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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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  • A61K40/00—Cellular immunotherapy
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  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/32—T-cell receptors [TCR]
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  • A61K40/42—Cancer antigens
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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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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