAGCAGTGGG GAAATGATTTTTCTTATTTA TCAGGGGTCT TATGACCAGC AAAATGCAAC AGAAGGTCGC TACTCATTGA ATTTCCAGAAGGCAAGAAAA TCCGCCAACC TTGTCATCTC CGCTTCACAA CTGGGGGACT CAGCAATGTA CTTCTGTGCAATGAGAGACC CACCCATGTT TTCTGGTGGC TACAATAAGC TGATTTTTGG AGCAGGGACC AGGCTGGCTGTACACCCGTA TATCCAGAAC CCTGACCCTG CG

[0054] SEQ ID NO: 13 Sequence:

[0055] VTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSPGTGSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGRAKRSGSG

[0056] SEQ ID NO: 14 Sequence:

[0057] GTTACGCAGA CACCAAGACA CCTGGTCATG GGAATGACAA ATAAGAAGTC TTTGAAATGTGAACAACATC TGGGTCATAA CGCTATGTAT TGGTACAAGC AAAGTGCTAA GAAGCCACTG GAGCTCATGTTTGTCTACAG TCTTGAAGAA CGGGTTGAAA ACAACAGTGT GCCAAGTCGC TTCTCACCTG AATGCCCCAACAGCTCTCAC TTATTCCTTC ACCTACACAC CCTGCAGCCA GAAGACTCGG CCCTGTATCT CTGCGCCAGCAGCCCCGGGA CAGGGTCTTA CGAGCAGTAC TTCGGGCCGG GCACCAGGCT CACGGTCACA GAGGACCTGAAAAACGTGTT CCCACCCGAG GTCGCTGTGT TTGAGCCATC AGAAGCAGAG ATCTCCCACA CCCAAAAGGCCACACTGGTA TGCCTGGCCA CAGGCTTCTA CCCCGACCAC GTGGAGCTGA GCTGGTGGGT GAATGGGAAGGAGGTGCACA GTGGGGTCAG CACAGACCCG CAGCCCCTCA AGGAGCAGCC CGCCCTCAAT GACTCCAGATACTGCCTGAG CAGCCGCCTG AGGGTCTCGG CCACCTTCTG GCAGAACCCC CGCAACCACT TCCGCTGTCAAGTCCAGTTC TACGGGCTCT CGGAGAATGA CGAGTGGACC CAGGATAGGG CCAAACCCGT CACCCAGATCGTCAGCGCCG AGGCCTGGGG TAGAGCAGAC TGTGGCTTCA CCTCCGAGTC TTACCAGCAA GGGGTCCTGTCTGCCACCAT CCTCTATGAG ATCTTGCTAG GGAAGGCCAC CTTGTATGCC GTGCTGGTCA GTGCCCTCGTGCTGATGGCT ATGGTCAAGA GAAAGGATTC CAGAGGCCGG GCCAAGCGGT CCGGATCCGG A

[0058] In another preferred embodiment of the invention, a novel artificial disulfide bond may be introduced between Thr48 of the α-chain constant region and Ser57 of the β-chain constant region. Therefore, the TCR of the present invention may contain an artificial disulfide bond formed by cysteine ​​residues introduced between the residues of its α and β-chain constant regions. It should be noted that the TCR of the present invention may or may not contain the artificial disulfide bond introduced above, and may contain both the TRAC constant region sequence and the TRBC1 or TRBC2 constant region sequence. Preferably, the cysteine ​​residues of the artificial disulfide bond replace one or more sites selected from: Thr48 of TRAC and Ser57 of TRBC1 or TRBC2; Leu50 of TRAC and Ser57 of TRBC1 or TRBC2.

[0059] Furthermore, the TCR of the present invention can also be a heterozygous TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are expressed more efficiently in human T cells than human TCRs. Therefore, the TCR of the present invention can comprise a heterozygous TCR consisting of a human variable region and a mouse constant region.

[0060] It should be understood that the amino acid names in this article are represented by internationally accepted single or three English letters. The correspondence between the single and three English letters in the amino acid names is as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0061] The nucleotide sequence encoding the CDR region of the α chain of the TCR molecule of this invention is as follows:

[0062] CDR1α-acatatgacaccagtgatccaagttatggtctattc (SEQ ID NO: 15)

[0063] CDR2α-atttatcaggggtctttatgaccagcaaaatgcaacagaaggtcgctactcattgaatttccagaaggcaagaaaatccgccaac (SEQ ID NO: 16)

[0064] CDR3α-tgtgcaatgagagacccacccatgttttctggtggctacaataagctgattttt (SEQ IDNO: 17)

[0065] The nucleotide sequence encoding the CDR region of the β chain of the TCR molecule of this invention is as follows:

[0066] CDR1β-gaacaacatctgggtcataacgctatgtat (SEQ ID NO: 18)

[0067] CDR2β-tttgtctacagtcttgaagaacgggttgaaaacaacagtgtgccaagtcgcttctcacctgaatgccccaacagctctcacttattc (SEQ ID NO: 19)

[0068] CDR3β-tgcgccagcagccccgggacagggtcttacgagcagtacttc (SEQ ID NO: 20) The amino acid sequence of the TCRα chain with the leader sequence of this invention is as shown in SEQ ID NO: 21: METLLGLLILWLQLQWVSSKQEITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRDPPMFSGGYNKLIFGAGTRLAVHPYIQNPDPA

[0069] The nucleotide sequence of the TCRα chain with the leader sequence is shown in SEQ ID NO: 22:

[0070] ATGGAGACCC TCTTGGGCCT GCTTATCCTT TGGCTGCAGC TGCAATGGGT GAGCAGCAAACAGGAGATAA CTCAAACCCA ACCAGGAATG TTCGTGCAGG AAAAGGAGGC TGTGACTCTG GACTGCACATATGACACCAG TGATCCAAGT TATGGTCTAT TCTGGTACAA GCAGCCCAGC AGTGGGGAAA TGATTTTTCTTATTTATCAG GGGTCTTATG ACCAGCAAAA TGCAACAGAA GGTCGCTACT CATTGAATTT CCAGAAGGCAAGAAAATCCG CCAACCTTGT CATCTCCGCT TCACAACTGG GGGACTCAGC AATGTACTTC TGTGCAATGAGAGACCCACC CATGTTTTCT GGTGGCTACA ATAAGCTGAT TTTTGGAGCA GGGACCAGGC TGGCTGTACACCCGTATATC CAGAACCCTG ACCCTGCG

[0071] The amino acid sequence of TCR β chain with leader sequence is shown in SEQ ID NO: 23:

[0072] MSIGLLCCAALSLLWAGPVNAGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSPGTGSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGRAKRSGSG

[0073] The nucleotide sequence of TCR β chain with leader sequence is shown in SEQ ID NO: 24:

[0074] ATGAGCATCG GCCTCCTGTG CTGTGCAGCC TTGTCTCTCC TGTGGGCAGG TCCAGTGAATGCTGGTGTTA CGCAGACACC AAGACACCTG GTCATGGGAA TGACAAATAA GAAGTCTTTG AAATGGAACAACATCTGGG TCATAACGCT ATGTATTGGT ACAAGCAAAG TGCTAAGAAG CCACTGGAGC TCATGTTTGTCTACAGTCTT GAAGAACGGG TTGGAAACAA CAGTGTGCCA AGTCGCTTCT CACCTGAATG CCCCAACAGCTCTCACTTAT TCCTTCACCT ACACACCCTG CAGCCAGAAG ACTCGGCCCT GTATCTCTGGC GCCAGCAGCCCCGGGACAGG GTCTTACGAG CAGCTCACG GTCACAGAGG ACCTGAAAAACGTGTTTCCCCA CCCGAGGTCG CTGTGTTTGA GCCATCAGAA GCAGAGATCT CCCACACCCA AAAAGCCACACTGGTATGCC TGGCCACAGG CTTCTACCCC GACCACGTGG AGCTGAGCTG GTGGGTGAAT GGGAAGGAGGTGCACAGTGG GGTCAGCACA GACCCGCAGC CCCTCAAGGA GCAGCCCGCC CTCAATGACT CCAGATACTGCCTGAGCAGC CGCCTGAGGG TCTCGGCCAC CTTCTGGCAG AACCCCCGCA ACCACTTCCG CTGTCAAGTCCAGTTCTACG GGCTCTCGGA GAATGACGAG TGGACCCAGG ATAGGGCCAA ACCCGTCACC CAGATCGTCAGCGCCGAGGC CTGGGGTAGA GCAGACTGTG GCTTCACCTC CGAGTCTTAC CAGCAAGGGG TCCTGTCTGCCACCATCCTC TATGAGATCT TGCTAGGGAA GGCCACCTTG TATGCCGTGC TGGTCAGTGC CCTCGTGCTGATGGCTATGG TCAAGAGAAAGGATTCCAGA GGCCGGGCCA AGCGGTCCGG ATCCGGA

[0075] The nucleotide sequence of the nucleic acid molecule of the present invention is double-stranded. The nucleic acid molecule can be DNA or RNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but is capable of encoding a polypeptide of the TCR of the present invention. For example, the nucleotide sequence of a nucleic acid molecule encoding the variable region of the TCR α chain of the present invention contains SEQ ID NO: 8, and / or the nucleotide sequence of a nucleic acid molecule encoding the variable region of the TCR β chain of the present invention contains SEQ ID NO: 12.

[0076] It should be understood that due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Therefore, the nucleic acid sequence encoding the TCR of this invention can be the same as the nucleic acid sequence shown in this invention or a degenerate variant. As one example of this invention, a "degenerate variant" refers to a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 7, but differs from the sequence of SEQ ID NO: 8.

[0077] Nucleotide sequences can be codon-optimized. Different cells utilize specific codons differently, and codons in the sequence can be changed to increase expression levels depending on the cell type. Codon selection tables for mammalian cells and many other organisms are well known to those skilled in the art.

[0078] The TCR of the present invention comprises, in particular, T cells. Preferably, the T cells are T cells derived from a tumor patient, typically derived from peripheral blood mononuclear cells (PBMCs), and may be from a mixed cell population containing CD4+ helper T cells and / or CD8+ cytotoxic T cells. Typically, the T cells can be activated with antibodies (such as anti-CD3 and / or anti-CD28 antibodies) to facilitate integration by electroporation of the CRISPR-Cas9 editing vector encoding the TCR molecule of the present invention and stable expression of the glioblastoma-specific TCR. The present invention also includes cells transduced by the nucleic acid or vector of the present invention; preferably, the cells are T cells or stem cells; more preferably, the cells are T cells or stem cells derived from a patient.

[0079] The main advantages of this invention are: the TCR of this invention can specifically recognize and bind to the HLA-A*0201 and GB antigen peptide complex, therefore, the GB-specific T cells of this invention can be used to treat patients with HLA-A*0201 and GB-related tumors. Furthermore, we utilize CRISPR-Cas9 technology to achieve precise genome editing simply and efficiently. Using an electroporation system, adding a dsDNA template, and inserting the TCR sequence into the TRAC gene locus of primary T cells, the dsDNA gene knock-in efficiency is higher than that of lentiviral transduction. Because T cells transduced with the TCR of this invention can not only be specifically activated and expanded by target cells presenting GB antigens, but also produce antigen-specific immune cytokines and kill tumor cells expressing GB antigens, it has stronger tumor specificity and lower toxicity than traditional therapies. Especially when the tumor has progressed to an advanced stage and spread, and traditional therapies are ineffective, by introducing the GB-specific T cells of this invention into the patient, these GB-specific T cells can kill metastatic tumor cells through circulation in the body.

[0080] The present invention will be further illustrated by the following specific embodiments. However, it should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0081] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Attached Figure Description

[0082] Figure 1 This indicates that the TCR of the present invention was obtained through single-cell transcriptome sequencing combined with immunomics sequencing analysis.

[0083] Figure 2 This diagram illustrates an HDR template in which the TCR of the present invention uses a CRISPR / Cas9 editing system to replace the endogenous TCR with a GB-TCR sequence.

[0084] Figure 3 This invention represents the results of flow cytometry analysis of proliferating T cells after co-culturing GB-TCR-T cells with U251-HLA-A*0201.

[0085] Figure 4 This indicates the results of flow cytometry analysis of tumor cells after co-culturing GB-TCR-T cells with U251-HLA-A*0201 according to the present invention.

[0086] Figure 5This indicates the results of flow cytometry detection of apoptotic tumor cells after co-culturing GB-TCR-T cells with U251-HLA-A*0201 according to the present invention.

[0087] Figure 6 This indicates that the GB-TCR-T cells of the present invention can produce GB-specific INF-γ after being stimulated by U251-HLA-A*0201.

[0088] Figure 7 This invention represents an evaluation of the tumor-killing effect of GB-TCR-T cells and U251-HLA-A*0201 under different effector-to-target ratios (E / T).

[0089] Figure 8 The results of in vivo mouse imaging demonstrate the functional verification of GB-TCR-T cells and U251-LUC-HLA-A*0201 in vivo according to the present invention.

[0090] Figure 9 This indicates the change in average body weight in mice during in vivo functional verification of the GB-TCR-T cells and U251-LUC-HLA-A*0201 of the present invention. Detailed Implementation

[0091] The present invention will be further described in detail below with reference to specific implementation schemes, but the embodiments of the present invention are not limited thereto.

[0092] This invention provides a TCR nucleic acid molecule encoding the GB antigen and a scheme comprising editing the exogenous GB-TCR to an endogenous TRCA chain site using a CRISPR / Cas9 system. This invention also provides the TCR, the nucleic acid molecule, and a scheme for preparing a drug for treating GB-related tumors for cell immunotherapy.

[0093] Example 1: Obtaining the GB-specific T cell receptor sequence

[0094] Using single-cell transcriptome sequencing combined with immunomics analysis, the detected cells were clustered and grouped in a dimensionality-reduced manner. TCR sequencing was then mapped back to the cell population to identify the top 10 TCR sequences. TCRs that specifically bind to GB-related antigens were identified, primarily TCR receptors expressed on CD8+ effector T cells. Figure 1 The antigen can form a complex with HLA-A*0201 and be presented together to the cell surface.

[0095] Example 2: Construction of TCR-design and Construction of Target Cells

[0096] A single guide RNA (sgRNA) sequence with a scaffold sequence was added. According to literature reports, adding some modifications to both ends of the sgRNA can significantly improve its stability and editing efficiency. Our sgRNA was modified with an 80nt scaffold sequence and modifications at both ends. The 80nt sgRNA scaffold sequence can be found in the literature (Sanjana NE et al., Nat Methods., 2014, 11(8):783-784). Usually, four Ts are added afterward as a transcription termination signal. For modifications at both ends, please refer to the literature of Basila et al. The homology-directed repair (HDR) template was designed and sent to GenScript to synthesize dsDNA (GenCRISPR dsDNA) (SEQ ID NO: 25).

[0097] SEQ ID NO: 25: (HDR)

[0098]

[0099] Unmodified sgRNA nucleotide sequences, such as SEQ ID NO: 26: AGAGTCTCTCAGCTGGTACA, and modified sgRNAs:

[0100] mA*mG*mA*rGrUrCrUrCrUrCrArGrCrUrGrGrUrArCrArGrUrUrUrUrArGrArGrCrUrArGrArArArArArArArGrCrUrUrUrArArArGrCrUrUrUrArArArGrGrCrUrUrArGrUrUrArArGrGrCrUrUrUrArGrCrUrUrGrCr ... Purchase the corresponding Cas9 (GenCrispro eSpCas9-N-NLS) protein. Construct the editing system as follows: Figure 2 As shown. The tumor cells of U251 are the HLA-A*0201 cell line. The human glioma cells-green fluorescent marker (U251-GreenFluorescent Proteins, U251-GFP) that we constructed earlier were used as the target cell line for subsequent TCR-T function experiments.

[0101] Example 3: Preparation of an electroporation system for GB-specific TCR-T cells

[0102] Peripheral blood was collected from healthy volunteers, and human peripheral blood mononuclear cells (PBMCs) were obtained by separation using lymphocyte separation tubes. The cell density was adjusted to 1×10⁻⁶. 6 Cells / ml were collected, and OKT-3 antibody (30 ng / ml) and IL-2 (600 U / ml) were added to the cell culture medium to activate T cells. NEPA21 electrotransfection was performed. T cells were centrifuged, resuspended in EP buffer: Opti-MEM medium (Invitrogen), and added to EP tubes. After washing 3-4 times with EP buffer, the cell volume was prepared according to the required amount. It is essential to ensure that the cell suspension is free of antibiotics and serum residues, otherwise the transfection efficiency will be greatly reduced. A small amount of suspension was taken for cell counting and concentration determination.

[0103] Preparation of electroporation mixture: Add sgRNA and Cas9 protein to a sterile, DNase / RNase-free 1.5 ml centrifuge tube according to the specified dosage, mix thoroughly, and incubate at room temperature for 10 minutes to form an RNP mixture. Then add the dsDNA HDR to the mixture, ensuring gentle mixing, and incubate at room temperature for approximately 2 minutes.

[0104] Electroporation steps: Prepare preheated culture medium and culture plate → Aliquot T cells and the transfection mixture into an electroporation cuvette → Set electroporation parameters → Remove air bubbles from the electroporation cuvette → Place the electroporation cuvette into the cavity of the cuvette → Measure and record the resistance value → Execute the electroporation program (record parameters such as current and energy, which can be used for troubleshooting) → Remove the electroporation cuvette and aspirate all the cell suspension into the culture plate for continued culture. 48 hours after electroporation, RNA is extracted and detected as successfully edited before proceeding with subsequent functional experiments for verification.

[0105] Example 4: Flow cytometry detection of the killing effect of GB-TCR-T cells on target cells

[0106] The constructed U251-GFP target cells were cultured in 500 μl of 10% FBS in RPMI 1640 medium in a 48-well round-bottom plate at a concentration of 1×10⁻⁶ cells / well. 5 target cells and 1×10 6 CellsGB-TCR-T cells were cultured in a mixed culture (temperature: 37℃, O2 concentration: 95%, CO2 concentration: 5%, pH: 7.2-7.4, under controlled osmotic pressure, free from contamination and toxicity), with unedited T cells as a control. After 24 hours, all cells from each well were aspirated into 1.5ml centrifuge tubes for flow cytometry staining.

[0107] Example 5: Analysis of GB-specific TCR-T cell function by flow cytometry

[0108] Figure 3 The study showed that after co-culturing GB-TCR-T cells with U251-GFP target cells, the number of GB-TCR-T cells was significantly higher than that of the control T cell group. The green box indicates the T cell count. Figure 4 The results showed that without GB-TCR-T cells, both control T cells and U251-GFP target cells survived well. However, with the addition of GB-TCR-T cells, the positive target cells U251-GFP were significantly killed, while the U251 tumor cells in the control T cells still survived well. This indicates that the GB-TCR-T cells of this invention can effectively kill glioma cells, and GB-TCR-T can kill more tumor cells. The black box indicates the count of GB tumor cells. We stained the dead tumor cells with Zombie NIR. Figure 5The proportion of tumor cells undergoing apoptosis in GB-TCR-T cells was significantly higher than that in the control T cell group, with the upper right quadrant representing tumor cell apoptosis. Figure 6 The study demonstrated that U251-GFP cells could stimulate GB-TCR-T cells to secrete IFN-γ, while control T cells co-cultured with U251-GFP cells secreted less IFN-γ. T cells transduced with this TCR were able to secrete more IFN-γ after recognizing target cells, thereby killing the target cells.

[0109] Example 6: Detection of GB-specific TCR-T cell function by flow cytometry

[0110] After 48 hours of electroporation, T cell counts were performed, and the effector-to-target ratio (the ratio of effector T cells to target cells) was 10:1, 3:1, 1:1, and 0.33:1. 4-6 hours before flow cytometry analysis, 0.5 μL of brefeldtin A (BFA) (2-50 μg / ml) and 0.5 μL of monensin (working concentration range 1-5 μM) were added to the culture system. Figure 7 The study showed that under different effector-to-target ratios (10:1, 3:1, 1:1, 0.33:1), the tumor-killing effect of T cells was significantly stronger when GB-TCR-T cells were co-cultured with U251-GFP under the same effector-to-target ratios than that of control T cells, and the apoptosis was more pronounced.

[0111] Example 7: In vitro investigation of GB-specific TCR-T cell function in mice via in situ tumorigenesis

[0112] First, mice were prepared to establish the animal model. Fifteen immunodeficient mice (NOD / SCID) (5-6 weeks old, weighing 16-20g) were raised in a specific pathogen-free (SPF) environment. Tumor cells were prepared. Human glioma cells labeled with luciferase (U251-Luc) were implanted into immunodeficient mice, effectively mimicking the significant pathological features of human brain glioma (GB). U251-Luc cells were cultured in 1640 medium supplemented with 10% (v / v) fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 U / mL) at 37°C under 5% CO2 and saturated humidity. Cells in the logarithmic growth phase were harvested and adjusted to a density of 1.0 × 10⁶ cells / mL. 6A single-cell suspension of cells / mL was prepared. The prepared transplanted cells were mixed 1:1 with Matrigel and drawn up with a 1mL syringe. Matrigel provides a nutrient environment for tumor cells, promoting their growth. In a clean bench, mice were anesthetized with 0.01ml / g of 1% pentobarbital intraperitoneally (after routine disinfection and draping, the injection site was determined (1mm anterior to the fontanelle and 2.5mm to the right)). A 50μL microsyringe was used to draw up the tumor cell suspension and inserted vertically and slowly into the mouse brain parenchyma. After gentle injection, the needle was left in place for 5 minutes and then slowly withdrawn. After disinfection, the mice were placed in an SPF environment for continued rearing. The growth status of the mice was observed and recorded daily.

[0113] Approximately 4-5 weeks later, in vivo imaging was performed to assess tumor formation in mice. Mice with similar tumor sizes were randomly divided into three groups of three: a control group (blank) receiving only PBS to exclude the influence of T cells themselves; a Tcell group (control group) containing T cells without TCR gene editing; and a GB-TCR-T cell group (experimental group) containing TCR gene-edited T cells. 1.0 × 10⁻⁶ T cells were injected into the tail vein of the mice. 7 T cell suspension. Every 7 days after T cell injection, in vivo imaging was performed to assess changes in intracranial tumor volume. Mice were injected intraperitoneally with the anesthetic aphthylamine (prepared at a concentration of 1.2%-2.5%; a 100% stock solution is 10g aphthylamine mixed with 10mL tert-amyl alcohol, diluted with sterile distilled water to 1.2%-2.5% before use; mouse dosage: 125-400mg / kg). 2-5 minutes later, each mouse was injected with a 15mg / mL solution of Potassium Luciferin, the substrate for luciferase, at a concentration of 10μL / g body weight. The mice were then allowed free movement for 10-15 minutes to allow the substrate to fully circulate in the bloodstream to the desired location and react with luciferase. The reaction was performed after the mice were completely anesthetized and immobile. Figure 8 In vivo imaging results in mice showed that, compared to the blank and control groups, the experimental group exhibited durable regression of glioblastoma after GB-TCR-T cell immunotherapy. Furthermore, mouse body weight was measured every 2–3 days after the start of T cell injection. Figure 9 The results showed that, compared with the control group, mice treated with TCR-T cell immunotherapy did not show a significant trend of weight loss, suggesting that GB-TCR-T cell immunotherapy can effectively control the tumor burden of progressive glioblastoma in mice.

Claims

1. A T-cell receptor, characterized in that, The T cell receptor includes a variable region of the TCRα chain and a variable region of the TCRβ chain; The variable region of the TCRα chain includes CDR1α, CDR2α and CDR3α, with sequences SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively. The variable region of the TCRβ chain includes CDR1β, CDR2β and CDR3β, with sequences SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. The T-cell receptor is able to recognize the GB antigen complex that binds to HLA-A*0201.

2. The T cell receptor according to claim 1, characterized in that, The T cell receptor is an αβ heterodimer, and also includes the TCR α chain constant region TRAC and the TCR β chain constant region TRBC1; the α chain amino acid sequence of the TCR is SEQ ID NO: 9, and the β chain amino acid sequence of the TCR is SEQ ID NO:

13.

3. A nucleic acid molecule encoding a T-cell receptor, characterized in that, It comprises a nucleic acid sequence encoding the T cell receptor of claim 1 or 2 or its complementary sequence, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the TCR.

4. A carrier, characterized in that, The carrier contains the nucleic acid molecule as described in claim 3.

5. An isolated host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 3 or the vector of claim 4.

6. A T cell, characterized in that, The T cell contains the nucleic acid molecule of claim 3 or the vector of claim 4.

7. The T cell according to claim 6, characterized in that, The T cells were derived from T cells from patients with glioblastoma.

8. A pharmaceutical composition, characterized in that, The composition contains the T cells as described in claim 6 or 7.

9. The use of the T cell receptor of claim 1 or 2, the nucleic acid molecule encoding the T cell receptor of claim 3, the vector of claim 4, the isolated host cell of claim 5, or the T cell of any one of claims 6-7 in the preparation of any of the following products: i) Application in the preparation of TCR-T products; ii) Use in the preparation of pharmaceutical compositions for treating glioblastoma; iii) Application in the preparation of diagnostic agents for glioblastoma; iiii) Application in the preparation of glioblastoma antigen detection products.

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