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WO2025189363A1 - Récepteur de lymphocytes t (tcr) et son utilisation - Google Patents

Récepteur de lymphocytes t (tcr) et son utilisation

Info

Publication number
WO2025189363A1
WO2025189363A1 PCT/CN2024/081278 CN2024081278W WO2025189363A1 WO 2025189363 A1 WO2025189363 A1 WO 2025189363A1 CN 2024081278 W CN2024081278 W CN 2024081278W WO 2025189363 A1 WO2025189363 A1 WO 2025189363A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
tcr
cells
amino acid
chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/081278
Other languages
English (en)
Chinese (zh)
Inventor
陈琳
王晓玲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Medical University
Original Assignee
Guangzhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Medical University filed Critical Guangzhou Medical University
Priority to PCT/CN2024/081278 priority Critical patent/WO2025189363A1/fr
Publication of WO2025189363A1 publication Critical patent/WO2025189363A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present application relates to the field of medical technology, and in particular to TCRs that specifically recognize A1101-restricted KRAS G12V mutations and A1101-restricted KRAS G12D mutations and uses thereof.
  • KRAS mutations are common in patients with metastatic colorectal cancer, with G12D mutations accounting for 45% and G12V mutations for 30%.
  • KRAS a member of the RAS family, is a GTPase protein encoded by genes that participate in epidermal growth factor receptor (EGFR) signaling, regulating cell growth, differentiation, proliferation, and survival. RAS mutations result in defective GTPase activity, leading to overactivation of the RAF-MAPK cell signaling cascade. Tumors with KRAS mutations are generally more aggressive and have a worse prognosis. Studies have shown that patients with KRAS colorectal cancer are resistant to EGFR monoclonal antibodies (cetuximab or panitumumab).
  • NCCN guidelines recommend RAS genetic testing for all patients with metastatic colorectal cancer, and EGFR monoclonal antibodies should only be used in patients with wild-type RAS.
  • KRAS was once considered an "undruggable target.” While molecular biology has made significant advances in the study of RAS protein mutations and signaling pathways in recent years, the development of targeted drugs remains challenging. In the direction of chemical drug development, due to the smooth structure of RAS protein, the hydrophobic pockets on its surface for binding small molecules are not obvious; in the direction of biological drug development, antibody drugs need to penetrate the cell membrane to target RAS protein, so the drug delivery efficiency is very low.
  • T cell immunotherapy refers to the use of specific T cells targeting tumor antigens to kill tumor cells. It is a highly personalized tumor immunotherapy method. Due to the presence of the local immunosuppressive microenvironment of the tumor, the patient's own T cells have limited ability to kill tumors. Therefore, people have attempted to improve their tumor-killing ability by genetically modifying T cells.
  • TCR-T and CAR-T are both genetically modified cell therapy drugs. After the transferred T cell receptor (T cell receptor, TCR) or chimeric antigen receptor (CAR) gene binds to the corresponding target, it can activate T cells and use the granzymes, perforins, cytokines, etc. released by T cells to eliminate tumor cells.
  • TCR-T targets cell surface membrane proteins
  • TCR-T targets antigen short peptide-major histocompatibility complex (pMHC).
  • TCRs are restricted by the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • Known TCR targets are limited, and most are A0201, a target prevalent in Western populations.
  • NY-ESO-1 and HPV E6/E7 currently sought after by domestic and international TCR-T companies, can only target patients with the A0201 genotype. Therefore, developing TCR-T drugs targeting the more common HLA-A1101 genotype in the Chinese population will benefit more Chinese patients.
  • this application relates to the following aspects:
  • TCR T cell receptor
  • variable region of the ⁇ chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; and/or
  • the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 3 or SEQ ID NO: 4; and/or
  • the amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 5 or SEQ ID NO: 6, and the T cell receptor (TCR) binds to the KRAS G12V mutation.
  • CDR3 complementarity determining region 3
  • TCR T cell receptor
  • TCR T cell receptor
  • variable region of the ⁇ chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 8; and/or
  • variable region of the ⁇ chain further includes a second leader sequence
  • the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, and/or the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16,
  • the ⁇ chain further comprises an ⁇ constant region, and/or the ⁇ chain further comprises a ⁇ constant region.
  • the constant region is a mouse constant region or a human constant region.
  • TCR T cell receptor
  • the TCR is human
  • the TCR is monoclonal;
  • the TCR is single chain
  • the TCR comprises two chains;
  • the TCR is in a cell-bound form or a soluble form, preferably a soluble form;
  • the TCR binds to the antigen short peptide-HLA-A1101 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO:1.
  • nucleic acid molecule wherein the nucleic acid molecule comprises a nucleotide sequence encoding the TCR according to any one of items 1 to 4, or the ⁇ chain or ⁇ chain of the TCR.
  • nucleic acid molecule according to item 5, wherein the nucleotide sequence encoding the ⁇ chain is the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18; and/or
  • the nucleotide sequence encoding the ⁇ chain is the nucleotide sequence shown in SEQ ID NO:19 or SEQ ID NO:20.
  • a vector wherein the vector comprises the nucleic acid molecule according to item 5 or 6.
  • the vector is a viral vector, preferably a retroviral vector;
  • the viral vector is a lentiviral vector.
  • An engineered cell comprising the TCR of any one of items 1-4, the nucleic acid molecule of any one of items 5-6, or the vector of any one of items 7-8.
  • the engineered cell is a cell line
  • the engineered cells are primary cells obtained from a subject, preferably a mammalian subject, preferably a human;
  • the engineered cells are T cells or NK cells, preferably, the T cells are T cells isolated from peripheral blood;
  • the T cells are CD8+ or CD4+.
  • a method for producing the engineered cell according to any one of items 9 to 10, comprising introducing the nucleic acid molecule according to any one of items 5 to 6 or the vector according to any one of items 7 to 8 into a cell in vitro or ex vivo.
  • a pharmaceutical composition comprising the T cell receptor (TCR) of any one of items 1 to 4, the nucleic acid molecule of any one of items 5 to 6, the vector of any one of items 7 to 8, or the engineered cell of any one of items 9 to 10;
  • TCR T cell receptor
  • it further comprises a pharmaceutically acceptable carrier or adjuvant.
  • TCR T cell receptor
  • variable region of the ⁇ chain comprises the complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33; and/or
  • the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 34 or SEQ ID NO: 35; and/or
  • the amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 36 or SEQ ID NO: 37, and the T cell receptor (TCR) binds to the KRAS G12D mutation.
  • CDR3 complementarity determining region 3
  • TCR T cell receptor
  • TCR T cell receptor
  • the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO:40 or SEQ ID NO:41; and/or
  • the amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO:42 or SEQ ID NO:43.
  • TCR T cell receptor
  • variable region of the ⁇ chain further comprises a first leader sequence
  • variable region of the ⁇ chain further includes a second leader sequence
  • the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 44 or SEQ ID NO: 45, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44 or SEQ ID NO: 45, and/or the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 46 or SEQ ID NO: 47, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 46 or SEQ ID NO: 47,
  • the ⁇ chain further comprises an ⁇ constant region, and/or the ⁇ chain further comprises a ⁇ constant region.
  • the constant region is a mouse constant region or a human constant region.
  • TCR T cell receptor
  • the TCR is human
  • the TCR is monoclonal;
  • the TCR is single chain
  • the TCR comprises two chains;
  • the TCR is in a cell-bound form or a soluble form, preferably a soluble form;
  • the TCR binds to the antigen short peptide-HLA-A1101 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO:32.
  • nucleic acid molecule wherein the nucleic acid molecule comprises a nucleotide sequence encoding the TCR according to any one of items 15 to 18, or the ⁇ chain or ⁇ chain of the TCR.
  • nucleic acid molecule according to item 19, wherein the nucleotide sequence encoding the ⁇ chain is the nucleotide sequence shown in SEQ ID NO: 48 or SEQ ID NO: 49; and/or
  • the nucleotide sequence encoding the ⁇ chain is the nucleotide sequence shown in SEQ ID NO:50 or SEQ ID NO:51.
  • a vector wherein the vector comprises the nucleic acid molecule according to item 19 or 20.
  • the vector is a viral vector, preferably a retroviral vector;
  • the viral vector is a lentiviral vector.
  • An engineered cell comprising the TCR of any one of items 15-18, the nucleic acid molecule of any one of items 19-20, or the vector of any one of items 21-22.
  • the engineered cells are primary cells obtained from a subject, preferably a mammalian subject, preferably a human;
  • the engineered cells are T cells or NK cells, preferably, the T cells are T cells isolated from peripheral blood;
  • the T cells are CD8+ or CD4+.
  • a method for producing the engineered cell of any one of items 23-24 comprising introducing the nucleic acid molecule of any one of items 19-20 or the vector of any one of items 7-8 into the cell in vitro or ex vivo.
  • a pharmaceutical composition comprising the T cell receptor (TCR) of any one of items 15-18, the nucleic acid molecule of any one of items 19-20, the vector of any one of items 21-22, or the engineered cell of any one of items 23-24;
  • TCR T cell receptor
  • TCR T cell receptor
  • the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, or prostate cancer.
  • T cell receptor (TCR) of the present application can specifically recognize the A1101-restricted-KRAS G12V mutation.
  • T cells (TCR-T) transduced with the TCR of the present application can bind to the antigen short peptide KRAS G12V -HLA-A1101 complex, specifically kill tumor cells for tumor antigens, and can be used to treat malignant tumors carrying the KRAS G12V mutation.
  • the T cell receptor (TCR) of the present application can specifically recognize the A1101-restricted-KRAS G12D mutation.
  • T cells (TCR-T) transduced with the TCR of the present application can bind to the antigen short peptide KRAS G12D -HLA-A1101 complex, specifically kill tumor cells for tumor antigens, and can be used to treat malignant tumors carrying the KRAS G12D mutation.
  • T cells transduced with the TCR of the present application can be specifically activated by tumor cells expressing A11 and KRAS G12V mutations; and have good specificity, only recognizing KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutations.
  • T cells transduced with the TCR of the present application can be specifically activated by tumor cells expressing A11 and KRAS G12D mutations; and have good specificity, only recognizing KRAS G12D mutations, but not wild-type KRAS and KRAS G12V mutations.
  • FIG. 1 shows that the TCR described in this application can be correctly expressed in the Jurkat T cell line.
  • FIG2 shows that T cells transduced with the TCR described in the present application can respond to target cells loaded with a short peptide of the KRAS G12V antigen.
  • FIG. 5 shows that the TCR of the present application has good specificity and only recognizes the KRAS G12V mutation, but not the wild-type KRAS and KRAS G12D mutation.
  • FIG. 6 shows that the TCR described in this application can be correctly expressed in the Jurkat T cell line.
  • FIG7 shows that T cells transduced with the TCR described in the present application can respond to target cells loaded with a short peptide of the KRAS G12D antigen.
  • FIG9 shows the release of interferon-gamma after T cells transduced with the TCR described herein were co-incubated with tumor cells expressing A11 and KRAS G12D mutations.
  • FIG10 shows that the TCR of the present application has good specificity and only recognizes the KRAS G12D mutation, but not the wild-type KRAS and KRAS G12V mutation.
  • the amino acid sequence shown in SEQ ID NO:1 is: DRVSQS; the amino acid sequence shown in SEQ ID NO:2 is: TSENNYY; the amino acid sequence shown in SEQ ID NO:3 is: IYSNGD; the amino acid sequence shown in SEQ ID NO:4 is: QEAYKQQN; the amino acid sequence shown in SEQ ID NO:5 is: AAVSGGSYIPT; the amino acid sequence shown in SEQ ID NO:6 is: AFMNGETSGSRLT.
  • variable region of the beta chain comprises a complementarity determining region 1 (CDR1) with an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 8; and/or a complementarity determining region 2 (CDR2) with an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10; and/or a complementarity determining region 3 (CDR3) with an amino acid sequence as shown in SEQ ID NO: 11 or SEQ ID NO: 12,
  • amino acid sequence shown in SEQ ID NO:10 is: ANQGSEA; the amino acid sequence shown in SEQ ID NO:11 is: ASSVGGLAGELLETQY; the amino acid sequence shown in SEQ ID NO:12 is: SVIPHGLYEQY.
  • variable region of the ⁇ chain further includes a first leader sequence; and/or the variable region of the ⁇ chain further includes a second leader sequence.
  • first leader sequence of the variable region of the ⁇ chain and the second leader sequence of the variable region of the ⁇ chain are well known to those skilled in the art.
  • the first leader sequence of the variable region of the ⁇ chain can use an amino acid sequence such as a leader sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22
  • the second leader sequence of the variable region of the ⁇ chain can use an amino acid sequence such as a leader sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24.
  • amino acid sequence represented by SEQ ID NO:21 is: MKSLRVLLVILWLQLSWVWSQ;
  • the amino acid sequence represented by SEQ ID NO:22 is: MTRVSLLWAVVVSTCLESGM;
  • amino acid sequence represented by SEQ ID NO:23 is: MGFRLLCCVAFCLLGAGPV;
  • the amino acid sequence represented by SEQ ID NO:24 is: MLSLLLLLLGLGSVF;
  • the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, and/or the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16,
  • amino acid sequence shown in SEQ ID NO: 13 is:
  • amino acid sequence shown in SEQ ID NO: 14 is:
  • amino acid sequence shown in SEQ ID NO: 15 is:
  • amino acid sequence shown in SEQ ID NO: 16 is:
  • the amino acid sequence of the ⁇ chain variable region has at least 90% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14.
  • the amino acid sequence of the ⁇ chain variable region has at least 90% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16.
  • the ⁇ chain further comprises an ⁇ constant region, and/or the ⁇ chain further comprises a ⁇ constant region, preferably, the constant region is a mouse constant region or a human constant region.
  • the amino acid sequence of the mouse ⁇ constant region is as shown in SEQ ID NO:25; and/or the amino acid sequence of the mouse ⁇ constant region is as shown in SEQ ID NO:26, that is, the constant regions of the ⁇ chains of the above-mentioned TCRs can all have the same constant region, and similarly, the constant regions of the ⁇ chains of all TCRs can also have the same constant region.
  • amino acids shown in SEQ ID NO: 25 are:
  • amino acids shown in SEQ ID NO: 26 are:
  • the constant region of the TCR can contain a short linker sequence in which cysteine residues form a disulfide bond, thereby connecting the two chains of the TCR.
  • the TCR can have additional cysteine residues in each of the ⁇ and ⁇ chains, such that the TCR contains two disulfide bonds in the constant region.
  • the amino acid sequence of the ⁇ chain variable region of the TCR is as shown in SEQ ID NO: 13 and the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 15, or the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 14 and the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO: 16.
  • an artificial disulfide bond is introduced between the residues of the constant regions of the ⁇ chain and the ⁇ chain of the TCR.
  • the positions of the disulfide bonds that can be introduced are well known to those skilled in the art.
  • the TCR is isolated or purified or recombinant.
  • the TCR is human.
  • the TCR is monoclonal.
  • the TCR is single chain.
  • the TCR comprises two chains.
  • TCRs can be obtained from biological sources, such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, for example, TCRs can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals, such as typically from human.
  • biological sources such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, for example, TCRs can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals, such as typically from human.
  • the TCR may be in a cell-bound form or a soluble form, preferably a soluble form.
  • the soluble form of the TCR refers to a TCR that has mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present application.
  • the present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR or the ⁇ chain or ⁇ chain of the TCR.
  • the nucleotide sequence encoding the ⁇ chain is a nucleotide sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18; and/or the nucleotide sequence encoding the ⁇ chain is a nucleotide sequence as shown in SEQ ID NO: 19 or SEQ ID NO: 20.
  • the nucleotide sequence shown in SEQ ID NO: 17 is:
  • the nucleotide sequence shown in SEQ ID NO: 18 is:
  • the nucleotide sequence shown in SEQ ID NO: 19 is:
  • the nucleotide sequence shown in SEQ ID NO:20 is:
  • the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain are codon-optimized.
  • codon optimization involves balancing the percentage of selected codons with the abundance of disclosed human transfer RNAs so that none of them is overloaded or restricted. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Codon usage differences between transfected genes and host cells may affect protein expression and immunogenicity of nucleic acid constructs.
  • codons are selected to select those codons that are balanced with human usage frequency.
  • the redundancy of amino acid codons is such that different codons encode a single amino acid.
  • the resulting mutation may be a silent mutation so that the codon change does not affect the amino acid sequence.
  • the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.
  • the present application provides a vector comprising the nucleic acid molecule described above.
  • one or more nucleic acids encoding one or both chains of the TCR are cloned into one or more suitable expression vectors.
  • the expression vectors can be any suitable recombinant expression vectors and can be used to transform or transfect any Suitable hosts. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses.
  • the vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), taking into account whether the vector is DNA-based or RNA-based.
  • the vector may also contain a non-natural promoter operably linked to the nucleotide sequence encoding the TCR.
  • the promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat sequence of the mouse stem cell virus, and other promoters known to those skilled in the art are also contemplated.
  • CMV cytomegalovirus
  • the vector is an expression vector, preferably a viral vector, preferably a retroviral vector, and more preferably a lentiviral vector.
  • the application also provides a host cell comprising the nucleic acid molecules described above, and in order to recombinantly produce TCR, the nucleic acid encoding TCR can be separated and inserted into one or more vectors to further clone and/or express in the host cell.
  • Conventional techniques can be used (for example, by using oligonucleotide probes that can bind specifically to the genes encoding the ⁇ chain and ⁇ chain of the TCR) to easily separate and sequence such nucleic acids.
  • a method for preparing TCR includes culturing a host cell comprising a nucleic acid encoding TCR as provided above under conditions suitable for expressing the TCR molecule, and optionally recovering the TCR from the host cell (or host cell culture medium).
  • host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations.
  • the present application also provides an engineered cell comprising the T cell receptor (TCR), the nucleic acid molecule or the vector.
  • TCR T cell receptor
  • the TCR is allogeneic to the cell.
  • the engineered cell is a cell line.
  • the engineered cell is a primary cell obtained from a subject, preferably a mammalian subject, preferably a human.
  • the engineered cells are T cells, preferably T cells isolated from peripheral blood.
  • the T cells are CD8+ or CD4+.
  • the engineered cell is a cell that is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate immunity or adaptive immunity, such as bone marrow or lymphoid cells (including lymphocytes, typically T cells and/or NK cells).
  • Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs).
  • Cell is typically primary cells, such as directly separated from a subject and/or separated and frozen from a subject.
  • cell includes one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells and subgroups thereof.
  • T cells and/or CD4+ and/or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adapted Regulatory T (Treg) cells, etc.
  • TN naive T
  • TEFF effector T cells
  • TCM stem cell memory T
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor infiltrating lymphocytes
  • immature T cells immature T cells
  • mature T cells mature T cells
  • helper T cells cytotoxic T cells
  • the engineered cells are natural killer (NK) cells, preferably, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and/or basophils.
  • NK natural killer
  • the present application provides a method for producing the engineered cells described above, which comprises introducing the nucleic acid molecule or the vector into cells in vitro or ex vivo.
  • the vector is a viral vector, and the introduction is performed by transduction.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned engineered cell.
  • TCR T cell receptor
  • it further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the pharmaceutically acceptable carrier or adjuvant refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject.
  • Pharmaceutically acceptable carriers or adjuvants include but are not limited to buffers, excipients, stabilizers or preservatives.
  • the pharmaceutical composition can utilize timed release, delayed release, and sustained release delivery systems so that delivery of the composition occurs before sensitization of the treated area and has sufficient time to cause sensitization.
  • Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician.
  • the present application provides the use of the aforementioned T cell receptor (TCR), the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned engineered cell or the aforementioned pharmaceutical composition in the preparation of a drug for treating malignant tumors.
  • TCR T cell receptor
  • the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, or prostate cancer.
  • the colorectal cancer is metastatic colorectal cancer.
  • T cell receptor (TCR) of the present application can specifically recognize the A1101-restricted-KRAS G12V mutation.
  • T cells (TCR-T) transduced with the TCR of the present application can bind to the antigen short peptide KRAS G12V -HLA-A1101 complex, specifically kill tumor cells against tumor antigens, and can be used to treat malignant tumors carrying the KRAS G12V mutation.
  • T cells transduced with the TCR of the present application can be specifically activated by tumor cells expressing A11 and KRAS G12V mutations; and have good specificity, recognizing only KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutations.
  • the present application provides a T cell receptor (TCR), wherein the TCR comprises an ⁇ chain containing a variable region and/or a ⁇ chain containing a variable region, the variable region of the ⁇ chain comprises a complementarity determining region 1 (CDR1) with an amino acid sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 33; and/or a complementarity determining region 2 (CDR2) with an amino acid sequence as shown in SEQ ID NO: 34 or SEQ ID NO: 35; and/or a complementarity determining region 3 (CDR3) with an amino acid sequence as shown in SEQ ID NO: 36 or SEQ ID NO: 37,
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • amino acid sequence shown in SEQ ID NO:32 is: TRDTTYY; the amino acid sequence shown in SEQ ID NO:33 is: SSVSVY; the amino acid sequence shown in SEQ ID NO:34 is: RNSFDEQN; the amino acid sequence shown in SEQ ID NO:35 is: YLSGSTLV; the amino acid sequence shown in SEQ ID NO:36 is: ALSEAAPGGSYIPT; the amino acid sequence shown in SEQ ID NO:37 is: AVIGNDYKLS.
  • variable region of the beta chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 38 or SEQ ID NO: 39; and/or an amino acid sequence as shown in SEQ ID NO: 40 or SEQ ID NO: 41. and/or the amino acid sequence of the complementarity determining region 3 (CDR3) as shown in SEQ ID NO: 42 or SEQ ID NO: 43,
  • CDR1 complementarity determining region 1
  • CDR3 complementarity determining region 3
  • amino acid sequence shown in SEQ ID NO:38 is: MDHEN; the amino acid sequence shown in SEQ ID NO:39 is: SNHLY; the amino acid sequence shown in SEQ ID NO:40 is: SYDVKM; the amino acid sequence shown in SEQ ID NO:41 is: FYNNEI;
  • amino acid sequence shown in SEQ ID NO:42 is: ASSLGPGQHNSPLH; the amino acid sequence shown in SEQ ID NO:43 is: ASSGTGGIEAF.
  • variable region of the ⁇ chain further includes a first leader sequence; and/or the variable region of the ⁇ chain further includes a second leader sequence.
  • first leader sequence of the variable region of the ⁇ chain and the second leader sequence of the variable region of the ⁇ chain are well known to those skilled in the art.
  • the first leader sequence of the variable region of the ⁇ chain can use an amino acid sequence such as a leader sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53
  • the second leader sequence of the variable region of the ⁇ chain can use an amino acid sequence such as a leader sequence shown in SEQ ID NO: 54 or SEQ ID NO: 55.
  • amino acid sequence shown in SEQ ID NO:44 is:
  • amino acid sequence shown in SEQ ID NO:45 is:
  • amino acid sequence shown in SEQ ID NO:46 is:
  • amino acid sequence shown in SEQ ID NO:47 is:
  • the amino acid sequence of the ⁇ chain variable region has at least 90% sequence identity with SEQ ID NO: 44 or SEQ ID NO: 45, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 44 or SEQ ID NO: 45.
  • An amino acid sequence having at least 90% sequence identity with SEQ ID NO:46 or SEQ ID NO:47 may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO:46 or SEQ ID NO:47.
  • the ⁇ chain further comprises an ⁇ constant region, and/or the ⁇ chain further comprises a ⁇ constant region, preferably, the constant region is a mouse constant region or a human constant region.
  • the amino acid sequence of the mouse ⁇ constant region is as shown in SEQ ID NO: 56; and/or the amino acid sequence of the mouse ⁇ constant region is as shown in SEQ ID NO: 57. That is, the constant regions of the ⁇ chains of the TCRs described above can all have the same constant region. Similarly, the constant regions of the ⁇ chains of all TCRs can also have the same constant region.
  • amino acids shown in SEQ ID NO:56 are:
  • amino acids shown in SEQ ID NO:57 are:
  • the constant region of the TCR can contain a short linker sequence in which cysteine residues form a disulfide bond, thereby connecting the two chains of the TCR.
  • the TCR can have additional cysteine residues in each of the ⁇ and ⁇ chains, such that the TCR contains two disulfide bonds in the constant region.
  • the amino acid sequence of the ⁇ chain variable region of the TCR is as shown in SEQ ID NO:44 and the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO:46, or the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO:45 and the amino acid sequence of the ⁇ chain variable region is as shown in SEQ ID NO:47.
  • an artificial disulfide bond is introduced between the residues of the constant regions of the ⁇ chain and the ⁇ chain of the TCR.
  • the positions of the disulfide bonds that can be introduced are well known to those skilled in the art.
  • the TCR is isolated or purified or recombinant.
  • the TCR is human.
  • the TCR is monoclonal.
  • the TCR is single chain.
  • the TCR comprises two chains.
  • TCRs can be obtained from biological sources, such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, for example, TCRs can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals, such as typically from human.
  • biological sources such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, for example, TCRs can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals, such as typically from human.
  • the TCR may be in a cell-bound form or a soluble form, preferably a soluble form.
  • the soluble form of the TCR refers to a TCR that has mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present application.
  • the present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR or the ⁇ chain or ⁇ chain of the TCR.
  • the nucleotide sequence encoding the ⁇ chain is a nucleotide sequence as shown in SEQ ID NO:48 or SEQ ID NO:49; and/or the nucleotide sequence encoding the ⁇ chain is a nucleotide sequence as shown in SEQ ID NO:50 or SEQ ID NO:51.
  • the nucleotide sequence shown in SEQ ID NO:48 is:
  • the nucleotide sequence shown in SEQ ID NO:49 is:
  • the nucleotide sequence shown in SEQ ID NO:50 is:
  • the nucleotide sequence shown in SEQ ID NO:51 is:
  • the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain is codon optimized.
  • codon optimization involves balancing the percentage of selected codons with the abundance of disclosed human transfer RNAs so that none of them is overloaded or restricted. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Differences in codon usage between transfected genes and host cells may affect protein expression and immunogenicity of nucleic acid constructs.
  • codons are selected to select those codons that are balanced with human usage frequency.
  • the redundancy of amino acid codons is such that different codons encode one amino acid.
  • codons for replacement when codons are changed, it may be desirable for the resulting mutation to be a silent mutation so that the codon change does not affect the amino acid sequence. Typically, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.
  • the present application provides a vector comprising the nucleic acid molecule described above.
  • one or more nucleic acids encoding one or both chains of the TCR described above are cloned into one or more suitable expression vectors, which can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host.
  • suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.
  • the vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), taking into account whether the vector is DNA-based or RNA-based.
  • the vector may also contain a non-natural promoter operably linked to the nucleotide sequence encoding the TCR.
  • the promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat sequence of the mouse stem cell virus, and other promoters known to those skilled in the art are also contemplated.
  • CMV cytomegalovirus
  • the vector is an expression vector, preferably a viral vector, preferably a retroviral vector, and more preferably a lentiviral vector.
  • the application also provides a host cell comprising the nucleic acid molecules described above, and in order to recombinantly produce TCR, the nucleic acid encoding TCR can be separated and inserted into one or more vectors to further clone and/or express in the host cell.
  • Conventional techniques can be used (for example, by using oligonucleotide probes that can bind specifically to the genes encoding the ⁇ chain and ⁇ chain of the TCR) to easily separate and sequence such nucleic acids.
  • a method for preparing TCR includes culturing a host cell comprising a nucleic acid encoding TCR as provided above under conditions suitable for expressing the TCR molecule, and optionally recovering the TCR from the host cell (or host cell culture medium).
  • host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations.
  • the present application also provides an engineered cell comprising the T cell receptor (TCR), the nucleic acid molecule or the vector.
  • TCR T cell receptor
  • the TCR is allogeneic to the cell.
  • the engineered cell is a cell line.
  • the engineered cell is a primary cell obtained from a subject, preferably a mammalian subject, preferably a human.
  • the engineered cells are T cells, preferably T cells isolated from peripheral blood.
  • the T cells are CD8+ or CD4+.
  • the engineered cell can be, for example, a cell colony or a genetically engineered cell expressing TCR, which is typically a eukaryotic cell, such as a mammalian cell, and typically a human cell.
  • the cell is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate immunity or adaptive immunity, such as bone marrow or lymphoid cells (including lymphocytes, typically T cells and/or NK cells).
  • Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSC).
  • Cells are typically primary cells, such as those directly separated from a subject and/or separated and frozen from a subject.
  • cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, or a combination thereof. cells and their subpopulations.
  • T cells and/or CD4+ and/or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, etc.
  • TN naive T
  • TEFF effector T cells
  • TCM stem cell memory T
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor infiltrating lymphocytes
  • immature T cells immature T cells
  • mature T cells mature T cells
  • helper T cells cytotoxic T cells
  • mucosal-associated invariant T (MAIT) cells naturally occurring and adaptive regulatory T (Treg
  • the engineered cells are natural killer (NK) cells, preferably, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and/or basophils.
  • NK natural killer
  • the present application provides a method for producing the engineered cells described above, which comprises introducing the nucleic acid molecule or the vector into cells in vitro or ex vivo.
  • the vector is a viral vector, and the introduction is performed by transduction.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned engineered cell.
  • TCR T cell receptor
  • it further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the pharmaceutically acceptable carrier or adjuvant refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject.
  • Pharmaceutically acceptable carriers or adjuvants include but are not limited to buffers, excipients, stabilizers or preservatives.
  • the pharmaceutical composition can utilize timed release, delayed release, and sustained release delivery systems so that delivery of the composition occurs before sensitization of the treated area and has sufficient time to cause sensitization.
  • Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician.
  • the present application provides the use of the aforementioned T cell receptor (TCR), the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned engineered cell or the aforementioned pharmaceutical composition in the preparation of a drug for treating malignant tumors.
  • TCR T cell receptor
  • the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, prostate cancer, etc.
  • the colorectal cancer is metastatic colorectal cancer.
  • T cell receptor (TCR) of the present application can specifically recognize the A1101-restricted-KRAS G12D mutation.
  • T cells (TCR-T) transduced with the TCR of the present application can bind to the antigen short peptide KRAS G12D -HLA-A1101 complex, specifically kill tumor cells against tumor antigens, and can be used to treat malignant tumors carrying the KRAS G12D mutation.
  • T cells transduced with the TCR of the present application can be specifically activated by tumor cells expressing A11 and KRAS G12D mutations; and have good specificity, recognizing only KRAS G12D mutations, but not wild-type KRAS and KRAS G12V mutations.
  • % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.
  • Example 1 Cloning of KRAS G12V antigen short peptide-specific T cells and TCR gene acquisition
  • VVVGAVGVGK synthetic peptide
  • the sorted single cells were used to amplify the TCR ⁇ chain and ⁇ chain respectively using a one-step RT-PCR kit (QIAGEN, catalog number 210212), and the PCR products were sequenced.
  • IMGT International Immunogenetics Information System
  • the nucleotide sequence of the ⁇ chain variable region of TCR059 is: SEQ ID NO: 28:
  • the nucleotide sequence of the ⁇ chain variable region of TCR059 is: SEQ ID NO: 29:
  • CDR1 complementarity determining region 1 (CDR1) of the ⁇ chain of TCR059 is: SEQ ID NO: 1: DRVSQS;
  • the amino acid sequence of the complementarity determining region 2 (CDR2) of the ⁇ chain of TCR059 is: SEQ ID NO: 3: IYSNGD;
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR059 is: SEQ ID NO: 5: AAVSGGSYIPT;
  • the amino acid sequence of the complementarity determining region 1 (CDR1) of the ⁇ chain of TCR059 is: SEQ ID NO:7: SGDLS;
  • the amino acid sequence of the complementarity determining region 2 (CDR2) of the ⁇ chain of TCR059 is: SEQ ID NO: 9: YYNGEE;
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR059 is: SEQ ID NO: 11: ASSVGGLAGELLETQY;
  • the nucleotide sequence of the ⁇ chain variable region of TCR076 is: SEQ ID NO: 30:
  • the nucleotide sequence of the ⁇ chain variable region of TCR076 is: SEQ ID NO: 31:
  • the amino acid sequence of the complementarity determining region 1 (CDR1) of the ⁇ chain of TCR076 is: SEQ ID NO: 2: TSENNYY;
  • CDR2 complementarity determining region 2
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR076 is: SEQ ID NO: 6: AFMNGETSGSRLT;
  • the amino acid sequence of the complementarity determining region 1 (CDR1) of the ⁇ chain of TCR076 is: SEQ ID NO: 8: SQVTM;
  • CDR2 complementarity determining region 2
  • ANQGSEA amino acid sequence of the complementarity determining region 2 of the ⁇ chain of TCR076
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR076 is: SEQ ID NO:12: SVIPHGLYEQY.
  • Example 2 Construction of VVVGAVGVGK antigen short peptide-specific TCR lentiviral vector and lentiviral packaging
  • VVVGAVGVGK TCR ⁇ and ⁇ chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene).
  • the ⁇ or ⁇ variable domains were cloned into a pLKO-based expression plasmid containing the mouse ⁇ or ⁇ constant region using standard methods using the Multi-Fragment Recombination Cloning Kit (Novozymes Biotech, catalog number C113).
  • the ligated plasmids were transformed into competent Escherichia coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and plated on LB/agar plates containing 100 ⁇ g/ml ampicillin.
  • VVVGAVGVGK TCR i.e., TCR059, TCR076, plasmid.
  • Assay culture medium 10% FBS (Lonsera, catalog number S711-001), DMEM (ThermoFisher, catalog number C11995500BT).
  • Example 3 Construction and functional characterization of a Jurkat cell line expressing a VVVGAVGVGK antigen peptide-specific TCR
  • NFAT Nuclear Factor of Activated T Cells
  • NFAT expression was measured using flow cytometry as a readout of T cell activation.
  • Assay medium 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT)
  • T2-A11 cells T2 cells were deposited with ATCC under the catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium at a concentration of 1.6 ⁇ 10 6 cells/mL. 50 ⁇ L was plated per well to yield 80,000 cells/well.
  • JK8NF Jurkat-CD8-NFAT
  • JK8NF cells Jurkat cells were deposited in ATCC with the deposit catalog number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006; 66(23): 11455-61, Front. Immunol. 11: 633) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, the transfection positive rate was determined to be approximately 100% by flow cytometry (the results are shown in FIG1 ). The concentration of the effector cells after expanded culture was adjusted to 1.6 ⁇ 10 6 cells/ml, and 50 ⁇ l was taken per well to obtain 80,000 cells/well.
  • VVVGAVGVGK The original concentration of 5 mg/ml short peptide (VVVGAVGVGK) was diluted to 400 ⁇ g/ml, and then diluted down in 10-fold ratio to 40 ⁇ g/ml, 4 ⁇ g/ml, 0.4 ⁇ g/ml, 0.04 ⁇ g/ml, 0.004 ⁇ g/ml, 0.0004 ⁇ g/ml, and 0.00004 ⁇ g/ml.
  • 50 ⁇ l was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 ⁇ g/ml, 10 ⁇ g/ml, 1 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, 0.001 ⁇ g/ml, 0.0001 ⁇ g/ml, and 0.00001 ⁇ g/ml, respectively.
  • the above-described method was used to examine the expression of NFAT in TCR-transduced T cells of the present invention in response to target cells loaded with the VVVGAVGVGK antigen peptide.
  • the NFAT expression level curve was plotted using Graphpad Prism8, and the results are shown in Figure 2.
  • T cells transduced with the TCR of the present application have a good activation response to target cells loaded with their specific short peptides.
  • Example 4 In vitro functional characterization of primary T cells expressing a VVVGAVGVGK antigen peptide-specific TCR using IFN gamma ELISPOT method
  • Assay medium ELISPOT kit (BD, catalog number 551849), 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT)
  • T cells The effector cells (T cells) of this experiment were T cells transduced with the TCR of the present application, and T cells of the same volunteer that were not transfected with the TCR of the present application were used as control T cells (TCR negative control group).
  • PBMCs peripheral blood mononuclear cells
  • T cells were stimulated with anti-CD3/CD28 magnetic beads and cultured in a 37°C, 5% CO2 incubator. After 24 hours, cell clumping was observed.
  • the cells were transduced with the TCR lentivirus obtained in Example 3 at an MOI (multiplicity of infection) of 2.
  • the cells were then expanded in 1640 medium containing 10% FBS and 200 IU/ml IL-2 until 3-4 days after transduction. TCR transfection efficiency was assessed by flow cytometry (the results are shown in Figure 3).
  • the effector cell concentration after expansion was adjusted to 5.0 ⁇ 104 positive cells/ml, and 100 ⁇ l was sampled from each well to yield 5,000 positive cells/well.
  • the number of TCR-T cells targeting T2-A11+KRAS G12V was 2000/well.
  • SW620 (purchased from ATCC, catalog number CCL-227), naturally express the G12V mutation and do not express A11. Therefore, SW620-A11 cells were constructed by overexpressing the A11 gene using a lentiviral vector. SW620-A11 cells served as one target cell group, and SW620 cells served as a negative control group. The cell count was 50,000 cells/well. Both groups were treated with an HLA class I neutralizing antibody (anti-HLA class I, clone W6/32, purchased from Biolegend, catalog number 311402) as a control group.
  • the T2-A11 cells used in this experiment were the target cell positive control group (T2 cells were deposited in ATCC with the collection catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). The number of cells used was 20,000 cells/well.
  • the original concentration of 5 mg/ml of the short peptide (VVVGAVGVGK) was diluted to 4 ⁇ g/ml, and 50 ⁇ l was taken to make the final concentration of the short peptide in the 96-well plate 1 ⁇ g/ml.
  • the corresponding target cells and effector cells and the corresponding short peptides were added and the plate was incubated overnight (37°C/5% CO2). The next day, the culture medium was discarded and the plate was washed twice with double distilled water and then three times with washing buffer. The plate was patted on a paper towel to remove Remove any remaining wash buffer. Then, dilute the detection antibody 1:400 in PBS containing 10% FBS and add 100 ⁇ l/well to each well. Incubate the plate at room temperature for 2 hours, then wash three times with wash buffer. Tap the plate gently on a paper towel to remove any excess wash buffer.
  • T cells expressing the VVVGAVGVGK-A1101 peptide-specific TCRs (TCR059 and TCR076) strongly activated the SW620-A11 cell line, while control T cells showed no activation.
  • Addition of HLA class I neutralizing antibodies inhibited TCR059 and TCR076 recognition of the SW620-A11 cell line.
  • NFAT Nuclear Factor of Activated T Cells
  • Flow cytometry was used to detect NFAT expression as a readout of T cell activation.
  • Assay culture medium 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT).
  • T2-A11 cells T2 cells were deposited with ATCC under the catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium at a concentration of 1.6 ⁇ 10 6 cells/mL. 50 ⁇ L was plated per well to yield 80,000 cells/well.
  • JK8NF Jurkat-CD8-NFAT
  • JK8NF cells Jurkat cells were deposited in ATCC with a deposit catalog number of TIB-152. JK8NF cells were constructed based on Jurkat cells according to Cancer Res 2006; 66(23): 11455-61, Front. Immunol. 11: 633) were added with the lentivirus carrying the TCR gene obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, The transfection positive rate was determined by flow cytometry to be approximately 100% (the results are shown in FIG1 ). The concentration of the effector cells after expansion culture was adjusted to 1.6 ⁇ 10 6 cells/ml, and 50 ⁇ l was taken from each well to obtain 80,000 cells/well.
  • the original concentration of 5 mg/ml short peptide (VVVGAVGVGK(G12V), VVVGAGGVGK(WT)VVVGADGVGK(G12D)) was diluted to 400 ⁇ g/ml, and then diluted down in 10-fold ratio to 40 ⁇ g/ml, 4 ⁇ g/ml, 0.4 ⁇ g/ml, 0.04 ⁇ g/ml, 0.004 ⁇ g/ml, 0.0004 ⁇ g/ml, and 0.00004 ⁇ g/ml.
  • 50 ⁇ l was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 ⁇ g/ml, 10 ⁇ g/ml, 1 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, 0.001 ⁇ g/ml, 0.0001 ⁇ g/ml, and 0.00001 ⁇ g/ml, respectively.
  • the above method was used to test the expression of NFAT in TCR-transduced T cells of the present application in response to target cells loaded with three antigenic peptides: KRAS G12V , KRAS WT , and KRAS G12D .
  • the expression level curve of NFAT was plotted using Graphpad Prism8, and the results are shown in FIG5 .
  • the TCR of the present application has good specificity and only recognizes the KRAS G12V mutation, but not the wild-type KRAS and KRAS G12D mutations.
  • the T cells transduced with the TCR in the present application can be specifically activated by tumor cells expressing the A11+KRAS G12V mutation complex; and have good specificity, only recognizing the KRAS G12V mutation, but not recognizing wild-type KRAS and KRAS G12D mutations.
  • VVVGADGVGK synthetic peptide
  • the sorted single cells were used to amplify the TCR ⁇ chain and ⁇ chain respectively using a one-step RT-PCR kit (QIAGEN, catalog number 210212), and the PCR products were sequenced.
  • IMGT International Immunogenetics Information System
  • the nucleotide sequence of the ⁇ chain variable region of TCR104 is: SEQ ID NO: 60:
  • the amino acid sequence of the complementarity determining region 1 (CDR1) of the ⁇ chain of TCR104 is: SEQ ID NO: 32: TRDTTYY;
  • CDR2 complementarity determining region 2
  • RNSFDEQN The amino acid sequence of the complementarity determining region 2 (CDR2) of the ⁇ chain of TCR104 is: SEQ ID NO: 34: RNSFDEQN;
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR104 is: SEQ ID NO: 36: ALSEAAPGGSYIPT;
  • the amino acid sequence of the complementarity determining region 1 (CDR1) of the ⁇ chain of TCR104 is: SEQ ID NO: 38: MDHEN;
  • the amino acid sequence of the complementarity determining region 2 (CDR2) of the ⁇ chain of TCR104 is: SEQ ID NO: 40: SYDVKM;
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR104 is: SEQ ID NO:42: ASSLGPGQHNSPLH;
  • the nucleotide sequence of the ⁇ chain variable region of TCR106 is: SEQ ID NO: 61:
  • the nucleotide sequence of the ⁇ chain variable region of TCR106 is: SEQ ID NO: 62:
  • the amino acid sequence of the complementarity determining region 2 (CDR2) of the ⁇ chain of TCR106 is: SEQ ID NO: 35: YLSGSTLV;
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR106 is: SEQ ID NO: 37: AVIGNDYKLS;
  • the amino acid sequence of the complementarity determining region 1 (CDR1) of the ⁇ chain of TCR106 is: SEQ ID NO: 39: SNHLY;
  • the amino acid sequence of the complementarity determining region 2 (CDR2) of the ⁇ chain of TCR106 is: SEQ ID NO:41: FYNNEI;
  • the amino acid sequence of the complementarity determining region 3 (CDR3) of the ⁇ chain of TCR106 is: SEQ ID NO:43: ASSGTGGIEAF.
  • Example 7 Construction of VVVGADGVGK antigen short peptide-specific TCR lentiviral vector and lentiviral packaging
  • VVVGADGVGK TCR ⁇ and ⁇ chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene).
  • the ⁇ or ⁇ variable domains were cloned into a pLKO-based expression plasmid containing the mouse ⁇ or ⁇ constant region using standard methods using the Multi-Fragment Recombination Cloning Kit (Novozymes Biotech, catalog number C113).
  • the ligated plasmids were transformed into competent Escherichia coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and plated on LB/agar plates containing 100 ⁇ g/ml ampicillin.
  • VVVGADGVGK TCR i.e., TCR104, TCR106
  • Assay medium 10% FBS (Lonsera, catalog number S711-001), DMEM (ThermoFisher, catalog number C11995500BT).
  • Example 8 Construction and functional identification of a Jurkat cell line expressing a VVVGADGVGK antigen peptide-specific TCR
  • NFAT Nuclear Factor of Activated T Cells
  • NFAT expression was measured using flow cytometry as a readout of T cell activation.
  • Assay culture medium 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT).
  • T2-A11 cells T2 cells were deposited with ATCC under the catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium at a concentration of 1.6 ⁇ 10 6 cells/mL. 50 ⁇ L was plated per well to yield 80,000 cells/well.
  • JK8NF Jurkat-CD8-NFAT
  • JK8NF cells Jurkat cells were deposited in ATCC with the deposit catalog number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006; 66(23): 11455-61, Front. Immunol. 11: 633) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, the transfection positive rate was determined to be approximately 100% by flow cytometry (the results are shown in FIG1 ). The concentration of the effector cells after expanded culture was adjusted to 1.6 ⁇ 10 6 cells/ml, and 50 ⁇ l was taken per well to obtain 80,000 cells/well.
  • VVVGADGVGK The original concentration of 5 mg/ml short peptide (VVVGADGVGK) was diluted to 400 ⁇ g/ml, and then diluted down in 10-fold ratio to 40 ⁇ g/ml, 4 ⁇ g/ml, 0.4 ⁇ g/ml, 0.04 ⁇ g/ml, 0.004 ⁇ g/ml, 0.0004 ⁇ g/ml, and 0.00004 ⁇ g/ml.
  • 50 ⁇ l was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 ⁇ g/ml, 10 ⁇ g/ml, 1 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, 0.001 ⁇ g/ml, 0.0001 ⁇ g/ml, and 0.00001 ⁇ g/ml, respectively.
  • the above-described method was used to examine the expression of NFAT in TCR-transduced T cells of the present invention in response to target cells loaded with the VVVGADGVGK antigen peptide.
  • the NFAT expression level curve was plotted using Graphpad Prism8, and the results are shown in Figure 2.
  • T cells transduced with the TCR of the present application have a good activation response to target cells loaded with their specific short peptides.
  • Example 9 In vitro functional characterization of primary T cells expressing a VVVGADGVGK antigen peptide-specific TCR using IFN gamma ELISPOT method
  • Assay medium ELISPOT kit (BD, catalog number 551849), 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT).
  • T cells The effector cells (T cells) of this experiment were T cells transduced with the TCR of the present application, and T cells of the same volunteer that were not transfected with the TCR of the present application were used as control T cells (TCR negative control group).
  • Peripheral blood of volunteers was subjected to density gradient centrifugation to obtain peripheral blood mononuclear cells.
  • Peripheral blood mononuclear cells were placed in 24-well plates at a concentration of 5.0 ⁇ 10 5 /500 ⁇ l per well, and a total of 1 ⁇ 10 6 cells were collected.
  • Anti-CD3/CD28 magnetic beads were used to spike the cells.
  • T cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, cell clumping was observed and transduced with the TCR lentivirus obtained in Example 2 at an MOI (multiplicity of infection) of 2.
  • Cells were then expanded in 1640 medium containing 10% FBS and 200 IU/ml IL-2 until 3-4 days after transduction. TCR transfection efficiency was assessed by flow cytometry (the results are shown in Figure 3). The concentration of the expanded effector cells was adjusted to 5.0 ⁇ 104 positive cells/ml, and 100 ⁇ l was aspirated per well to obtain 5,000 positive cells/well.
  • the number of TCR-T cells targeting T2-A11+KRAS G12D was 2000/well.
  • panc0813 (purchased from ATCC, catalog number CRL-2551), naturally express the G12D mutation and do not express A11. Therefore, panc0813-A11 cells were constructed by overexpressing the A11 gene using a lentiviral vector. Panc0813-A11 cells served as one target cell group, and panc0813 cells served as a negative control group. The cell count was 50,000 cells per well. Both groups were treated with an HLA class I neutralizing antibody (anti-HLA I, clone W6/32, purchased from Biolegend, catalog number 311402) as a control group.
  • the T2-A11 cells used in this experiment were the target cell positive control group (T2 cells were deposited in ATCC with the collection catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). The number of cells used was 20,000 cells/well.
  • the original concentration of 5 mg/ml of the short peptide (VVVGADGVGK) was diluted to 4 ⁇ g/ml, and 50 ⁇ l was taken to make the final concentration of the short peptide in the 96-well plate 1 ⁇ g/ml.
  • the corresponding target and effector cells and the corresponding peptide were then added, and the plates were incubated overnight (37°C/5% CO2). The next day, the medium was discarded, and the plates were washed twice with double-distilled water and three times with wash buffer, patting gently on a paper towel to remove any residual wash buffer.
  • the detection antibody was then diluted 1:400 in PBS containing 10% FBS, and 100 ⁇ l/well was added to each well. The plates were incubated at room temperature for 2 hours, then washed three times with wash buffer, patting gently on a paper towel to remove any excess wash buffer.
  • TCR104 and TCR106 T cells expressing the VVVGADGVGK-A1101 peptide-specific TCR, strongly activated the panc0813-A11 cell line, while control T cells showed no activation.
  • Addition of HLA class I neutralizing antibodies inhibited TCR104 and TCR106 recognition of the panc0813-A11 cell line.
  • NFAT Nuclear Factor of Activated T Cells
  • VVVGADGVGK antigen short peptide-specific TCR had no cross-reactivity to wild-type KRAS and the KRAS G12D mutation.
  • Flow cytometry was used to detect NFAT expression as a readout of T cell activation.
  • Assay culture medium 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT).
  • T2-A11 cells T2 cells were deposited with ATCC under the catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium at a concentration of 1.6 ⁇ 10 6 cells/mL. 50 ⁇ L was plated per well to yield 80,000 cells/well.
  • JK8NF Jurkat-CD8-NFAT
  • JK8NF cells Jurkat cells were deposited in ATCC with the deposit catalog number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006; 66(23): 11455-61, Front. Immunol. 11: 633) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, the transfection positive rate was determined to be approximately 100% by flow cytometry (the results are shown in FIG1 ). The concentration of the effector cells after expanded culture was adjusted to 1.6 ⁇ 10 6 cells/ml, and 50 ⁇ l was taken per well to obtain 80,000 cells/well.
  • the original concentration of 5 mg/ml short peptides (VVVGADGVGK(G12D), VVVGAGGVGK(WT), VVVGAVGVGK(G12V)) was diluted to 400 ⁇ g/ml, and then diluted down in 10-fold ratio to 40 ⁇ g/ml, 4 ⁇ g/ml, 0.4 ⁇ g/ml, 0.04 ⁇ g/ml, 0.004 ⁇ g/ml, 0.0004 ⁇ g/ml, and 0.00004 ⁇ g/ml.
  • 50 ⁇ l was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 ⁇ g/ml, 10 ⁇ g/ml, 1 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, 0.001 ⁇ g/ml, 0.0001 ⁇ g/ml, and 0.00001 ⁇ g/ml, respectively.
  • the above method was used to test the expression of NFAT in TCR-transduced T cells of the present application in response to target cells loaded with three antigenic peptides: KRAS G12D , KRAS WT , and KRAS G12D .
  • the expression level curve of NFAT was plotted using Graphpad Prism8, and the results are shown in FIG5 .
  • the TCR of the present application has good specificity and only recognizes the KRAS G12D mutation, but not the wild-type KRAS and KRAS G12D mutation.
  • the T cells transduced with the TCR in the present application can be specifically activated by tumor cells expressing the A11+KRAS G12D mutation complex; and have good specificity, only recognizing the KRAS G12D mutation, but not recognizing wild-type KRAS and KRAS G12V mutations.

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Abstract

L'invention concerne un récepteur de lymphocytes T (TCR) et son utilisation, le TCR contenant une chaîne α contenant une région variable et/ou une chaîne β contenant une région variable. Le TCR pouvant reconnaître de manière spécifique une mutation KRASG12V limitée par A1101 et une mutation KRASG12D limitée par A1101. Un lymphocyte T transduisant le TCR (TCR-T) peut se lier à un complexe peptide court antigène-HLA-A1101, et peut être utilisé pour traiter des tumeurs malignes portant la mutation KRASG12V et la mutation KRASG12D.
PCT/CN2024/081278 2024-03-12 2024-03-12 Récepteur de lymphocytes t (tcr) et son utilisation Pending WO2025189363A1 (fr)

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