WO2024093056A1 - Kras_g12v突变抗原特异性tcr及其与cd8共表达重定向cd4 t细胞 - Google Patents

Kras_g12v突变抗原特异性tcr及其与cd8共表达重定向cd4 t细胞 Download PDF

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WO2024093056A1
WO2024093056A1 PCT/CN2023/078124 CN2023078124W WO2024093056A1 WO 2024093056 A1 WO2024093056 A1 WO 2024093056A1 CN 2023078124 W CN2023078124 W CN 2023078124W WO 2024093056 A1 WO2024093056 A1 WO 2024093056A1
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amino acid
seq
chain
cdr3 amino
cdr2
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French (fr)
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彭松明
安多
钟山
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新景智源生物科技(苏州)有限公司
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)

Definitions

  • the present invention generally relates to the field of immunology. Specifically, the present invention relates to a T cell receptor (hereinafter also abbreviated as TCR) that specifically binds to a KRAS_G12V mutant antigen, a genetically engineered cell expressing the TCR, and a method for preparing the genetically engineered cell.
  • TCR T cell receptor
  • the present invention also relates to co-expressing exogenous CD8 molecules with the TCR gene in T cells to enhance the function of T cells.
  • the present invention provides the use of the TCR and the genetically engineered cell in detecting, preventing and/or treating cancers associated with the KRAS_G12V mutant antigen.
  • the RAS gene is the first human oncogene discovered, and the RAS protein it encodes is at the center of many important cell signaling networks.
  • the RAS gene is the most commonly mutated oncogene in human cancer.
  • RAS protein activation caused by RAS gene mutations has been found in about 1/5 of all human tumors.
  • the KRAS protein encoded by the KRAS gene (Kirsten rat sarcoma viral oncogene homolog) is a small GTPase that belongs to the RAS superprotein family.
  • KRAS protein switches between inactive and activated states.
  • KRAS protein binds to guanine nucleoside diphosphate (GDP)
  • KRAS protein binds to guanine nucleoside triphosphate (GTP)
  • GTP guanine nucleoside triphosphate
  • KRAS gene In human cancer, the KRAS gene is one of the most famous oncogenes in the field of oncology and was once considered an "undruggable" target. KRAS gene mutations occur in nearly 90% of pancreatic cancer, 30-40% of colon cancer, 17% of endometrial cancer, 15-20% of lung cancer (including lobular lung cancer), as well as bile duct cancer, cervical cancer, bladder cancer, etc.
  • KRAS mutants which target KRAS mutants through allosteric sites, reducing the affinity of KRAS mutants to GTP and achieving the purpose of "locking" the activity of KRAS mutants.
  • Amgen's sotorasib (AMG510) for the treatment of patients with non-small cell lung cancer (NSCLC) carrying KRAS_G12C mutations is a KRAS_G12C inhibitor;
  • Mirati Therapeutics' MRTX1257 is also a KRAS_G12C inhibitor and is currently in the preclinical development stage.
  • KRAS_G12V mutations There are currently no relevant therapeutic drugs for other KRAS mutations, such as KRAS_G12V mutation.
  • KRAS_G12V mutant antigens there are currently no relevant therapeutic drugs for other KRAS mutations, such as KRAS_G12V mutation.
  • TCR-T cells specific immune cells, such as TCR-T cells, for KRAS_G12V mutant antigens to effectively detect, prevent and treat cancers associated with KRAS_G12V mutant antigens.
  • TCR T cell receptor
  • the present invention provides an isolated or purified T cell receptor (TCR) that specifically binds to the KRAS_G12V mutant antigen.
  • TCR comprises an ⁇ chain and a ⁇ chain, wherein the ⁇ chain and the ⁇ chain each comprise three complementarity determining regions (CDRs), and the amino acid sequence of CDR3 of the ⁇ chain is selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48 and variants having 1 or 2 amino acid residues changed from the sequence, and the amino acid sequence of CDR3 of the ⁇ chain is selected from SEQ ID NO: 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224 and variants having 1 or 2 amino acid residues changed from the sequence.
  • CDRs complementarity determining regions
  • amino acid sequence of CDR3 of the TCR ⁇ chain and the amino acid sequence of CDR3 of the ⁇ chain of the present invention are:
  • the amino acid sequence of the three complementarity determining regions (CDRs) comprised by the ⁇ chain and the amino acid sequence of the three CDRs comprised by the ⁇ chain of the TCR of the present invention is:
  • the TCR of the invention comprises an alpha chain sequence as set forth in SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, or 175, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and SEQ ID NO: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377 or the beta chain sequence shown in 379, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
  • the present invention provides a T cell receptor fusion protein or a T cell receptor conjugate, which comprises the TCR described in the first aspect of the present invention and other biologically active molecules, wherein the other biologically active molecules are, for example, antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable markers, and there is or is no linker between the TCR and the other biologically active molecules.
  • the other biologically active molecules are, for example, antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable markers, and there is or is no linker between the TCR and the other biologically active molecules.
  • the present invention also provides nucleic acids encoding the TCR ⁇ chain and/or ⁇ chain of the present invention.
  • the present invention provides a vector, preferably a plasmid, a shuttle plasmid, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector and/or a homologous recombination repair (HDR) vector, which comprises one or more nucleic acids as described above.
  • a vector preferably a plasmid, a shuttle plasmid, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector and/or a homologous recombination repair (HDR) vector, which comprises one or more nucleic acids as described above.
  • HDR homologous recombination repair
  • the present invention provides an engineered cell transformed with the above vector and expressing the TCR described in the first aspect of the present invention.
  • the present invention provides a method for preparing TCR-T cells using a targeting strategy that does not use a viral vector to express the exogenous TCR of the present invention.
  • the present invention provides a method for editing the genome of a human cell, the method comprising inserting the following nucleic acid sequence into a target region of exon 1 of an endogenous T cell receptor (TCR) ⁇ chain constant region gene in a human cell, the nucleic acid sequence comprising from N-terminus to C-terminus:
  • TCR T cell receptor
  • first cleavable linker polypeptide and the second cleavable linker polypeptide are the same or different viral 2A peptides.
  • the cells expressing exogenous TCR prepared by the method have high binding affinity to the VVVGAVGVGK-HLA-A*11:01 complex and/or VVGAVGVGK-HLA-A*11:01 and have a strong in vitro killing effect on SW620 (HLA-A*11:01 overexpression, KRAS G12V+) cells.
  • the method for preparing cells expressing exogenous TCR is implemented by knocking out endogenous TCR and knocking in exogenous TCR using CRISPR/Cas9 technology and homologous recombination technology.
  • the invention provides methods and engineered cells for improving cell therapy.
  • the present invention co-expresses exogenous TCR and CD8aa molecules in T cells. In some embodiments, the present invention co-expresses exogenous TCR and CD8ab molecules in T cells.
  • CD8aa molecules and/or CD8ab molecules with TCR genes in CD8+ and CD4+ T cells By co-expressing CD8aa molecules and/or CD8ab molecules with TCR genes in CD8+ and CD4+ T cells, the functions of CD8+ and CD4+ T cells are beneficially affected.
  • CD4+ T cells are reprogrammed into multifunctional hybrid T cells, which have both cytotoxic effector functions and natural helper functions.
  • the present invention provides the use of the TCR described in the first aspect and the engineered cells obtained in the second and third aspects in detecting, preventing and/or treating cancers associated with the KRAS_G12V mutant antigen.
  • Figure 1A shows a diagram of the targeting strategy for knocking exogenous TCR into the TRAC site using gRNA002.
  • Figure 1B shows a diagram of the targeting strategy for knocking out TRBC1 and TRBC2 loci using gRNA004.
  • FIG. 2 shows a schematic diagram of the results of flow cytometry detection of TCR gene editing efficiency.
  • the flow cytometry data analysis is a cell distribution diagram of 4 quadrants (Q1, Q2, Q3, Q4), where Q2 is a cell population that has completed endogenous TCR knockout (knock-out; KO) and exogenous TCR knock-in (knock-in; KI) and expressed nwTCR; Q3 is a wild-type T cell that has not undergone gene editing; Q4 is a KO cell that has completed endogenous TCR knockout.
  • Figures 3A-3P illustrate the flow cytometry results of CD4+T cells and CD8+T cells electroporated with different nwTCRs stained with pMHC tetramers.
  • the CD4+T cells and CD8+T cells electroporated with nwTCR-0125, nwTCR-0126, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, nwTCR-2241, nwTCR-2308 and nwTCR-2563 are exemplified by VVVGAVGVGK-HLA-A*1 labeled with 1:01 tetramer staining results; the CD4+T cells and CD8+T cells electroporated and transfected with nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2595 and nwT
  • Figures 4A-4H show the binding affinity test results and EC50 values of T cells expressing each nwTCR to the short peptide VVVGAVGVGK (SEQ ID NO: 381) or VVGAVGVGK (SEQ ID NO: 382) presented by HLA-A*11:01.
  • Figures 4A, 4C, 4D and 4G show the binding affinity to the short peptide VVVGAVGVGK (SEQ ID NO: 381);
  • Figures 4B, 4E, 4F and 4H show the binding affinity to the short peptide VVGAVGVGK (SEQ ID NO: 382).
  • Figures 5A-5E show the in vitro killing effect of T cells expressing each nwTCR on SW620 (HLA-A*11:01 overexpression, KRAS G12V+) cell line (colorectal cancer (CRC) cell line) as target cells.
  • "Blank” in the figure means that there are only target cells and no T cells expressing any nwTCR are added.
  • Figure 6 illustrates the fluorescence imaging results of T cells expressing nwTCR-2404 killing SW620 (HLA-A*11:01 overexpression, KRAS G12V+) cells.
  • the “Blank” in the figure indicates that there are only target cells and no T cells expressing nwTCR-2404 are added.
  • Figure 7 illustrates real-time analysis data of T cells expressing nwTCR-2404 killing SW620 (HLA-A*11:01 overexpression, KRAS G12V+) cells.
  • the results show that gene-edited T cells kill SW620 (HLA-A*11:01 overexpression
  • the “Blank” in the figure indicates that there are only target cells and no T cells expressing any nwTCR are added.
  • FIG8A shows a diagram of the targeting strategy of nwTCR-CD8a.
  • FIG8B shows a diagram of the targeting strategy of nwTCR-CD8ab.
  • FIG9A shows the flow cytometry results of CD4+T cells and CD8+T cells electroporated and transfected with nwTCR-1708 stained with pMHC tetramer.
  • FIG. 9B shows the flow cytometry results of CD4+T cells electroporated with nwTCR-1708-CD8a and CD8+T cells stained with pMHC tetramer.
  • FIG9C shows the flow cytometry results of CD4+T cells and CD8+T cells electroporated and transfected with nwTCR-1708-CD8ab and stained with pMHC tetramer.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” is used, unless otherwise indicated, the situation consisting of the stated elements, integers or steps is also covered.
  • an antibody variable region “comprising” a specific sequence it is also intended to cover the antibody variable region consisting of the specific sequence.
  • RAS protein family belongs to a large family of small GTPases. RAS proteins can be constitutively activated due to single amino acid mutations. Mutated RAS protein products are involved in signal transduction in the early stages of tumor formation in many human cancers.
  • a variety of human cancers e.g., lung cancer (e.g., lung adenocarcinoma), ovarian cancer (e.g., epithelial ovarian cancer), pancreatic cancer, prostate cancer, endometrial cancer, and colorectal cancer express mutated RAS proteins.
  • KRAS protein KRAS protein
  • the upstream of KRAS is regulated by the epidermal growth factor receptor EGFR family.
  • the EGFR signal can activate the SOS protein, thereby regulating the activation of KRAS.
  • the inactivation and activation state transitions of the KRAS protein in the cell are determined by the molecules it binds to.
  • the guanine nucleotide exchange factor GEF catalyzes KARS to bind to GTP and activate KRAS; while the GTPase activating protein GAP can promote the hydrolysis of GTP bound to KRAS into GDP leads to KRAS inactivation.
  • KRAS Activated KRAS regulates its downstream signaling pathways such as MAPK and PI3K that are related to cell proliferation, cell migration and other functions. KRAS mutations cause it to continuously bind to GTP and remain activated, leading to continuous activation of downstream signaling pathways, thereby promoting tumorigenesis.
  • antigen is any molecule that can be specifically detected by an organism's immune system.
  • KRAS_G12V mutant antigen refers to a KRAS protein with a G12V mutation, which can be specifically detected by the immune system of an organism.
  • G12V or “G12V mutation” are used interchangeably and refer to a KRAS protein in which the glycine at position 12 is replaced by valine.
  • T cell receptor is a protein on the surface of T cells that is responsible for the specific recognition of antigenic peptides bound to MHC (major histocompatibility complex). When TCR binds to antigenic peptides and MHC, T lymphocytes are activated through signal transduction and enter the subsequent immune response process.
  • TCR genes in the human genome two encoding light chain TCRs: TRA gene encodes TCR ⁇ , TRG gene encodes TCR ⁇ ; two encoding heavy chain TCRs: TRB gene encodes TCR ⁇ , TRD gene encodes TCR ⁇ . Heavy chain TCR and light chain TCR form heterodimers to form a complete TCR.
  • TCR ⁇ / ⁇ and TCR ⁇ / ⁇ of which 95% of T cells express TCR ⁇ / ⁇ , called ⁇ T cells; 5% of T cells express TCR ⁇ / ⁇ , called ⁇ / ⁇ T cells. This ratio changes during individual development and in diseased states (such as leukemia), and also varies between species.
  • the mature heavy chain TCR gene is composed of four gene segments: variable region (V), variable region (D), joining region (J) and constant region (C) (VDJC), while the light chain TCR lacks the D region (VJC).
  • V variable region
  • D variable region
  • J joining region
  • C constant region
  • VJC constant region
  • Both heavy chain and light chain TCR have three complementary determining regions (CDRs), which play a major role in antigen recognition. Among them, CDR1 and CDR2 are relatively conservative and are responsible for recognizing MHC; CDR3 is the main CDR responsible for recognizing antigens.
  • the TCR gene is the most complex gene in the human genome and also the gene with the highest degree of variation.
  • Human peripheral blood contains approximately 2x10 16 -10 18 types of T cells expressing different TCRs. This complexity mainly comes from three factors: (i) Compositional diversity: The VDJC/VJC structure of mature TCR is generated through complex rearrangement. There are 65-100 V gene segments, 2 D gene segments and 13 J gene segments in the genome.
  • connection mobility During the rearrangement process, non-templated nucleotides are often randomly inserted or deleted in the connection region of VD and DJ, further increasing the diversity of the CDR3 region;
  • Somatic mutation The mutation frequency of the D region of T cells is about 1000 times that of normal.
  • polynucleotide or “nucleic acid” used interchangeably herein refers to a chain of nucleotides of any length, and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a chain by a DNA or RNA polymerase.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequence or nucleic acid sequence for optimal alignment or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package is used (available at http://www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).
  • APC antigen presenting cell
  • MHC major histocompatibility complex
  • T cells can recognize these complexes using their T cell receptors (TCR). APCs process and present antigens to T cells.
  • guide RNA refers to an RNA specific to a target DNA, which can form a complex with a Cas protein and bring the Cas protein to the target DNA, so that the Cas protein introduces a double-strand break at the site of the target DNA.
  • the guide RNA can be composed of two RNAs, namely, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), or the guide RNA can be a single-stranded guide RNA (sgRNA) generated by fusing the necessary parts of crRNA and tracrRNA.
  • Ribonucleoprotein is a complex formed by Cas9 protein and gRNA with gene editing function.
  • the CRISPR/Cas9 gene editing system mainly consists of two parts: the Cas9 protein, which acts as a "wrench”, and the CRISPR guide RNA, which acts as a "thread nail".
  • the guide RNA is responsible for locating the target site and recruiting and activating the Cas9 protein; the Cas9 protein is responsible for cutting the target DNA.
  • nucleic acid, protein or vector when applied to, for example, a cell, nucleic acid, protein or vector, means that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the alteration of a native nucleic acid or protein.
  • target site refers to any DNA sequence in the target genome that is to be modified or repaired.
  • the DNA sequence near the target site allows the integration of exogenous sequences at the target site, and the integration includes but is not limited to gene knock-in (KI).
  • the target DNA sequence is a double-stranded DNA sequence, including but not limited to, a DNA sequence in the chromosome genome of a cell, a DNA sequence outside the chromosome genome of a cell (such as a mitochondrial genome), a DNA sequence of a plasmid, a virus, etc.
  • site-directed recombination refers to the integration of an exogenous sequence into a specific target site in a non-random manner, including integration into the 5' upstream, 3' downstream, or between target sites of a specific target site.
  • exogenous DNA sequence refers to a DNA sequence that is expected to be site-specifically recombined into a target site.
  • the exogenous DNA sequence may be a sequence that does not exist or is altered at the target site.
  • donor DNA or “donor nucleic acid sequence” refers to a polynucleotide comprising a polynucleotide sequence of interest to be expressed, which is inserted into a target site in the target genome.
  • the donor DNA further comprises a sequence homologous to the genomic sequence (also referred to as a "homologous arm”).
  • homologous means a similar DNA sequence. Homologous arms are sufficient for homologous recombination with homologous genomic sequences. For example, homologous arms can comprise at least 50-3500 or more bases in length.
  • HDR homologous directed DNA repair
  • HDR vectors can refer to vectors used for electroporation transfection using CRISPR/Cas9 and homologous recombination technology.
  • HDR efficiency can refer to the gene knock-in efficiency of electroporation transfection using CRISPR/Cas9 and homologous recombination technology.
  • a synonymous mutation is a neutral mutation.
  • the genetic code is degenerate, that is, there is usually more than one codon that determines an amino acid, and the substitution of the third nucleotide in a triplet codon often does not change the composition of the amino acid. Although the third nucleotide in the triplet codon mutates, the encoded amino acid does not change.
  • This mutation is a synonymous mutation.
  • vector means a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell.
  • examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors.
  • the vector may contain a nucleic acid sequence that allows the gene or sequence of interest to replicate in the host cell, such as a replication initiation region.
  • the vector may also contain one or more selectable marker genes and other genetic elements known to those skilled in the art.
  • the vector is preferably an expression vector comprising a nucleic acid according to the present invention, and the nucleic acid is operably connected to a sequence that allows the nucleic acid to be expressed.
  • operably linked refers to a functional connection between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein so as to perform an overall function. Genetic recombination techniques well known in the art can be used to prepare an operative connection with a recombinant vector, and enzymes well known in the art can be used for site-specific DNA cleavage and connection.
  • engineered cell refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of these cells.
  • Engineered cells include "transfected cells", which include primary transfected cells and progeny derived therefrom, without considering the number of passages. Progeny may not be completely identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as the cells screened or selected in the initial transfected cells are included herein.
  • subject refers to an animal, preferably a mammal, and more preferably a human, that needs to alleviate and/or treat KRAS_G12V mutant antigen-related cancer.
  • Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats.
  • Adoptive Cell Transfer Therapy refers to the separation of immune-active cells from the subject or patient, activation and amplification, gene editing and other treatments in vitro, and then back into the patient's body to kill the target cells.
  • T cell receptors (TCR) of the present invention and nucleic acids encoding TCR
  • the wild-type human KRAS protein is 188 amino acid residues long, with a molecular weight of about 21.6KD, and glycine is at position 12.
  • glycine is at position 12.
  • 83% are mutations at position 12, and the most common mutation is the mutation of glycine at position 12 to valine (also referred to as G12V in this article).
  • the present invention provides an isolated or purified TCR having antigenic specificity for a KRAS peptide having a G12V mutation presented by a human leukocyte antigen (HLA) class I molecule.
  • the KRAS peptide having a G12V mutation presented by a human leukocyte antigen (HLA) class I molecule has any length suitable for binding to any HLA class I molecule.
  • the KRAS peptide with a G12V mutation has a length of about 9 to about 10 amino acid residues, including any consecutive about 9 to about 10 amino acid residues with a G12V mutation in a KRAS protein.
  • the TCR of the present invention has antigenic specificity for a KRAS peptide with a G12V mutation, wherein the mutated KRAS peptide has a length of about 9 amino acid residues or about 10 amino acid residues.
  • KRAS peptides with a G12V mutation that can be recognized by the TCR of the present invention are a short peptide VVVGAVGVGK (SEQ ID NO: 381) of the amino acid sequence of KRAS from position 7 to position 16 (also referred to herein as "KRAS_G12V_7-16 peptide”); and a short peptide VVGAVGVGK (SEQ ID NO: 382) of the amino acid sequence of KRAS from position 8 to position 16 (also referred to herein as "KRAS_G12V_8-16 peptide”).
  • T cell receptor is a molecule present on the surface of T cells that is responsible for recognizing antigen peptide-MHC complexes (i.e., pMHC).
  • the specific binding of TCR to antigen peptide-MHC complexes triggers T cell activation through a series of biochemical events mediated by related enzymes, co-receptors, and auxiliary molecules.
  • TCR heterodimers are composed of ⁇ and ⁇ chains, while in 5% of T cells, TCR heterodimers are composed of ⁇ and ⁇ chains.
  • Each chain of the TCR is a member of the immunoglobulin superfamily, having an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a cell membrane-spanning region (i.e., a transmembrane region), and a short cytoplasmic tail at the C-terminus.
  • Ig immunoglobulin
  • C Ig constant
  • a cell membrane-spanning region i.e., a transmembrane region
  • cytoplasmic tail at the C-terminus.
  • each variable domain has three hypervariable regions or complementary determining regions (CDRs), of which CDR3 in each variable domain is the primary CDR responsible for recognizing processed antigens. It is believed that CDR2 recognizes MHC molecules.
  • the constant domain of TCR consists of a short linker sequence in which cysteine residues form disulfide bonds, creating a connection between the TCR ⁇ and ⁇ chains.
  • TCR and CD3 form a TCR/CD3 complex.
  • the formation process of the TCR/CD3 complex usually proceeds in the following order: first, the three peptide chains of CD3 ⁇ , ⁇ and ⁇ form two heterodimers of ⁇ - ⁇ and ⁇ - ⁇ to form a stable complex core, and TCR ⁇ (or TCR ⁇ ) binds to it, and then ⁇ - ⁇ or ⁇ - ⁇ dimers bind to the TCR ⁇ (or TCR ⁇ )/CD3 ⁇ complex, and finally transfer to the surface of T cells.
  • the signal is transmitted from TCR to the cell through the TCR/CD3 complex.
  • the signal from the TCR/CD3 complex is enhanced by the simultaneous binding of MHC to a specific co-receptor.
  • this co-receptor is the CD4 molecule, which is specific for class II MHC, and in cytotoxic T cells, this co-receptor is CD8, which is specific for class I MHC.
  • T cell receptor has the conventional meaning in the art and is used to refer to a molecule capable of recognizing a peptide presented by an MHC molecule.
  • the molecule is a heterodimer of two chains ⁇ and ⁇ (or optionally ⁇ and ⁇ ).
  • the TCR of the present invention provides specific affinity recognition for KRAS_G12V mutant antigen.
  • KRAS_G12V mutant antigen is degraded by proteasomes in cells into short peptides of 8 to 10 amino acids in length, for example, KRAS_G12V_7-16 peptide shown in SEQ ID NO:381 and/or KRAS_G12V_8-16 peptide shown in SEQ ID NO:382.
  • These short peptides are presented on the cell surface by MHC class I as peptide/MHC complexes (pMHC).
  • pMHC peptide/MHC complexes
  • the present invention provides isolated or purified T cell receptor (TCR) alpha chain and/or beta chain.
  • TCR T cell receptor
  • the TCR of the present invention can be a hybrid TCR comprising sequences derived from more than one species. For example, considering that murine TCR can be expressed more effectively than human TCR in human T cells, TCR can comprise human variable regions and murine constant regions.
  • the TCR of the present invention comprises an ⁇ chain and a ⁇ chain, wherein the ⁇ chain and the ⁇ chain each comprise three complementarity determining regions (CDRs), and the amino acid sequence of the TCR ⁇ chain CDR3 which is primarily responsible for antigen recognition is selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48 and variants having 1 or 2 amino acid residue changes with the sequence, and the amino acid sequence of the ⁇ chain CDR3 is selected from SEQ ID NO: 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224 and variants having 1 or 2 amino acid residue changes with the sequence.
  • CDRs complementarity determining regions
  • the TCR of the present invention comprises an ⁇ chain and a ⁇ chain
  • the amino acid sequence of the three complementarity determining regions (CDRs) comprised by the ⁇ chain and the amino acid sequence of the three CDRs comprised by the ⁇ chain are:
  • the TCR of the present invention comprises an alpha chain sequence as shown in SEQ ID NO:145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 or 175, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and a beta chain sequence as shown in SEQ ID NO:349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377 or 379, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
  • the constant region of the TCR of the present invention is a mouse constant region.
  • the amino acid residue changes in the TCR variants of the present invention are substitutions, additions or deletions of amino acid residues in the ⁇ chain sequence shown in any SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 or 175, or in the ⁇ chain sequence shown in any SEQ ID NO: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377 or 379, provided that the TCR variant still retains or improves the ability to bind to the epitope peptide-MHC complex of the KRAS_G12V mutant antigen.
  • the substitution is a conservative substitution. Examples of conservative substitutions are given in Table A below.
  • Amino acids can be grouped according to common side chain properties:
  • Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.
  • the TCR of the present invention is capable of recognizing and binding to epitope peptides of mutated KRAS protein presented by HLA class I molecules, triggering an immune response.
  • the HLA class I molecule is any HLA-A molecule, for example, the HLA class I molecule is an HLA-A11 molecule.
  • the HLA-A11 molecule can be any HLA-A11 molecule.
  • Examples of HLA-A11 molecules include, but are not limited to, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, or HLA-A*11:04.
  • the HLA class I molecule is an HLA-A*11:01 molecule.
  • the HLA-A*11:01 molecule is the most common HLA-A molecule in Asians.
  • the present invention also relates to nucleic acids encoding the TCR of the present invention or portions thereof, such as one or more CDRs; one or more variable regions; an alpha chain; or a beta chain, etc.
  • the nucleic acid may be double-stranded or single-stranded, and may be RNA or DNA.
  • the nucleic acid sequence may be codon-optimized to achieve high expression in mammalian production cells. Codon selection for mammalian cells and a variety of other organisms is well known in the art. Codon optimization may also include removal of mRNA unstable motifs and hidden splice sites.
  • the TCR of the present invention can be modified by various methods (e.g., gene fusion, chemical conjugation, etc.) so that the TCR can be connected to other biologically active molecules.
  • the TCR that can be connected to other biologically active molecules can be a TCR heterodimer or a soluble form thereof, more preferably a soluble, single-chain TCR.
  • the other biologically active molecules can be various biologically active effectors, such as antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable markers, etc. There may or may not be a linker between the TCR and other biologically active molecules.
  • the TCR fusion protein is a fusion of a TCR with an antibody, including a complete antibody (e.g., IgG, IgM, or IgA class) or a fragment thereof (e.g., Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabody; single-chain antibody (e.g., scFv); single-domain antibody); and multispecific antibodies (e.g., bispecific antibodies).
  • a complete antibody e.g., IgG, IgM, or IgA class
  • a fragment thereof e.g., Fv, Fab, Fab', Fab'-SH, F(ab') 2
  • diabody single-chain antibody (e.g., scFv); single-domain antibody)
  • multispecific antibodies e.g., bispecific antibodies.
  • the TCR fusion protein is a fusion of a TCR with a cytokine, such as an interleukin (eg, IL-2), a chemokine (eg, MIP-1 ⁇ ), or a growth factor (eg, GCSF).
  • a cytokine such as an interleukin (eg, IL-2), a chemokine (eg, MIP-1 ⁇ ), or a growth factor (eg, GCSF).
  • the TCR conjugate is a covalent linking of the TCR to a cytotoxic agent, such as doxorubicin.
  • a TCR conjugate is a TCR covalently linked to a radioactive substance, such as I 125 .
  • a TCR conjugate is a TCR covalently linked to a detectable label, such as a fluorescent label.
  • T cell receptor fusion proteins or T cell receptor conjugates of the present invention can be used in various applications, including in vivo detection of cells and/or imaging of cells or tissues, and therapeutic uses, such as killing target cells or target tissues expressing KRAS_G12V mutant antigens with specific binding TCR in vivo or in vitro.
  • the present invention also relates to a vector comprising a nucleic acid encoding the TCR of the present invention.
  • the vector is a pUC57-Simple vector (purchased from GenScript Biotech Co., Ltd.).
  • a pUC57-HA vector is used, which is a vector optimized on the basis of the pUC57-Simple vector. It only retains the Ori and Amp sequences of the pUC57-Simple vector, then replaces the Amp sequence with the Kana sequence, and adds the left and right homology arm (HA) sequences (about 800bp) of the TRAC site.
  • HA homology arm
  • the vector transfers the nucleic acid encoding the TCR of the present invention into cells, such as T cells, NK cells, stem cells, e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs), so that the engineered cells express a TCR specific for the KRAS_G12V mutant antigen.
  • cells such as T cells, NK cells, stem cells, e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs), so that the engineered cells express a TCR specific for the KRAS_G12V mutant antigen.
  • the KRAS_G12V mutant antigen-specific TCR refers to a TCR that can specifically bind to and immunologically recognize the G12V mutant KRAS with high affinity. For example, after about 1 ⁇ 10 4 to about 1 ⁇ 10 5 T cells expressing TCRs are co-cultured with antigen-presenting cells such as T2 cells or K562 cells pulsed with KRAS with G12V mutations and overexpressing HLA class I molecules, the secretion of IFN- ⁇ is induced with an EC50 of about 1 ⁇ 10 -7 M or less (e.g., 1 ⁇ 10 -8 M or less, 1 ⁇ 10 -9 M or less, 1 ⁇ 10 -10 M or less), then the TCR is considered to have antigen specificity for the G12V mutant KRAS.
  • the HLA class I molecule can be any HLA class I molecule described herein (e.g., HLA-A*11:01 molecule).
  • the vector allows for sustained high-level expression of the introduced exogenous TCR in engineered cells (e.g., engineered T cells), and the introduced exogenous TCR can successfully compete with the endogenous TCR for a limited pool of CD3 molecules.
  • the vector optionally comprises genes for CD3- ⁇ , CD3- ⁇ , CD3- ⁇ , and/or CD3- ⁇ .
  • the vector comprises genes for CD3- ⁇ .
  • one or more separate vectors encoding CD3 genes can also be provided for co-transfer into cells with exogenous TCR encoding vectors.
  • the vector form is not limited to homologous recombination repair (HDR) vectors, and can also be viral vectors.
  • the viral vector can be a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a retroviral vector, or a baculoviral vector, which is used to edit the cell genome.
  • Genome editing technology refers to a technology that inserts, deletes or replaces nucleic acids in the cell's genomic DNA.
  • T cells that have been genetically modified using gene editing technology for primary human T cells have shown excellent efficacy in clinical trials of a variety of adoptive immunotherapy drugs.
  • chimeric antigen receptors (CAR) or T cell receptors (TCR) are often used to transform primary human T cells to achieve recognition of certain specific target epitopes. These modified T cells can specifically kill specific target cells.
  • TCR gene editing methods can be roughly divided into two categories according to the gene integration method.
  • One is the random integration of genes: including lentivirus (LV) system, adeno-associated virus (AAV) system, transposon system, etc.
  • the other is precise gene editing methods: including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) technology, etc.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR clustered regularly interspaced short palindromic repeats
  • CRISPR technology recognizes and edits DNA through the guidance of gRNA, and inserts large gene fragments at a specific point through homologous recombination, which has the advantages of easy operation and stronger scalability.
  • the target TCR can be introduced into cells using viral vectors.
  • the method of introducing exogenous TCR ⁇ / ⁇ genes into cells based on viral vectors does not knock out the endogenous TCR of the cells, which may cause mismatching of the exogenous TCR ⁇ chain and ⁇ chain.
  • Non-viral vectors can also be used to introduce the target TCR into cells, and the exogenous TCR ⁇ / ⁇ genes can be precisely integrated into specific genomic sites of the cells.
  • non-viral vector-based gene editing methods can knock out the endogenous T cell receptor ⁇ chain and ⁇ chain of human T cells through CRISPR/Cas9 technology and homologous recombination technology, and knock in exogenous target T cell receptor ⁇ and ⁇ chain encoding nucleotides at the TRAC gene exon.
  • endogenous TCR expression is destroyed, and endogenous TCR promoters are used to express exogenous target TCR ⁇ and ⁇ .
  • exogenous target T cell receptor ⁇ and ⁇ chain encoding nucleotides are knocked into exon 1 of the endogenous TRAC gene, and the exogenous knock-in fragment does not need to add the TRAC gene, thereby reducing the length of the gene knock-in fragment and reducing the difficulty of gene knock-in.
  • the technology of expressing TCR using non-viral vectors can be used as a fast, simple and low-cost way to introduce exogenous TCR ⁇ / ⁇ genes into cells.
  • TCR is a dimer, composed of a combination of TCR ⁇ chain and TCR ⁇ chain.
  • the TCR ⁇ chain gene is formed by the rearrangement of TRAV, TRAJ and TRAC genes, where TRAV and TRAJ genes contain multiple sequences respectively, and there are differences between the multiple sequences. During rearrangement, only one sequence can be randomly selected for expression. If TRAV and TRAJ genes are selected as knockout sites, it is difficult to avoid the generation of any random TCR ⁇ chain gene, while there is only one TRAC gene. By knocking out the TRAC gene, any random TCR ⁇ chain gene can be knocked out, so TRAC is suitable as a knockout site.
  • the TCR ⁇ chain gene is formed by the rearrangement of TRBV, TRBJ, TRBD and TRBC genes, where TRBV and TRBJ genes contain multiple sequences respectively, and there are differences between the multiple sequences, and they are not suitable as knockout sites.
  • TRBC gene contains TRBC1 and TRBC2, both of which contain partially identical sequences.
  • the common sequence can be selected as a knockout site, and any random TCR ⁇ gene can be knocked out by knocking out the common sequence.
  • one or more of the endogenous TRAC gene, the endogenous TRBC1 gene and/or the TRBC2 gene are knocked out. In some embodiments, the endogenous TRAC gene and the endogenous TRBC1 and TRBC2 genes are knocked out simultaneously, thereby obtaining a higher endogenous TCR knockout efficiency and reducing the risk of mismatch between the chains of the exogenous TCR and the endogenous TCR that may be caused by the expression of the endogenous TCR.
  • Nuclease-based genome editing tools can be used to target the disruption of endogenous TRAC and TRBC genes by inducing double-strand breaks and DNA repair via nonhomologous end joining (NHEJ). These tools include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), megaTAL nucleases, and CRISPR/CRISPR-associated protein 9 (CRISPR/Cas9).
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR/CRISPR-associated protein 9 CRISPR/CRISPR-associated protein 9
  • the exogenous TCR ⁇ / ⁇ gene knock-in site is selected as the endogenous TRAC site.
  • the endogenous TCR promoter of human T cells can be used to express the exogenous TCR ⁇ / ⁇ gene of the present invention (also called the "nwTCR" gene) without the need to add the TRAC gene, thereby reducing the size of the knock-in fragment and improving the efficiency of gene editing.
  • the expression construct of nwTCR is cloned into a targeting vector (e.g., pUC57-S vector), and the nwTCR is site-specifically knocked into the constant region of the TCR ⁇ chain by designing homology arms, and the expression is regulated by the transcriptional regulatory sequence of the locus. Since the regulation level of the endogenous promoter at the knock-in site is better than that at other sites, the continuous and stable expression of the nwTCR gene is ensured.
  • a targeting vector e.g., pUC57-S vector
  • the present invention provides engineered cells expressing exogenous TCRs.
  • engineered cells expressing TCRs are prepared from cells derived from blood, bone marrow, lymph or lymphoid organs, e.g., lymphocytes, including but not limited to T cells, NK cells, or stem cells, e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs).
  • lymphocytes including but not limited to T cells, NK cells, or stem cells, e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs).
  • stem cells e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs).
  • the cells are typically primary cells, e.g., cells isolated directly from a subject and/or isolated from a subject and frozen.
  • the cells can be allogeneic cells and/or autologous cells.
  • CRISPR/Cas9 and homologous recombination technology are used to electroporate and transfect CD3/CD28 activated primary cells (e.g., sorted CD4+ T cells and CD8+ T cells) using RNPs and plasmids to prepare engineered TCR cells.
  • sgRNAs are designed against the endogenous TRAC gene, and sgRNAs are designed against the endogenous TRBC1 gene and TRBC2 gene.
  • the Cas9 protein is guided by sgRNA to bind to a specific site in the target genome, and the Cas9 protein cuts the specific site.
  • sgRNA For double-strand breaks formed by the endogenous TRAC gene caused by RNP, homologous recombination can occur in the presence of donor DNA with homologous arms, thereby achieving site-specific insertion of the target nwTCR gene.
  • the specific site of the target genome to which the sgRNA guides the Cas9 protein to bind is located in exon 1 of the TRAC gene, and the Cas9 protein cuts the specific site.
  • the designed and verified sgRNA recognition sequence and PAM sequence that efficiently target it comprise the nucleotide sequence shown in TCAGGGTTCTGGATATCTGT-GGG (the sgRNA recognition sequence shown in SEQ ID NO: 383-the PAM sequence shown in SEQ ID NO: 385, and "-" is used to distinguish the CRISPR/Cas9 recognition site and the PAM sequence).
  • the specific site of the target genome to which sgRNA guides Cas9 protein binding is located in exon 1 of TRBC1 and TRBC2 genes, and Cas9 protein cuts the specific site.
  • the designed and verified sgRNA recognition sequence and PAM sequence that efficiently target it include the nucleotide sequence shown in CTGCCTGAGCAGCCGCCTGA-GGG (the sgRNA recognition sequence shown in SEQ ID NO: 384 - the PAM sequence shown in SEQ ID NO: 385, and "-" is used to distinguish the CRISPR/Cas9 recognition site and the PAM sequence).
  • the CRISPR/Cas system may include Cas components in the form of proteins or in the form of nucleic acids encoding Cas proteins.
  • the Cas protein may be any Cas protein as long as it has endonuclease or nickase activity when complexed with a guide RNA.
  • the Cas protein is a Cas9 protein or a variant or a functional fragment thereof.
  • the Cas protein can be a protein isolated from an organism such as a Streptococcus sp., preferably Streptococcus pyogenes, or a recombinant protein, but is not limited thereto.
  • the Cas protein comprises Cas9 derived from Streptococcus pyogenes, such as Cas9 having the amino acid sequence shown in SEQ ID NO: 405.
  • the Cas protein comprises an amino acid sequence having at least 50% homology to the amino acid sequence shown in SEQ ID NO: 405, preferably having at least 60, 70, 80, 90, 95, 97, 98 or 99% homology to the amino acid sequence shown in SEQ ID NO: 405, but is not limited thereto.
  • the Cas protein encoding nucleic acid may be in the form of a vector, such as a plasmid comprising a Cas encoding sequence under a promoter such as CMV or CAG.
  • the Cas protein is Cas9
  • the Cas9 encoding sequence may be derived from Streptococcus, preferably from Streptococcus pyogenes.
  • the Cas9 encoding nucleic acid may comprise a nucleotide sequence encoding SEQ ID NO: 405.
  • the Cas9 encoding nucleic acid may comprise a nucleotide sequence having at least 50% homology to the nucleotide sequence encoding SEQ ID NO: 405, preferably a nucleotide sequence having at least 60, 70, 80, 90, 95, 97, 98 or 99% homology to the nucleotide sequence encoding SEQ ID NO: 405, but is not limited thereto.
  • the donor DNA sequentially comprises a 5' homology arm, a sequence encoding a cleavable linker polypeptide, an exogenous TCR ⁇ / ⁇ gene or a functional fragment thereof, and a 3' homology arm. After the sequence encoding the cleavable linker polypeptide is expressed, the cleavable linker polypeptide is cleaved.
  • the cleavable linker polypeptide sequence comprises a 2A ribosomal skipping element such as T2A, E2A, P2A, and F2A.
  • the donor DNA is located in a targeting vector.
  • the basic targeting vector as a backbone is not particularly limited, and only needs to have a prokaryotic replication origin and a selection marker for vector propagation in bacteria.
  • a sequence encoding a cleavable linker polypeptide and a signal peptide sequence are connected to the N-termini of the exogenous TCR ⁇ chain gene and the exogenous TCR ⁇ chain gene in the targeting vector, respectively.
  • the targeting vector for knocking in the nwTCR gene sequence comprises the following structure in an operative connection: 5'-2A ribosomal jumping element-SP-TCR ⁇ -2A ribosomal jumping element-SP-TRAV-TRAJ-3'
  • SP is the signal peptide coding sequence.
  • the targeting vector, RNP complex and cells for knocking in the nwTCR gene sequence are mixed and the delivery step of the nwTCR gene sequence to the cell is implemented.
  • the delivery step is selected from: electroporation, transfection, deformation of the cell membrane by physical means, lipid nanoparticles (LNP), virus-like particles (VLP) and sonication.
  • the delivery step includes electroporation.
  • the engineered cells are primary cells.
  • the engineered cell is an isolated cell, wherein the isolated cell is isolated from a subject.
  • the engineered cells are ex vivo cultured cells.
  • ex vivo cultured cells include stimulated cells.
  • stimulated cells include cytokine stimulated T cells, optionally, wherein cytokine stimulated T cells include CD3 stimulated T cells, CD28 stimulated T cells or CD3 and CD28 stimulated T cells.
  • cytokine stimulated T cells are cultured in the presence of IL7, IL15 or a combination thereof.
  • cytokine stimulated T cells are cultured in the presence of IL2.
  • the engineered cells are stem cells, e.g., hematopoietic stem cells (HSCs). Transferring the nwTCR gene to HSCs does not result in expression of the TCR on the cell surface because stem cells do not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, the initiation of CD3 expression will result in expression of the introduced nwTCR on the surface of thymocytes.
  • HSCs hematopoietic stem cells
  • TCR gene-modified stem cells are a continuous source of mature T cells with desired antigen specificity. Therefore, nwTCR gene-modified stem cells produce T cells expressing the TCR of the present invention after differentiation.
  • the present invention provides a method for preventing or treating cancer associated with KRAS_G12V mutant antigen, comprising administering an engineered cell of the present invention, a TCR nucleic acid of the present invention, a vector or a pharmaceutical composition to a subject in need thereof.
  • the method comprises administering a polynucleotide encoding a TCR.
  • the method comprises administering a vector comprising a polynucleotide encoding a TCR.
  • the method comprises administering an effective amount of an engineered cell of the present invention.
  • the engineered cells of the present invention, the TCR nucleic acids of the present invention, the vectors or the pharmaceutical compositions of the present invention are used to prevent or treat cancer associated with the KRAS_G12V mutant antigen.
  • the TCR of the present invention can specifically bind to the KRAS_G12V mutant antigen, thereby mediating an immune response against target cells expressing the KRAS_G12V mutant antigen.
  • the treatment or prevention may include treatment or prevention of one or more symptoms of the cancer being treated or prevented, including promoting tumor regression, delaying the onset of cancer or its symptoms, preventing or delaying the recurrence of cancer or its symptoms.
  • the present invention also provides a method for detecting the presence of cancer in a mammal.
  • the method comprises: (i) contacting a sample comprising one or more cells from a mammal with any of the TCR of the present invention, a cell population expressing the TCR of the present invention, or a pharmaceutical composition comprising a cell population expressing the TCR of the present invention as described herein, thereby forming a complex; and (ii) The complex is detected, wherein the complex indicates that a mammal has cancer.
  • the contact can be implemented in vitro or in vivo in a mammal. In one embodiment, the contact is implemented in vitro.
  • the complex can be detected in a variety of ways known in the art.
  • the TCR of the present invention or a cell colony expressing the TCR of the present invention is labeled with a detectable marker, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particles (e.g., gold particles).
  • a detectable marker such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particles (e.g., gold particles).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • an enzyme e.g., alkaline phosphatas
  • the present invention also provides a method for inducing anti-tumor immunity, wherein the tumor is a KRAS_G12V mutant antigen-associated tumor, and the method comprises administering an effective amount of the engineered cells of the present invention to a subject.
  • the present invention provides a method for inducing an immune response in a subject, comprising administering an effective amount of an engineered cell of the present invention.
  • the immune response is a T cell-mediated immune response.
  • the T cell-mediated immune response is directed to one or more target cells.
  • the engineered immune cell comprises a TCR of the present invention.
  • the target cell is a cancer cell associated with a KRAS_G12V mutant antigen.
  • donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy).
  • donor stem cells to be differentiated into T cells for T cell therapy are obtained from a non-patient subject.
  • T cells can be administered in a therapeutically effective amount.
  • a therapeutically effective amount of T cells can be at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 cells, or at least about 10 10 cells/kg body weight.
  • the cancer mentioned in the various methods of the present invention can be any cancer, including but not limited to: acute lymphocytic cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, brain cancer, glioma, nasopharyngeal cancer, eye cancer, oral cancer, cervical cancer, esophageal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, lung cancer, bone cancer, breast cancer, gastrointestinal tumors, colon cancer, small intestine cancer, colorectal cancer, rectal cancer, gastric cancer, skin cancer, melanoma, multiple myeloma, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, ureteral cancer, bladder cancer, penis cancer, testicular cancer, pancreatic cancer, prostate cancer, kidney cancer, soft tissue cancer and thyroid cancer.
  • the cancer is lung cancer, pancre
  • the invention also provides methods and engineered cells for improving cell therapy.
  • CD8 molecules are type I transmembrane glycoproteins expressed on the cell surface in the form of homodimers composed of two CD8a chains (also referred to herein as "CD8aa”) and/or in the form of heterodimers composed of one CD8a chain and one CD8b chain (also referred to herein as "CD8ab”)
  • the present invention co-expresses exogenous TCR and CD8aa molecules in T cells.
  • the non-viral gene editing method based on CRISPR/Cas9 technology uses nucleic acids encoding the nwTCR of the present invention and nucleic acids encoding the CD8a chain to perform gene editing on CD8+T cells/CD4+T cells, indicating that the co-expression of exogenous nwTCR and CD8aa molecules in CD8+T cells/CD4+T cells can enhance the binding of TCR-T cells to pMHC molecules.
  • exogenous nwTCR and CD8aa molecules are co-expressed in CD8+T cells, since they can be The increase in CD8aa molecules used by exogenous nwTCR is expected to improve the TCR-specific cytotoxicity (including its continuous killing ability) and in vivo anti-tumor function of CD8+ T cells.
  • exogenous nwTCR and CD8aa molecules are co-expressed in CD4+ T cells, along with the expression of endogenous CD4 molecules, CD4+ T cells exhibit a hybrid phenotype, which is expected to recognize antigens with similar affinity to natural CD8+ T cells and kill target cells, showing cytotoxic effector function; while retaining the natural helper function of CD4+ T cells.
  • the present invention co-expresses exogenous TCR and CD8ab molecules in T cells.
  • the nucleic acid encoding the nwTCR of the present invention is used to perform gene editing on CD8+ T cells/CD4+ T cells, indicating that the co-expression of exogenous nwTCR and CD8ab molecules in CD8+ T cells/CD4+ T cells can enhance the binding of TCR-T cells to pMHC molecules.
  • CD4+ T cells When exogenous nwTCR and CD8ab molecules are co-expressed in CD4+ T cells, accompanied by the expression of endogenous CD4 molecules, CD4+ T cells exhibit a hybrid phenotype, which is expected to recognize antigens with an affinity similar to that of natural CD8+ T cells and kill target cells, exhibiting cytotoxic effector function; while retaining the natural auxiliary function of CD4+ T cells.
  • CD8aa molecules and/or CD8ab molecules are co-expressed with TCR genes in CD8+ and CD4+ T cells, it has a beneficial effect on the function of CD8+ and CD4+ T cells.
  • CD4+ T cells can be reprogrammed into multifunctional hybrid T cells through MHC class I TCR and CD8 molecules, which have both cytotoxic effector function and natural helper function.
  • Dendritic cells (DC cells) from cancer patients with HLA-A*11:01 genotype and expressing KRAS_G12V were pulsed in vitro with KRAS_G12V_7-16 peptide and KRAS_G12V_8-16 peptide resuspended in DMSO, and co-cultured with CD8+ T cells sorted from the patient's peripheral blood for 10 days.
  • the negative control was pulsed in vitro with DMSO and co-cultured with CD8+ T cells sorted from the patient's peripheral blood for 10 days.
  • TCRs antigen-specific T cell receptors
  • the antigen-specific T cell receptors (TCRs) on these 16 T cell clones that specifically bind to the KRAS_G12V_7-16 epitope peptide and the KRAS_G12V_8-16 epitope peptide were named nwTCR-0125, nwTCR-0126, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, and nwTCR-2241, respectively.
  • nwTCR-2308, nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2563, nwTCR-2595 and nwTCR-2629, and the amino acid sequences of the paired TCR ⁇ and ⁇ chains on these 16 T cell clones were determined on a single-cell basis using high-throughput paired TCR sequencing.
  • the codon frequencies used in different organisms are different. Therefore, the codon sequences of the coding nucleotides of the TCR ⁇ chain and ⁇ chain amino acid sequences were optimized to increase the expression of TCR when expressed in eukaryotic cells.
  • the nucleotide sequences of 16 TCRs that specifically recognize the KRAS_G12V_7-16 epitope peptide and the KRAS_G12V_8-16 epitope peptide were obtained after codon optimization.
  • Table 1A and Table 1B list the amino acid sequence information and nucleotide sequence information of the ⁇ chain and ⁇ chain of 16 TCRs expressed by the cloned T cell lines generated by sequencing, respectively.
  • This example describes the preparation and characterization of KRAS_G12V mutant antigen-specific TCR-T cells by knocking out the TCR gene in primary T cells by CRISPR/Cas9 technology and knocking in the KRAS_G12V mutant antigen-specific TCR gene by homologous recombination technology.
  • CD4/CD8 T cells A mixture of CD4 T cells and CD8 T cells (also referred to herein as "CD4/CD8 T cells”) was enriched and sorted from peripheral blood mononuclear cells (PBMC, purchased from Shanghai Sai Li Biotechnology Co., Ltd., Donor: S2001095).
  • PBMC peripheral blood mononuclear cells
  • the enriched and sorted CD4/CD8 T cells were aliquoted and frozen (5x10 6 cells/cryotube) for future use.
  • T cell activator Miltenyi T cell TransACT Miltenyi catalog number: 130-111-160
  • T cell culture medium e.g., RPMI 1640, FBS, L-glutamine, non-essential amino acids, sodium pyruvate, HEPES buffer, 2-mercaptoethanol, and optionally IL2
  • T cell culture medium e.g., RPMI 1640, FBS, L-glutamine, non-essential amino acids, sodium pyruvate, HEPES buffer, 2-mercaptoethanol, and optionally IL2
  • gRNAs used were gRNA002 and gRNA004 (see Table 2), the target site of gRNA002 was located in exon 1 of the endogenous TRAC gene ( Figure 1A); the target site of gRNA004 was located in exon 1 of the endogenous TRBC1 and TRBC2 genes ( Figure 1B).
  • Cas9 enzyme was purchased from GenScript Biotech Co., Ltd., catalog number: Z03469.
  • the backbone of the targeting vector (also called HDR vector) is the pUC57-HA vector, which is an optimized vector based on the pUC57-Simple vector. It only retains the Ori and Amp sequences of the pUC57-Simple vector, then replaces the Amp sequence with the Kana sequence, and adds the left and right homologous arm (HA) sequences of the TRAC site (about 800bp).
  • the sequence to be knocked in (KI) can be constructed between the left and right HAs.
  • the KI sequence construct structure of nwTCR includes: 2A or its variant-SP-TCR ⁇ -2A or its variant-SP-TRAV-TRAJ, where 2A is a ribosomal skipping element; SP is a signal peptide; 4 synonymous mutant bases are introduced into the TRBC gene in the targeting vector, which are the mutation of the coding nucleotide of TRBC S77 from AGC to TCC and the mutation of the coding nucleotide of S78 from AGC to TCC.
  • nwTCR-0126 the KI sequence construct sequence is as shown in SEQ ID NO:386, wherein: HA is the homology arm (5’HA is as shown in SEQ ID NO:387, 3’HA is as shown in SEQ ID NO:391; 2A or its variants are as shown in SEQ ID NO:388 and SEQ ID NO:390 respectively; SP is the signal peptide sequence shown in SEQ ID NO:389; TCR ⁇ is the nucleotide sequence of the complete TRB (TCR ⁇ chain) of nwTCR-0126 (SEQ ID NO:352), wherein the coding nucleotide of TRBC S77 is mutated from AGC to TCC and the coding nucleotide of S78 is mutated from AGC to TCC, thereby introducing 4 homologous residues into the TRBC gene.
  • HA is the homology arm
  • 5’HA is as shown in SEQ ID NO:387
  • 3’HA is as shown in SEQ ID NO:391
  • TRAV is the nucleotide sequence of the nwTCR-0126TRAV gene (SEQ ID NO: 54); and TRAJ is the nucleotide sequence of the nwTCR-0126TRAJ gene (SEQ ID NO: 56).
  • KI sequence constructs of nwTCR-0125, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, nwTCR-2241, nwTCR-2308, nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2563, nwTCR-2595, and nwTCR-2629 were prepared.
  • the sgRNA of Example 2.2 was fully mixed with the Cas9 enzyme and incubated at room temperature for 10 min to prepare RNP.
  • the targeting vector containing the KI TCR sequence prepared in Example 2.2 was fully mixed with the incubated RNPs and T cells of a specified concentration (about 1.25E6 T cells/electroporation tube) prepared in Example 2.1 to knock out (KO) endogenous TCR and knock in (KI) exogenous TCR.
  • electroporation transfection instrument (Celetrix; catalog number: CTX-1500A LE) for cell electroporation transfection.
  • the conditions of electroporation transfection were 480-560V, 20ms.
  • the cells were left to rest for 15 minutes before the electroporation transfected T cells were taken out and transferred to pre-warmed culture medium (ImmunoCult TM -XF T Cell Expansion Medium, Stemcell Company Catalog Number: 10981). After culturing the cells for 5 days, flow cytometry characterization was performed on Day 7.
  • Example 2.3 The cell suspension obtained in Example 2.3 was thoroughly mixed, the cells were counted, and an appropriate amount of cells were collected for staining with two labeled peptide-MHC (HLA-A*11:01) tetramers (VVVGAVGVGK-HLA-A*11:01 tetramer and VVGAVGVGK-HLA-A*11:01 tetramer, which may also be referred to as pMHC).
  • HLA-A*11:01 two labeled peptide-MHC
  • the peptide-MHC (HLA-A*11:01) tetramer staining solution and LIVE/DEAD TM Fixable Near-IR containing the two markers were prepared in advance and purchased from Invitrogen, catalog number: L10119; CD4-FITC was purchased from BioLegend, catalog number: 357406; CD8-PerCP-cy5.5 was purchased from BioLegend, catalog number: 344710; anti-human TCR ⁇ / ⁇ -BV510 antibody was purchased from BioLegend, catalog number: 306734.
  • the collected cells were stained with the two labeled peptide-MHC (HLA-A*11:01) tetramer stains, washed, and characterized by flow cytometry.
  • FIG3A-3P illustrate flow cytometry results of CD4+T cells and CD8+T cells electroporated with different nwTCRs and stained with pMHC tetramers.
  • nwTCR-0125, nwTCR-0126, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, nwTCR-2241, nwTCR-2308 and nwTCR-2563 are exemplified with the staining results of the labeled VVVGAVGVGK-HLA-A*11:01 tetramer;
  • nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2595 and nwTCR-2629 are exemplified with the staining results of the labeled VVGAVGVGK-HLA-A*11:01
  • FIG. 1 A schematic diagram of the results of flow cytometry detection of TCR gene editing efficiency is shown in Figure 2.
  • CD8+T cells knocked into each nwTCR can bind to the peptide-MHC complex (pMHC) tetramer; there are significant differences in the binding of CD4+T cells knocked into each TCR to the peptide-MHC complex (pMHC) tetramer.
  • pMHC peptide-MHC complex
  • the CD8+T cells expressing each nwTCR can specifically bind to the peptide-MHC complex (pMHC) tetramer; and when each nwTCR is knocked into CD4+T cells, the CD4+T cells expressing each nwTCR have the following situation: Generally, when the affinity of nwTCR and MHC is strong enough, TCR can bind to MHC molecules without the assistance of CD8 molecules. For example, it is believed that nwTCR-0127 has a stronger binding ability to MHC molecules than nwTCR-1708. Therefore, they will have differences in the pMHC tetramer staining of CD4+ T cells.
  • pMHC peptide-MHC complex
  • each nwTCR is edited separately After being introduced into CD4+ T cells and expressed, nwTCRs with strong affinity can still bind to MHC class I antigens, while nwTCRs with weak affinity also have weak ability to bind to MHC class I antigens.
  • the KRAS_G12V mutant antigen-specific TCR-T cells of Example 2 were selectively activated using TransACT activator (Miltenyi catalog number: 130-111-160). The TCR-T cells were cultured until Day 14. On Day 14, in vitro functional studies were performed on each TCR-T cell.
  • the TCR-T cell affinity detection method is implemented as follows. Antigen presenting cells (T2 cells or K562 cells overexpressing HLA-A*11:01) are collected, the cells are counted, and an appropriate amount of culture medium (such as RPMI-1640 culture medium, purchased from Gibco, catalog number: 22400089; FBS, purchased from Gibco, catalog number: 10099141C) is added to resuspend the cells to a cell density of 1E6 cells/mL, and 1 mL of the cell suspension is added to each well of a 24-well plate.
  • culture medium such as RPMI-1640 culture medium, purchased from Gibco, catalog number: 22400089
  • FBS purchased from Gibco, catalog number: 10099141C
  • the peptide solution to be tested (the peptide is KRAS_G12V_7-16 peptide shown in SEQ ID NO: 381 and/or KRAS_G12V_8-16 peptide shown in SEQ ID NO: 382) was diluted to 10 -10 -10 -3 M, and 10ul of the diluted peptide solution was added to the corresponding wells of the 24-well plate, and incubated in an incubator (37°C, 5% CO 2 ) for 2 hours, and the incubated antigen-presenting cells were collected and washed, and 100ul of 1E6/mL antigen-presenting cells were taken to the corresponding wells of the 96-well plate.
  • the nwTCR-T cells to be tested were collected, and an appropriate amount of T cell culture medium (purchased from STEMCELL, catalog number: 10981) was added to the cell density of 1E6/mL, and 100ul of the cell suspension was added to the corresponding wells of the 96-well plate.
  • Each nwTCR-T cell was co-cultured with antigen presenting cells (37°C, 5% CO 2 ) for 16 h, and the cell supernatant was collected and the IFN- ⁇ concentration was detected using an ELISA kit (purchased from Biolegend, catalog number: 430104).
  • the binding affinity of T cells expressing each nwTCR to the short peptide represented by SEQ ID NO: 381 or SEQ ID NO: 382 presented by HLA-A*11:01 was detected by detecting the release level of IFN- ⁇ .
  • Figures 4A-4H show the binding affinity test results and EC50 values of T cells expressing each nwTCR for the short peptide VVVGAVGVGK (SEQ ID NO: 381) or VVGAVGVGK (SEQ ID NO: 382) presented by HLA-A*11:01, among which Figures 4A, 4C, 4D and 4G are the binding affinities with the short peptide VVVGAVGVGK (SEQ ID NO: 381); Figures 4B, 4E, 4F and 4H are the binding affinities with the short peptide VVGAVGVGK (SEQ ID NO: 382).
  • FIGS. 4A-4H show that after T2 cells presenting peptide-MHC complexes were co-incubated with T cells expressing each nwTCR, T cells expressing each nwTCR were detected to specifically bind to the peptide-MHC complex, resulting in IFN- ⁇ release.
  • the experimental results using 9-mer and 10-mer short peptides in Figures 4A-4F show that T cells expressing each nwTCR can specifically bind to pMHC complexes presenting 9-mer and 10-mer short peptides, respectively.
  • the killing detection method of each TCR-T cell on the target cell was implemented as follows.
  • the target cells SW620 (HLA-A*11:01 overexpression, KRAS G12V+) cell line (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were collected, and the cells were counted and then incubated with target cell culture medium (RPMI-1640 culture medium, purchased from Gibco, catalog number: 22400089, FBS, The target cells were resuspended in an E-plate (purchased from Gibco, catalog number: 10099141C) to a cell density of 1E6 cells/mL.
  • target cell culture medium RPMI-1640 culture medium, purchased from Gibco, catalog number: 22400089, FBS
  • An E-plate (obtained from Agilent, catalog number: 300600890) was prepared, 100 ⁇ L of the mixed target cell suspension was added to the corresponding well, and then placed in an RTCA real-time cell analyzer (purchased from Agilent, model: xCELLigence RTCA DP) for overnight detection.
  • RTCA real-time cell analyzer purchased from Agilent, model: xCELLigence RTCA DP
  • TCR-T cell Collect each TCR-T cell to be tested, count the cells, add an appropriate amount of T cell culture medium (purchased from STEMCELL, catalog number: 10981) to resuspend the cells. Take out the E-plate inoculated with target cells, add T cell suspension, put the E-Plate back into the RTCA analyzer for detection, and obtain the cell index for 72 hours. Each independent experiment was performed three times. The interval slope was automatically calculated using RTCA software to evaluate the rate of change of the cell index. In order to demonstrate the effect of the treatment, the cell index was standardized to an equal value at the standardized time point.
  • T cell culture medium purchased from STEMCELL, catalog number: 10981
  • nwTCR-T cells to be tested, add an appropriate amount of T cell culture medium (purchased from STEMCELL, catalog number: 10981) to a cell density of 1E6/mL, add 100 ⁇ L of the nwTCR-T cell suspension to be tested to the wells of the 96-well plate, and mix with the SW620 (HLA-A*11:01 overexpression, KRAS G12V+) cells in the wells of the 96-well plate. Add 1 ⁇ L of ethidium bromide (1mg/mL) solution to the cell mixture, mix well, and place the cell culture plate in a real-time fluorescence imaging system (BioTek Lionheart) for cell killing characterization experiments. Target cells specifically recognized by T cells will be stained with ethidium bromide after entering apoptosis, showing red fluorescence signals.
  • T cell culture medium purchased from STEMCELL, catalog number: 10981
  • SW620 HLA-A*11:01 overexpression, KRAS G12V+
  • FIG. 6 illustrates the results of the initial co-incubation of nwTCR-2424-expressing T cells with SW620 cells (0h) and 18 hours after co-incubation (18h).
  • the red fluorescent signal at 18 hours after co-incubation (18h) indicates the specific killing of SW620 cells by nwTCR-2424-expressing T cells.
  • This example describes the use of CRISPR/Cas9 and homologous recombination technology to co-express exogenous TCR and CD8 molecules on the surface of CD4+ T cell membranes, thereby redirecting CD4+ T cells.
  • this gene editing method can enhance the binding of TCR-T cells to pMHC molecules.
  • T cells can be obtained commercially (e.g., frozen human peripheral blood CD4+CD45RA+ T cells, Stem Cell Technology, catalog number 70029) or prepared from a single sample of leukocytes (Day 0).
  • CD4/CD8 T cells were enriched and sorted from leukocyte aliquots.
  • the enriched and sorted CD4/CD8 T cells were aliquoted and frozen (5x10 6 cells/cryotube) for future use.
  • the targeting strategy and targeting vector of TCR are the same as those in the above embodiment 2.2.
  • the TCR sequence is nwTCR-1708
  • the KI amino acid sequence of nwTCR-1708 is: 2A or its variant-SP-TCR ⁇ -2A or its variant-SP-TRAV-TRAJ (SEQ ID NO: 392)
  • the KI nucleotide sequence of nwTCR-1708 is: 2A or its variant-SP-TCR ⁇ -2A or its variant-SP-TRAV-TRAJ (SEQ ID NO: 393).
  • CD8+ T cells expressing nwTCR-1708 can bind to the pMHC molecule of the amino acid sequence of KRAS G12V from position 7 to position 16 (KRAS_G12V_7-16 peptide), but CD4+ T cells expressing nwTCR-1708 cannot bind to the pMHC molecule presenting the KRAS_G12V_7-16 peptide.
  • the purpose of selecting pMHC tetramers that do not bind to CD4+ T cells expressing TCR after gene editing in this example is to show that even for such nwTCR, the binding of CD4 T cells to pMHC molecules after gene editing of nwTCR can be enhanced by introducing CD8 molecules into CD4 T cells. Therefore, the specific nwTCR used in this example can be replaced by any other nwTCR of the present invention and can achieve the effect of enhancing the binding of CD4 T cells to pMHC molecules after gene editing of nwTCR.
  • KI amino acid sequence of nwTCR-1708-CD8a is: 2A or its variant-CD8a-2A or its variant-SP-TCR ⁇ -2A or its variant-SP-TRAV-TRAJ (SEQ ID NO: 394).
  • the KI nucleotide sequence of nwTCR-1708-CD8a is: 2A or its variant-CD8a-2A or its variant-SP-TCR ⁇ -2A or its variant-SP-TRAV-TRAJ (SEQ ID NO:395).
  • FIG. 8A The targeting strategy diagram of nwTCR-CD8a is shown in Figure 8A.
  • KI amino acid sequence of nwTCR-1708-CD8ab is: 2A or its variant-CD8a-2A or its variant-CD8b-2A or its variant-SP-TCR ⁇ -2A or its variant-SP-TRAV-TRAJ (SEQ ID NO: 398).
  • the KI nucleotide sequence of nwTCR-1708-CD8ab is: 2A or its variant-CD8a-2A or its variant-CD8b-2A or its variant-SP-TCR ⁇ -2A or its variant-SP-TRAV-TRAJ (SEQ ID NO:399).
  • nwTCR-CD8ab The targeting strategy diagram of nwTCR-CD8ab is shown in Figure 8B.
  • the sgRNA designed and synthesized in Example 2.2 was fully mixed with the Cas9 enzyme and incubated at room temperature for 10 min to prepare RNP.
  • the three targeting vectors prepared in Example 4.2 i.e., the targeting vectors for knocking in nwTCR-1708, nwTCR-1708-CD8a, or nwTCR-1708-CD8ab, were fully mixed with the incubated RNPs and the specified concentration of T cells (1.25E6/electric shock tube) prepared in Example 4.1, respectively, to knock out (KO) endogenous TCR and knock in (KI) exogenous TCR, TCR and CD8a, or TCR and CD8ab.
  • T cells (1.25E6/electric shock tube
  • the mixed solution was loaded into an electroporation transfection instrument (Celetrix; catalog number: CTX-1500A LE) for cell electroporation transfection.
  • the conditions of electroporation transfection were 480-560V, 20ms.
  • the cells were left to stand for 15 minutes before being taken out and transferred to a pre-warmed culture medium (ImmunoCult TM -XF T Cell Expansion Medium, Stemcell Company Catalog Number: 10981). After culturing the cells for 5 days, flow cytometry characterization was performed on Day 7.
  • a pre-warmed culture medium ImmunoCult TM -XF T Cell Expansion Medium, Stemcell Company Catalog Number: 10981.
  • Example 4.3 The cell suspension obtained in Example 4.3 was thoroughly mixed, and an appropriate amount of cells were collected after cell counting, and then stained with the labeled peptide-MHC (HLA-A11:01) tetramer (VVVGAVGVGK-HLA-A11:01).
  • the peptide-MHC tetramer stain solution and LIVE/DEAD TM Fixable Near-IR containing specific antigens were prepared in advance and purchased from Invitrogen, catalog number: L10119; CD4-FITC was purchased from BioLegend, catalog number: 357406; CD8-PerCP-cy5.5 was purchased from BioLegend, catalog number: 344710; anti-human TCR ⁇ / ⁇ -BV510 antibody was purchased from BioLegend, catalog number: 306734.
  • CD8+ T cells edited with nwTCR-1708 can bind to specific antigen MHC tetramers, while CD4+ T cells edited with nwTCR-1708 cannot bind to specific antigen MHC tetramers due to the lack of assistance from CD8 molecules ( Figure 9A).
  • CD8a molecules or CD8ab molecules were co-edited with nwTCR-1708 into primary T cells (the primary T cells contained CD4+ T cells and CD8+ T cells), flow cytometry characterization showed that the proportion of CD8+CD4+T cells increased. These cells were CD4+ T cells expressing exogenous CD8 molecules. With the assistance of exogenous CD8 molecules, these CD4+ T cells could specifically bind to the labeled peptide-MHC (HLA-A11:01) tetramer (VVVGAVGVGK-HLA-A11:01) ( Figures 9B and 9C).
  • the overall gene editing efficiency of T cells is improved (Table 3), where the calculation formula for gene editing efficiency (GE%) is: the percentage of cells expressing only CD8+ in the living T cell population ⁇ the percentage of cells expressing only CD8+ in tetramer staining + the percentage of cells expressing only CD4+ in the living T cell population ⁇ the percentage of cells expressing only CD4+ in tetramer staining + the percentage of cells expressing CD8+ and CD4+ in the living T cell population ⁇ the percentage of cells expressing CD8+ and CD4+ in tetramer staining.
  • GE% the percentage of cells expressing only CD8+ in the living T cell population ⁇ the percentage of cells expressing only CD8+ in tetramer staining + the percentage of cells expressing only CD4+ in the living T cell population ⁇ the percentage of cells expressing CD8+ and CD4+ in tetramer staining.

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Abstract

本发明涉及特异性结合KRAS_G12V突变抗原的T细胞受体（TCR）、包含所述TCR的融合蛋白或缀合物、编码所述TCR的核酸及包含其的工程化细胞，以及制备所述工程化细胞的方法。本发明还涉及将外源性CD8分子与所述TCR基因共表达于T细胞以增强T细胞的功能。本发明提供了所述TCR和所述基因工程化细胞在检测、预防和/或治疗KRAS_G12V突变抗原相关的癌症中的用途。

Description

KRAS_G12V突变抗原特异性TCR及其与CD8共表达重定向CD4 T细胞 技术领域
本发明总体上涉及免疫学领域。具体地，本发明涉及特异性结合KRAS_G12V突变抗原的T细胞受体(下文中也缩写为TCR)、表达所述TCR的基因工程化细胞、以及制备所述基因工程化细胞的方法。本发明还涉及将外源性CD8分子与所述TCR基因共表达于T细胞以增强T细胞的功能。本发明提供了所述TCR和所述基因工程化细胞在检测、预防和/或治疗KRAS_G12V突变抗原相关的癌症中的用途。
背景技术
RAS基因是第一个被发现的人类肿瘤基因(oncogene)，其编码的RAS蛋白在许多重要的细胞信号网络上处于中心位置。RAS基因是人类癌症中最常出现突变的致癌基因。目前已经在所有人类肿瘤的大约1/5中发现了RAS基因突变引起的RAS蛋白激活。
KRAS基因(Kirsten大鼠肉瘤病毒癌基因同源物)编码的KRAS蛋白是一种小GTP酶，属于RAS超蛋白家族。
在细胞内，KRAS蛋白在失活和激活状态之间转变，其中当KRAS蛋白与鸟嘌呤核苷二磷酸(GDP)结合时，KRAS蛋白处于失活状态；当KRAS蛋白与鸟嘌呤核苷三磷酸(GTP)结合时，KRAS蛋白处于激活状态，并且可以激活下游信号通路。大部分细胞中的KRAS蛋白处于失活状态。
在KRAS基因的突变中，97％是第12号或者第13号氨基酸残基发生了突变。其中一种主要的突变是G12V。结构学研究表明。当KRAS发生G12V突变后，会通过破坏GAP活性而使KRAS蛋白一直保持与GTP结合，将KRAS蛋白锁定在具有酪氨酸激酶活性状态，并不断激活下游信号通路(如PI3K信号通路、MAPK信号通路、PI3K和Ral-GEFs信号通路等)。这些下游的信号通路打开之后，就会刺激细胞增殖、迁移，最终促成肿瘤发生。
在人类癌症中，KRAS基因为肿瘤学领域最著名的致癌基因之一，曾被认为是“不可成药(undruggable)”的靶点。KRAS基因突变出现在接近90％的胰腺癌中，30-40％的结肠癌中，17％的子宫内膜癌中，15-20％的肺癌(包括小叶性肺癌)中，以及胆管癌、宫颈癌、膀胱癌等。
近年来，针对KRAS突变体，研究开发了共价抑制剂，其通过变构位点靶向KRAS突变体，使得KRAS突变体与GTP的亲和力降低而达到“锁死”KRAS突变体活性的目的。例如，安进公司的用于治疗携带KRAS_G12C突变的非小细胞肺癌(NSCLC)患者的sotorasib(AMG510)，为KRAS_G12C抑制剂；MiratiTherapeutics公司的MRTX1257，也为一种KRAS_G12C抑制剂，目前处于临床前开发阶段。
针对其他KRAS突变，例如，KRAS_G12V突变目前尚无相关治疗性药物。本领域需要开发出针对KRAS_G12V突变抗原的特异性免疫细胞，例如，TCR-T细胞，来有效检测、预防和治疗KRAS_G12V突变抗原相关的癌症。
发明内容
本发明人通过锐意研究，获得了能与KRAS_G12V突变抗原特异性结合的T细胞受体(TCR)，并制备了重组表达该TCR的淋巴细胞，由此，能够通过TCR与KRAS_G12V突变抗原特异性结合来检测哺乳动物中KRAS_G12V突变抗原相关的癌症存在；和/或能够通过体内介导针对表达KRAS_G12V突变抗原的靶细胞的免疫应答来杀伤表达KRAS_G12V突变抗原的癌细胞，从而满足了上述需求。
因此，在一个方面，本发明提供了分离的或纯化的T细胞受体(TCR)，其与KRAS_G12V突变抗原特异性结合，优选地，所述TCR包含α链和β链，其中所述α链和β链各包含三个互补决定区(CDR)，并且α链的CDR3的氨基酸序列选自SEQ ID NO:3、6、9、12、15、18、21、24、27、30、33、36、39、42、45、48和与所述序列具有1个或2个氨基酸残基改变的变体，β链的CDR3的氨基酸序列选自SEQ ID NO:179、182、185、188、191、194、197、200、203、206、209、212、215、218、221、224和与所述序列具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCRα链的CDR3的氨基酸序列和β链的CDR3的氨基酸序列是：
(i)SEQ ID NO:3所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:179所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(ii)SEQ ID NO:6所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:182所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(iii)SEQ ID NO:9所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:185所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(iv)SEQ ID NO:12所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:188所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(v)SEQ ID NO:15所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:191所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(vi)SEQ ID NO:18所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:194所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(vii)SEQ ID NO:21所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:197所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(viii)SEQ ID NO:24所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:200所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(ix)SEQ ID NO:27所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:203所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(x)SEQ ID NO:30所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:206所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xi)SEQ ID NO:33所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:209所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xii)SEQ ID NO:36所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:212所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xiii)SEQ ID NO:39所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:215所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xiv)SEQ ID NO:42所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:218所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xv)SEQ ID NO:45所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:221所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；或
(xvi)SEQ ID NO:48所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:224所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCR的α链包含的三个互补决定区(CDR)的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
(i)SEQ ID NO:1、2、3所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:177、178、179所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(ii)SEQ ID NO:4、5、6所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:180、181、182所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(iii)SEQ ID NO:7、8、9所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:183、184、185所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(iv)SEQ ID NO:10、11、12所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:186、187、188所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(v)SEQ ID NO:13、14、15所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:189、190、191所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(vi)SEQ ID NO:16、17、18所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:192、193、194所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(vii)SEQ ID NO:19、20、21所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:195、196、197所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(viii)SEQ ID NO:22、23、24所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:198、199、200所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(ix)SEQ ID NO:25、26、27所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:201、202、203所示的 β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(x)SEQ ID NO:28、29、30所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:204、205、206所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(xi)SEQ ID NO:31、32、33所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:207、208、209所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(xii)SEQ ID NO:34、35、36所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:210、211、212所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(xiii)SEQ ID NO:37、38、39所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:213、214、215所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(xiv)SEQ ID NO:40、41、42所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:216、217、218所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
(xv)SEQ ID NO:43、44、45所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:219、220、221所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；或
(xvi)SEQ ID NO:46、47、48所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:222、223、224所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体。
在一些实施方案中，本发明的TCR包含SEQ ID NO:145、147、149、151、153、155、157、159、161、163、165、167、169、171、173或175所示的α链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:349、351、353、355、357、359、361、363、365、367、369、371、373、375、377 或379所示的β链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一些实施方案中，本发明提供了T细胞受体融合蛋白或T细胞受体缀合物，其包含本发明第一方面所述的TCR和其他生物活性分子，其中所述其他生物活性分子是例如抗体、细胞因子、细胞毒性剂、酶、放射性物质、可检测标记，其中所述TCR和其他生物活性分子之间具有或不具有接头。
本发明还提供了编码本发明TCRα链和/或β链的核酸。
此外，本发明提供了载体，优选地为质粒、穿梭质粒、噬菌粒、粘粒、表达载体、逆转录病毒载体、腺病毒载体和/或同源重组修复(HDR)载体，其包含一个或多个如上所述的核酸。
在第二方面，本发明提供了用以上载体转化并表达本发明第一方面所述TCR的工程化细胞。
在一些实施方案中，本发明提供了一种不使用病毒载体表达本发明的外源性TCR的打靶策略来制备TCR-T细胞的方法。
在一些实施方案中，本发明提供了一种编辑人细胞基因组的方法，所述方法包括将以下核酸序列插入人细胞中内源性T细胞受体(TCR)α链恒定区基因的外显子1的靶区域中，所述核酸序列从N端到C端包含：
(i)编码第一可裂解接头多肽的序列；
(ii)编码本发明第一方面所述TCR的β链的序列；
(iii)编码第二可裂解接头多肽的序列；
(iv)编码本发明第一方面所述TCR的α链可变区的序列；
其中，所述第一可裂解接头多肽和所述第二可裂解接头多肽是相同或不同的病毒2A肽。
通过所述方法制备的表达外源性TCR的细胞与VVVGAVGVGK-HLA-A*11:01复合物和/或VVGAVGVGK-HLA-A*11:01有很高的结合亲和力以及对SW620(HLA-A*11:01过表达，KRAS G12V+)细胞具有很强的体外杀伤作用。
在一些实施方案中，制备表达外源性TCR的细胞的方法是通过采用CRISPR/Cas9技术和同源重组技术，敲除内源性TCR和敲入外源性TCR实施的。
在第三方面，本发明提供了改善细胞疗法的方法和工程化细胞。
在一些实施方案中，本发明将外源性TCR和CD8aa分子在T细胞中进行了共表达。又在一些实施方案中，本发明将外源性TCR和CD8ab分子在T细胞中进行了共表达。
通过将CD8aa分子和/或CD8ab分子与TCR基因共表达于CD8+和CD4+ T细胞，对CD8+和CD4+ T细胞的功能产生了有益的作用。特别地，通过将MHC I类TCR和CD8分子共表达于CD4+ T细胞，将CD4+ T细胞重编程为多功能杂化T细胞，其同时具有细胞毒性效应功能和天然的辅助功能。
在第四方面，本发明提供了第一方面所述的TCR、第二方面和第三方面所获得的工程化细胞在检测、预防和/或治疗KRAS_G12V突变抗原相关的癌症中的用途。
附图说明
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1A显示了采用gRNA002将外源性TCR敲入TRAC位点的打靶策略图。
图1B显示了采用gRNA004基因敲除TRBC1和TRBC2位点的打靶策略图。
图2显示了流式细胞术检测TCR基因编辑效率的结果示意图。流式细胞术数据分析为4个象限(Q1、Q2、Q3、Q4)的细胞分布图，其中，Q2是完成了内源性TCR敲除(knock-out；KO)和外源性TCR敲入(knock-in；KI)，表达了nwTCR的细胞群；Q3是未进行基因编辑的野生型T细胞；Q4是完成了内源性TCR敲除的KO细胞。
图3A-图3P例示了电穿孔转染不同nwTCR的CD4+T细胞、CD8+T细胞用pMHC四聚体染色的流式细胞术结果。其中，对电穿孔转染nwTCR-0125、nwTCR-0126、nwTCR-0127、nwTCR-1708、nwTCR-1862、nwTCR-2162、nwTCR-2241、nwTCR-2308和nwTCR-2563的CD4+T细胞、CD8+T细胞例示的是与标记的VVVGAVGVGK-HLA-A*11:01四聚体的染色结果；对电穿孔转染nwTCR-2310、nwTCR-2390、nwTCR-2392、nwTCR-2424、nwTCR-2561、nwTCR-2595和nwTCR-2629的CD4+T细胞、CD8+T细胞例示的是与标记的VVGAVGVGK-HLA-A*11:01四聚体的染色结果。
图4A-图4H显示了分别表达各nwTCR的T细胞对HLA-A*11:01呈递的VVVGAVGVGK(SEQ ID NO:381)或VVGAVGVGK(SEQ ID NO:382)所示短肽的结合亲和力检测实验结果和EC50值。其中图4A、图4C、图4D和图4G是与VVVGAVGVGK(SEQ ID NO:381)所示短肽的结合亲和力；图4B、图4E、图4F和图4H是与VVGAVGVGK(SEQ ID NO:382)所示短肽的结合亲和力。
图5A-图5E显示了显示了表达各nwTCR的T细胞对作为靶细胞的SW620(HLA-A*11:01过表达，KRAS G12V+)细胞系(结直肠癌(CRC)细胞系)的体外杀伤效果。图中的“Blank”表示只有靶细胞且未添加表达任一nwTCR的T细胞。
图6例示了表达nwTCR-2404的T细胞杀伤SW620(HLA-A*11:01过表达，KRAS G12V+)细胞的荧光成像结果。图中的“Blank”表示只有靶细胞且未添加表达nwTCR-2404的T细胞。
图7例示了表达nwTCR-2404的T细胞杀伤SW620(HLA-A*11:01过表达，KRAS G12V+)细胞的实时分析数据。结果显示经基因编辑的T细胞对SW620(HLA-A*11:01过表 达，KRAS G12V+)细胞有特异性杀伤效果。图中的“Blank”表示只有靶细胞且未添加表达任一nwTCR的T细胞。
图8A显示了nwTCR-CD8a的打靶策略图。
图8B显示了nwTCR-CD8ab的打靶策略图。
图9A显示了电穿孔转染nwTCR-1708的CD4+T细胞、CD8+T细胞用pMHC四聚体染色的流式细胞术结果。
图9B显示了电穿孔转染nwTCR-1708-CD8a的CD4+T细胞、CD8+T细胞用pMHC四聚体染色的流式细胞术结果。
图9C显示了电穿孔转染nwTCR-1708-CD8ab的CD4+T细胞、CD8+T细胞用pMHC四聚体染色的流式细胞术结果。
具体实施方式
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
I.定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
“RAS蛋白家族”属于小GTPase的大家族。RAS蛋白可以因为单个氨基酸突变而组成型活化。突变的RAS蛋白产物参与许多人类癌症的肿瘤形成早期的信号转导。多种人类癌症(例如，肺癌(如，肺腺癌)、卵巢癌(如，上皮卵巢癌)、胰腺癌、前列腺癌、子宫内膜癌和结肠直肠癌)表达突变的RAS蛋白。
“Kirsten大鼠肉瘤病毒癌基因同源物(Kirsten rat sarcoma viral oncogene homolog，也简称为：KRAS蛋白)”是RAS蛋白家族中的重要一员，KRAS上游由表皮生长因子受体EGFR家族调控，EGFR信号能够激活SOS蛋白，从而调控KRAS的激活。细胞内KRAS蛋白的失活和激活状态转变由其结合的分子决定。鸟嘌呤核苷酸交换因子GEF催化KARS结合GTP，激活KRAS；而GTP酶激活蛋白GAP能够促进与KRAS结合的GTP水解为 GDP，从而导致KRAS失活。激活的KRAS调控其下游MAPK和PI3K等与细胞增殖、细胞迁移等功能有关的信号通路。KRAS突变会导致其持续结合GTP，保持激活状态，导致下游信号通路持续激活，从而促进肿瘤发生。
术语“抗原”是能够被生物体的免疫系统特异性检测的任何分子。
“KRAS_G12V突变抗原”是指具有G12V突变的KRAS蛋白，其能够被生物体的免疫系统特异性检测。“G12V”或“G12V突变”可互换地使用，指KRAS蛋白第12位的甘氨酸由缬氨酸代替。
T细胞受体(T cell receptor，TCR)是T细胞表面负责特异性识别与MHC(主要组织相容性复合体)结合的抗原肽的蛋白。当TCR与抗原肽和MHC结合时，T淋巴细胞通过信号转导被激活，进入后续的免疫应答过程。人类基因组中有4个TCR基因：两个编码轻链TCR：TRA基因编码TCRα，TRG基因编码TCRγ；两个编码重链TCR：TRB基因编码TCRβ，TRD基因编码TCRδ。重链TCR和轻链TCR形成异源二聚体，组成完整的TCR。在人类中存在两种TCR：TCRα/β和TCRγ/δ，其中95％的T细胞表达TCRα/β，称为αβT细胞；5％的T细胞表TCRγ/δ，称为γ/δT细胞。该比例在个体发育过程中和患病状态(例如白血病)中发生变化，物种之间也有所不同。
成熟的重链TCR基因由可变区(V)、多变区(D)、连接区(J)和恒定区(C)四部分基因片段组成(VDJC)，轻链TCR则缺少D区(VJC)。重链和轻链TCR都具有3个互补决定区(CDR)，在抗原识别中起主要作用，其中CDR1和CDR2相对保守，负责识别MHC；CDR3是负责识别抗原的主要CDR。
TCR基因是人类基因组中复杂度最高的基因，也是变异程度最高的基因。人外周血中大概含有2x1016－1018种表达不同TCR的T细胞。这个复杂度主要源自3个因素：(i)组成的多样性：成熟TCR的VDJC/VJC结构，是通过复杂的重排生成的。基因组中有65－100种V基因片段、2种D基因片段和13种J基因片段，TCR重组时需要从上述三种片段中各选一个，这赋予了TCR高度的多样性；(ii)连接的机动性：在重排的过程中，在V-D及D-J的连接区经常有非模板的核苷酸的随机插入或删除，进一步增加了CDR3区的多样性；(iii)体细胞突变：T细胞D区的突变频率约为正常的1000倍。
如本领域已知，在本文中可交换使用的“多核苷酸”或“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位 置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是同一的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
术语“抗原呈递细胞”或“APC”是指其表面上呈递与主要组织相容性复合体(MHC)复合的外来抗原的免疫系统细胞，例如辅助细胞(例如，B-细胞、树突细胞等)。T细胞可以使用其T细胞受体(TCR)识别这些复合体。APC加工抗原且将抗原呈递给T细胞。
术语“引导RNA(guide RNA，gRNA)”指特异于靶DNA的RNA，其可以与Cas蛋白质形成复合体并将Cas蛋白质带至靶DNA，从而Cas蛋白质在靶DNA的位点处引入双链断裂。在本发明中，引导RNA可以由两个RNA，即CRISPR RNA(crRNA)和反式激活crRNA(tracrRNA)组成，或者引导RNA可以是通过融合crRNA和tracrRNA的必要部分而产生的一条单链引导RNA(single guide RNA，sgRNA)。
核糖核蛋白复合物(Ribonucleoprotein，RNP)是Cas9蛋白和gRNA复合形成的具有基因编辑功能的复合物。
CRISPR/Cas9基因编辑系统主要由两部分组成：相当于“扳手”作用的Cas9蛋白与相当于“螺纹钉”作用的CRISPR引导RNA。引导RNA负责对靶位点进行定位，并招募和激活Cas9蛋白；Cas9蛋白则负责切割靶DNA。
术语“重组”，当用于例如细胞、核酸、蛋白质或载体时，表示该细胞、核酸、蛋白质或载体已通过引入异源核酸或蛋白质、或改变天然核酸或蛋白质而被修饰。
术语“靶位点”指在目标基因组中任何一段欲加以改造或修复的DNA序列。靶位点附近的DNA序列允许外源序列在靶位点处的整合，所述整合包括但不限于基因敲入(knock-in，KI)。在具体实施方式中，目标DNA序列是双链的DNA序列，包括但不限于，细胞的染色体基因组中的DNA序列、细胞染色体基因组外的DNA序列(例如线粒体基因组)、质粒、病毒等的DNA序列。
在本发明中，术语“定点重组”是指，将外源序列通过非随机的方式整合到特定的靶位点处，包括整合到某特定靶位点的5’上游、3’下游、或者靶位点之间。
在本发明中，术语“外源DNA序列”是指，期望被定点重组到靶位点处的DNA序列。外源DNA序列可以是靶位点处不存在或被改变的序列。
术语“供体DNA”或“供体核酸序列”是指包含待表达的目的多核苷酸序列的多核苷酸，该目的多核苷酸序列被插入目标基因组中的靶位点处。在某些实施方案中，供体DNA进一步包含与基因组序列同源的序列(也被称为“同源臂”)。“同源”意指类似的DNA序列。同源臂足以发生与同源基因组序列的同源重组。例如，同源臂可以包含至少50-3500或更多个碱基长度。
术语“同源定向DNA修复(Homology directed repair，HDR)”是基于同源重组的修复，可用于特异性地将供体DNA模板(编码目的序列)高效地插入目标基因组位点，是细胞DNA双链损伤后启动的一种修复途径。只有当细胞核内存在与损伤DNA同源的DNA片段时，HDR才能发生。HDR载体可以指采用CRISPR/Cas9和同源重组技术电穿孔转染用的载体。HDR效率可以指采用CRISPR/Cas9和同源重组技术电穿孔转染的基因敲入效率。
同义突变(synonymous mutation)是一种中性突变，遗传密码是简并性的，即，决定一个氨基酸的密码子大多不止一个，三联体密码子中第三个核苷酸的置换往往不会改变氨基酸的组成。虽然三联体密码子中第三个核苷酸发生了突变，但编码的氨基酸没有改变，这种突变是同义突变。
如本文所用，“载体(vector)”表示一种构建体，其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、质粒、粘粒或噬菌体载体。载体可以包含允许所关注的基因或序列在宿主细胞中复制的核酸序列，诸如复制起始区。载体还可以包含一种或多种可选择的标志物基因和本领域技术人员已知的其他遗传元件。载体优选地是包含根据本发明的核酸的表达载体，所述核酸与容许所述核酸表达的序列有效连接。
术语“有效连接”是指核酸表达调节序列和编码目的蛋白的核酸序列之间的功能性连接，以便执行总体功能。可使用本领域众所周知的基因重组技术制备与重组载体的有效连接，并使用本领域众所周知的酶进行位点特异性DNA切割和连接。
在本发明中，术语“工程化细胞”是指已经引入外源性核酸的细胞，包括这些细胞的子代。工程化细胞包括“转染的细胞”，其包括原代转染细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与亲代细胞可能不完全相同，但可能含有突变。本文包括与在初始转染的细胞中筛选或选择的细胞具有相同功能或生物学活性的突变子代。
在本文中，“受试者”、“个体”指需要缓解和/或治疗KRAS_G12V突变抗原相关癌症的动物，优选哺乳动物，更优选是人。哺乳动物还包括但不限于农场动物、竞赛动物、宠物、灵长类、马、犬、猫、小鼠和大鼠。
过继性免疫细胞治疗(Adoptive Cell Transfer Therapy,ACT)，是指从受试者或患者体内分离出具有免疫活性的细胞，在体外进行激活扩增、基因编辑等处理之后，再回输到患者体内，从而实现对靶细胞的杀伤。
II.本发明的T细胞受体(TCR)和编码TCR的核酸
野生型人KRAS蛋白长为188个氨基酸残基，分子量约是21.6KD，其第12位是甘氨酸。在KRAS的基因突变中，83％是第12位氨基酸残基发生了突变，其中最主要的一种突变是第12位的甘氨酸突变为缬氨酸(本文中也简称为G12V)。
本发明提供了分离的或纯化的TCR，其对由人白细胞抗原(HLA)I类分子呈递的具有G12V突变的KRAS肽具有抗原特异性。所述由人白细胞抗原(HLA)I类分子呈递的具有G12V突变的KRAS肽具有适合于结合任何HLA I类分子的任何长度。
在一些实施方案中，所述具有G12V突变的KRAS肽具有约9个至约10个氨基酸残基的长度，其包括KRAS蛋白中具有G12V突变的任何连续的约9个至约10个氨基酸残基。在一些实施方案中，本发明的TCR对具有G12V突变的KRAS肽具有抗原特异性，所述突变的KRAS肽具有约9个氨基酸残基或约10个氨基酸残基的长度。可以被本发明的TCR识别的具有G12V突变的KRAS肽的实例是KRAS第7位到第16位氨基酸序列的短肽VVVGAVGVGK(SEQ ID NO:381)(文中也简称为“KRAS_G12V_7-16肽”)；和KRAS第8位到第16位氨基酸序列的短肽VVGAVGVGK(SEQ ID NO:382)(文中也简称为“KRAS_G12V_8-16肽”)。
T细胞受体(TCR)是存在于T细胞表面的分子，其负责识别抗原肽-MHC复合物(即，pMHC)。TCR与抗原肽-MHC复合物的特异性结合引发T细胞通过一系列由相关酶、共受体和辅助性分子介导的生化事件而活化。在95％的T细胞中，TCR异二聚体由α和β链组成，而在5％的T细胞中，TCR异二聚体由γ和δ链组成。
TCR的每一条链均属于免疫球蛋白超家族的成员，具有一个N端免疫球蛋白(Ig)可变(V)结构域、一个Ig恒定(C)结构域、跨细胞膜区域(即，跨膜区)以及在C末端的短胞质尾。在TCRα链和β链的可变结构域中，各可变结构域具有三个高变区或互补决定区(CDR)，其中各可变结构域中的CDR3是负责识别经加工的抗原的主要CDR。认为CDR2识别MHC分子。
TCR的恒定结构域由短的连接序列组成，其中的半胱氨酸残基形成二硫键，在TCRα链和β链之间产生连接。
在T细胞成熟过程中，TCR与CD3形成TCR/CD3复合体。TCR/CD3复合体形成过程通常是按以下顺序进行的；首先CD3γ、δ和ε三种肽链通过形成γ-ε和δ-ε两种异源二聚体成为稳定的复合物核心，TCRαβ(或TCRγδ)与之结合，随后ζ-ζ或ζ-η二聚体与TCRαβ(或TCRγδ)/CD3γεδε复合物结合，最后转移到T细胞表面。通过TCR/CD3复合体将信号从TCR传导至细胞内。
来自TCR/CD3复合体的信号通过MHC与特异性共受体的同时结合而增强。在辅助T细胞中，这一共受体是CD4分子，所述CD4分子对II类MHC是特异性的；而在细胞毒性T细胞中，这一共受体是CD8，所述CD8分子对I类MHC是特异性的。
在本文中，术语“T细胞受体”具有本领域的常规意义，用于表示能够识别由MHC分子呈递的肽的分子。该分子是两条链α和β(或任选地γ和δ)的异二聚体。
本发明的TCR提供对KRAS_G12V突变抗原的特异性亲和识别。KRAS_G12V突变抗原在细胞内被蛋白酶体降解为8到10个氨基酸长的短肽，例如，SEQ ID NO:381所示的KRAS_G12V_7-16肽和/或SEQ ID NO:382所示的KRAS_G12V_8-16肽。这些短肽被MHC I类以肽/MHC复合物(pMHC)呈递在细胞表面。已证明一些pMHC与各种癌症有关，因此，可以成为TCR治疗的潜在靶标。
本发明提供了分离的或纯化的T细胞受体(TCR)α链和/或β链。本发明的TCR可以是包含衍生自超过一种物种的序列的杂合TCR。例如，考虑到鼠科TCR在人T细胞中能够比人TCR更有效地表达，TCR可包含人可变区和鼠科恒定区。
在一个实施方案中，本发明的TCR包含α链和β链，其中所述α链和β链各包含三个互补决定区(CDR)，且其中主要负责抗原识别的TCRα链CDR3的氨基酸序列选自SEQ ID NO:3、6、9、12、15、18、21、24、27、30、33、36、39、42、45、48和与所述序列具有1个或2个氨基酸残基改变的变体，β链CDR3的氨基酸序列选自SEQ ID NO:179、182、185、188、191、194、197、200、203、206、209、212、215、218、221、224和与所述序列具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCR包含α链和β链，所述α链包含的三个互补决定区(CDR)的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
(i)SEQ ID NO:3所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:179所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(ii)SEQ ID NO:6所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:182所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(iii)SEQ ID NO:9所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:185所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(iv)SEQ ID NO:12所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:188所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(v)SEQ ID NO:15所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:191所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(vi)SEQ ID NO:18所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:194所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(vii)SEQ ID NO:21所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:197所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(viii)SEQ ID NO:24所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:200所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(ix)SEQ ID NO:27所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:203所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(x)SEQ ID NO:30所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:206所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xi)SEQ ID NO:33所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:209所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xii)SEQ ID NO:36所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:212所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xiii)SEQ ID NO:39所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:215所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xiv)SEQ ID NO:42所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:218所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
(xv)SEQ ID NO:45所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:221所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；或
(xvi)SEQ ID NO:48所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:224所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCR包含SEQ ID NO:145、147、149、151、153、155、157、159、161、163、165、167、169、171、173或175所示的α链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:349、351、353、355、357、359、361、363、365、367、369、371、373、375、377或379所示的β链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。优选地，本发明的TCR的恒定区是小鼠恒定区。
在一些实施方案中，本发明的TCR变体中所述氨基酸残基的改变是任一SEQ ID NO:145、147、149、151、153、155、157、159、161、163、165、167、169、171、173或175所示的α链序列、任一SEQ ID NO:349、351、353、355、357、359、361、363、365、367、369、371、373、375、377或379所示的β链序列中的氨基酸残基的取代、添加或缺失，条件是该TCR变体仍保留了或改善了与KRAS_G12V突变抗原的表位肽-MHC复合物结合的能力。在一个实施方案中，所述取代是保守性取代。下表A中给出了保守性取代的例子。
表A

氨基酸可以根据常见的侧链特性分组：
(1)疏水性：正亮氨酸、Met、Ala、Val、Leu；Ile；
(2)中性亲水：Cys、Ser、Thr、Asn；Gln；
(3)酸性：Asp、Glu；
(4)碱性：His、Lys、Arg；
(5)影响链方向的残基：Gly、Pro；
(6)芳族：Trp、Tyr、Phe。
非保守性取代将使这些分类之一的成员交换为另一个分类的成员。
在一些实施方案中，本发明的TCR能够识别并结合由HLA I类分子呈递的突变的KRAS蛋白的表位肽，引发免疫应答。
在一些实施方案中，所述HLA I类分子是任何HLA-A分子，例如，HLA I类分子是HLA-A11分子。所述HLA-A11分子可以是任何HLA-A11分子。HLA-A11分子的实例包括但不限于HLA-A*11:01、HLA-A*11:02、HLA-A*11:03或HLA-A*11:04。优选地，HLA I类分子是HLA-A*11:01分子。HLA-A*11:01分子是亚洲人中最常见的HLA-A分子。
本发明还涉及编码本发明TCR或其部分的核酸，所述TCR部分例如，一个或多个CDR；一个或多个可变区；α链；或β链等。该核酸可以是双链或单链的，并且可以是RNA或DNA。该核酸序列可以是经密码子优化的，以实现哺乳动物生产细胞中的高表达。哺乳动物细胞以及多种其他生物的密码子选择是本领域公知的。密码子优化还可包括移除mRNA不稳定基序和隐藏的剪接位点。
本发明的TCR可经多种方法(例如，基因融合、化学共轭等)进行修饰，从而使TCR与其他生物活性分子连接。可与其他生物活性分子连接的TCR可以是TCR异二聚体或其可溶性型式，更优选地为可溶性、单链TCR。所述其他生物活性分子可以是各种生物活性效应物，例如抗体、细胞因子、细胞毒性剂、酶、放射性物质、可检测标记等。所述TCR和其他生物活性分子之间具有或不具有接头。
在一些实施方案中，TCR融合蛋白是将TCR与抗体融合，所述抗体包括完整抗体(例如IgG、IgM或IgA类)或者其片段(例如Fv，Fab，Fab’，Fab’-SH，F(ab’)2；双体抗体(diabody)；单链抗体(例如scFv)；单结构域抗体)；以及多特异性抗体(例如双特异性抗体)。
在一些实施方案中，TCR融合蛋白是将TCR与细胞因子融合，所述细胞因子例如白细胞介素(例如IL-2)、趋化因子(例如MIP-1β)、生长因子(例如GCSF)。
在一些实施方案中，TCR缀合物是将TCR共价连接至细胞毒性剂，例如阿霉素。
在一些实施方案中，TCR缀合物是将TCR共价连接至放射性物质，例如I125。
在一些实施方案中，TCR缀合物是将TCR共价连接至可检测标记，例如荧光标记物。
本发明的T细胞受体融合蛋白或T细胞受体缀合物可用于各种应用，包括体内检测细胞和/或使细胞或组织成像，以及治疗用途，例如在体内或体外杀死具有特异性结合TCR的表达KRAS_G12V突变抗原的靶细胞或靶组织。
III.包含编码本发明TCR的核酸的载体
本发明还涉及包含编码本发明TCR的核酸的载体。在一个实施方案中，所述载体是pUC57-Simple载体(购自金斯瑞生物科技有限公司)。在又一个实施方案中，使用了pUC57-HA载体，所述pUC57-HA载体是在pUC57-Simple载体基础上优化后的载体。其仅保留了pUC57-Simple载体的Ori和Amp序列，然后将Amp序列替换为Kana序列，并添加TRAC位点的左右同源臂(HA)序列(800bp左右)。
载体将编码本发明TCR的核酸转移至细胞中，诸如T细胞、NK细胞、干细胞，例如多能干细胞、诱导的多能干细胞(iPSC)中，使得所述经工程化的细胞表达KRAS_G12V突变抗原特异的TCR。
所述KRAS_G12V突变抗原特异的TCR是指这样的TCR，其能够以高亲和力特异性结合且免疫性识别G12V突变的KRAS。例如，约1×104至约1×105个表达TCR的T细胞与经G12V突变的KRAS脉冲的且HLA I类分子过表达的抗原呈递细胞例如T2细胞或K562细胞共培养后，以TCR的EC50约1×10-7M或更少(例如，1×10-8M或更少、1×10-9M或更少、1×10-10M或更少)引起分泌IFN-γ，则认为TCR对G12V突变的KRAS具有抗原特异性。HLA I类分子可以是本文所述的任何HLA I类分子(例如HLA-A*11:01分子)。
优选地，载体使得在工程化的细胞(例如，工程化的T细胞)中持续高水平表达引入的外源性TCR，且引入的外源性TCR可成功地与内源TCR竞争有限的CD3分子池。备选地，增加CD3分子供应也可增加基因修饰细胞中的外源性TCR表达。因此载体任选地包含CD3-γ、CD3-δ、CD3-ε和/或CD3-ζ的基因。在一个实施方案中，载体包含CD3-ζ的基因。另外，也可提供一个或多个编码CD3基因的单独的载体用于与外源性TCR编码载体共转移入细胞中。
载体形式不限于同源重组修复(HDR)载体，也可以是病毒载体。病毒载体可以是慢病毒载体、腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、逆转录病毒载体、杆状病毒载体，用来实施对细胞基因组的编辑。
基因组的编辑技术是指在细胞基因组DNA中进行核酸插入、缺失或者替换的一项技术。利用基因编辑技术对于人原代T细胞的基因改造后的T细胞，已经在多种过继性免疫治疗药物的临床试验中展现了优异的疗效。其中，嵌合抗原受体(Chimeric Antigen Receptors，CAR)或者T细胞受体(T Cell Receptor，TCR)常被用于改造人的原代T细胞，从而实现对某些特定靶点表位(epitope)的识别。这些经改造后的T细胞可以对特定的靶细胞起到特异性杀伤作用。
常见的TCR基因编辑手段根据基因整合方式可以大致分为两类，一类是基因的随机整合：包括慢病毒(Lenti Virus，LV)体系，腺相关病毒(Adeno-Associated Virus，AAV)体系，转座子(Transposon)体系等。另一类是精准的基因编辑手段：包括锌指核酸酶(ZFN)，转录激活样效应因子核酸酶(TALEN)和成簇规律间隔短回文重复(CRISPR)技术等。其中，CRISPR技术通过gRNA的导向来识别及编辑DNA，通过同源重组的方式进行大基因片段的定点插入，具有易于操作，具有更强的可拓展性等优势。
IV.工程化细胞的制备
可以使用病毒载体将目的TCR导入细胞。但是，基于病毒载体将外源性TCRα/β基因导入细胞的方法没有敲除细胞内源性的TCR，可能造成外源性TCRα链和β链的错配，即使通过修饰二硫键或换用鼠源恒定区可以减少外源性TCRα链和β链的错配问题，但病毒载体随机插入到细胞基因组中，这仍会带来破坏其他基因的潜在风险。
也可以使用非病毒载体将目的TCR导入细胞，将外源性TCRα/β基因精确整合到细胞的特定基因组位点。在一些实施方案中，非基于病毒载体的基因编辑方法可以通过CRISPR/Cas9技术和同源重组技术敲除人源T细胞的内源性T细胞受体α链和β链，并在TRAC基因外显子处敲入外源性目的T细胞受体α和β链编码核苷酸。由此，既破坏了内源性的TCR表达，又采用内源性TCR启动子来表达外源性目的TCRα和β。
在一个实施方案中，在内源性TRAC基因外显子1处敲入外源性目的T细胞受体α和β链编码核苷酸，且该外源性敲入片段不用添加TRAC基因，由此减少了基因敲入的片段长度，降低了基因敲入的难度。相比于采用病毒载体表达TCR的技术，可以将采用非病毒载体方式表达TCR的技术作为一种快速、简便、低成本的外源性TCRα/β基因导入细胞方式。
IV.1敲除位点的选择
TCR是一个二聚体，由TCRα链和TCRβ链组合而成。TCRα链基因是由TRAV、TRAJ和TRAC基因重排而成，其中TRAV和TRAJ基因分别含有多个序列，且该多个序列之间有差异，重排时只能分别随机选择其中一个序列表达。如果选择TRAV和TRAJ基因作为敲除位点，则很难避免任何随机TCRα链基因的产生，而TRAC基因只有一个，通过敲除该TRAC基因可以敲除任何随机TCRα链基因，因此TRAC适合作为敲除位点。TCRβ链基因是由TRBV、TRBJ、TRBD和TRBC基因重排而成，其中TRBV和TRBJ基因分别含有多个序列，且该多个序列之间有差异，不适合作为敲除位点。TRBC基因包含TRBC1和TRBC2，两者含有部分相同序列，可以选择该共同序列作为敲除位点，通过敲除该共同序列而敲除任何随机TCRβ基因。
在一些实施方案中，敲除了内源性TRAC基因、内源性TRBC1基因和/或TRBC2基因中的一者或多者。在一些实施方案中，同时敲除了内源性TRAC基因和内源性TRBC1和TRBC2基因，由此可以获得更高的内源性TCR敲除效率，降低了内源性TCR表达可能导致的外源性TCR和内源性TCR的链之间的错配风险。
可以使用基于核酸酶的基因组编辑工具，通过非同源端连接(NHEJ)诱导双链断裂和DNA修复来靶向破坏内源性TRAC基因和TRBC基因。这些工具包括大范围的核酸酶(meganucleases)、锌指核酸酶(ZFNs)、转录激活因子样效应核酸酶(TALENs)、megaTAL核酸酶，以及CRISPR/CRISPR相关蛋白9(CRISPR/Cas9)。
IV.2敲入位点的选择
由于内源性TRAC基因唯一且所有的TCRα表达均需要TRAC基因，将外源性TCRα/β基因敲入位点选择为内源性TRAC位点，由此，在消除内源性TCR的同时，可以采用人源T细胞的内源性TCR启动子来表达本发明的外源性TCRα/β基因(也称为“nwTCR”基因)且不用额外添加TRAC基因，从而减少敲入片段的大小，有利于提高基因编辑的效率。
在一些实施方案中，将nwTCR的表达构建体克隆于打靶载体(例如pUC57-S载体)，通过设计同源臂使nwTCR定点敲入到TCRα链恒定区，并受该基因座的转录调控序列调控表达。由于该敲入位点处其内源启动子的调控水平优于其他位点，确保了nwTCR基因的持续稳定表达。
IV.3工程化细胞
本发明提供了表达外源性TCR的工程化细胞。
在一些实施方案中，自来源于血液、骨髓、淋巴或淋巴器官的细胞，例如，淋巴细胞或干细胞制备表达TCR的工程化细胞，所述淋巴细胞包括但不限于T细胞、NK细胞，所述干细胞例如多能干细胞、诱导的多能干细胞(iPSC)。
细胞通常是原代细胞，例如直接从受试者分离和/或从受试者分离并冷冻的细胞。细胞可以是同种异体细胞和/或自体细胞。
在一些实施方案中，通过CRISPR/Cas9和同源重组技术，采用RNP和质粒的方式来电穿孔转染CD3/CD28激活后的原代细胞(例如，分选的CD4+ T细胞和CD8+T细胞)，由此制备工程化的TCR细胞。
在一些实施方案中，设计了针对内源性TRAC基因的sgRNA，并设计了针对内源性TRBC1基因和TRBC2基因的sgRNA。
通过sgRNA引导Cas9蛋白结合于目标基因组的特异性位点，Cas9蛋白切割该特异性位点。对于使用RNP所致内源性TRAC基因形成的双链断裂，在具有同源臂的供体DNA存在的条件下可以发生同源重组，由此实现目的nwTCR基因的定点插入。
在一个具体实施方案中，sgRNA引导Cas9蛋白结合的目标基因组的特异性位点位于TRAC基因的外显子1，Cas9蛋白切割该特异性位点，所设计并经验证的高效靶向其的sgRNA识别序列和PAM序列包含TCAGGGTTCTGGATATCTGT-GGG所示的核苷酸序列(为SEQ ID NO:383所示的sgRNA识别序列-SEQ ID NO:385所示的PAM序列，“-”用来区分隔开CRISPR/Cas9识别位点和PAM序列)。
在一个具体实施方案中，sgRNA引导Cas9蛋白结合的目标基因组的特异性位点位于TRBC1和TRBC2基因的外显子1，Cas9蛋白切割所述特异性位点，所设计并经验证的高效靶向其的sgRNA识别序列和PAM序列包含CTGCCTGAGCAGCCGCCTGA-GGG所示的核苷酸序列(为SEQ ID NO:384所示的sgRNA识别序列-SEQ ID NO:385所示的PAM序列，“-”用来区分隔开CRISPR/Cas9识别位点和PAM序列)。
CRISPR/Cas系统中可包含蛋白质形式或编码Cas蛋白质的核酸形式的Cas成分。
在本发明中，Cas蛋白质可以是任何Cas蛋白质，只要当其与引导RNA复合时具有核酸内切酶或切口酶活性即可。
优选地，Cas蛋白质是Cas9蛋白质或其变体或其功能片段。
Cas蛋白质可以是从生物体如链球菌属物种(Streptococcus sp.)，优选化脓性链球菌(Streptococcus pyogens)中分离的蛋白质、或重组蛋白质，但并不限于此。
在一个实施方案中，Cas蛋白质包含源自化脓性链球菌的Cas9，例如具有SEQ ID NO：405所示氨基酸序列的Cas9。
在另一个实施方案中，Cas蛋白质包含与SEQ ID NO：405所示的氨基酸序列具有至少50％同源性的氨基酸序列，优选地与SEQ ID NO：405所示的氨基酸序列具有至少60、70、80、90、95、97、98或99％的同源性，但不限于此。
就本发明而言，Cas蛋白质编码核酸可以是载体形式，如包含在启动子如CMV或CAG下的Cas编码序列的质粒。当Cas蛋白质是Cas9时，Cas9编码序列可源自链球菌属，优选源自化脓性链球菌。例如，Cas9编码核酸可以包含编码SEQ ID NO：405的核苷酸序列。此外，Cas9编码核酸可包含与编码SEQ ID NO：405的核苷酸序列具有至少50％同源性的核苷酸序列，优选地与编码SEQ ID NO：405的核苷酸序列具有至少60、70、80、90、95、97、98或99％同源性的核苷酸序列，但不限于此。
在一个实施方案中，供体DNA中依次包含5’同源臂、编码可裂解接头多肽的序列、外源性TCRα/β基因或其功能片段和3’同源臂。所述编码可裂解接头多肽的序列在表达后，可裂解接头多肽被裂解。在一些实施方案中，可裂解接头多肽序列包含2A核糖体跳跃元件例如T2A、E2A、P2A和F2A。
在一个实施方案中，供体DNA位于打靶载体中。不特别地限制作为骨架的基础打靶载体，只需具备用于细菌中的载体增殖的原核复制起点和选择标记即可。
在一个优选的实施方案中，为了增加外源性TCRα/β基因或其片段的表达，在打靶载体中分别在外源性TCRα链基因和外源性TCRβ链基因的N端连接编码可裂解接头多肽的序列和信号肽序列。
在一个具体实施方案中，用于敲入nwTCR基因序列的打靶载体包含有效连接的以下结构：5’-2A核糖体跳跃元件-SP-TCRβ-2A核糖体跳跃元件-SP-TRAV-TRAJ-3’
其中：
SP为信号肽编码序列。
将用于敲入nwTCR基因序列的打靶载体、RNP复合体和细胞混合并实施nwTCR基因序列向细胞的递送步骤。在一些实施方案中，递送步骤选自：电穿孔、转染、通过物理手段使细胞膜变形、脂质纳米颗粒(LNP)、病毒样颗粒(VLP)和声处理。在一些实施方案中，递送步骤包括电穿孔。
在一些实施方案中，工程化的细胞是原代细胞。
在一些实施方案中，工程化的细胞是分离的细胞，其中分离的细胞是从受试者分离的。
在一些实施方案中，工程化的细胞是离体培养的细胞。在一些实施方案中，离体培养的细胞包括经刺激的细胞。在一些实施方案中，经刺激的细胞包括细胞因子刺激的T细胞，任选地，其中细胞因子刺激的T细胞包括CD3刺激的T细胞、CD28刺激的T细胞或CD3和CD28刺激的T细胞。在一些实施方案中，细胞因子刺激的T细胞是在IL7、IL15或其组合的存在下培养的。在一些实施方案中，细胞因子刺激的T细胞是在IL2的存在下培养的。
在一些实施方案中，工程化的细胞是干细胞，例如，造血干细胞(HSC)。将nwTCR基因转移至HSC不会导致在细胞表面表达TCR，因为干细胞不表达CD3分子。然而，当干细胞分化为迁移至胸腺的淋巴前体细胞(lymphoid precursor)时，CD3表达的启动将导致在胸腺细胞的表面表达该引入的nwTCR。这一方法的优点是成熟T细胞一旦产生，其仅表达引入的nwTCR，而表达很少的或不表达内源TCR链，因为引入的nwTCR链的表达抑制了内源TCR基因片段重排形成功能性TCRα和β基因。这一方法的其他益处是，TCR基因修饰的干细胞是具有期望的抗原特异性的成熟T细胞的持续来源。因此，nwTCR基因修饰的干细胞在分化后产生表达本发明TCR的T细胞。
V.检测、预防或治疗KRAS_G12V突变抗原相关的癌症的方法
本发明提供了一种预防或治疗KRAS_G12V突变抗原相关的癌症的方法，其包括对有需要的受试者施用本发明的工程化细胞、本发明的TCR核酸、载体或药物组合物。在一些实施方案中，该方法包括给予编码TCR的多核苷酸。在一些实施方案中，该方法包括施用包含编码TCR的多核苷酸的载体。在一些实施方案中，该方法包括施用有效量的本发明的工程化细胞。
在一些实施方案中，使用本发明的工程化细胞、本发明的TCR核酸、载体或药物组合物预防或治疗KRAS_G12V突变抗原相关的癌症。不受任何理论的束缚，认为本发明的TCR能够特异性结合KRAS_G12V突变抗原，由此，介导针对表达KRAS_G12V突变抗原的靶细胞的免疫应答。
所述治疗或预防可以包括对被治疗或预防的癌症的一种或多种症状的治疗或预防，包括促进肿瘤消退、延迟癌症或其症状的发作、预防或延迟癌症或其症状的复发。
本发明还提供了检测哺乳动物中癌症存在的方法。该方法包括：(i)使包含来自哺乳动物的一个或多个细胞的样品与本文所述的本发明TCR、表达本发明TCR的细胞群体、或包含表达本发明TCR的细胞群体的药物组合物中的任一种接触，从而形成复合物；以及(ii) 检测复合物，其中检测到复合物指示哺乳动物存在癌症。所述接触可以于哺乳动物的体外或体内实施。在一个实施方案中，所述接触是在体外实施的。可以通过本领域已知的多种方式来检测所述复合物。在一些实施方案中，将本发明的TCR或者表达本发明TCR的细胞群体用可检测的标记物进行标记，所述可检测标记物是例如放射性同位素、荧光团(例如异硫氰酸荧光素(FITC)、藻红素(PE))、酶(例如碱性磷酸酶、辣根过氧化物酶)和元素颗粒(例如金颗粒)。
本发明还提供了诱导抗肿瘤免疫的方法，其中所述肿瘤是KRAS_G12V突变抗原相关的肿瘤，所述方法包括给予受试者有效量的本发明的工程化细胞。
本发明提供了在受试者中诱导免疫应答的方法，包括给予有效量的本发明的工程化细胞。在一些实施方案中，免疫应答是T细胞介导的免疫应答。在一些实施方案中，T细胞介导的免疫应答针对一种或多种靶细胞。在一些实施方案中，工程化免疫细胞包含本发明的TCR。在一些实施方案中，靶细胞是KRAS_G12V突变抗原相关的癌细胞。
在一些实施方案中，用于T细胞治疗的供体T细胞从患者获得(例如，用于自体T细胞治疗)。在其它实施方案中，待分化为T细胞用于T细胞治疗的供体干细胞是从非患者的受试者获得的。
T细胞可以治疗有效量施用。例如，治疗有效量的T细胞可为至少约104个细胞、至少约105个细胞、至少约106个细胞、至少约107个细胞、至少约108个细胞、至少约109个细胞或至少约1010个细胞/kg体重。
对于本发明的各种方法中提及的癌症可以是任何癌症，包括但不限于：急性淋巴细胞性癌症、急性骨髓样白血病、慢性淋巴细胞性白血病、慢性骨髓样癌症、霍奇金淋巴瘤、非霍奇金淋巴瘤、脑癌、胶质瘤、鼻咽癌、眼癌、口腔癌、颈癌、食管癌、肝癌、肝内胆管癌、胆囊癌、肺癌、骨癌、乳腺癌、胃肠道肿瘤、结肠癌、小肠癌、结肠直肠癌、直肠癌、胃癌、皮肤癌、黑色素瘤、多发性骨髓瘤、宫颈癌、子宫内膜癌、子宫癌、卵巢癌、输尿管癌、膀胱癌、阴茎癌、睾丸癌、胰腺癌、前列腺癌、肾癌、软组织癌和甲状腺癌。优选地，所述癌症是肺癌、胰腺癌、结肠直肠癌、子宫内膜癌、卵巢癌或前列腺癌。
VI.改善细胞疗法的方法和工程化细胞
本发明还提供了改善细胞疗法的方法和工程化细胞。
天然的CD8+细胞表达CD8分子。CD8分子是以两条CD8a链组成的同二聚体形式(本文中也简称为“CD8aa”)和/或以一条CD8a链和一条CD8b链组成的异二聚体形式(本文中也简称为“CD8ab”)表达于细胞表面的I型跨膜醣蛋白。
在一些实施方案中，本发明将外源性TCR和CD8aa分子在T细胞中进行了共表达。基于CRISPR/Cas9技术的非病毒基因编辑方法，采用编码本发明nwTCR的核酸和编码CD8a链的核酸对CD8+T细胞/CD4+ T细胞进行基因编辑，表明外源性nwTCR和CD8aa分子在CD8+T细胞/CD4+ T细胞中共表达，能够增强TCR-T细胞与pMHC分子的结合。当外源性nwTCR和CD8aa分子在CD8+ T细胞中共表达时，由于CD8+ T细胞中能够被 外源性nwTCR使用的CD8aa分子增加，预期会改善CD8+ T细胞的TCR特异性细胞毒性(包括其连续杀伤能力)和体内抗肿瘤功能。当外源性nwTCR和CD8aa分子在CD4+ T细胞中共表达时，伴随着内源性CD4分子的表达，CD4+ T细胞表现出杂化表型，预期其以与天然CD8+ T细胞相似的亲和力识别抗原，并杀死靶细胞，表现出细胞毒性效应功能；同时保留CD4+ T细胞的天然辅助功能。
又在一些实施方案中，本发明将外源性TCR和CD8ab分子在T细胞中进行了共表达。基于CRISPR/Cas9技术的非病毒基因编辑方法，采用编码本发明nwTCR的核酸、编码CD8a链的核酸和编码CD8b链的核酸对CD8+ T细胞/CD4+ T细胞进行基因编辑，表明外源性nwTCR和CD8ab分子在CD8+ T细胞/CD4+ T细胞中共表达，能够增强TCR-T细胞与pMHC分子的结合。当外源性nwTCR和CD8ab分子在CD8+ T细胞中共表达时，由于CD8+T细胞中能够被外源性nwTCR使用的CD8ab分子增加，预期会改善CD8+ T细胞的TCR特异性细胞毒性(包括其连续杀伤能力)和体内抗肿瘤功能。当外源性nwTCR和CD8ab分子在CD4+ T细胞中共表达时，伴随着内源性CD4分子的表达，CD4+ T细胞表现出杂化表型，预期其以与天然CD8+ T细胞相似的亲和力识别抗原，并杀死靶细胞，表现出细胞毒性效应功能；同时保留CD4+ T细胞的天然辅助功能。
因此，将CD8aa分子和/或CD8ab分子与TCR基因共表达于CD8+和CD4+ T细胞时，对CD8+和CD4+ T细胞的功能具有有益的作用。可以通过MHC I类TCR和CD8分子将CD4+ T细胞重编程为多功能杂化T细胞，其同时具有细胞毒性效应功能和天然的辅助功能。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
通过参考以下实施例将更容易地理解本文一般地描述的本发明，这些实施例是以举例说明的方式提供的，并且不旨在限制本发明的范围。这些实施例并不旨在表示下面的实验是全部进行了的实验或仅进行了的实验。
实施例1.识别KRAS_G12V突变抗原的T细胞和TCR的生成和克隆
化学合成KRAS第12位氨基酸由G突变为V后的附近序列，即，KRAS第7位到第16位氨基酸序列的短肽VVVGAVGVGK(SEQ ID NO:381)(文中也简称为“KRAS_G12V_7-16肽”)；和KRAS第8位到第16位氨基酸序列的短肽VVGAVGVGK(SEQ ID NO:382)(文中也简称为“KRAS_G12V_8-16肽”)。
分别使用重悬于DMSO中的KRAS_G12V_7-16肽和KRAS_G12V_8-16肽体外脉冲刺激来源于HLA-A*11:01基因型且表达KRAS_G12V的癌症患者的树突状细胞(DC细胞)，并与该患者外周血中分选出的CD8+ T细胞共培养10天。阴性对照为使用DMSO体外脉冲刺激该患者DC细胞并与该患者外周血中分选出的CD8+ T细胞共培养10天。
然后，检测培养上清中细胞因子IFN-γ的释放量和CD8+ T细胞上CD137的表达，分选出释放IFN-γ且CD137表达的反应性T细胞。使用基于流式细胞术的染色来评估分选出的释放IFN-γ且CD137表达的反应性T细胞与肽-MHC(HLA-A*11:01)四聚体(VVVGAVGVGK-HLA-A*11:01和VVGAVGVGK-HLA-A*11:01)的结合；含有无关肽的四聚体用作阴性对照。用两种四聚体(分别为VVVGAVGVGK-HLA-A*11:01四聚体和VVGAVGVGK-HLA-A*11:01四聚体)对T细胞进行流式细胞术染色增加了T细胞特异性的置信度。
筛选到16个具有所需的高亲和力的特异性T细胞克隆。将这16个T细胞克隆上的与KRAS_G12V_7-16表位肽和KRAS_G12V_8-16表位肽特异性结合的抗原特异性T细胞受体(TCR)分别命名为nwTCR-0125、nwTCR-0126、nwTCR-0127、nwTCR-1708、nwTCR-1862、nwTCR-2162、nwTCR-2241、nwTCR-2308、nwTCR-2310、nwTCR-2390、nwTCR-2392、nwTCR-2424、nwTCR-2561、nwTCR-2563、nwTCR-2595和nwTCR-2629，使用高通量配对TCR测序，在单细胞基础上确定了这16个T细胞克隆上配对的TCRα链和β链的氨基酸序列。
由于多个核苷酸可以翻译成同一个氨基酸，在不同生物体中使用密码子频率是不同的，因此，对TCRα链和β链氨基酸序列的编码核苷酸进行了密码子序列优化，旨在于真核细胞中表达时增加TCR的表达量。获得了通过密码子优化后的特异性识别KRAS_G12V_7-16表位肽和KRAS_G12V_8-16表位肽的16个TCR的核苷酸序列。
表1A和表1B分别列出了测序生成的克隆T细胞系所表达的16个TCR的α链和β链的氨基酸序列信息以及核苷酸序列信息。
表1A.KRAS_G12V突变抗原特异性TCRα链的氨基酸和核苷酸序列

表1B.KRAS_G12V突变抗原特异性TCRβ链的氨基酸和核苷酸序列
实施例2.自T细胞制备KRAS_G12V突变抗原特异性TCR-T细胞
本实施例描述了通过CRISPR/Cas9技术敲除原代T细胞中的TCR基因并通过同源重组技术敲入KRAS_G12V突变抗原特异性TCR基因，由此制备并表征KRAS_G12V突变抗原特异性TCR-T细胞。
2.1 T细胞的分选和激活
从外周血单个核细胞(PBMC，购自：上海赛笠生物科技有限公司，Donor：S2001095)中富集和分选CD4T细胞和CD8T细胞的混合物(文中也称为“CD4/CD8T细胞”)。将富集和分选的CD4/CD8T细胞等分并冷冻(5x106个细胞/冻存管)以便将来使用。
根据需要解冻冻存管，并且通过在T细胞培养基(例如，RPMI 1640、FBS、L-谷氨酰胺、非必需氨基酸、丙酮酸钠、HEPES缓冲液、2-巯基乙醇和任选的IL2)中加入1:100倍稀释的T细胞激活剂Miltenyi T cell TransACT(Miltenyi目录号：130-111-160)对分选得到的T细胞进行激活，培养细胞约48小时(2天)后用于电穿孔转染。
2.2打靶策略和打靶载体的制备
gRNA采用gRNA002和gRNA004(见表2)，gRNA002的打靶位点位于内源性TRAC基因的外显子1(图1A)；gRNA004的打靶位点位于内源性TRBC1和TRBC2基因的外显子1(图1B)。Cas9酶购自金斯瑞生物科技有限公司，目录号：Z03469。
表2.TRAC和TRBC基因对应的sgRNA
打靶载体(也称为HDR载体)采用的骨架为pUC57-HA载体，是在pUC57-Simple载体基础上优化后的载体。其仅保留了pUC57-Simple载体的Ori和Amp序列，然后将Amp序列替换为Kana序列，并添加TRAC位点的左右同源臂(HA)序列(800bp左右)。可以将待基因敲入(KI)的序列构建到左右HA之间。nwTCR的KI序列构建体结构包含：2A或其变体-SP-TCRβ-2A或其变体-SP-TRAV-TRAJ，其中2A为核糖体跳跃元件；SP为信号肽；对打靶载体中TRBC基因中引入了4个同义突变碱基，分别为将TRBC S77的编码核苷酸由AGC突变为TCC和将S78的编码核苷酸由AGC突变为TCC。对于nwTCR-0126，其KI序列构建体序列如SEQ ID NO:386所示，其中：HA为同源臂(5’HA如SEQ ID NO:387所示，3’HA如SEQ ID NO:391所示；2A或其变体分别如SEQ ID NO:388和SEQ ID NO:390所示；SP为SEQ ID NO:389所示信号肽序列；TCRβ为nwTCR-0126完整TRB(TCRβ链)的核苷酸序列(SEQ ID NO:352)，其中将TRBC S77的编码核苷酸由AGC突变为TCC和将S78的编码核苷酸由AGC突变为TCC，由此在TRBC基因上引入了4个同义突变碱基；TRAV为nwTCR-0126TRAV基因的核苷酸序列(SEQ ID NO:54)；TRAJ为nwTCR-0126TRAJ基因的核苷酸序列(SEQ ID NO:56)。类似地，制备了nwTCR-0125、nwTCR-0127、nwTCR-1708、nwTCR-1862、nwTCR-2162、nwTCR-2241、nwTCR-2308、nwTCR-2310、nwTCR-2390、nwTCR-2392、nwTCR-2424、nwTCR-2561、nwTCR-2563、nwTCR-2595和nwTCR-2629的KI序列构建体。
2.3.电穿孔转染(Day 2)
将实施例2.2的sgRNA与Cas9酶充分混合，室温孵育10min，制备RNP。
将实施例2.2制备的含有KI TCR序列的打靶载体与孵育好的RNP、实施例2.1制备的指定浓度的T细胞(约1.25E6个T细胞/电击管)进行充分混合，以用于敲除(KO)内源性TCR和敲入(KI)外源性TCR。
将上述混合液装载到电穿孔转染仪(Celetrix；目录号：CTX-1500A LE)中进行细胞电穿孔转染，电穿孔转染的条件为480-560V，20ms。
电穿孔转染结束后，静置细胞15min后再取出经电穿孔转染的T细胞，转移至预温的培养基中(ImmunoCultTM-XF T Cell Expansion Medium，Stemcell公司目录号：10981)。培养细胞5天后，于Day 7进行流式细胞术表征。
2.4.nwTCR表达的流式细胞术分析(Day 7)
充分混匀自实施例2.3.获得的细胞悬液，进行细胞计数，收集适量细胞进行两种标记的肽-MHC(HLA-A*11:01)四聚体(分别为VVVGAVGVGK-HLA-A*11:01四聚体和VVGAVGVGK-HLA-A*11:01四聚体，它们也可简称为pMHC)染色。
提前配制好含有所述两种标记的肽-MHC(HLA-A*11:01)四聚体染液以及LIVE/DEADTM Fixable Near-IR，购自Invitrogen，目录号：L10119；CD4-FITC购自BioLegend，目录号：357406；CD8-PerCP-cy5.5购自BioLegend，目录号：344710；抗人TCRα/β-BV510抗体购自BioLegend，目录号：306734。
对收集的细胞进行所述两种标记的肽-MHC(HLA-A*11:01)四聚体染液染色，洗涤后，用流式细胞术进行表征。
图3A-图3P例示了电穿孔转染不同nwTCR的CD4+T细胞、CD8+T细胞用pMHC四聚体染色的流式细胞术结果。其中，对nwTCR-0125、nwTCR-0126、nwTCR-0127、nwTCR-1708、nwTCR-1862、nwTCR-2162、nwTCR-2241、nwTCR-2308和nwTCR-2563例示的是与标记的VVVGAVGVGK-HLA-A*11:01四聚体的染色结果；对nwTCR-2310、nwTCR-2390、nwTCR-2392、nwTCR-2424、nwTCR-2561、nwTCR-2595和nwTCR-2629例示的是与标记的VVGAVGVGK-HLA-A*11:01四聚体的染色结果。可以看出，Day7的细胞分为三群:
1)未进行基因编辑的野生型T细胞(Q3)；
2)完成了内源性TCR敲除的KO细胞(Q4)；
3)完成了KO和KI，表达了nwTCR的细胞群(Q2)。
流式细胞术检测TCR基因编辑效率的结果示意图如图2所示。
由图3A-图3P可见，分别敲入各nwTCR的CD8+T细胞均能够与肽-MHC复合物(pMHC)四聚体结合；分别敲入各TCR的CD4+T细胞与肽-MHC复合物(pMHC)四聚体的结合存在显著性差异。这是因为如实施例1所示，本发明中的各nwTCR均是利用CD8+T细胞进行筛选得到的nwTCR，因此，当分别敲入各nwTCR至CD8+T细胞后，表达各nwTCR的CD8+T细胞均能够与肽-MHC复合物(pMHC)四聚体特异性结合；而当分别敲入各nwTCR至CD4+T细胞后，表达各nwTCR的CD4+T细胞存在如下情形：一般地，当nwTCR与MHC的亲和力足够强的时候，TCR可以在不借助CD8分子协助的情况下结合MHC分子，例如，认为nwTCR-0127与MHC分子的结合能力较nwTCR-1708更强。因而它们在CD4+ T细胞的pMHC四聚体染色上会有差异。因此，将各nwTCR分别编辑 到CD4+ T细胞中并表达后，亲和力强的nwTCR仍然可以结合MHC I类抗原，而亲和力弱的nwTCR结合MHC I类抗原的能力也弱。
实施例3.KRAS_G12V突变抗原特异性TCR-T细胞的体外功能性研究
在Day7对实施例2的KRAS_G12V突变抗原特异性TCR-T细胞使用TransACT激活剂(Miltenyi目录号：130-111-160)进行选择性激活。继续培养TCR-T细胞至Day14。在Day14对各TCR-T细胞进行了体外功能性研究。
3.1各TCR-T细胞与肽结合的亲和力检测
如下实施TCR-T细胞亲和力检测方法。收集抗原呈递细胞(HLA-A*11:01过表达的T2细胞或K562细胞)，进行细胞计数，加适量培养基(如RPMI-1640培养基，购自Gibco，目录号：22400089；FBS，购自Gibco，目录号：10099141C)重悬细胞至细胞密度为1E6个/mL，分别加1mL细胞悬液到24孔板的每孔中。将待检测多肽溶液(所述多肽是SEQ ID NO:381所示的KRAS_G12V_7-16肽和/或SEQ ID NO:382所示的KRAS_G12V_8-16肽)进行梯度稀释到10-10-10-3M，并分别加10ul稀释后的多肽溶液到24孔板对应孔中，在培养箱(37℃，5％CO2)中孵育2h后，将孵育好的抗原呈递细胞收集洗涤，取100ul 1E6/mL的抗原呈递细胞至96孔板对应孔中。收集待检测的nwTCR-T细胞，加适量T细胞培养基(购自STEMCELL，目录号：10981)至细胞密度为1E6/mL，加100ul细胞悬液至中96孔板对应孔中。将各nwTCR-T细胞分别与抗原呈递细胞共培养(37℃，5％CO2)16h后，收集细胞上清用ELISA试剂盒(购自Biolegend，目录号：430104)检测IFN-γ浓度。通过检测IFN-γ的释放水平来检测表达各nwTCR的T细胞对HLA-A*11:01呈递的SEQ ID NO:381或SEQ ID NO:382所示短肽的结合亲和力。
图4A-图4H显示了分别表达各nwTCR的T细胞对HLA-A*11:01呈递的VVVGAVGVGK(SEQ ID NO:381)或VVGAVGVGK(SEQ ID NO:382)所示短肽的结合亲和力检测实验结果和EC50值，其中图4A、图4C、图4D和图4G是与VVVGAVGVGK(SEQ ID NO:381)所示短肽的结合亲和力；图4B、图4E、图4F和图4H是与VVGAVGVGK(SEQ ID NO:382)所示短肽的结合亲和力。
图4A-图4H的结果表明，呈递肽-MHC复合物的T2细胞与表达各nwTCR的T细胞共孵育后，检测到表达各nwTCR的T细胞与肽-MHC复合物特异性结合，导致了IFN-γ释放。另外，考虑到MHC呈递多肽时，9-mer、10-mer的短肽都有可能被MHC呈递，在图4A-图4F使用9-mer、10-mer的短肽进行的实验结果表明，表达各nwTCR的T细胞能够特异性结合分别呈递9-mer、10-mer短肽的pMHC复合物。
3.2各TCR-T细胞对靶细胞的杀伤
如下实施各TCR-T细胞对靶细胞的杀伤检测方法。收集靶细胞(为SW620(HLA-A*11:01过表达，KRAS G12V+)细胞系(购自：南京科佰生物科技有限公司)，细胞计数后用靶细胞培养基(RPMI-1640培养基，购自Gibco，目录号：22400089，FBS， 购自Gibco，目录号：10099141C)重悬靶细胞至细胞密度为1E6个细胞/mL。准备E-plate(获自Agilent，目录号：300600890)，在对应孔中加入100μL混合均匀的靶细胞悬液后，将其放入至RTCA实时细胞分析仪(real-time cell analyzer)(购自Agilent，型号：xCELLigence RTCA DP)中，过夜检测。
收集待检测的各TCR-T细胞，细胞计数后加适量T细胞培养基(购自STEMCELL，目录号：10981)重悬细胞。取出上述接种有靶细胞的E-plate，加入T细胞悬液，将E-Plate放回到RTCA分析仪中检测，得到72小时的细胞指数。每一个独立的实验分三次进行。利用RTCA软件自动计算区间斜率，评价细胞指数的变化率。为了证明处理的效果，在标准化时间点将细胞指数标准化为相等的值。
各TCR-T细胞对SW620(HLA-A*11:01过表达，KRAS G12V+)细胞的体外杀伤结果如图5A-图5E所示，结果表明了各TCR-T细胞对作为靶细胞的SW620(HLA-A*11:01过表达，KRAS G12V+)细胞系显示出显著的体外杀伤效果。
收集待检测的nwTCR-T细胞，加适量T细胞培养基(购自STEMCELL，目录号：10981)至细胞密度为1E6/mL，加100μL待检测的nwTCR-T细胞悬液至96孔板的孔中，与96孔板孔中的SW620(HLA-A*11:01过表达，KRAS G12V+)细胞混合。向该细胞混合液中添加1μL溴化乙锭(1mg/mL)溶液，混合均匀后，将细胞培养板置于实时荧光成像系统(BioTek Lionheart)中进行细胞杀伤表征实验。被T细胞特异性识别的靶细胞在进入凋亡状态后，会被溴化乙锭染色，呈现出红色的荧光信号。
结果表明，表达nwTCR的各T细胞均能使得SW620(HLA-A*11:01过表达，KRAS G12V+)细胞被溴化乙锭染色。图6例示了表达nwTCR-2424的T细胞与SW620细胞开始共孵育时(0h)和共孵育18小时时(18h)的结果，共孵育18小时时(18h)红色的荧光信号表明了表达nwTCR-2424的T细胞对于SW620细胞的特异性杀伤。
对细胞杀伤实时分析数据的分析结果显示，表达nwTCR的各T细胞均对于SW620(HLA-A*11:01过表达，KRAS G12V+)细胞存在特异性杀伤。图7例示了表达nwTCR-2424的T细胞对于SW620细胞的特异性杀伤。
实施例4.外源性TCR与CD8分子的共表达
本实施例描述了通过采用CRISPR/Cas9和同源重组技术将外源性TCR和CD8分子共表达于CD4+ T细胞膜表面，从而将CD4+ T细胞重定向。并且，这种基因编辑方法可以增强TCR-T细胞与pMHC分子的结合。
4.1 T细胞的分选和激活
可以商业获得T细胞(例如，冷冻人外周血CD4+CD45RA+T细胞，Stem Cell Technology，目录号70029)或者自白细胞单采样本制备T细胞(Day 0)。
对于自白细胞单采样本制备T细胞，从白细胞单采样本中富集和分选CD4/CD8T细胞。将富集和分选的CD4/CD8T细胞等分并冷冻(5x106个细胞/冻存管)以便将来使用。
4.2打靶策略和打靶载体制备
TCR的打靶策略和打靶载体同上述实施例2.2。当TCR序列采用nwTCR-1708时，nwTCR-1708的KI氨基酸序列为：2A或其变体-SP-TCRβ-2A或其变体-SP-TRAV-TRAJ(SEQ ID NO:392)，nwTCR-1708的KI核苷酸序列为：2A或其变体-SP-TCRβ-2A或其变体-SP-TRAV-TRAJ(SEQ ID NO:393)，由上图3D可见，表达nwTCR-1708的CD8+ T细胞能与KRAS G12V第7到第16位氨基酸序列(KRAS_G12V_7-16肽)的pMHC分子结合，但表达nwTCR-1708的CD4+ T细胞不能与呈递KRAS_G12V_7-16肽的pMHC分子结合。本实施例选择pMHC四聚体不与基因编辑后表达TCR的CD4+ T细胞结合旨在表明，即使对于这样的nwTCR，通过在CD4T细胞中引入CD8分子，可以增强基因编辑nwTCR后的CD4T细胞与pMHC分子的结合，因此，本实施例中使用的特定nwTCR可以用任何本发明的其他nwTCR替换且能够实现增强基因编辑nwTCR后的CD4T细胞与pMHC分子的结合的作用。
为了使CD4+ T细胞能与KRAS G12V第7到第16位氨基酸序列(KRAS_G12V_7-16肽)的pMHC分子结合，进一步构建了如下构建体，并插入pUC57-HA打靶载体(也称为HDR载体)：
nwTCR-1708-CD8a的KI氨基酸序列为：2A或其变体-CD8a-2A或其变体-SP-TCRβ-2A或其变体-SP-TRAV-TRAJ(SEQ ID NO:394)。
nwTCR-1708-CD8a的KI核苷酸序列为：2A或其变体-CD8a-2A或其变体-SP-TCRβ-2A或其变体-SP-TRAV-TRAJ(SEQ ID NO:395)。
nwTCR-CD8a的打靶策略图如图8A所示。
nwTCR-1708-CD8ab的KI氨基酸序列为：2A或其变体-CD8a-2A或其变体-CD8b-2A或其变体-SP-TCRβ-2A或其变体-SP-TRAV-TRAJ(SEQ ID NO:398)。
nwTCR-1708-CD8ab的KI核苷酸序列为：2A或其变体-CD8a-2A或其变体-CD8b-2A或其变体-SP-TCRβ-2A或其变体-SP-TRAV-TRAJ(SEQ ID NO:399)。
nwTCR-CD8ab的打靶策略图如图8B所示。
4.3.电穿孔转染(Day 2)
将实施例2.2设计并合成的sgRNA与Cas9酶充分混合，室温孵育10min，制备RNP。
将实施例4.2制备的三种打靶载体，即，用于敲入nwTCR-1708、nwTCR-1708-CD8a、或nwTCR-1708-CD8ab的打靶载体，分别与孵育好的RNP、实施例4.1制备的指定浓度的T细胞(1.25E6/电击管)进行充分混合，以用于敲除(KO)内源性TCR和敲入(KI)外源性TCR、TCR和CD8a、或者TCR和CD8ab。
将上述混合液装载到电穿孔转染仪(Celetrix；目录号：CTX-1500A LE)中进行细胞电穿孔转染，电穿孔转染的条件为480-560V，20ms。
电穿孔转染结束后，静置细胞15min后再取出经电穿孔转染的细胞，转移至预温的培养基中(ImmunoCultTM-XF T Cell Expansion Medium，Stemcell公司目录号：10981)。培养细胞5天后，于Day 7进行流式细胞术表征。
4.4.nwTCR表达的流式细胞术分析(Day 7)
充分混匀自实施例4.3获得的细胞悬液，细胞计数后收集适量细胞，进行标记的肽-MHC(HLA-A11:01)四聚体(VVVGAVGVGK-HLA-A11:01)染色。
提前配制好含有特异性抗原的标记的肽-MHC四聚体染液以及LIVE/DEADTM Fixable Near-IR，购自Invitrogen，目录号：L10119；CD4-FITC购自BioLegend，目录号：357406；CD8-PerCP-cy5.5购自BioLegend，目录号：344710；抗人TCRα/β-BV510抗体购自BioLegend，目录号：306734。
对收集的细胞进行标记的肽-MHC四聚体染液染色，洗涤后，用流式细胞术进行表征。结果如下所示。在流式细胞图中，Day7的细胞分为三群:
1)未进行基因编辑的野生型T细胞(Q3)；
2)完成了内源性TCR敲除的KO细胞(Q4)；
3)完成了KO和KI，表达了nwTCR的细胞群(Q2)。
编辑了nwTCR-1708的CD8+ T细胞可以结合特异性抗原MHC四聚体，而编辑了nwTCR-1708的CD4+ T细胞因为缺少CD8分子的辅助，无法结合特异性抗原MHC四聚体(图9A)。
当将CD8a分子或者CD8ab分子与nwTCR-1708共同编辑入原代T细胞后(所述原代T细胞包含CD4+ T细胞和CD8+T细胞)，通过流式细胞术表征，可以看出，CD8+CD4+T细胞的比例增加，这部分细胞是表达了外源CD8分子的CD4+ T细胞，这部分CD4+ T细胞在外源CD8分子的辅助作用下，可以特异性结合标记的肽-MHC(HLA-A11:01)四聚体(VVVGAVGVGK-HLA-A11:01)(图9B和图9C)。
同时，得益于本方法可以重定向CD4+ T细胞，使得对于T细胞整体的基因编辑效率提高(表3)，其中基因编辑效率(GE％)的计算公式为：活的T细胞群体中仅表达CD8+的细胞百分数X四聚体染色中仅表达CD8+的细胞百分数+活的T细胞群体中仅表达CD4+的细胞百分数X四聚体染色中仅表达CD4+的细胞百分数+活的T细胞群体中表达CD8+和CD4+的细胞百分数X四聚体染色中表达CD8+和CD4+的细胞百分数。
表3.CD8分子共表达的T细胞中各群细胞的基因编辑效率。

以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。
示例性序列
































Claims (12)

  1. 分离的或纯化的T细胞受体，所述T细胞受体也简称为TCR，其特征在于，与KRAS_G12V突变抗原特异性结合，所述TCR包含α链和β链，其中所述α链和β链各包含三个互补决定区，所述互补决定区也简称为CDR，并且α链的CDR3的氨基酸序列选自SEQ ID NO:3、6、9、12、15、18、21、24、27、30、33、36、39、42、45、48和与所述序列具有1个或2个氨基酸残基改变的变体，β链的CDR3的氨基酸序列选自SEQ ID NO:179、182、185、188、191、194、197、200、203、206、209、212、215、218、221、224和与所述序列具有1个或2个氨基酸残基改变的变体。
  2. 根据权利要求1所述的TCR，其中，所述α链的CDR3的氨基酸序列和β链的CDR3的氨基酸序列是：
    (i)SEQ ID NO:3所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:179所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (ii)SEQ ID NO:6所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:182所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (iii)SEQ ID NO:9所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:185所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (iv)SEQ ID NO:12所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:188所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (v)SEQ ID NO:15所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:191所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (vi)SEQ ID NO:18所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:194所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (vii)SEQ ID NO:21所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:197所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (viii)SEQ ID NO:24所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:200所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (ix)SEQ ID NO:27所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:203所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (x)SEQ ID NO:30所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:206所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (xi)SEQ ID NO:33所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:209所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (xii)SEQ ID NO:36所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:212所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (xiii)SEQ ID NO:39所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:215所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (xiv)SEQ ID NO:42所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:218所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    (xv)SEQ ID NO:45所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:221所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；或
    (xvi)SEQ ID NO:48所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:224所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；
    优选地，所述α链的CDR3的氨基酸序列和β链的CDR3的氨基酸序列是：
    (i)SEQ ID NO:3所示的α链CDR3氨基酸序列；以及SEQ ID NO:179所示的β链CDR3氨基酸序列；
    (ii)SEQ ID NO:6所示的α链CDR3氨基酸序列；以及SEQ ID NO:182所示的β链CDR3氨基酸序列；
    (iii)SEQ ID NO:9所示的α链CDR3氨基酸序列；以及SEQ ID NO:185所示的β链CDR3氨基酸序列；
    (iv)SEQ ID NO:12所示的α链CDR3氨基酸序列；以及SEQ ID NO:188所示的β链CDR3氨基酸序列；
    (v)SEQ ID NO:15所示的α链CDR3氨基酸序列；以及SEQ ID NO:191所示的β链CDR3氨基酸序列；
    (vi)SEQ ID NO:18所示的α链CDR3氨基酸序列；以及SEQ ID NO:194所示的β链CDR3氨基酸序列；
    (vii)SEQ ID NO:21所示的α链CDR3氨基酸序列；以及SEQ ID NO:197所示的β链CDR3氨基酸序列；
    (viii)SEQ ID NO:24所示的α链CDR3氨基酸序列；以及SEQ ID NO:200所示的β链CDR3氨基酸序列；
    (ix)SEQ ID NO:27所示的α链CDR3氨基酸序列；以及SEQ ID NO:203所示的β链CDR3氨基酸序列；
    (x)SEQ ID NO:30所示的α链CDR3氨基酸序列；以及SEQ ID NO:206所示的β链CDR3氨基酸序列；
    (xi)SEQ ID NO:33所示的α链CDR3氨基酸序列；以及SEQ ID NO:209所示的β链CDR3氨基酸序列；
    (xii)SEQ ID NO:36所示的α链CDR3氨基酸序列；以及SEQ ID NO:212所示的β链CDR3氨基酸序列；
    (xiii)SEQ ID NO:39所示的α链CDR3氨基酸序列；以及SEQ ID NO:215所示的β链CDR3氨基酸序列；
    (xiv)SEQ ID NO:42所示的α链CDR3氨基酸序列；以及SEQ ID NO:218所示的β链CDR3氨基酸序列；
    (xv)SEQ ID NO:45所示的α链CDR3氨基酸序列；以及SEQ ID NO:221所示的β链CDR3氨基酸序列；
    (xvi)SEQ ID NO:48所示的α链CDR3氨基酸序列；以及SEQ ID NO:224所示的β链CDR3氨基酸序列。
  3. 根据权利要求2所述的TCR，其中，所述α链包含的三个CDR的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
    (i)SEQ ID NO:1、2、3所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:177、178、179所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (ii)SEQ ID NO:4、5、6所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:180、181、182所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (iii)SEQ ID NO:7、8、9所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:183、184、185所示的β链 CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (iv)SEQ ID NO:10、11、12所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:186、187、188所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (v)SEQ ID NO:13、14、15所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:189、190、191所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (vi)SEQ ID NO:16、17、18所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:192、193、194所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (vii)SEQ ID NO:19、20、21所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:195、196、197所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (viii)SEQ ID NO:22、23、24所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:198、199、200所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (ix)SEQ ID NO:25、26、27所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:201、202、203所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (x)SEQ ID NO:28、29、30所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:204、205、206所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (xi)SEQ ID NO:31、32、33所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:207、208、209所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (xii)SEQ ID NO:34、35、36所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:210、211、212所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (xiii)SEQ ID NO:37、38、39所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:213、214、215所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (xiv)SEQ ID NO:40、41、42所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:216、217、218所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    (xv)SEQ ID NO:43、44、45所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:219、220、221所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；或
    (xvi)SEQ ID NO:46、47、48所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:222、223、224所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；
    优选地，所述α链包含的三个CDR的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
    (i)SEQ ID NO:1、2、3所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:177、178、179所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (ii)SEQ ID NO:4、5、6所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:180、181、182所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (iii)SEQ ID NO:7、8、9所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:183、184、185所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (iv)SEQ ID NO:10、11、12所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:186、187、188所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (v)SEQ ID NO:13、14、15所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:189、190、191所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (vi)SEQ ID NO:16、17、18所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:192、193、194所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (vii)SEQ ID NO:19、20、21所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:195、196、197所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (viii)SEQ ID NO:22、23、24所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:198、199、200所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (ix)SEQ ID NO:25、26、27所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:201、202、203所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (x)SEQ ID NO:28、29、30所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:204、205、206所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (xi)SEQ ID NO:31、32、33所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:207、208、209所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (xii)SEQ ID NO:34、35、36所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:210、211、212所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (xiii)SEQ ID NO:37、38、39所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:213、214、215所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (xiv)SEQ ID NO:40、41、42所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:216、217、218所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (xv)SEQ ID NO:43、44、45所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:219、220、221所示的β链CDR1、CDR2、CDR3氨基酸序列；
    (xvi)SEQ ID NO:46、47、48所示的α链CDR1、CDR2、CDR3氨基酸序列；以及SEQ ID NO:222、223、224所示的β链CDR1、CDR2、CDR3氨基酸序列。
  4. 根据权利要求1至3中任一项所述的TCR，其中，所述TCR还包含恒定区，例如，所述恒定区是小鼠恒定区；
    优选地，所述TCR包含SEQ ID NO:145、147、149、151、153、155、157、159、161、163、165、167、169、171、173或175所示的α链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:349、351、353、355、357、359、361、363、365、367、369、371、373、375、377或379所示的β链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    更优选地，所述TCR包含SEQ ID NO:145所示的α链序列和SEQ ID NO:349所示的β链序列；
    所述TCR包含SEQ ID NO:147所示的α链序列和SEQ ID NO:351所示的β链序列；
    所述TCR包含SEQ ID NO:149所示的α链序列和SEQ ID NO:353所示的β链序列；
    所述TCR包含SEQ ID NO:151所示的α链序列和SEQ ID NO:355所示的β链序列；
    所述TCR包含SEQ ID NO:153所示的α链序列和SEQ ID NO:357所示的β链序列；
    所述TCR包含SEQ ID NO:155所示的α链序列和SEQ ID NO:359所示的β链序列；
    所述TCR包含SEQ ID NO:157所示的α链序列和SEQ ID NO:361所示的β链序列；
    所述TCR包含SEQ ID NO:159所示的α链序列和SEQ ID NO:363所示的β链序列；
    所述TCR包含SEQ ID NO:161所示的α链序列和SEQ ID NO:365所示的β链序列；
    所述TCR包含SEQ ID NO:163所示的α链序列和SEQ ID NO:367所示的β链序列；
    所述TCR包含SEQ ID NO:165所示的α链序列和SEQ ID NO:369所示的β链序列；
    所述TCR包含SEQ ID NO:167所示的α链序列和SEQ ID NO:371所示的β链序列；
    所述TCR包含SEQ ID NO:169所示的α链序列和SEQ ID NO:373所示的β链序列；
    所述TCR包含SEQ ID NO:171所示的α链序列和SEQ ID NO:375所示的β链序列；
    所述TCR包含SEQ ID NO:173所示的α链序列和SEQ ID NO:377所示的β链序列；
    所述TCR包含SEQ ID NO:175所示的α链序列和SEQ ID NO:379所示的β链序列。
  5. 核酸分子，其特征在于，编码权利要求1－4中任一项所述的TCR，优选地，所述核酸分子是密码子优化的、编码权利要求1－4中任一项所述的TCR的核苷酸序列。
  6. 载体，其特征在于，包含权利要求5所述的核酸分子，所述载体优选地为质粒、穿梭质粒、噬菌粒、粘粒、表达载体；更优选地为同源重组修复(HDR)载体或病毒载体，例如，慢病毒载体、腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、逆转录病毒载体、杆状病毒载体。
  7. T细胞受体融合蛋白或T细胞受体缀合物，其包含权利要求1－4中任一项所述的TCR和其他生物活性分子，其中所述其他生物活性分子是例如抗体、细胞因子、细胞毒性剂、酶、放射性物质、可检测标记，其中所述TCR和其他生物活性分子之间具有或不具有接头。
  8. 一种工程化细胞，其特征在于，表达权利要求1－4中任一项所述的TCR，优选地，所述工程化细胞是工程化的T细胞、工程化的NK细胞；或者所述工程化细胞是工程化的干细胞，例如，所述工程化细胞是工程化的人CD4+T细胞或工程化的人CD8+T细胞，或者是工程化的人CD4+T细胞和工程化的人CD8+T细胞的混合细胞群；或者，所述工程化细胞是工程化的造血干细胞。
  9. 一种工程化的人CD4+T细胞和/或工程化的人CD8+T细胞，其特征在于，表达权利要求1－4中任一项所述的TCR并表达外源CD8a或者CD8ab。
  10. 产生权利要求9所述的T细胞的方法，所述方法包括用外源CD8a或者CD8ab；以及权利要求1－4中任一项所述的TCR转染CD4+T细胞和/或CD8+T细胞的步骤，例 如，所述外源CD8a或者CD8ab以及权利要求1－4中任一项所述的TCR在所述CD4+T细胞和/或CD8+T细胞中从相同载体表达，例如，其中表达所述外源CD8a或者CD8ab的构建体以及表达权利要求1－4中任一项所述TCR的构建体被2A元件或IRES元件分开。
  11. 药物组合物，其特征在于，包含权利要求8所述的工程化细胞和/或权利要求9所述的工程化的人CD4+T细胞和/或工程化的人CD8+T细胞。
  12. 根据权利要求11的药物组合物的用途，其特征在于，用于制备治疗具有KRAS_G12V突变的疾病(例如，肿瘤)的药物。
PCT/CN2023/078124 2022-11-04 2023-02-24 Kras_g12v突变抗原特异性tcr及其与cd8共表达重定向cd4 t细胞 WO2024093056A1 (zh)

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CN112300269A (zh) * 2020-09-29 2021-02-02 中国科学院微生物研究所 Kras突变特异性t细胞受体筛选及抗肿瘤用途
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WO2021083363A1 (zh) * 2019-11-01 2021-05-06 香雪生命科学技术（广东）有限公司 一种识别Kras G12V的高亲和力TCR

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