KRASG12D mutant antigen specificity TCR and redirected CD4 T cell co-expressed by KRASG12D mutant antigen specificity TCR and CD8

Abstract

The present invention relates to a T cell receptor (TCR) that specifically binds to a KRASG12D mutant antigen, a fusion protein or conjugate comprising the TCR, a nucleic acid encoding the TCR and an engineered cell comprising the same, and a method for preparing the engineered cell. The invention also relates to co-expression of an exogenous CD8 molecule and the TCR gene in a T cell to enhance the function of the T cell. The invention provides application of the TCR and the genetically engineered cell in detection, prevention and / or treatment of cancers related to a KRASG12D mutant antigen, and also provides application of the TCR and the genetically engineered cell in detection, prevention and / or treatment of the cancers related to the KRASG12D mutant antigen.

Description

Technical Field

[0001] 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 the KRAS_G12D mutant antigen, a genetically engineered cell expressing the TCR, and a method for preparing the genetically engineered cell. The present invention also relates to co-expressing an exogenous CD8 molecule with the TCR gene in a T cell to enhance the function of the T cell. The present invention provides the use of the TCR and the genetically engineered cell in detecting, preventing, and / or treating cancers related to the KRAS_G12D mutant antigen. Background Art

[0002] The RAS gene is the first human oncogene discovered, and the RAS protein encoded by it is at the center of many important cellular signaling networks. The RAS gene is the most frequently mutated oncogene in human cancers. Currently, RAS protein activation caused by RAS gene mutations has been found in approximately 1 / 5 of all human tumors.

[0003] The KRAS protein encoded by the KRAS gene (Kirsten rat sarcoma viral oncogene homolog) is a small GTPase and belongs to the RAS superprotein family.

[0004] Inside the cell, the KRAS protein transitions between an inactivated and an activated state. When the KRAS protein binds to guanosine diphosphate (GDP), the KRAS protein is in an inactivated state; when the KRAS protein binds to guanosine triphosphate (GTP), the KRAS protein is in an activated state and can activate downstream signaling pathways. The KRAS protein in most cells is in an inactivated state.

[0005] When the KRAS gene mutates, the resulting mutant KRAS protein (e.g., a mutant with a single amino acid substitution) keeps the KRAS protein bound to GTP all the time by disrupting GAP activity, locks the KRAS protein in a state with tyrosine kinase activity, and continuously activates downstream signaling pathways (such as the PI3K signaling pathway, the MAPK signaling pathway, the PI3K and Ral-GEFs signaling pathways, etc.). After these downstream signaling pathways are turned on, they will stimulate cell proliferation and migration, and ultimately contribute to tumorigenesis.

[0006] Among all KRAS mutations, G12D (35%), G12V (29%) and G12C (21%) mutations are the most prevalent. Compared with KRAS_G12C, KRAS_G12D only differs in the type of amino acid mutation at the same codon. KRAS_G12D is a codon mutation of the 12th amino acid residue of KRAS that introduces aspartic acid instead of cysteine, directly showing differences in epidemiology. KRAS_G12C is the most common KRAS mutation in NSCLC patients, while KRAS_G12D is the most common mutation in colorectal cancer and pancreatic cancer. In China, about 37.6% of colon cancer patients have KRAS mutations, and nearly 32.2% of these patients have the KRAS G12D mutation subtype, which usually occurs in patients under 65 years old. Pancreatic cancer is also known as the "king of cancers" and has limited treatment options. Therefore, there is a need in the art to develop specific immune cells against the KRAS_G12D mutant antigen, such as TCR-T cells, to effectively detect, prevent and treat cancers related to the KRAS_G12D mutant antigen, bringing new treatment options to more patients. Summary of the Invention

[0007] The present invention provides a T cell receptor (TCR) capable of specifically binding to the KRAS_G12D mutant antigen, and prepares lymphocytes recombinantly expressing the TCR. Thus, it is possible to detect the presence of cancers related to the KRAS_G12D mutant antigen in mammals by the specific binding of the TCR to the KRAS_G12D mutant antigen; and / or kill cancer cells expressing the KRAS_G12D mutant antigen by mediating an immune response against target cells expressing the KRAS_G12D mutant antigen in vivo, thereby meeting the above needs.

[0008] Therefore, in one aspect, the present invention provides an isolated or purified T cell receptor (TCR) that specifically binds to the KRAS_G12D mutant antigen. Preferably, the TCR comprises an α chain and a β chain, wherein each of the α chain and the β chain comprises three complementarity-determining regions (CDRs), and the amino acid sequence of CDR3 of the α chain is selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 and variants having 1 or 2 amino acid residue changes from the said sequences, and the amino acid sequence of CDR3 of the β chain is selected from SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140 and variants having 1 or 2 amino acid residue changes from the said sequences.

[0009] In one embodiment, the amino acid sequences of CDR3 of the TCR α chain and the CDR3 of the β chain of the present invention are:

[0010] (i) The α-chain CDR3 amino acid sequence shown in SEQ ID NO: 3 or a variant having 1 or 2 amino acid residue changes from said sequence; and the β-chain CDR3 amino acid sequence shown in SEQ ID NO: 113 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0011] (ii) The α-chain CDR3 amino acid sequence shown in SEQ ID NO: 6 or a variant having 1 or 2 amino acid residue changes from said sequence; and the β-chain CDR3 amino acid sequence shown in SEQ ID NO: 116 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0012] (iii) The α-chain CDR3 amino acid sequence shown in SEQ ID NO: 9 or a variant having 1 or 2 amino acid residue changes from said sequence; and the β-chain CDR3 amino acid sequence shown in SEQ ID NO: 119 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0013] (iv) The α-chain CDR3 amino acid sequence shown in SEQ ID NO: 12 or a variant having 1 or 2 amino acid residue changes from said sequence; and the β-chain CDR3 amino acid sequence shown in SEQ ID NO: 122 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0014] (v) The α-chain CDR3 amino acid sequence shown in SEQ ID NO: 15 or a variant having 1 or 2 amino acid residue changes from said sequence; and the β-chain CDR3 amino acid sequence shown in SEQ ID NO: 125 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0015] (vi) The α-chain CDR3 amino acid sequence shown in SEQ ID NO: 18 or a variant having 1 or 2 amino acid residue changes from said sequence; and the β-chain CDR3 amino acid sequence shown in SEQ ID NO: 128 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0016] (vii) The α-chain CDR3 amino acid sequence shown in SEQ ID NO: 21 or a variant having 1 or 2 amino acid residue changes from said sequence; and the β-chain CDR3 amino acid sequence shown in SEQ ID NO: 131 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0017] (viii) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO:24 or a variant having 1 or 2 amino acid residue changes from said sequence; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO:134 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0018] (ix) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO:27 or a variant having 1 or 2 amino acid residue changes from said sequence; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO:137 or a variant having 1 or 2 amino acid residue changes from said sequence;

[0019] (x) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO:30 or a variant having 1 or 2 amino acid residue changes from said sequence; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO:140 or a variant having 1 or 2 amino acid residue changes from said sequence.

[0020] In one embodiment, the amino acid sequences of the three complementarity-determining regions (CDRs) of the α-chain of the TCR of the present invention and the amino acid sequences of the three CDRs of the β-chain are:

[0021] (i) The amino acid sequences of the α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:1, 2, 3 or variants having 1 or 2 amino acid residue changes from said sequences respectively; and the amino acid sequences of the β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:111, 112, 113 or variants having 1 or 2 amino acid residue changes from said sequences respectively;

[0022] (ii) The amino acid sequences of the α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:4, 5, 6 or variants having 1 or 2 amino acid residue changes from said sequences respectively; and the amino acid sequences of the β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:114, 115, 116 or variants having 1 or 2 amino acid residue changes from said sequences respectively;

[0023] (iii) The amino acid sequences of the α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:7, 8, 9 or variants having 1 or 2 amino acid residue changes from said sequences respectively; and the amino acid sequences of the β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:117, 118, 119 or variants having 1 or 2 amino acid residue changes from said sequences respectively;

[0024] (iv) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 10, 11, and 12, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 120, 121, and 122, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences;

[0025] (v) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 13, 14, and 15, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 123, 124, and 125, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences;

[0026] (vi) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 16, 17, and 18, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 126, 127, and 128, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences;

[0027] (vii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 19, 20, and 21, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 129, 130, and 131, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences;

[0028] (viii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 22, 23, and 24, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 132, 133, and 134, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences;

[0029] (ix) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 25, 26, and 27, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 135, 136, and 137, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences;

[0030] (x) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 28, 29, and 30, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 138, 139, and 140, or variants having 1 or 2 amino acid residue changes respectively compared with the said sequences.

[0031] In some embodiments, the TCR of the present invention comprises an α-chain sequence shown in SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a β-chain sequence shown in SEQ ID NO: 219, 221, 223, 225, 227, 229, 231, 233, 235, or 237, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0032] In some embodiments, 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 bioactive molecules, wherein the other bioactive molecules are, for example, antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable labels, and there is or is not a linker between the TCR and the other bioactive molecules.

[0033] The present invention also provides nucleic acids encoding the TCR α-chain and / or β-chain of the present invention.

[0034] In addition, the present invention provides vectors, preferably plasmids, shuttle plasmids, phagemids, cosmids, expression vectors, retroviral vectors, adenoviral vectors, and / or homologous recombination repair (HDR) vectors, which comprise one or more nucleic acids as described above.

[0035] In a second aspect, the present invention provides engineered cells transformed with the above vectors and expressing the TCR described in the first aspect of the present invention.

[0036] In some embodiments, the present invention provides a method for preparing TCR-T cells by a targeting strategy that expresses the exogenous TCR of the present invention without using a viral vector.

[0037] In some embodiments, 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 the endogenous T cell receptor (TCR) α-chain constant region gene in a human cell, the nucleic acid sequence comprising, from the N-terminus to the C-terminus:

[0038] (i) a sequence encoding a first cleavable linker polypeptide;

[0039] (ii) a sequence encoding the β-chain of the TCR according to the first aspect of the present invention;

[0040] (iii) a sequence encoding a second cleavable linker polypeptide;

[0041] (iv) a sequence encoding the variable region of the α-chain of the TCR according to the first aspect of the present invention;

[0042] wherein the first cleavable linker polypeptide and the second cleavable linker polypeptide are the same or different viral 2A peptides.

[0043] The cells expressing the exogenous TCR prepared by the method have a high binding affinity for the VVVGADGVGK-HLA-A*11:01 complex and / or VVVGADGVGK-HLA-A*11:01, and

[0044] both have a strong in vitro killing effect on the CL-40 cell line (HLA-A*11:01+, KRAS G12D+) and the SNU-601 cell line (HLA-A*11:01+, KRAS G12D+).

[0045] In some embodiments, the method for preparing cells expressing an exogenous TCR is implemented by using the CRISPR / Cas9 technology and homologous recombination technology to knock out the endogenous TCR and knock in the exogenous TCR.

[0046] In a third aspect, the present invention provides a method for improving cell therapy and engineered cells.

[0047] In some embodiments, the present invention co-expresses an exogenous TCR and a CD8ab molecule in T cells.

[0048] Co-expressing the CD8ab molecule and the TCR gene in CD8+ and CD4+ T cells has beneficial effects on the functions of CD8+ and CD4+ T cells. In particular, by co-expressing the MHC class I TCR and the CD8 molecule in CD4+ T cells, the CD4+ T cells are reprogrammed into multifunctional hybrid T cells that simultaneously possess cytotoxic effector functions and natural helper functions.

[0049] In a fourth aspect, 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 the detection, prevention, and / or treatment of cancers related to the KRAS_G12D mutant antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The preferred embodiments of the present invention described in the following detailed description will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the currently preferred embodiments are shown in the figures. However, it should be understood that the present invention is not limited to the exact arrangements and means shown in the figures.

[0051] Figure 1A A targeting strategy diagram showing the knock-in of exogenous TCR into the TRAC locus using gRNA002 is shown.

[0052] Figure 1B A targeting strategy diagram showing the gene knockout of TRBC1 and TRBC2 loci using gRNA004 is shown.

[0053] Figure 2 A schematic diagram showing the results of detecting the TCR gene editing efficiency by flow cytometry (Day 7) is shown. The flow cytometry data analysis is a cell distribution diagram of 4 quadrants (Q1, Q2, Q3, Q4). Among them, Q2 is the cell population that has completed the knockout (KO) of the endogenous TCR and the knock-in (KI) of the exogenous TCR and expresses the nwTCR; Q3 is the wild-type T cells that have not undergone gene editing; Q4 is the KO cells that have completed the knockout of the endogenous TCR.

[0054] Figures 3A - 3J Illustrates the flow cytometry results (Day 7) of CD4+ T cells and CD8+ T cells transfected with different nwTCRs by electroporation and stained with pMHC tetramer (VVVGADGVGK-HLA-A11:01 tetramer).

[0055] Figure 4A Shows the experimental results of detecting the binding affinity and EC50 values of T cells expressing each nwTCR to the short peptide shown in SEQ ID NO:239 presented by HLA-A*11:01.

[0056] Figure 4BThe experimental results of the binding affinity assay of T cells expressing each nwTCR to the short peptide shown in SEQ ID NO:240 presented by HLA-A*11:01 and the EC50 values are shown.

[0057] Figure 5A - Figure 5C The real-time fluorescence imaging results of the specific killing of the CL-40 (KRAS G12D+) cell line by T cells expressing each nwTCR are shown (when the cells start co-incubation, it is 0 h, and the target cells killed after 18 h show a red fluorescence signal). "Blank" in the figure indicates that there are only target cells and T cells expressing any nwTCR are not added.

[0058] Figure 5D - Figure 5F The real-time fluorescence imaging results of the specific killing of the SNU-601 (KRAS G12D+) cell line by T cells expressing each nwTCR are shown (when the cells start co-incubation, it is 0 h, and the target cells killed after 18 h show a red fluorescence signal). "Blank" in the figure indicates that there are only target cells and T cells expressing any nwTCR are not added.

[0059] Figure 6A - Figure 6C The real-time analysis data of the killing of CL-40 cells by T cells expressing each nwTCR are shown. The results show that the gene-edited T cells have a specific killing effect on CL-40 cells. Figure 6A - Figure 6C "Blank" in

[0060] Figure 6D - Figure 6F The real-time analysis data of the killing of SNU-601 cells by T cells expressing each nwTCR are shown. The results show that the gene-edited T cells have a specific killing effect on SNU-601 cells. Figure 6D - Figure 6F "Blank" in

[0061] Figure 7 The targeting strategy diagram of nwTCR-CD8ab is shown.

[0062] Figures 8A - 8FFlow cytometry results of CD4+ T cells, CD8+ T cells, and CD8+CD4+ T cells stained with pMHC tetramers that were transfected with nwTCR-2985, nwTCR-2985-CD8ab, nwTCR-4536, nwTCR-4536-CD8ab, nwTCR-6670-2, and nwTCR-6670-2-CD8ab by electroporation (Day 7). Detailed implementation mode

[0063] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be changed. In addition, the terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive.

[0064] I. Definitions

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0066] The term "about", when used in conjunction with a numerical value, means a numerical value that encompasses a range with a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0067] The term "and / or", when used to connect two or more alternatives, should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0068] As used herein, the term "comprising" or "including" means including the stated elements, integers, or steps, but not excluding any other elements, integers, or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, the case consisting of the stated elements, integers, or steps is also covered. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to cover an antibody variable region consisting of that specific sequence.

[0069] The "RAS protein family" belongs to the large family of small GTPases. RAS proteins can be constitutively activated due to single amino acid mutations. The mutant RAS protein products are involved in signal transduction in the early stages of tumor formation in many human cancers. Multiple human cancers (e.g., lung cancer (such as, lung adenocarcinoma), ovarian cancer (such as, epithelial ovarian cancer), pancreatic cancer, prostate cancer, endometrial cancer, and colorectal cancer) express mutant RAS proteins.

[0070] "Kirsten rat sarcoma viral oncogene homolog (also simply referred to as: KRAS protein)" is an important member of the RAS protein family. The upstream of KRAS is regulated by the epidermal growth factor receptor (EGFR) family. EGFR signals can activate the SOS protein, thereby regulating the activation of KRAS. The inactivation and activation state transition of the intracellular KRAS protein are determined by the molecules it binds to. Guanine nucleotide exchange factor (GEF) catalyzes the binding of KARS to GTP, activating KRAS; while GTPase-activating protein (GAP) can promote the hydrolysis of GTP bound to KRAS into GDP, resulting in the inactivation of KRAS. Activated KRAS regulates downstream signaling pathways such as MAPK and PI3K, which are related to functions such as cell proliferation and cell migration. KRAS mutations can lead to its continuous binding to GTP, maintaining an activated state, resulting in the continuous activation of downstream signaling pathways, thereby promoting tumorigenesis.

[0071] The term "antigen" refers to any molecule that can be specifically detected by the immune system of an organism.

[0072] "KRAS_G12D mutant antigen" refers to the KRAS protein with a G12D mutation, which can be specifically detected by the immune system of an organism. "G12D" or "G12D mutation" are used interchangeably and refer to the substitution of glycine at position 12 of the KRAS protein by aspartic acid.

[0073] The T cell receptor (TCR) is a protein on the surface of T cells responsible for specifically recognizing antigen peptides bound to MHC (major histocompatibility complex). When TCR binds to antigen peptides and MHC, T lymphocytes are activated through signal transduction and enter the subsequent immune response process. There are 4 TCR genes in the human genome: two encoding light-chain TCRs: the TRA gene encodes TCRα and the TRG gene encodes TCRγ; two encoding heavy-chain TCRs: the TRB gene encodes TCRβ and the TRD gene encodes TCRδ. Heavy-chain TCR and light-chain TCR form a heterodimer to form a complete TCR. In humans, there are two types of TCRs: TCRα / β and TCRγ / δ. Among them, 95% of T cells express TCRα / β, called αβ T cells; 5% of T cells express TCRγ / δ, called γ / δ T cells. This ratio changes during ontogeny and in diseased states (such as leukemia) and also varies between species.

[0074] The mature heavy-chain TCR gene consists of four gene segments: variable region (V), diversity region (D), joining region (J), and constant region (C) (VDJC), while the light-chain TCR lacks the D region (VJC). Both the heavy-chain and light-chain TCRs have three complementarity-determining regions (CDRs) that play a major role in antigen recognition. Among them, CDR1 and CDR2 are relatively conserved and are responsible for recognizing MHC; CDR3 is the main CDR responsible for recognizing antigens.

[0075] The TCR gene is the most complex gene in the human genome and also the gene with the highest degree of variation. There are approximately 2x10 16 -10 18 T cells expressing different TCRs in human peripheral blood. This complexity mainly stems from three factors: (i) combinatorial diversity: the VDJC / VJC structure of mature TCRs is generated through complex rearrangements. There are 65 - 100 V gene segments, 2 D gene segments, and 13 J gene segments in the genome. When TCR recombinates, one segment needs to be selected from each of the above three segments, which endows TCR with a high degree of diversity; (ii) junctional flexibility: during the rearrangement process, random insertions or deletions of non-template nucleotides often occur in the V-D and D-J junction regions, further increasing the diversity of the CDR3 region; (iii) somatic mutations: the mutation frequency in the D region of T cells is approximately 1000 times that of normal cells.

[0076] As is known in the art and used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0077] The calculation of sequence identity between sequences is performed as follows.

[0078] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being 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. Subsequently, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecule is identical at this position.

[0079] Sequence comparison between two sequences and calculation of the percentage identity can be achieved using mathematical algorithms. In a preferred embodiment, the Needleman and

[0080] Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com) is used, with a Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, with the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage identity between two nucleotide sequences. A particularly preferred set of parameters (and a set of parameters that should be used unless otherwise specified) 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.

[0081] The percentage identity between two amino acid sequences or nucleotide sequences can also be determined using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4), using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0).

[0082] The term "antigen-presenting cell" or "APC" refers to an immune system cell that presents foreign antigens complexed with the major histocompatibility complex (MHC) on its surface, such as accessory cells (e.g., B-cells, dendritic cells, etc.). T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0083] The term "guide RNA (gRNA)" refers to an RNA specific to a target DNA that 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. In the present invention, the guide RNA can consist of two RNAs, namely, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), or the guide RNA can be a single-guide RNA (sgRNA) generated by fusing the essential parts of crRNA and tracrRNA.

[0084] A ribonucleoprotein complex (RNP) is a complex formed by the Cas9 protein and gRNA that has gene editing function.

[0085] The CRISPR / Cas9 gene editing system mainly consists of two parts: the Cas9 protein that acts as a "wrench" and the CRISPR guide RNA that acts as a "screw nail". The guide RNA is responsible for localizing the target site and recruiting and activating the Cas9 protein; the Cas9 protein is responsible for cleaving the target DNA.

[0086] The term "recombinant", when used for, e.g., 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 alteration of a native nucleic acid or protein.

[0087] The term "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 an exogenous sequence at the target site, and the integration includes but is not limited to gene knock-in (KI). In a specific embodiment, the target DNA sequence is a double-stranded DNA sequence, including but not limited to, the DNA sequence in the chromosomal genome of a cell, the DNA sequence outside the chromosomal genome of a cell (such as mitochondrial genome), plasmid, viral DNA sequence, etc.

[0088] In the present invention, the term "site-directed recombination" means that an exogenous sequence is integrated into a specific target site in a non-random manner, including integration upstream of the 5' of a specific target site, downstream of the 3', or between target sites.

[0089] In the present invention, the term "exogenous DNA sequence" means a DNA sequence that is expected to be site-directed recombined into a target site. The exogenous DNA sequence can be a sequence that does not exist or is altered at the target site.

[0090] The term "donor DNA" or "donor nucleic acid sequence" refers to a polynucleotide containing a polynucleotide sequence of interest to be expressed, and the polynucleotide sequence of interest is inserted into a target site in the target genome. In certain embodiments, the donor DNA further contains a sequence homologous to the genomic sequence (also referred to as "homologous arm"). "Homologous" means a similar DNA sequence. The homologous arm is sufficient to undergo homologous recombination with the homologous genomic sequence. For example, the homologous arm can contain at least 50 - 3500 or more base lengths.

[0091] The term "Homology directed repair (HDR)" is repair based on homologous recombination, which can be used to specifically and efficiently insert a donor DNA template (encoding a target sequence) into a target genomic locus, and is a repair pathway initiated after double-strand DNA damage in cells. HDR can only occur when there is a DNA fragment homologous to the damaged DNA in the cell nucleus. An HDR vector can refer to a vector used for electroporation transfection using CRISPR / Cas9 and homologous recombination techniques. HDR efficiency can refer to the gene knock-in efficiency of electroporation transfection using CRISPR / Cas9 and homologous recombination techniques.

[0092] A synonymous mutation is a neutral mutation. The genetic code is degenerate, that is, there are usually more than one codon that determines an amino acid, and a substitution of the third nucleotide in a triplet codon often does not change the amino acid composition. Although the third nucleotide in the triplet codon has mutated, the encoded amino acid has not changed, and this mutation is a synonymous mutation.

[0093] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids or phage vectors. A vector can contain nucleic acid sequences that allow the gene or sequence of interest to replicate in a host cell, such as an origin of replication. A vector can 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 containing the nucleic acid according to the present invention, the nucleic acid being operably linked to a sequence that permits expression of the nucleic acid.

[0094] The term "operably linked" refers to a functional linkage between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein, so as to perform an overall function. An operable linkage with a recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes well known in the art.

[0095] In the present invention, the term "engineered cell" refers to a cell into which exogenous nucleic acid has been introduced, including progeny of these cells. Engineered cells include "transfected cells", which include primary transfected cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly the same as the parental cell in terms of nucleic acid content, but may contain mutations. This includes mutant progeny that have the same function or biological activity as the cells screened or selected in the initially transfected cells.

[0096] As used herein, "subject", "individual" refers to an animal in need of alleviation and / or treatment of KRAS_G12D mutation antigen-related cancer, preferably a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats.

[0097] Adoptive Cell Transfer Therapy (ACT) refers to isolating immunologically active cells from a subject or patient, activating and expanding them in vitro, performing gene editing and other treatments, and then re-infusing them into the patient to achieve killing of target cells.

[0098] II. T cell receptors (TCRs) of the present invention and nucleic acids encoding TCRs

[0099] The wild-type human KRAS protein is 188 amino acid residues long and has a molecular weight of approximately 21.6 KD. The 12th position is glycine. Among the gene mutations of KRAS, 83% are mutations at the 12th amino acid residue, and one of the mutations is the mutation of glycine at the 12th position to aspartic acid (also simply referred to as G12D in this article).

[0100] The present invention provides an isolated or purified TCR that is antigen-specific for a KRAS peptide with a G12D mutation presented by a human leukocyte antigen (HLA) class I molecule. The KRAS peptide with a G12D mutation presented by a human leukocyte antigen (HLA) class I molecule has any length suitable for binding to any HLA class I molecule.

[0101] In some embodiments, the KRAS peptide with a G12D mutation has a length of about 9 to about 10 amino acid residues, which includes any continuous about 9 to about 10 amino acid residues in the KRAS protein with a G12D mutation. In some embodiments, the TCR of the present invention is antigen-specific for a KRAS peptide with a G12D mutation, and the mutated KRAS peptide has a length of about 9 amino acid residues or about 10 amino acid residues. Examples of KRAS peptides with a G12D mutation that can be recognized by the TCR of the present invention are the short peptide VVVGADGVGK (SEQ ID NO: 239) of the amino acid sequence from the 7th to the 16th position of KRAS (also simply referred to as "KRAS_G12D_7-16 peptide" in the text); and the short peptide VVGADGVGK (SEQ ID NO: 240) of the amino acid sequence from the 8th to the 16th position of KRAS (also simply referred to as "KRAS_G12D_8-16 peptide" in the text).

[0102] The T cell receptor (TCR) is a molecule present on the surface of T cells that is responsible for recognizing the antigen peptide-MHC complex (i.e., pMHC). Specific binding of the TCR to the antigen peptide-MHC complex triggers T cell activation through a series of biochemical events mediated by associated enzymes, co-receptors, and accessory molecules. In 95% of T cells, the TCR heterodimer consists of α and β chains, while in 5% of T cells, the TCR heterodimer consists of γ and δ chains.

[0103] Each chain of the TCR belongs to the members of the immunoglobulin superfamily and has an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane region (i.e., transmembrane domain), and a short cytoplasmic tail at the C-terminus. In the variable domains of the TCR α and β chains, each variable domain has three hypervariable regions or complementarity-determining regions (CDRs), where CDR3 in each variable domain is the main CDR responsible for recognizing the processed antigen. It is thought that CDR2 recognizes MHC molecules.

[0104] The constant domain of the TCR consists of short linker sequences in which cysteine residues form disulfide bonds, creating a link between the TCR α and β chains.

[0105] During T cell maturation, the TCR forms a TCR / CD3 complex with CD3. The process of forming the TCR / CD3 complex generally proceeds in the following order: first, the three peptide chains of CD3γ, δ, and ε become a stable complex core by forming two heterodimers, γ-ε and δ-ε. The TCRαβ (or TCRγδ) binds to it. Subsequently, the ζ-ζ or ζ-η dimer binds to the TCRαβ (or TCRγδ) / CD3γεδε complex, and finally, it is transferred to the surface of T cells. Signals are conducted from the TCR to the cell interior through the TCR / CD3 complex.

[0106] Signals from the TCR / CD3 complex are enhanced by the simultaneous binding of MHC to specific co-receptors. In helper T cells, this co-receptor is the CD4 molecule, which is specific for class II MHC; while in cytotoxic T cells, this co-receptor is CD8, which is specific for class I MHC.

[0107] In this article, the term "T cell receptor" has its conventional meaning in the art and is used to denote a molecule capable of recognizing peptides presented by MHC molecules. This molecule is a heterodimer of two chains, α and β (or optionally γ and δ).

[0108] The TCR of the present invention provides specific affinity recognition of the KRAS_G12D mutant antigen. The KRAS_G12D mutant antigen is degraded by the proteasome in the cell into short peptides 8 to 10 amino acids in length, for example, the KRAS_G12D_7-16 peptide shown in SEQ ID NO:239 and / or the KRAS_G12D_8-16 peptide shown in SEQ ID NO:240. These short peptides are presented on the cell surface by MHC class I as peptide / MHC complexes (pMHC). Some pMHCs have been shown to be associated with various cancers and can thus be potential targets for TCR therapy.

[0109] The present invention provides isolated or purified T cell receptor (TCR) α chain and / or β chain. The TCR of the present invention can be a chimeric TCR comprising sequences derived from more than one species. For example, considering that murine TCRs can be expressed more efficiently in human T cells than human TCRs, the TCR can comprise a human variable region and a murine constant region.

[0110] In one embodiment, the TCR of the present invention comprises an α chain and a β chain, wherein each of the α chain and the β chain comprises three complementarity-determining regions (CDRs), and wherein the amino acid sequence of the TCRα chain CDR3 that is mainly responsible for antigen recognition is selected from SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30 and variants having 1 or 2 amino acid residue changes from said sequences, and the amino acid sequence of the β chain CDR3 is selected from SEQ ID NO:113, 116, 119, 122, 125, 128, 131, 134, 137, 140 and variants having 1 or 2 amino acid residue changes from said sequences.

[0111] In one embodiment, the TCR of the present invention comprises an α chain and a β chain, and the amino acid sequences of the three complementarity-determining regions (CDRs) comprised by the α chain and the amino acid sequences of the three CDRs comprised by the β chain are:

[0112] (i) the amino acid sequences of the α chain CDR1, CDR2, CDR3 shown in SEQ ID NO:1, 2, 3 or variants having 1 or 2 amino acid residue changes from said sequences respectively; and the amino acid sequences of the β chain CDR1, CDR2, CDR3 shown in SEQ ID NO:111, 112, 113 or variants having 1 or 2 amino acid residue changes from said sequences respectively;

[0113] (ii) the amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 4, 5, 6, or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 114, 115, 116, or variants having 1 or 2 amino acid residue changes respectively from the said sequences;

[0114] (iii) the amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 7, 8, 9, or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 117, 118, 119, or variants having 1 or 2 amino acid residue changes respectively from the said sequences;

[0115] (iv) the amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 10, 11, 12, or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 120, 121, 122, or variants having 1 or 2 amino acid residue changes respectively from the said sequences;

[0116] (v) the amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 13, 14, 15, or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 123, 124, 125, or variants having 1 or 2 amino acid residue changes respectively from the said sequences;

[0117] (vi) the amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 16, 17, 18, or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 126, 127, 128, or variants having 1 or 2 amino acid residue changes respectively from the said sequences;

[0118] (vii) the amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 19, 20, 21, or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 129, 130, 131, or variants having 1 or 2 amino acid residue changes respectively from the said sequences;

[0119] (viii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 22, 23, 24, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 132, 133, 134, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences;

[0120] (ix) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 25, 26, 27, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 135, 136, 137, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences;

[0121] (x) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 28, 29, 30, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 138, 139, 140, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences.

[0122] In one embodiment, the TCR of the present invention comprises an α-chain sequence shown in SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith; and a β-chain sequence shown in SEQ ID NO: 219, 221, 223, 225, 227, 229, 231, 233, 235, or 237, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith. Preferably, the constant region of the TCR of the present invention is a murine constant region.

[0123] In some embodiments, the alteration of the amino acid residues in the TCR variants of the present invention is a substitution, addition, or deletion of an amino acid residue in the α-chain sequence shown in any of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, or in the β-chain sequence shown in any of SEQ ID NO: 219, 221, 223, 225, 227, 229, 231, 233, 235, or 237, provided that the TCR variant still retains or improves the ability to bind to the epitope peptide-MHC complex of the KRAS_G12D mutant antigen. In one embodiment, the substitution is a conservative substitution. Examples of conservative substitutions are given in Table A below.

[0124] Table A

[0125] Original residue Exemplary substitution Preferred substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu, Val; Met; Ala; Phe; norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; norleucine Leu

[0126] Amino acids can be grouped according to common side-chain properties:

[0127] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu; Ile;

[0128] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn; Gln;

[0129] (3) Acidic: Asp, Glu;

[0130] (4) Basic: His, Lys, Arg;

[0131] (5) Residues affecting chain direction: Gly, Pro;

[0132] (6) Aromatic: Trp, Tyr, Phe.

[0133] A non-conservative substitution would exchange a member of one of these classes for a member of another class.

[0134] In some embodiments, the TCR of the present invention is capable of recognizing and binding to an epitope peptide of a mutant KRAS protein presented by an HLA class I molecule, thereby triggering an immune response.

[0135] In some embodiments, 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. Preferably, 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 among Asians.

[0136] 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; the α chain; or the β chain, etc. The nucleic acid can be double-stranded or single-stranded and can be RNA or DNA. The nucleic acid sequence can be codon-optimized for high expression in mammalian production cells. Codon usage in mammalian cells and many other organisms is well known in the art. Codon optimization can also include removing mRNA instability motifs and hidden splice sites.

[0137] The TCR of the present invention can be modified by various methods (e.g., gene fusion, chemical conjugation, etc.) to link the TCR to other bioactive molecules. The TCR that can be linked to other bioactive molecules can be a TCR heterodimer or its soluble form, more preferably a soluble, single-chain TCR. The other bioactive molecules can be various bioactive effectors, such as antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable labels, etc. There may or may not be a linker between the TCR and the other bioactive molecules.

[0138] In some embodiments, the TCR fusion protein is a fusion of the TCR with an antibody, which includes intact antibodies (e.g., IgG, IgM, or IgA classes) or fragments 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).

[0139] In some embodiments, the TCR fusion protein is a fusion of the TCR with a cytokine, such as interleukin (e.g., IL-2), chemokine (e.g., MIP-1β), growth factor (e.g., GCSF).

[0140] In some embodiments, the TCR conjugate is a covalent linkage of the TCR to a cytotoxic agent, such as doxorubicin.

[0141] In some embodiments, the TCR conjugate is a covalent linkage of a TCR to a radioactive substance, such as I 125 .

[0142] In some embodiments, the TCR conjugate is a covalent linkage of a TCR to a detectable label, such as a fluorescent label.

[0143] The T cell receptor fusion protein or T cell receptor conjugate 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 the KRAS_G12D mutant antigen that specifically binds to the TCR in vivo or in vitro.

[0144] III. Vectors Containing Nucleic Acids Encoding the TCR of the Present Invention

[0145] The present invention also relates to vectors containing nucleic acids encoding the TCR of the present invention. In one embodiment, the vector is a pUC57-Simple vector (purchased from GenScript Biotech Corporation). In yet another embodiment, the pUC57-HA vector is used, and the pUC57-HA vector 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 (about 800 bp) of the TRAC locus.

[0146] The vector transfers the nucleic acid encoding the TCR of the present invention into cells, such as T cells, NK cells, stem cells, such as pluripotent stem cells, induced pluripotent stem cells (iPSCs), such that the engineered cells express a TCR specific for the KRAS_G12D mutant antigen.

[0147] The TCR specific for the KRAS_G12D mutant antigen refers to a TCR that can specifically bind with high affinity and immunologically recognize G12D-mutated KRAS. For example, after co-culturing about 1×10 4 to about 1×10 5 T cells expressing the TCR with antigen-presenting cells, such as T2 cells or K562 cells, pulsed with G12D-mutated KRAS and overexpressing HLA class I molecules, the EC50 of the TCR is about 1×10 -4 M or less (e.g., 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8If the TCR elicits the secretion of IFN-γ in the presence of G12D-mutated KRAS (e.g., at a concentration of M or less), the TCR is considered to be antigen-specific for the G12D-mutated KRAS. The HLA class I molecule can be any HLA class I molecule described herein (e.g., the HLA-A*11:01 molecule).

[0148] Preferably, the vector enables 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 the limited pool of CD3 molecules. Alternatively, increasing the supply of CD3 molecules can also increase the expression of the exogenous TCR in gene-modified cells. Thus, the vector optionally contains the genes for CD3-γ, CD3-δ, CD3-ε, and / or CD3-ζ. In one embodiment, the vector contains the gene for CD3-ζ. Additionally, one or more separate vectors encoding CD3 genes can be provided for co-transfer into cells with the exogenous TCR-encoding vector.

[0149] The vector form is not limited to homologous recombination repair (HDR) vectors and can also be viral vectors. Viral vectors can be lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, baculovirus vectors, which are used to implement the editing of the cell genome.

[0150] Genome editing technology refers to a technology for inserting, deleting, or replacing nucleic acids in the genomic DNA of cells. Genetically modified T cells obtained by using gene editing technology on human primary T cells have shown excellent efficacy in clinical trials of various adoptive immunotherapy drugs. Among them, chimeric antigen receptors (CARs) or T cell receptors (TCRs) are often used to modify human primary T cells to achieve the recognition of certain specific target epitopes. These modified T cells can specifically kill specific target cells.

[0151] Common TCR gene editing methods can be roughly divided into two categories according to the gene integration method. One category is random gene integration, including lentivirus (LV) systems, adeno-associated virus (AAV) systems, transposon systems, etc. The other category is precise gene editing methods, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) technology, etc. Among them, the CRISPR technology uses the guidance of gRNA to recognize and edit DNA, and performs site-directed insertion of large gene fragments through homologous recombination, which has the advantages of being easy to operate and having stronger expandability.

[0152] IV. Preparation of Engineered Cells

[0153] A viral vector can be used to introduce the target TCR into cells. However, the method of introducing exogenous TCRα / β genes into cells based on viral vectors does not knock out the endogenous TCR in cells, which may cause mismatching of exogenous TCRα and β chains. Even though the mismatching problem of exogenous TCRα and β chains can be reduced by modifying disulfide bonds or using murine constant regions, the random insertion of viral vectors into the cell genome still poses a potential risk of disrupting other genes.

[0154] Alternatively, a non-viral vector can be used to introduce the target TCR into cells, precisely integrating the exogenous TCRα / β genes into specific genomic loci of the cells. In some embodiments, a gene editing method not based on viral vectors can knock out the endogenous T cell receptor α and β chains of human T cells through CRISPR / Cas9 technology and homologous recombination technology, and knock in the coding nucleotides of exogenous target T cell receptor α and β chains at the exon of the TRAC gene. Thus, both the expression of endogenous TCR is disrupted, and the endogenous TCR promoter is used to express exogenous target TCRα and β.

[0155] In one embodiment, the coding nucleotides of exogenous target T cell receptor α and β chains are knocked into exon 1 of the endogenous TRAC gene, and the exogenous knock-in fragment does not add the TRAC gene, thereby reducing the length of the gene knock-in fragment and the difficulty of gene knock-in. Compared with the technology of expressing TCR using viral vectors, the technology of expressing TCR using non-viral vectors can be used as a fast, simple, and low-cost method for introducing exogenous TCRα / β genes into cells.

[0156] IV.1 Selection of Knockout Sites

[0157] The TCR is a dimer composed of a TCRα chain and a TCRβ chain. The TCRα chain gene is rearranged from TRAV, TRAJ, and TRAC genes. Among them, TRAV and TRAJ genes each contain multiple sequences, and there are differences between these multiple sequences. Only one sequence can be randomly selected for expression during rearrangement. If the TRAV and TRAJ genes are selected as knockout sites, it is difficult to avoid the generation of any random TCRα chain genes. However, there is only one TRAC gene. By knocking out this TRAC gene, any random TCRα chain gene can be knocked out. Therefore, TRAC is suitable as a knockout site. The TCRβ chain gene is rearranged from TRBV, TRBJ, TRBD, and TRBC genes. Among them, TRBV and TRBJ genes each contain multiple sequences, and there are differences between these multiple sequences, so they are not suitable as knockout sites. The TRBC gene contains TRBC1 and TRBC2, and the two contain some identical sequences. This common sequence can be selected as the knockout site to knock out any random TCRβ gene by knocking out this common sequence.

[0158] In some embodiments, one or more of the endogenous TRAC gene, endogenous TRBC1 gene, and / or 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 exogenous TCR and the endogenous TCR chains caused by the expression of the endogenous TCR.

[0159] Nuclease-based genome editing tools can be used to target and disrupt the endogenous TRAC gene and TRBC gene by inducing double-strand breaks and DNA repair through non-homologous 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).

[0160] IV.2 Selection of knock-in sites

[0161] Since the endogenous TRAC gene is unique and the expression of all TCRα requires the TRAC gene, the exogenous TCRα / β gene knock-in site is selected as the endogenous TRAC site. Thus, while eliminating the endogenous TCR, the endogenous TCR promoter of human T cells can be used to express the exogenous TCRα / β gene (also referred to as the "nwTCR" gene) of the present invention without the need to additionally add the TRAC gene, thereby reducing the size of the knock-in fragment and facilitating the improvement of gene editing efficiency.

[0162] In some embodiments, the expression construct of nwTCR is cloned into a targeting vector (such as the pUC57-HA vector), and by designing homologous arms, nwTCR is site-specifically knocked into the constant region of the TCRα chain and is regulated by the transcriptional regulatory sequences of this locus for expression. Since the regulatory level of its endogenous promoter at this knock-in site is superior to other sites, the continuous and stable expression of the nwTCR gene is ensured.

[0163] IV.3 Engineered cells

[0164] The present invention provides engineered cells expressing exogenous TCR.

[0165] In some embodiments, engineered cells expressing TCR are prepared from cells derived from blood, bone marrow, lymph, or lymphoid organs, such as lymphocytes or stem cells, where the lymphocytes include but are not limited to T cells, NK cells, and the stem cells are, for example, pluripotent stem cells, induced pluripotent stem cells (iPSCs).

[0166] The cells are usually primary cells, such as cells directly isolated from a subject and / or cells isolated from a subject and frozen. The cells can be allogeneic cells and / or autologous cells.

[0167] In some embodiments, primary cells (such as sorted CD4+ T cells and CD8+ T cells) activated by CD3 / CD28 are electroporated and transfected by means of RNP and plasmid through CRISPR / Cas9 and homologous recombination techniques, thereby preparing engineered TCR cells.

[0168] In some embodiments, sgRNAs targeting the endogenous TRAC gene are designed, and sgRNAs targeting the endogenous TRBC1 gene and TRBC2 gene are designed.

[0169] Guided by the sgRNA, the Cas9 protein binds to a specific site on the target genome, and the Cas9 protein cleaves this specific site. For the double-strand break formed in the endogenous TRAC gene caused by the use of RNP, homologous recombination can occur in the presence of donor DNA with homologous arms, thereby achieving the site-specific insertion of the target nwTCR gene.

[0170] In a specific embodiment, the specific site on the target genome where the sgRNA guides the Cas9 protein to bind is exon 1 of the TRAC gene, the Cas9 protein cleaves this specific site, and the designed and verified highly efficient sgRNA recognition sequence and PAM sequence targeting it contain the nucleotide sequence shown as TCAGGGTTCTGGATATCTGT-GGG (sgRNA recognition sequence - PAM sequence, "-" is used to distinguish between the CRISPR / Cas9 recognition site and the PAM sequence).

[0171] In a specific embodiment, the specific site on the target genome where the sgRNA guides the binding of the Cas9 protein is exon 1 of the TRBC1 and TRBC2 genes. The Cas9 protein cleaves the specific site, and the designed and validated highly efficient sgRNA recognition sequence and PAM sequence targeting it contain the nucleotide sequence shown as CTGCCTGAGCAGCCGCCTGA-GGG (sgRNA recognition sequence - PAM sequence, "-" is used to distinguish and separate the CRISPR / Cas9 recognition site and the PAM sequence).

[0172] The CRISPR / Cas system may contain a Cas component in the form of a protein or a nucleic acid encoding a Cas protein.

[0173] In the present invention, the Cas protein can be any Cas protein as long as it has endonuclease or nickase activity when complexed with the guide RNA.

[0174] Preferably, the Cas protein is Cas9 protein or its variant or its functional fragment.

[0175] The Cas protein can be a protein isolated from an organism such as a Streptococcus sp., preferably Streptococcus pyogens, or a recombinant protein, but is not limited thereto.

[0176] In one embodiment, the Cas protein comprises Cas9 derived from Streptococcus pyogenes, such as Cas9 having the amino acid sequence shown in SEQ ID NO: 254.

[0177] In another embodiment, the Cas protein comprises an amino acid sequence having at least 50% homology with the amino acid sequence shown in SEQ ID NO: 254, preferably having at least 60, 70, 80, 90, 95, 97, 98, or 99% homology with the amino acid sequence shown in SEQ ID NO: 254, but is not limited thereto.

[0178] For the present invention, the Cas protein-encoding nucleic acid can be in the form of a vector, such as a plasmid containing a Cas-encoding sequence under a promoter such as CMV or CAG. When the Cas protein is Cas9, the Cas9-encoding sequence can be derived from the genus Streptococcus, preferably from Streptococcus pyogenes. For example, the Cas9-encoding nucleic acid can contain a nucleotide sequence encoding SEQ ID NO:254. In addition, the Cas9-encoding nucleic acid can contain a nucleotide sequence having at least 50% homology with the nucleotide sequence encoding SEQ ID NO:254, preferably a nucleotide sequence having at least 60, 70, 80, 90, 95, 97, 98, or 99% homology with the nucleotide sequence encoding SEQ ID NO:254, but not limited thereto.

[0179] In one embodiment, the donor DNA sequentially contains a 5' homologous arm, a sequence encoding a cleavable linker polypeptide, an exogenous TCRα / β gene or a functional fragment thereof, and a 3' homologous arm. After the sequence encoding the cleavable linker polypeptide is expressed, the cleavable linker polypeptide is cleaved. In some embodiments, the cleavable linker polypeptide sequence contains a 2A ribosome skipping element such as T2A, E2A, P2A, and F2A or variants thereof.

[0180] In one embodiment, the donor DNA is located in a targeting vector. The basic targeting vector used as the backbone is not particularly limited, as long as it has a prokaryotic origin of replication and a selection marker for vector propagation in bacteria.

[0181] In a preferred embodiment, in order to increase the expression of the exogenous TCRα / β gene or its fragment, a sequence encoding a cleavable linker polypeptide and a signal peptide sequence are respectively ligated to the N-terminus of the exogenous TCRα chain gene and the exogenous TCRβ chain gene in the targeting vector.

[0182] In a specific embodiment, the targeting vector for knocking in the nwTCR gene sequence contains the following structures effectively linked: 2A ribosome skipping element - SP - TCRβ - 2A ribosome skipping element - SP - TRAV - TRAJ

[0183] Wherein, SP is a signal peptide encoding sequence.

[0184] The targeting vector for knocking in the nwTCR gene sequence, the RNP complex, and the cells are mixed and the step of delivering the nwTCR gene sequence into the cells is carried out. In some embodiments, the delivery step is selected from: electroporation, transfection, deforming the cell membrane by physical means, lipid nanoparticles (LNP), virus-like particles (VLP), and sonication. In some embodiments, the delivery step includes electroporation.

[0185] In some embodiments, the engineered cells are primary cells.

[0186] In some embodiments, the engineered cells are isolated cells, where the isolated cells are isolated from a subject.

[0187] In some embodiments, the engineered cells are ex vivo cultured cells. In some embodiments, the ex vivo cultured cells include stimulated cells. In some embodiments, the stimulated cells include cytokine-stimulated T cells, optionally, where the cytokine-stimulated T cells include CD3-stimulated T cells, CD28-stimulated T cells, or CD3- and CD28-stimulated T cells. In some embodiments, the cytokine-stimulated T cells are cultured in the presence of IL7, IL15, or a combination thereof. In some embodiments, the cytokine-stimulated T cells are cultured in the presence of IL2.

[0188] In some embodiments, the engineered cells are stem cells, e.g., hematopoietic stem cells (HSCs). Transfer of the nwTCR gene into HSCs does not result in TCR expression on the cell surface because stem cells do not express CD3 molecules. However, when the stem cells differentiate into lymphoid precursors that migrate to the thymus, initiation of CD3 expression will result in expression of the introduced nwTCR on the surface of thymocytes. An advantage of this method is that once mature T cells are generated, they express only the introduced nwTCR and little or no endogenous TCR chains because expression of the introduced nwTCR chains inhibits rearrangement of endogenous TCR gene segments to form functional TCRα and β genes. Another benefit of this method is that the TCR gene-modified stem cells are a continuous source of mature T cells with the desired antigen specificity. Thus, the nwTCR gene-modified stem cells give rise to T cells expressing the TCR of the present invention upon differentiation.

[0189] V. Methods for Detecting, Preventing, or Treating Cancer Associated with KRAS_G12D Mutation Antigen

[0190] The present invention provides a method for preventing or treating cancer associated with KRAS_G12D mutation antigen, which comprises administering to a subject in need thereof the engineered cells, the TCR nucleic acids, vectors, or pharmaceutical compositions of the present invention. In some embodiments, the method comprises administering a polynucleotide encoding a TCR. In some embodiments, the method comprises administering a vector comprising a polynucleotide encoding a TCR. In some embodiments, the method comprises administering an effective amount of the engineered cells of the present invention.

[0191] In some embodiments, the engineered cells of the present invention, the TCR nucleic acids, vectors or pharmaceutical compositions of the present invention are used to prevent or treat cancers associated with the KRAS_G12D mutant antigen. Without being bound by any theory, it is believed that the TCR of the present invention can specifically bind to the KRAS_G12D mutant antigen, and thereby mediate an immune response against target cells expressing the KRAS_G12D mutant antigen.

[0192] The treatment or prevention may include treating or preventing 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.

[0193] The present invention also provides a method for detecting the presence of cancer in a mammal. The method includes: (i) contacting a sample containing one or more cells from the mammal with any one of the TCR of the present invention described herein, a cell population expressing the TCR of the present invention, or a pharmaceutical composition containing a cell population expressing the TCR of the present invention, thereby forming a complex; and (ii) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal. The contacting can be carried out in vitro or in vivo of the mammal. In one embodiment, the contacting is carried out in vitro. The complex can be detected by various ways known in the art. In some embodiments, the TCR of the present invention or the cell population expressing the TCR of the present invention is labeled with a detectable marker, and the detectable marker is, for example, a radioisotope, a fluorophore (such as fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (such as alkaline phosphatase, horseradish peroxidase), and elemental particles (such as gold particles).

[0194] The present invention also provides a method for inducing anti-tumor immunity, wherein the tumor is a tumor associated with the KRAS_G12D mutant antigen, and the method includes administering to a subject an effective amount of the engineered cells of the present invention.

[0195] The present invention provides a method for inducing an immune response in a subject, including administering an effective amount of the engineered cells of the present invention. In some embodiments, the immune response is a T cell-mediated immune response. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered immune cells contain the TCR of the present invention. In some embodiments, the target cells are cancer cells associated with the KRAS_G12D mutant antigen.

[0196] In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor stem cells to be differentiated into T cells for T cell therapy are obtained from a subject other than the patient.

[0197] T cells can be administered in a therapeutically effective amount. For example, 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.

[0198] The cancers 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 lymphoma, non-Hodgkin 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, penile cancer, testicular cancer, pancreatic cancer, prostate cancer, kidney cancer, soft tissue cancer and thyroid cancer. Preferably, the cancer is lung cancer, pancreatic cancer, colorectal cancer, endometrial cancer, ovarian cancer or prostate cancer.

[0199] VI. Methods for improving cell therapy and engineered cells

[0200] The present invention also provides methods for improving cell therapy and engineered cells.

[0201] Native CD8+ cells express the CD8 molecule. The CD8 molecule is a type I transmembrane glycoprotein expressed on the cell surface in the form of a homodimer composed of two CD8a chains (also simply referred to as "CD8aa" herein) and / or in the form of a heterodimer composed of one CD8a chain and one CD8b chain (also simply referred to as "CD8ab" herein).

[0202] In some embodiments, the present invention co-expresses exogenous TCR and CD8aa molecules in T cells. A non-viral gene editing method based on the 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 exogenous nwTCR and CD8aa molecules are co-expressed in CD8+ T cells / CD4+ T cells, which can enhance the binding of TCR-T cells to pMHC molecules. When the exogenous nwTCR and CD8aa molecules are co-expressed in CD8+ T cells, due to the increase in CD8aa molecules available for the exogenous nwTCR in CD8+ T cells, it is expected to improve the TCR-specific cytotoxicity (including its continuous killing ability) and anti-tumor function in vivo of CD8+ T cells. When the exogenous nwTCR and CD8aa molecules are co-expressed in CD4+ T cells, along with the expression of endogenous CD4 molecules, the CD4+ T cells exhibit a hybrid phenotype, and it is expected that they can recognize antigens with an affinity similar to that of natural CD8+ T cells, kill target cells, and exhibit cytotoxic effector functions; meanwhile, retain the natural helper function of CD4+ T cells.

[0203] In some other embodiments, the present invention co-expresses exogenous TCR and CD8ab molecules in T cells. A non-viral gene editing method based on the CRISPR / Cas9 technology uses nucleic acids encoding the nwTCR of the present invention, nucleic acids encoding the CD8a chain, and nucleic acids encoding the CD8b chain to perform gene editing on CD8+ T cells / CD4+ T cells, indicating that the exogenous nwTCR and CD8ab molecules are co-expressed in CD8+ T cells / CD4+ T cells, which can enhance the binding of TCR-T cells to pMHC molecules. When the exogenous nwTCR and CD8ab molecules are co-expressed in CD8+ T cells, due to the increase in CD8ab molecules available for the exogenous nwTCR in CD8+ T cells, it is expected to improve the TCR-specific cytotoxicity (including its continuous killing ability) and anti-tumor function in vivo of CD8+ T cells. When the exogenous nwTCR and CD8ab molecules are co-expressed in CD4+ T cells, along with the expression of endogenous CD4 molecules, the CD4+ T cells exhibit a hybrid phenotype, and it is expected that they can recognize antigens with an affinity similar to that of natural CD8+ T cells, kill target cells, and exhibit cytotoxic effector functions; meanwhile, retain the natural helper function of CD4+ T cells.

[0204] Thus, co-expression of CD8aa molecule and / or CD8ab molecule with TCR gene in CD8+ and CD4+ T cells has beneficial effects on the functions of CD8+ and CD4+ T cells. CD4+ T cells can be reprogrammed into multifunctional hybrid T cells with both cytotoxic effector function and natural helper function through MHC class I TCR and CD8 molecules.

[0205] The following examples are described to assist in understanding the present invention. The examples are not intended and should not in any way be construed as limiting the scope of the present invention.

[0206] Examples

[0207] The present invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the scope of the invention. These examples are not intended to represent that the following experiments are all the experiments conducted or the only experiments conducted.

[0208] Example 1. Generation and cloning of T cells and TCRs that recognize KRAS_G12D mutant antigen

[0209] Chemically synthesize the adjacent sequence after the 12th amino acid of KRAS is mutated from G to D, that is, the short peptide VVVGADGVGK (SEQ ID NO: 239) of the 7th to 16th amino acid sequence of KRAS (also abbreviated as "KRAS_G12D_7-16 peptide" in the text); and the short peptide VVGADGVGK (SEQ ID NO: 240) of the 8th to 16th amino acid sequence of KRAS (also abbreviated as "KRAS_G12D_8-16 peptide" in the text).

[0210] Dendritic cells (DC cells) from cancer patients with HLA-A*11:01 genotype and expressing KRAS_G12D mutant antigen were pulsed and stimulated in vitro with KRAS_G12D_7-16 peptide and KRAS_G12D_8-16 peptide resuspended in DMSO respectively, and co-cultured with CD8+ T cells sorted from the peripheral blood of this patient for 10 days. The negative control was to pulse and stimulate the patient's DC cells with DMSO in vitro and co-culture them with CD8+ T cells sorted from the peripheral blood of this patient for 10 days.

[0211] Then, the release amount of cytokine IFN-γ in the culture supernatant and the expression of CD137 on CD8+ T cells were detected, and reactive T cells that released IFN-γ and expressed CD137 were sorted out. Flow cytometry-based staining was used to evaluate the binding of the sorted reactive T cells that released IFN-γ and expressed CD137 to peptide-MHC (HLA-A*11:01) tetramers (VVVGADGVGK-HLA-A*11:01 and VVGADGVGK-HLA-A*11:01); tetramers containing irrelevant peptides were used as negative controls. Flow cytometry staining of T cells with two tetramers (VVVGADGVGK-HLA-A*11:01 tetramer and VVGADGVGK-HLA-A*11:01 tetramer respectively) increased the confidence in T cell specificity.

[0212] Ten specific T cell clones with the required high affinity were screened out. The antigen-specific T cell receptors (TCRs) on these 10 T cell clones that specifically bind to the KRAS_G12D_7-16 epitope peptide and the KRAS_G12D_8-16 epitope peptide were named nwTCR-0884-2, nwTCR-2462, nwTCR-2918, nwTCR-2985, nwTCR-3178, nwTCR-4412, nwTCR-4536, nwTCR-5963, nwTCR-6670-2, and nwTCR-6673 respectively. Using high-throughput paired TCR sequencing, the amino acid sequences of the paired TCR α and β chains on these 10 T cell clones were determined on a single-cell basis.

[0213] Since multiple nucleotides can translate into the same amino acid and the codon frequencies are different in different organisms, the coding nucleotides of the TCR α and β chain amino acid sequences were codon-optimized, aiming to increase the expression level of TCR when expressed in eukaryotic cells. The nucleotide sequences of 10 TCRs that specifically recognize the KRAS_G12D_7-16 epitope peptide and the KRAS_G12D_8-16 epitope peptide were obtained through codon optimization.

[0214] Table 1A and Table 1B list the amino acid sequence information and nucleotide sequence information of the α and β chains of the 10 TCRs expressed by the sequenced cloned T cell lines respectively.

[0215] Table 1A. Amino acid and nucleotide sequences of the KRAS_G12D mutant antigen-specific TCR α chain

[0216]

[0217]

[0218] Table 1 Amino acid and nucleotide sequences of the TCR β-chain specific for the KRAS_G12D mutant antigen

[0219] Name Amino acid sequence Nucleotide sequence nwTCR - 0884 - 2 full - length TRB (TCR β - chain) SEQ ID NO:219 SEQ ID NO:220 nwTCR - 2462 full - length TRB (TCR β - chain) SEQ ID NO:221 SEQ ID NO:222 nwTCR - 2918 full - length TRB (TCR β - chain) SEQ ID NO:223 SEQ ID NO:224 nwTCR - 2985 full - length TRB (TCR β - chain) SEQ ID NO:225 SEQ ID NO:226 nwTCR - 3178 full - length TRB (TCR β - chain) SEQ ID NO:227 SEQ ID NO:228 nwTCR - 4412 full - length TRB (TCR β - chain) SEQ ID NO:229 SEQ ID NO:230 nwTCR - 4536 full - length TRB (TCR β - chain) SEQ ID NO:231 SEQ ID NO:232 nwTCR - 5963 full - length TRB (TCR β - chain) SEQ ID NO:233 SEQ ID NO:234 nwTCR - 6670 - 2 full - length TRB (TCR β - chain) SEQ ID NO:235 SEQ ID NO:236 nwTCR - 6673 full - length TRB (TCR β - chain) SEQ ID NO:237 SEQ ID NO:238

[0220] Example 2 Preparation of KRAS_G12D mutant antigen-specific TCR-T cells from T cells

[0221] This example describes the preparation and characterization of KRAS_G12D mutant antigen-specific TCR-T cells by knocking out the TCR gene in primary T cells using CRISPR / Cas9 technology and knocking in the KRAS_G12D mutant antigen-specific TCR gene using homologous recombination technology.

[0222] 2.1 Sorting and activation of T cells

[0223] A mixture of CD4 T cells and CD8 T cells (also referred to as "CD4 / CD8 T cells" in the text) was enriched and sorted from peripheral blood mononuclear cells (PBMCs, purchased from: Shanghai Saili Biotechnology Co., Ltd., Donor: S2001095). The enriched and sorted CD4 / CD8 T cells were aliquoted and frozen (5x10 6 cells / vial) for future use.

[0224] The frozen vials were thawed as needed, and the sorted T cells were activated by adding a 1:100-fold diluted T cell activator, Miltenyi T cell TransACT (Miltenyi catalog number: 130-111-160), to the T cell medium (e.g., RPMI 1640, FBS, L-glutamine, non-essential amino acids, sodium pyruvate, HEPES buffer, 2-mercaptoethanol, and optionally IL2). The cells were cultured for approximately 48 hours (2 days) and then used for electroporation transfection.

[0225] 2.2 Targeting strategy and preparation of targeting vectors

[0226] The gRNAs used were gRNA002 and gRNA004 (see Table 2). The targeting site of gRNA002 was located in exon 1 of the endogenous TRAC gene ( Figure 1A ); the targeting site of gRNA004 was located in exon 1 of the endogenous TRBC1 and TRBC2 genes ( Figure 1B ). The Cas9 enzyme was purchased from GenScript Biotech Corporation, catalog number: Z03469.

[0227] Table 2 sgRNAs corresponding to the TRAC and TRBC genes

[0228]

[0229] The backbone of the targeting vector (also known as the 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 (about 800 bp) of the TRAC locus. The sequence to be knocked-in (KI) can be constructed between the left and right HAs. The KI sequence construct of nwTCR contains: 2A or its variant - SP - TCRβ - 2A or its variant - SP - TRAV - TRAJ, where 2A or its variant is a ribosome skipping element; SP is a signal peptide; 4 synonymous mutation bases are introduced into the TRBC gene in the targeting vector, specifically, the coding nucleotide of TRBC S77 is mutated from AGC to TCC and the coding nucleotide of S78 is mutated from AGC to TCC. For nwTCR-4536, the nucleotide sequence of its KI construct is shown in SEQ ID NO:260, where: 2A or its variant encodes any of the amino acid sequences shown in SEQ ID NO:246, 248, 250, and SEQ ID NO:252; SP encodes the signal peptide sequence shown in SEQ ID NO:244; TCRβ is the nucleotide sequence (SEQ ID NO:232) of the complete TRB (TCRβ chain) of nwTCR-4536, where 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 synonymous mutation bases into the TRBC gene; TRAV is the nucleotide sequence (SEQ ID NO:56) of the TRAV gene of nwTCR-4536; TRAJ is the nucleotide sequence (SEQ ID NO:58) of the TRAJ gene of nwTCR-4536. Similarly, KI sequence constructs of nwTCR-0884-2, nwTCR-2462, nwTCR-2918, nwTCR-2985, nwTCR-3178, nwTCR-4412, nwTCR-5963, nwTCR-6670-2, and nwTCR-6673 were prepared.

[0230] 2.3. Electroporation transfection (Day 2)

[0231] Fully mix the sgRNA in Example 2.2 with Cas9 enzyme and incubate at room temperature for 10 min to prepare RNP.

[0232] The targeting vector containing the KI TCR sequence prepared in Example 2.2 was thoroughly mixed with the pre-incubated RNP and the T cells at the specified concentration prepared in Example 2.1 (approximately 1.25E6 T cells / electroporation tube) for knocking out (KO) the endogenous TCR and knocking in (KI) the exogenous TCR.

[0233] The above mixture was loaded into an electroporation transfection instrument (Celetrix; catalog number: CTX-1500A LE) for cell electroporation transfection, and the conditions for electroporation transfection were 480 - 560 V and 20 ms.

[0234] After the electroporation transfection was completed, the cells were allowed to stand for 15 min, and then the electroporated T cells were taken out and transferred to pre-warmed medium (ImmunoCult TM -XF T Cell Expansion Medium, Stemcell catalog number: 10981). After culturing the cells for 5 days, flow cytometry characterization was performed on Day 7.

[0235] 2.4. Flow cytometry analysis of nwTCR expression (Day 7)

[0236] The cell suspension obtained from Example 2.3 was thoroughly mixed, cell counting was performed, and an appropriate amount of cells was collected for staining with peptide-MHC (HLA-A*11:01) tetramers labeled with two labels (VVVGADGVGK-HLA-A*11:01 tetramer and VVGADGVGK-HLA-A*11:01 tetramer, which can also be abbreviated as pMHC for short).

[0237] Prepare in advance a staining solution containing the peptide-MHC (HLA-A*11:01) tetramers labeled with the two labels and LIVE / DEAD TM Fixable Near-IR, 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 TCRα / β-BV510 antibody was purchased from BioLegend, catalog number: 306734.

[0238] The collected cells were stained with the peptide-MHC (HLA-A*11:01) tetramer staining solution labeled with the two labels, washed, and characterized by flow cytometry.

[0239] Figures 3A - 3JIllustrated are the staining results of pMHC tetramers (labeled VVVGADGVGK-HLA-A*11:01 tetramers) of different nwTCR binding markers expressed on CD4+ and CD8+ T cells after transfection of CD4+ and CD8+ T cells with different nwTCRs by electroporation. It can be seen that the cells on Day 7 are divided into three populations:

[0240] 1) Wild-type T cells without gene editing (Q3);

[0241] 2) KO cells (Q4) that have completed endogenous TCR knockout;

[0242] 3) The cell population (Q2) that has completed KO and KI and expresses nwTCR.

[0243] The schematic diagram of the results of detecting TCR gene editing efficiency by flow cytometry is as shown in Figure 2 shown.

[0244] As can be seen from Figures 3A - 3J it, CD8+ T cells transfected with each nwTCR can bind to peptide-MHC complex (pMHC) tetramers; there are significant differences in the binding of CD4+ T cells transfected with each TCR to peptide-MHC complex (pMHC) tetramers. This is because, as shown in Example 1, each nwTCR in the present invention is an nwTCR screened using CD8+ T cells. Therefore, when each nwTCR is transfected into CD8+ T cells respectively, the CD8+ T cells expressing each nwTCR can specifically bind to peptide-MHC complex (pMHC) tetramers; while when each nwTCR is transfected into CD4+ T cells respectively, the CD4+ T cells expressing each nwTCR are as follows: Generally, when the affinity of nwTCR for MHC is strong enough, TCR can bind to MHC molecules without the assistance of CD8 molecules. For example, it is considered that the binding ability of nwTCR-5963 to MHC molecules is stronger than that of nwTCR-4536. Therefore, there will be differences in the pMHC tetramer staining of CD4+ T cells. Therefore, after each nwTCR is edited into CD4+ T cells and expressed respectively, the nwTCR with strong affinity can still bind to MHC class I antigens, while the ability of the nwTCR with weak affinity to bind to MHC class I antigens is also weak.

[0245] Example 3. In vitro functional study of KRAS_G12D mutant antigen-specific TCR-T cells

[0246] On Day 7, the KRAS_G12D mutant antigen-specific TCR-T cells of Example 2 were selectively activated using the TransACT activator (Miltenyi catalog number: 130-111-160). The TCR-T cells were continuously cultured until Day 14. On Day 14, in vitro functional studies were performed on each TCR-T cell.

[0247] 3.1 Detection of the binding affinity of each TCR-T cell to the peptide

[0248] The TCR-T cell affinity detection method was implemented as follows. Antigen-presenting cells (T2 cells overexpressing HLA-A*11:01 or K562 cells) were collected, cell counts were performed, and an appropriate amount of culture medium (such as RPMI-1640 medium, purchased from Gibco, catalog number: 22400089; FBS, purchased from Gibco, catalog number: 10099141C) was added to resuspend the cells to a cell density of 1E6 cells / mL. 1 mL of the cell suspension was added to each well of a 24-well plate. The polypeptide solution to be tested (the polypeptide is the KRAS_G12D_7-16 peptide shown in SEQ ID NO: 239 and / or the KRAS_G12D_8-16 peptide shown in SEQ ID NO: 240) was serially diluted to 10 -12 -10 -5 M, and 10 μL of the diluted polypeptide solution was added to the corresponding wells of the 24-well plate. After incubation in an incubator (37 °C, 5% CO2) for 2 h, the incubated antigen-presenting cells were collected and washed, and 100 μL of 1E6 / mL antigen-presenting cells was taken into the corresponding wells of a 96-well plate. The nwTCR-T cells to be tested were collected, an appropriate amount of T cell medium (purchased from STEMCELL, catalog number: 10981) was added to a cell density of 1E6 / mL, and 100 μL of the cell suspension was added to the corresponding wells of the 96-well plate. Each nwTCR-T cell was co-cultured with the antigen-presenting cells (37 °C, 5% CO2) for 16 h, and then the cell supernatant was collected and the IFN-γ concentration was detected using an ELISA kit (purchased from Biolegend, catalog number: 430104). The binding affinity of the T cells expressing each nwTCR to the short peptides shown in SEQ ID NO: 239 or SEQ ID NO: 240 presented by HLA-A*11:01 was detected by detecting the release level of IFN-γ.

[0249] Figure 4A and Figure 4B shows the experimental results of the detection of the binding affinity of the T cells expressing each nwTCR to the short peptides shown in SEQ ID NO: 239 or SEQ ID NO: 240 presented by HLA-A*11:01, where Figure 4A is the binding affinity to the short peptide shown in SEQ ID NO: 239;Figure 4B is the binding affinity with the short peptide shown in SEQ ID NO: 240.

[0250] Figure 4A and Figure 4B The results of and show that after the T2 cells presenting the peptide-MHC complex were co-incubated with the T cells expressing each nwTCR, specific binding of the T cells expressing each nwTCR to the peptide-MHC complex was detected, leading to the release of IFN-γ. Additionally, considering that when MHC presents polypeptides, short peptides of 9-mer and 10-mer may both be presented by MHC, in Figure 4A and Figure 4B The experimental results using 9-mer and 10-mer short peptides show that the T cells expressing each nwTCR can specifically bind to the pMHC complexes presenting 9-mer and 10-mer short peptides respectively.

[0251] 3.2 Detection of the killing of target cells by each TCR-T cell through real-time fluorescence imaging

[0252] Collect the target cell lines CL-40 cell line (HLA-A*11:01+, KRAS G12D+; purchased from Nanjing Kebai Biotechnology Co., Ltd.) and SNU-601 cell line (HLA-A*11:01+, KRAS G12D+; purchased from Nanjing Kebai Biotechnology Co., Ltd.). After cell counting, resuspend each target cell with the target cell medium (RPMI-1640 medium, purchased from Gibco, catalog number: 22400089, FBS, purchased from Gibco, catalog number: 10099141C) to a cell density of 1E6 cells / mL. Take 100 μL of 1E6 / mL antigen-presenting cells into the wells of a 96-well plate.

[0253] Collect the nwTCR-T cells to be detected, add an appropriate amount of T cell medium (purchased from STEMCELL, catalog number: 10981) to a cell density of 1E6 / mL, add 100 μL of the nwTCR-T cell suspension to be detected into the wells of the 96-well plate, and mix with the target cells in the wells of the 96-well plate. Add 1 μL of ethidium bromide (1 mg / mL) solution to the cell mixture (i.e., add ethidium bromide at 0 h). After mixing evenly, 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 and show a red fluorescence signal after entering the apoptotic state.

[0254] Figure 5A - Figure 5CThe real-time fluorescence imaging results of the specific killing of CL-40 (KRAS G12D+) cell line by T cells expressing nwTCR-0884-2, nwTCR-2462 or nwTCR-2918 are shown respectively (0 h when the cells start co-incubation, and the target cells killed after 18 h show red fluorescence signals). "Blank" in the figure indicates that there are only target cells and T cells expressing any nwTCR are not added.

[0255] Figure 5D - Figure 5F The real-time fluorescence imaging results of the specific killing of SNU-601 (KRAS G12D+) cell line by T cells expressing nwTCR-2985, nwTCR-4412, nwTCR-4536, nwTCR-5963, nwTCR-3178, nwTCR-6670-2 and nwTCR-6673 are shown respectively (0 h when the cells start co-incubation, and the target cells killed after 18 h show red fluorescence signals). "Blank" in the figure indicates that there are only target cells and T cells expressing any nwTCR are not added.

[0256] 3.3 Detection of the killing of target cells by each TCR-T cell by real-time cell analyzer

[0257] Collect the target cell lines CL-40 cell line and SNU-601 cell line. After cell counting, resuspend each target cell with target cell medium (RPMI-1640 medium, purchased from Gibco, catalog number: 22400089, FBS, purchased from Gibco, catalog number: 10099141C) to a cell density of 1E6 cells / mL. Prepare an E-plate (obtained from Agilent, catalog number: 300600890). After adding 100 μL of the well-mixed target cell suspension to the corresponding wells, place it into a RTCA real-time cell analyzer (purchased from Agilent, model: xCELLigence RTCA DP) for overnight detection.

[0258] Collect each TCR-T cell to be detected. After cell counting, resuspend the cells with an appropriate amount of T cell medium (purchased from STEMCELL, catalog number: 10981). Take out the above E-plate inoculated with target cells, add the T cell suspension, and put the E-Plate back into the RTCA analyzer for detection to obtain the cell index for 72 hours. Each independent experiment is carried out three times. Use the RTCA software to automatically calculate the interval slope and evaluate the change rate of the cell index. To prove the effect of the treatment, the cell index is normalized to equal values at the standardized time points. The in vitro killing results of each TCR-T cell against target cells are as Figures 6A - 6F shown.

[0259] Figure 6A - Figure 6C Shows the real-time analysis data of T cells expressing each nwTCR killing CL-40 cells. The results show that the gene-edited T cells have a specific killing effect on CL-40 cells. Figure 6A - Figure 6C "Blank" in [] represents only CL-40 cells without adding T cells expressing any nwTCR.

[0260] Figure 6D - Figure 6F Shows the real-time analysis data of T cells expressing each nwTCR killing SNU-601 cells. The results show that the gene-edited T cells have a specific killing effect on SNU-601 cells. Figure 6D - Figure 6F "Blank" in [] represents only SNU-601 cells without adding T cells expressing any nwTCR.

[0261] Therefore, each TCR-T cell shows a significant in vitro killing effect on HLA-A*11:01+ and KRAS G12D+ target cells.

[0262] Example 4. Co-expression of exogenous TCR and CD8 molecule

[0263] This example describes redirecting CD4+ T cells by co-expressing exogenous TCR and CD8 molecule on the surface of CD4+ T cell membrane using CRISPR / Cas9 and homologous recombination technologies. Moreover, this gene editing method can enhance the binding of TCR-T cells to pMHC molecules.

[0264] 4.1 Sorting and activation of T cells

[0265] T cells can be obtained commercially (e.g., cryopreserved human peripheral blood CD4+CD45RA+ T cells, Stem Cell Technology, catalog number 70029) or prepared from leukapheresis samples (Day 0).

[0266] For preparing T cells from leukapheresis samples, CD4 / CD8 T cells are enriched and sorted from the leukapheresis samples. The enriched and sorted CD4 / CD8 T cells are aliquoted and cryopreserved (5x10 6 cells / vial) for future use.

[0267] 4.2 Targeting strategy and preparation of targeting vector

[0268] The targeting strategy and targeting vector of TCR are the same as in Example 2.2 above.

[0269] When the TCR sequence is nwTCR-2985, Figure 8A It can be seen that the binding of CD8+ T cells expressing nwTCR-2985 to the pMHC molecule of the amino acid sequence at positions 7 to 16 of KRASG12D (KRAS_G12D_7-16 peptide) is stronger than the binding of CD4+ T cells expressing nwTCR-2985 to the pMHC molecule presenting the KRAS_G12D_7-16 peptide.

[0270] When the TCR sequence is nwTCR-4536, Figure 8C It can be seen that CD8+ T cells expressing nwTCR-4536 can specifically bind to the pMHC molecule of the amino acid sequence at positions 7 to 16 of KRAS G12D (KRAS_G12D_7-16 peptide), but CD4+ T cells expressing nwTCR-4536 hardly specifically bind to the pMHC molecule presenting the KRAS_G12D_7-16 peptide because of the lack of the assistance of CD8 molecules.

[0271] When the TCR sequence is nwTCR-6670-2, Figure 8E It can be seen that the binding of CD8+ T cells expressing nwTCR-6670-2 to the pMHC molecule of the amino acid sequence at positions 7 to 16 of KRAS G12D (KRAS_G12D_7-16 peptide) is stronger than the binding of CD4+ T cells expressing nwTCR-6670-2 to the pMHC molecule presenting the KRAS_G12D_7-16 peptide.

[0272] In order to redirect CD4+ T cells, the following constructs were further constructed and inserted into the pUC57-HA targeting vector (also known as the HDR vector):

[0273] The KI construct of nwTCR-4536-CD8ab is: 2A or its variant - CD8a - 2A or its variant - CD8b - 2A or its variant - SP - TCRβ - 2A or its variant - SP - TRAV - TRAJ, and its amino acid sequence and nucleotide sequence are shown in SEQ ID NO:261 and SEQ ID NO:262 respectively.

[0274] The KI construct of nwTCR-2985-CD8ab is: 2A or its variant - CD8a - 2A or its variant - CD8b - 2A or its variant - SP - TCRβ - 2A or its variant - SP - TRAV - TRAJ, and its construction method is similar to that of the KI construct of nwTCR-4536-CD8ab, but among them, TCRβ, TRAV, and TRAJ use TCRβ, TRAV, and TRAJ of nwTCR-2985 given in the sequence listing.

[0275] The KI construct of nwTCR-6670-2-CD8ab is: 2A or its variant - CD8a - 2A or its variant - CD8b - 2A or its variant - SP - TCRβ - 2A or its variant - SP - TRAV - TRAJ. Its construction method is similar to that of the KI construct of nwTCR-4536-CD8ab, but among them, for TCRβ, TRAV, and TRAJ, the TCRβ, TRAV, and TRAJ of nwTCR-6670-2 given in the sequence listing are used.

[0276] The targeting strategy diagram of nwTCR-CD8ab is as Figure 7 shown.

[0277] 4.3. Electroporation transfection (Day 2)

[0278] Fully mix the sgRNA designed and synthesized in Example 2.2 with Cas9 enzyme, and incubate at room temperature for 10 min to prepare RNP.

[0279] Mix the targeting vectors prepared in Example 4.2, namely the targeting vectors for knocking in nwTCR-2985-CD8ab, nwTCR-4536-CD8ab, and nwTCR-6670-2-CD8ab respectively, with the incubated RNP and the specified concentration of T cells (1.25E6 / cuvette) prepared in Example 4.1 fully, for knocking out (KO) endogenous TCR and knocking in (KI) exogenous TCR and CD8ab.

[0280] Load the above mixture into an electroporation transfection instrument (Celetrix; catalog number: CTX-1500A LE) for cell electroporation transfection. The conditions for electroporation transfection are 480 - 560 V and 20 ms.

[0281] After the electroporation transfection is completed, let the cells stand for 15 min and then take out the electroporated cells, and transfer them to pre-warmed medium (ImmunoCult TM -XF T Cell Expansion Medium, Stemcell catalog number: 10981). After culturing the cells for 5 days, perform flow cytometry characterization on Day 7.

[0282] 4.4. Flow cytometry analysis of nwTCR expression (Day 7)

[0283] Fully mix the cell suspension obtained from Example 4.3. After cell counting, collect an appropriate amount of cells and perform staining with labeled peptide - MHC (HLA-A11:01) tetramer (VVVGADGVGK - HLA-A11:01).

[0284] Prepare in advance a labeled peptide-MHC tetramer staining solution containing specific antigens and LIVE / DEAD TM Fixable Near-IR, purchased from Invitrogen, catalog number: L10119; CD4-FITC purchased from BioLegend, catalog number: 357406; CD8-PerCP-cy5.5 purchased from BioLegend, catalog number: 344710; anti-human TCRα / β-BV510 antibody purchased from BioLegend, catalog number: 306734.

[0285] Stain the collected cells with the labeled peptide-MHC tetramer staining solution, wash, and then characterize them by flow cytometry. The results are shown below. In the flow cytometry plot, the cells on Day 7 are divided into three populations:

[0286] 1) Wild-type T cells (Q3) without gene editing;

[0287] 2) KO cells (Q4) that have completed endogenous TCR knockout;

[0288] 3) A cell population (Q2) that has completed KO and KI and expresses nwTCR.

[0289] It can be seen from Figure 8B , Figure 8D and Figure 8F that when the CD8ab molecule is co-edited with nwTCR-2985, nwTCR-4536, or nwTCR-6670-2 into primary T cells (the primary T cells contain CD4+ T cells and CD8+ T cells), and characterized by flow cytometry, it can be seen that the proportion of CD8+CD4+ T cells increases. These cells are CD4+ T cells that express exogenous CD8 molecules. With the assistance of the exogenous CD8 molecules, these CD4+ T cells can specifically bind to the labeled peptide-MHC (HLA-A11:01) tetramer (VVVGADGVGK-HLA-A11:01).

[0290] In this example, it was selected that the pMHC tetramer hardly binds to CD4+ T cells expressing nwTCR-4536 after gene editing, aiming to show that even for such nwTCRs, by introducing CD8 molecules into CD4 T cells, the binding of CD4 T cells after gene editing of nwTCR to pMHC molecules can be enhanced. 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 after gene editing of nwTCR to pMHC molecules. This example also further illustrates the results after co-editing nwTCR-2985 and nwTCR-6670-2 into primary T cells with CD8ab molecules.

[0291] Meanwhile, thanks to the fact that this method can redirect CD4+ T cells, the overall gene editing efficiency of T cells is improved (Table 3), where the calculation formula for the gene editing efficiency (GE%) is: the percentage of cells expressing only CD8+ in the live T cell population × the percentage of cells expressing only CD8+ in the tetramer staining + the percentage of cells expressing only CD4+ in the live T cell population × the percentage of cells expressing only CD4+ in the tetramer staining + the percentage of cells expressing both CD8+ and CD4+ in the live T cell population × the percentage of cells expressing both CD8+ and CD4+ in the tetramer staining.

[0292] Table 3. Gene editing efficiency of each cell population in nwTCR and nwTCR-CD8ab cells

[0293]

[0294] The above describes the exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are only exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed in the text.

[0295] Sequence Listing

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Claims

1. An isolated or purified T cell receptor, also abbreviated as TCR, characterized in that, Specifically binds to the KRAS_G12D mutant antigen, wherein the TCR comprises an α-chain and a β-chain, each of the α-chain and the β-chain comprising three complementarity-determining regions, also abbreviated as CDRs, and the amino acid sequence of CDR3 of the α-chain is selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 and variants having one or two amino acid residue changes from said sequences, and the amino acid sequence of CDR3 of the β-chain is selected from SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140 and variants having one or two amino acid residue changes from said sequences.

2. The TCR according to claim 1, wherein, The amino acid sequences of CDR3 of the α-chain and CDR3 of the β-chain are: (i) The amino acid sequence of CDR3 of the α-chain shown in SEQ ID NO: 3 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of CDR3 of the β-chain shown in SEQ ID NO: 113 or a variant having one or two amino acid residue changes from said sequence; (ii) The amino acid sequence of CDR3 of the α-chain shown in SEQ ID NO: 6 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of CDR3 of the β-chain shown in SEQ ID NO: 116 or a variant having one or two amino acid residue changes from said sequence; (iii) The amino acid sequence of CDR3 of the α-chain shown in SEQ ID NO: 9 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of CDR3 of the β-chain shown in SEQ ID NO: 119 or a variant having one or two amino acid residue changes from said sequence; (iv) The amino acid sequence of CDR3 of the α-chain shown in SEQ ID NO: 12 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of CDR3 of the β-chain shown in SEQ ID NO: 122 or a variant having one or two amino acid residue changes from said sequence; (v) The amino acid sequence of CDR3 of the α-chain shown in SEQ ID NO: 15 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of CDR3 of the β-chain shown in SEQ ID NO: 125 or a variant having one or two amino acid residue changes from said sequence; (vi) The amino acid sequence of CDR3 of the α-chain shown in SEQ ID NO: 18 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of CDR3 of the β-chain shown in SEQ ID NO: 128 or a variant having one or two amino acid residue changes from said sequence; (vii) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 21 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 131 or a variant having one or two amino acid residue changes from said sequence; (viii) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 24 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 134 or a variant having one or two amino acid residue changes from said sequence; (ix) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 27 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 137 or a variant having one or two amino acid residue changes from said sequence; (x) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 30 or a variant having one or two amino acid residue changes from said sequence; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 140 or a variant having one or two amino acid residue changes from said sequence; Preferably, the amino acid sequences of the CDR3 of the α-chain and the CDR3 of the β-chain are: (i) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 3; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 113; (ii) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 6; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 116; (iii) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 9; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 119; (iv) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 12; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 122; (v) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 15; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 125; (vi) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 18; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 128; (vii) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 21; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 131; (viii) The amino acid sequence of the α-chain CDR3 shown in SEQ ID NO: 24; and the amino acid sequence of the β-chain CDR3 shown in SEQ ID NO: 134; (ix) The amino acid sequence of α-chain CDR3 shown in SEQ ID NO:27; and the amino acid sequence of β-chain CDR3 shown in SEQ ID NO:137; (x) The amino acid sequence of α-chain CDR3 shown in SEQ ID NO:30; and the amino acid sequence of β-chain CDR3 shown in SEQ ID NO:

140.

3. The TCR according to claim 2, wherein, The amino acid sequences of the three CDRs contained in the α-chain and the amino acid sequences of the three CDRs contained in the β-chain are: (i) The amino acid sequences of α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:1, 2, 3 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:111, 112, 113 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; (ii) The amino acid sequences of α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:4, 5, 6 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:114, 115, 116 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; (iii) The amino acid sequences of α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:7, 8, 9 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:117, 118, 119 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; (iv) The amino acid sequences of α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:10, 11, 12 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:120, 121, 122 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; (v) The amino acid sequences of α-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:13, 14, 15 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; and the amino acid sequences of β-chain CDR1, CDR2, CDR3 shown in SEQ ID NO:123, 124, 125 or variants having 1 or 2 amino acid residue changes respectively from the said sequences; (vi) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 16, 17, 18, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 126, 127, 128, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; (vii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 19, 20, 21, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 129, 130, 131, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; (viii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 22, 23, 24, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 132, 133, 134, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; (ix) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 25, 26, 27, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 135, 136, 137, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; (x) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 28, 29, 30, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 138, 139, 140, or variants having 1 or 2 amino acid residue changes respectively compared with said sequences; Preferably, the amino acid sequences of the three CDRs contained in the α-chain and the amino acid sequences of the three CDRs contained in the β-chain are: (i) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 1, 2, 3; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 111, 112, 113; (ii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 4, 5, 6; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO: 114, 115, 116; (iii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:7, 8, 9; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:117, 118, 119; (iv) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:10, 11, 12; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:120, 121, 122; (v) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:13, 14, 15; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:123, 124, 125; (vi) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:16, 17, 18; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:126, 127, 128; (vii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:19, 20, 21; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:129, 130, 131; (viii) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:22, 23, 24; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:132, 133, 134; (ix) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:25, 26, 27; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:135, 136, 137; (x) The amino acid sequences of α-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:28, 29, 30; and the amino acid sequences of β-chain CDR1, CDR2, and CDR3 shown in SEQ ID NO:138, 139, 140.

4. The TCR according to any one of claims 1 to 3, wherein, The TCR further comprises a constant region. For example, the constant region is a murine constant region; Preferably, the TCR comprises an α-chain sequence shown in SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and a β-chain sequence shown in SEQ ID NO: 219, 221, 223, 225, 227, 229, 231, 233, 235 or 237, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; More preferably, the TCR comprises an α-chain sequence shown in SEQ ID NO: 91 and a β-chain sequence shown in SEQ ID NO: 219; The TCR comprises an α-chain sequence shown in SEQ ID NO: 93 and a β-chain sequence shown in SEQ ID NO: 221; The TCR comprises an α-chain sequence shown in SEQ ID NO: 95 and a β-chain sequence shown in SEQ ID NO: 223; The TCR comprises an α-chain sequence shown in SEQ ID NO: 97 and a β-chain sequence shown in SEQ ID NO: 225; The TCR comprises an α-chain sequence shown in SEQ ID NO: 99 and a β-chain sequence shown in SEQ ID NO: 227; The TCR comprises an α-chain sequence shown in SEQ ID NO: 101 and a β-chain sequence shown in SEQ ID NO: 229; The TCR comprises an α-chain sequence shown in SEQ ID NO: 103 and a β-chain sequence shown in SEQ ID NO: 231; The TCR comprises an α-chain sequence shown in SEQ ID NO: 105 and a β-chain sequence shown in SEQ ID NO: 233; The TCR comprises an α-chain sequence shown in SEQ ID NO: 107 and a β-chain sequence shown in SEQ ID NO: 235; The TCR comprises an α-chain sequence shown in SEQ ID NO: 109 and a β-chain sequence shown in SEQ ID NO:

237.

5. A nucleic acid molecule, characterized in that, Encoding the TCR according to any one of claims 1-4, preferably, the nucleic acid molecule is codon-optimized and encodes a nucleotide sequence of the TCR according to any one of claims 1-4.

6. A vector, characterized in that, Comprising the nucleic acid molecule according to claim 5, the vector is preferably a plasmid, shuttle plasmid, phagemid, cosmid, expression vector; more preferably a homologous recombination repair (HDR) vector or a viral vector, such as a lentiviral vector, adenoviral vector, adeno-associated virus (AAV) vector, herpesvirus vector, retroviral vector, baculovirus vector.

7. A T cell receptor fusion protein or a T cell receptor conjugate, comprising the TCR according to any one of claims 1 - 4 and other bioactive molecules, wherein the other bioactive molecules are, for example, antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable labels, and there is or is not a linker between the TCR and the other bioactive molecules.

8. An engineered cell, characterized in that, Express the TCR described in any one of claims 1-4. Preferably, the engineered cell is an engineered T cell or an engineered NK cell; or the engineered cell is an engineered stem cell. For example, the engineered cell is an engineered human CD4+ T cell or an engineered human CD8+ T cell, or a mixed cell population of an engineered human CD4+ T cell and an engineered human CD8+ T cell; or, the engineered cell is an engineered hematopoietic stem cell.

9. An engineered human CD4+ T cell and / or an engineered human CD8+ T cell, characterized in that, Express the TCR described in any one of claims 1-4 and express exogenous CD8a or CD8ab.

10. A method for generating the T cells of claim 9, the method comprising transfecting CD4+ T cells and / or CD8+ T cells with exogenous CD8a or CD8ab; and the TCR of any one of claims 1-4. For example, the exogenous CD8a or CD8ab and the TCR of any one of claims 1-4 are expressed from the same vector in the CD4+ T cells and / or CD8+ T cells. For example, the construct expressing the exogenous CD8a or CD8ab and the construct expressing the TCR of any one of claims 1-4 are separated by a 2A element or an IRES element.

11. A pharmaceutical composition, characterized in that Comprise the engineered cell described in claim 8 and / or the engineered human CD4+ T cell described in claim 9 and / or the engineered human CD8+ T cell described in claim 9.

12. Use of the TCR of any one of claims 1-4, the engineered cell of claim 8 or 9, and the pharmaceutical composition of claim 11, characterized in that For preparing a medicament for treating a disease (such as a tumor) having a KRAS_G12D mutation.

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