Modified T cell receptor or fragment thereof targeting KRAS G12V and use thereof

Abstract

The present invention relates to the field of immunity, in particular to a modified T cell receptor targeting KRAS G12V or a fragment thereof and its use. The modified T cell receptor targeting KRAS G12V or a fragment thereof provided by the present invention comprises a TCR α chain variable region and a TCR β chain variable region, wherein the TCR α chain variable region comprises CDR1, CDR2 and CDR3 whose amino acid sequences are respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and the TCR β chain variable region comprises β chain CDR1, CDR2 and CDR3 whose amino acid sequences are respectively shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The TCR can bind to the target antigen peptide with high affinity, has good expression stability, and high membrane stability, and can mediate the specific killing effect of effector cells on antigen-positive target cells.

Description

Technical Field

[0001] The present invention relates to the field of immunity, in particular to T cell receptors (TCRs), and uses thereof, in particular in detecting, diagnosing, preventing, alleviating or treating KRAS G12V mutation-positive diseases and / or disorders. Background Art

[0002] The RAS gene family, which includes NRAS, HRAS, and KRAS, encodes a small, membrane-bound GTPase protein that plays a major role in signal transduction for cell growth regulation. RAS plays a central role in cellular signal transduction, regulating processes such as transcription and translation at the molecular level and cell proliferation, differentiation, aging, and apoptosis at the cellular level.

[0003] As early as 1982, researchers discovered mutated RAS genes in human bladder cancer cells, making RAS the first human tumor gene discovered. Among them, the KRAS gene has the greatest impact on human cancer. When activated, KRAS transmits upstream signals from cell surface receptors to downstream pathways to control normal cell function and proliferation. The most common activation mode of KRAS is point mutation, which is one of the most common mutations in solid tumors. The sites are almost all concentrated at codon 12 of exon 2, and the most common types are G12D, G12V, and G12C.

[0004] Despite widespread consensus on the crucial role of KRAS mutations in cancer, no KRAS-targeting drugs have yet been approved for marketing. In recent years, new drug candidates specifically targeting the KRAS G12C mutation have made some progress: small molecule drugs targeting KRAS can directly inhibit KRAS protein activity. KRAS protein is widely expressed, and indiscriminate inhibition of both wild-type and mutant KRAS proteins may result in unacceptable toxicity. Another approach to directly inhibiting KRAS activity is to interfere with KRAS binding to the lipid membrane periphery. This, again, is unlikely to distinguish between mutant and wild-type proteins, leading to potential toxicity. KRAS protein regulates numerous downstream effector molecules, primarily the RAF / MEK / ERK and PI3K / AKT / mTOR pathways. However, due to the numerous feedback mechanisms involved, clinical trials have shown limited efficacy of inhibitors of multiple downstream molecules.

[0005] Numerous research results suggest that the KRAS mutant antigen epitope is a target that can be efficiently recognized by TCRs. KRAS mutant antigen-specific TCRs are a promising approach for treating KRAS mutant solid tumors, and the development of KRAS mutant antigen-specific TCR-T products has important clinical significance. However, currently discovered wild-type KRAS mutant antigen-specific TCRs generally have low affinity and are difficult to effectively exert anti-tumor effects in vivo. Therefore, there is a need to develop high-affinity TCR products that specifically target KRAS mutant antigens. Summary of the Invention

[0006] The present invention uses the wild-type human KRAS G12V-specific TCR sequence as a template, uses in vitro mutagenesis to mutate the complementarity determining region (CDR) region of the TCR, and obtains high-affinity mutant clones by performing in vitro functional positive screening on the mutated TCR. At the same time, in vitro negative screening is performed to exclude mutant clones with non-specific activation, thereby obtaining a mutant TCR sequence with high affinity and no non-specific reaction.

[0007] Accordingly, in a first aspect, the present disclosure provides a modified T cell receptor or fragment thereof targeting KRAS G12V, comprising a T cell receptor (TCR) α chain variable region and a TCR β chain variable region, wherein the TCR α chain variable region comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and the TCR β chain variable region comprises β chain CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0008] In a second aspect, the present disclosure provides a nucleic acid encoding the T cell receptor or a fragment thereof according to the first aspect.

[0009] In a third aspect, the present disclosure provides a vector comprising the nucleic acid of the second aspect.

[0010] In a fourth aspect, the present disclosure provides a cell comprising the T cell receptor or a fragment thereof according to the first aspect, the nucleic acid according to the second aspect, or the vector according to the third aspect.

[0011] In a fifth aspect, the present disclosure provides use of the T cell receptor or fragment thereof described in the first aspect, the nucleic acid described in the second aspect, or the vector described in the third aspect in the preparation of a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12V-positive disease or condition.

[0012] In a sixth aspect, the present disclosure provides a drug comprising the T cell receptor or a fragment thereof according to the first aspect, the nucleic acid according to the second aspect, or the vector according to the third aspect.

[0013] The TCR provided by this invention possesses the ability to bind target antigen peptides with high affinity, significantly improving target cell recognition capabilities. It also exhibits excellent expression and membrane stability, ensuring its long-lasting and high efficacy in complex in vivo environments. By mediating the specific killing of antigen-positive target cells by effector cells, it can be used to treat various KRAS G12V-positive cancers, providing an important tool for cell therapy and demonstrating strong technical value and clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The EC50 values ​​of the affinity-optimized TCR sequences provided by the present invention and the parental TCR are shown.

[0015] Figure 2 This shows that the affinity-optimized TCR provided by the present invention has good membrane expression stability.

[0016] Figure 3 This shows that the affinity-optimized TCR provided by the present invention has efficient specific killing ability.

[0017] Figure 4 This shows that the affinity-optimized TCR-T provided by the present invention has a good specific killing effect on HLA-antigen peptide-matched target cells.

[0018] Figure 5 and Figure 6 The activation response of the TCR sequence provided by the present invention after co-incubation with non-target cells is shown. DETAILED DESCRIPTION

[0019] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For example, the terms used herein are as defined in Janeway CA Jr, Travers P, Walport M, et al., Immunobiology, Fifth Edition, New York: Garland Science (2001) and in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," Leuenberger, HGW, Nagel, B. and Kölbl, H., eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0020] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprising," "including," and "having" can be used interchangeably.

[0021] When the term "comprising" is used herein and in the appended claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising or containing at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists only of these embodiments.

[0022] The present disclosure provides a modified T cell receptor or fragment thereof targeting KRAS G12V, comprising a T cell receptor (TCR) α chain variable region and a TCR β chain variable region, wherein the TCR α chain variable region comprises CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NO: 1 (VSGYAW), SEQ ID NO: 2 (YITGDNLV) and SEQ ID NO: 3 (AVRDIEGAGNNRKLI), respectively; and the TCR β chain variable region comprises β chain CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NO: 4 (SGHNS), SEQ ID NO: 5 (FNNNVP) and SEQ ID NO: 6 (ASSEGFYTEAF), respectively.

[0023] The inventors of the present invention have found that by introducing a mutation in which the amino acid YAW replaces NPY at the CDR1 (CDR1α) of the parental TCR α chain, the modified TCR of the present invention is obtained, which has improved binding affinity to the KRAS G12V mutant antigen.

[0024] The term "parent TCR" as used herein refers to the HLA-A1101-restricted human TCR targeting the KRAS G12V mutant antigen previously developed and patented by the applicant of the present invention (KVN02, patent application number: 202310065643.3), which is incorporated herein by reference in its entirety.

[0025] The term "avidity" or "binding avidity" refers to the ability of T cells expressing the TCR of the present invention to respond to a given concentration of ligand in vitro, and is believed to be related to the in vivo effector capacity of cells expressing the TCR. By definition, cells expressing TCRs with high binding avidity respond to very low antigen doses in in vitro assays, while such cells with lower binding avidity require higher amounts of antigen before reaching an immune response similar to that of cells expressing TCRs with high avidity. Therefore, binding avidity can be considered as a quantitative determinant of the activation threshold of cells expressing the TCR. This is determined by exposing such cells to different amounts of cognate antigen in vitro. Cells with TCRs with high binding avidity respond to low antigen doses. In some embodiments, EC50 is used as a criterion for judging the affinity of a given TCR.

[0026] In some embodiments of the invention, the TCR provided herein is capable of binding to an epitope comprising the amino acid sequence VVVGAVGVGK (SEQ ID NO: 17) or a complex of the epitope and an MHC molecule.

[0027] The term "epitope" generally refers to a site on an antigen, typically a (poly)peptide that a binding domain recognizes. The term "binding domain" refers in its broadest sense to an "antigen binding site," i.e., a domain that characterizes a molecule that binds to / interacts with a specific epitope on an antigen target. An antigen target can comprise a single epitope, but typically comprises at least two epitopes, and depending on the size, conformation, and type of the antigen, the antigen target can comprise any number of epitopes. The term "epitope" generally includes linear epitopes and conformational epitopes. A linear epitope is a continuous epitope contained in the primary sequence of amino acids, and it typically includes at least 2 amino acids or more amino acids. A conformational epitope is formed by non-continuous amino acids that are arranged side by side by the folding of the target antigen, and particularly the target (poly)peptide.

[0028] As used herein, the terms "T cell receptor" or "TCR" are used interchangeably to refer to a native TCR or variants, fragments, and constructs thereof. The term thus includes heterodimers, as well as multimers and single-chain constructs comprising a TCR α chain and a TCR β chain; optionally comprising other domains and / or portions, as long as the TCR retains its ability to recognize an antigen target (preferably in complex with HLA-A*11).

[0029] In its native form, the TCR exists as a complex of several proteins on the surface of the T cell. The T cell receptor consists of two (separate) protein chains, which are produced by independent T cell receptor α and β (TCRα and TCRβ) genes and are called α chain and β chain. Each chain of the TCR has an N-terminal immunoglobulin-like (Ig)-variable (V) region / domain, an Ig-constant (C) region / domain, a transmembrane / cell membrane spanning region that anchors the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminus.

[0030] Antigen specificity is conferred by the variable regions of the α and β chains. Both variable regions of the TCR α and β chains contain three hypervariable or complementarity-determining regions (CDR1α / β, CDR2α / β, and CDR3α / β) surrounded by framework (FR) regions. CDR3 is the primary determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 primarily interact with the MHC molecules that present the antigenic peptide.

[0031] TCR recognizes antigenic peptides that are bound to major histocompatibility complex (MHC) molecules at the surface of antigen-presenting cells ("presented / displayed on MHC molecules"). Antigenic peptides presented on MHC molecules are also referred to herein as "complexes of epitopes and MHC molecules," "epitope-MHC complexes," or "target antigen peptide-MHC complexes." There are two different classes of MHC molecules: MHC I and MHC II, which present peptides from different cell compartments. MHC class I molecules are expressed on the surface of all nucleated cells in the human body and display peptides or protein fragments from intracellular compartments to cytotoxic T cells. In humans, MHC is also known as human leukocyte antigen (HLA). There are three main types of MHC class I: HLA-A, HLA-B, and HLA-C. Once the TCR binds to its specific epitope-MHC complex, the T cell is activated and exerts biological effector functions.

[0032] The TCRs provided herein can advantageously (specifically) recognize the KRAS G12V epitope or a complex of the epitope with an MHC molecule, such as a complex of the KRAS G12V epitope with HLA-A*11, and have a significantly improved binding affinity compared to their parental TCRs.

[0033] In some embodiments, the MHC molecule is of the HLA-A*11 type, e.g., HLA-A*11:01.

[0034] In some embodiments of the invention, the TCR alpha chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:7.

[0035] In some embodiments of the invention, the TCR β chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:8.

[0036] As used herein, the term "sequence identity" refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and ClustalW.

[0037] Thus, the amino acid sequence of SEQ ID NO: 7 or 8 can, for example, serve as a "subject sequence" or "reference sequence," while a TCR α chain or β chain variable region amino acid sequence that differs therefrom can serve as a "query sequence."

[0038] In some embodiments, the TCR α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the TCR β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the TCR α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, and the TCR β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0039] In some embodiments, the TCR α chain variable region is contained on a first polypeptide and the TCR β chain variable region is contained on a second, different polypeptide. In some embodiments, the TCR α chain variable region and the TCR β chain variable region are contained on a single polypeptide.

[0040] In some embodiments, the TCR is soluble or membrane-bound.

[0041] The TCR of the present invention can be provided in a soluble form, for example, in the form of a soluble TCR. Soluble TCR (sTCR) can be used as a diagnostic tool and a carrier or "adapter" for specifically targeting therapeutic agents or effector cells to, for example, cancer cells expressing an antigen target recognized by the soluble TCR. Soluble TCRs are typically fragments or constructs comprising TCR α chains and / or β chains or their variable regions or CDRs, and are optionally stabilized by disulfide bonds or covalently linked by suitable linkers. Typically, soluble TCRs do not include, for example, a transmembrane region.

[0042] The TCR of the present invention is provided in the form of a membrane-bound TCR. Typically, a membrane-bound TCR includes a transmembrane region to anchor it to the cell membrane.

[0043] In some embodiments, the TCR further comprises a TCR constant region or a fragment thereof.

[0044] As used herein, the term "constant region" can be a human constant region or derived from another species, thereby creating a "chimeric" TCR. For example, the human α and / or β chains can be replaced with their murine counterparts ("murinization"), which have been found to enhance surface expression of the human TCR by supporting preferential pairing of the TCR α and β chains and to enhance more stable binding to the CD3 co-receptor.

[0045] In some embodiments, the TCR constant region is a murine constant region or a human constant region.

[0046] It has been reported that the addition of disulfide bonds in the constant region can promote the correct pairing of TCR α and β chains (Kuball J et al. Blood. 2007 Mar 15; 109(6): 2331-8). Therefore, in some embodiments of the present invention, one or more cysteine ​​modifications are also added to the constant region to form a disulfide bond between the TCR α chain and the TCR β chain.

[0047] In some embodiments, the TCR constant region includes a TCR α chain constant region and / or a TCR β chain constant region; preferably, the TCR α chain constant region and / or the TCR β chain constant region contain at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between the TCR α chain and the TCR β chain.

[0048] In some embodiments, the cysteine ​​mutation is at one or more of the following positions: position 48 of the wild-type human TCR α chain constant region, position 48 of the wild-type mouse TCR α chain constant region, position 57 of the wild-type human TCR β chain constant region, and position 57 of the wild-type mouse TCR β chain constant region.

[0049] The sequence of the wild-type TCR constant region can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant domain sequence of the TCR molecule α chain is "TRAC*01", and the constant domain sequence of the TCR molecule β chain is "TRBC1*01" or "TRBC2*01".

[0050] To facilitate the description of cysteine ​​mutation positions, the positions of wild-type TCR constant region amino acid sequences in this disclosure are numbered according to the International Immunogenetics Information System (IMGT) nomenclature. For example, if an amino acid in the TCR α chain constant region (TRAC) is numbered as position 48 in IMGT, it will be described herein as amino acid position 48 in the TCR α chain constant region (TRAC); if an amino acid in the TCR β chain constant region (TRBC) is numbered as position 57 in IMGT, it will be described herein as amino acid position 57 in the TCR β chain constant region (TRBC), and so on. Herein, the position numbering of amino acid sequences in the variable regions TRAV and TRBV follows the position numbering listed in IMGT. For example, if an amino acid in TRAV is numbered as position 46 in IMGT, it will be described herein as amino acid position 46 in TRAV, and so on. In this disclosure, if the sequence position numbering of other amino acids is specifically indicated, the specific indication will be used.

[0051] In some embodiments, the TCR α chain constant region further comprises an LVL mutation or an LIV mutation, such that the constant region (and / or transmembrane region) comprises the amino acid sequence LLVIVLRIL. For example, when the TCR α chain comprises a human constant region, the human constant region may comprise an LVL mutation, such that the constant region (and / or transmembrane region) comprises the amino acid sequence LLVIVLRIL. When the TCR α chain comprises a murine constant region, the murine constant region may comprise an LIV mutation, such that the constant region (and / or transmembrane region) comprises the amino acid sequence LLVIVLRIL.

[0052] In some embodiments, the TCR alpha chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the TCR beta chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 10. In some embodiments, the TCR alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 9, and the TCR beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0053] In some embodiments, the fragment of the TCR constant region is the extracellular segment of the TCR constant region.

[0054] In some embodiments, the TCR is a TCR comprising an α chain and a β chain. In some cases, the TCR α chain and / or the β chain can include a leader sequence. For example, the leader sequence of the TCR α chain can have the amino acid sequence (MASAPISMLAMLFTLSGLR) shown in SEQ ID NO: 18. The leader sequence of the TCR β chain can have the amino acid sequence (MDSWTFCCVSLCILVAKHT) shown in SEQ ID NO: 19.

[0055] In some embodiments, the TCR further comprises a transmembrane region and / or a cytoplasmic region.

[0056] In some embodiments, the TCR comprises a TCR alpha chain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one selected from SEQ ID NO: 11; in some embodiments, the TCR comprises a TCR beta chain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one selected from SEQ ID NO: 12. In some embodiments, the TCR comprises the TCR alpha chain as set forth in SEQ ID NO: 11 and the TCR beta chain as set forth in SEQ ID NO: 12.

[0057] In some embodiments, the TCR further comprises an intracellular signaling domain. In some embodiments, the TCR further comprises one or more antigen binding domains that bind to other antigens or epitopes.

[0058] In some embodiments, the TCR is isolated or purified.

[0059] As used herein, the term "isolated or purified" means that the TCR has been identified, separated, and / or recovered from the components of its production environment, such that the "isolated or purified" TCR is free or substantially free of other contaminant components from its production environment that may interfere with its therapeutic or diagnostic use. Contaminant components may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Thus, an "isolated or purified" TCR can be prepared by at least one purification step that removes or substantially removes these contaminant components.

[0060] Nucleic Acids

[0061] The present disclosure provides nucleic acids encoding the TCRs of the present disclosure.

[0062] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 13, encoding a TCR alpha chain variable region, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14, encoding a TCR beta chain variable region.

[0063] In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a TCR α chain constant region and / or encoding a TCR β chain constant region. In some embodiments, the nucleic acid encoding the TCR α chain constant region has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the nucleic acid encoding the TCR β chain constant region has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21.

[0064] In some embodiments, the nucleic acid further comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15, encoding a TCR alpha chain, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16, encoding a TCR beta chain.

[0065] The present disclosure provides vectors comprising the nucleic acids of the present disclosure.

[0066] As used herein, the term "vector" is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material to a host cell, in which the nucleic acid molecule as a vector can be, for example, replicated and / or expressed. The term "vector" encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, slow viral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), bacteriophages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton." Engineered vectors are typically included in the starting point (if stable expression of polynucleotides is desired) for autonomous replication in the host cell, a selective marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector can additionally include a promoter, a genetic marker, a reporter gene, a targeting sequence, and / or a protein purification tag. As known to those skilled in the art, a large amount of suitable vectors are known to those skilled in the art, and many are commercially available. Examples of suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is incorporated herein by reference in its entirety.

[0067] In some embodiments, the vector is preferably selected from the group consisting of a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0068] The present disclosure provides a cell comprising a TCR, a nucleic acid, or a vector according to the present disclosure.

[0069] As used herein, the term "cell" refers to any type of cell capable of expressing the TCR of the present disclosure. The cell can be a eukaryotic cell, for example, a plant (without the potential to develop into a plant), an animal, a fungus, or an algae, or can be a prokaryotic cell, for example, a bacterium or a protozoan. The cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism, such as a human. The cell can be an adherent cell or a suspension cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of producing the TCR of the present disclosure, the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.

[0070] In some embodiments, the cell is selected from lymphocytes (e.g., T cells, NK cells), monocytes (e.g., PBMCs), and stem cells. The term "stem cell" as used herein is a stem cell for expressing the TCR (particularly TCR) of the present disclosure. For example, the stem cell can be a lymphoid progenitor cell, an induced pluripotent stem cell (iPSC), or a hematopoietic stem cell (HSC). In some embodiments, the stem cell does not include embryonic stem cells obtained by destroying a human embryo, and / or does not include totipotent stem cells for developing and forming an animal individual. Gene transfer to stem cells generally does not result in expression of TCR on the cell surface because CD3 molecules are not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, the expression of CD3 molecules will initiate expression of the introduced TCR molecules on the surface of thymocytes.

[0071] In some embodiments, the stem cell is a lymphoid progenitor cell or an induced pluripotent stem cell (iPSC).

[0072] In some embodiments, the cell is a T cell. The T cell can be any T cell, such as a cultured T cell, for example, a primary T cell or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or liquids. The T cell can also be enriched or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell separated from people. The T cell can be any type of T cell and can be a T cell at any stage of development, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD4+T cells, CD8+T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc. In some embodiments, the T cell does not express endogenous TCR.

[0073] In some embodiments, the method of preparing the cells of the present disclosure comprises the step of transducing or transfecting the cells with the vector of the present disclosure.

[0074] As used herein, the term "transfection" is a process in which a nucleic acid molecule or polynucleotide (including a vector) is intentionally introduced into a target cell. An example is RNA transfection, which is the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. This term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is typically used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening a transient pore or "hole" in the cell membrane to allow the uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with the cell membrane and deposit their cargo inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation.

[0075] In some embodiments, the method further comprises the step of expanding and / or activating the cells before or after the transduction or transfection.

[0076] The present disclosure provides use of a T cell receptor or a fragment thereof, a nucleic acid, or a vector provided by the present disclosure in preparing a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12V-positive disease or condition.

[0077] In some embodiments, the product is a drug, which comprises the T cell receptor or fragment thereof disclosed herein, the nucleic acid disclosed herein, or the vector disclosed herein.

[0078] In some embodiments, the drug is in the form of a conjugate comprising a TCR of the present disclosure and an active agent coupled or conjugated to the TCR.

[0079] In some embodiments, the active agent is selected from a detectable label, an immunostimulatory molecule, and a therapeutic agent. Preferably, the detectable label is selected from biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent, or chemiluminescent molecule. Preferably, the immunostimulatory molecule is selected from a cytokine (such as IL-2 and IFN-γ), a chemokine (such as IL-8), a platelet factor (such as platelet factor 4), and a complement initiator. Preferably, the therapeutic agent is selected from an immunomodulator, a radioactive compound, an enzyme, a chemotherapeutic agent, and a toxin. Other suitable therapeutic agents include small molecule cytotoxic agents, i.e., compounds with the ability to kill mammalian cells with a molecular weight of less than 700 daltons. Such compounds may also contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decay or are converted to release cytotoxic agents under physiological conditions. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, pyroximate, temozolomide, topotecan, trimetrexate glucuronide, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells; for example, ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA e and RNase; radionuclides, i.e., unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays while decaying, for example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225 and astatine-213; chelating agents that can be used to promote the binding of these radionuclides to molecules or polymers thereof; or heterologous protein domains, allologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0080] In some embodiments, the drug is in the form of a composition comprising the TCR, nucleic acid, vector, or cell of the present disclosure. Preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0081] The term "composition" particularly refers to a composition suitable for administration to humans. However, the term also generally encompasses compositions suitable for administration to non-human animals. The composition and its components (i.e., the active agent and optional carrier or excipient) are preferably pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutically acceptable compositions of the present invention may, for example, be sterile. Specifically, the term "pharmaceutically acceptable" may mean approved by a regulatory agency or other recognized pharmacopoeia for use in animals, more particularly in humans.

[0082] The term "excipient" includes fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavorings, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators. It is known to those skilled in the art that suitable excipients are selected to prepare compositions of the present invention. Exemplary carriers for compositions of the present invention include saline, buffered saline, glucose and water. Usually, the selection of suitable excipients especially depends on the desired dosage form of the activating agent used, the disease to be treated and the compositions.

[0083] Depending on the active agent employed (e.g., soluble TCR), the compositions of the present disclosure may be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, or in a form particularly suitable for the desired method of administration. Processes known in the art for producing pharmaceuticals are shown in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar-coating, grinding, emulsifying, encapsulating, embedding, or lyophilizing processes. Compositions comprising, for example, host cells or soluble TCRs as described herein are typically provided in liquid form and preferably contain a pharmaceutically acceptable buffer.

[0084] In some embodiments, the compositions of the present disclosure further comprise a second therapeutic agent, preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0085] Preferred examples of the second therapeutic agent include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, E), vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes and RNA enzymes; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, platelet factor 4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides.

[0086] In some embodiments, the product is a kit comprising the TCR or conjugate of the present disclosure, and the kit is used to detect the presence of a positive epitope in a test sample, wherein the epitope comprises a KRAS G12V mutation epitope.

[0087] In some embodiments, the kit is a kit for detecting (e.g., diagnosing) a KRAS G12V mutation-positive disease and / or condition in a subject, comprising a TCR or conjugate of the present disclosure.

[0088] In some embodiments, the conjugate comprises a detectable label. Examples of detectable labels include, but are not limited to, biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent, or chemiluminescent molecule; preferably, an enzyme or catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent, or chemiluminescent molecule. In some embodiments, the kit may further comprise instructions for use of the kit.

[0089] In embodiments of the present disclosure, the disease and / or condition may include a KRAS G12V mutation-positive disease and / or condition. In some embodiments, the KRAS G12V mutation may be an epitope of the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the KRAS G12V mutation-positive disease may include a tumor. In some embodiments, the tumor may include a solid tumor and / or a hematological tumor. In some embodiments, the tumor includes at least one of colorectal cancer, pancreatic cancer, lung adenocarcinoma, and endometrial cancer.

[0090] The present invention is further illustrated by the following specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional conditions in the art, such as those described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd ed.) (2001), CSHL Press, or according to the manufacturer's recommendations. Unless otherwise stated, the experimental materials and reagents used in the following examples were commercially available.

[0091] Example

[0092] Example 1 Screening of modified TCRs with enhanced affinity

[0093] Using the human KVA11-N02 sequence (202310065643.3) as a template, the inventors performed in vitro substitutions in the complementarity-determining regions (CDRs) of KVA11-N02. High-affinity substitution clones were obtained through in vitro positive screening of the substituted TCRs. This method identified a preferred TCR sequence with amino acid mutations at three sites in CDR1α, resulting in a modified TCR named KVA11-M02-321. Specific TCR information is shown in Table 1 below.

[0094] To increase the correct pairing and expression of exogenous TCRs on the surface of human T cells, the variable regions of the α and β chains of the TCR were fused to the murine TCR constant region, and the classic α chain C region "LIV" substitution was introduced into the murine TCR constant region (i.e., amino acid substitutions were introduced into the murine TCR α chain constant region so that the constant region contained the amino acid sequence LLVIVLRIL; shown in bold) and the classic α chain T48C and β chain S57C substitutions (shown in bold and underlined) were introduced into the murine TCR constant region.

[0095] The α and β chains of TCR (the N-termini of the α and β chains contain the signal peptide sequences MASAPISMLAMLFTLSGLR (SEQ ID NO: 18) and MDSWTFCCVSLCILVAKHT (SEQ ID NO: 19), respectively) were constructed in series into a lentiviral expression vector in the order of β-P2A-α through a 2A self-cleavage peptide (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 22)).

[0096] Table 1

[0097]

[0098]

[0099] In order to determine the degree of improvement in the affinity of the TCR provided by the present invention compared to the parent TCR, the parent TCR and the modified TCR sequence of the present invention were cloned into TCR tandem expression lentiviral vectors, the lentivirus was packaged, the reporter cell JKR9 was transduced, and then the TCR expression-positive cells were sorted. HLA-A*1101-positive T2 cells were loaded with different concentrations of KRASG12V target antigen peptide (SEQ ID NO: 17), and then co-incubated with the JKR9 reporter cells transduced with the parent TCR and the TCR of the present invention for activation. After 16 hours of activation, the reporter gene was stained and flow cytometry was performed. The activation results of the high-affinity TCR of the present invention on the reporter cells are shown in Figure 2. Figure 1 As shown. Figure 1 The EC50 value was calculated based on the results of , the EC50 value of KVA11-N02 was set to 1, and the EC50 enhancement fold of the substituted TCR relative to KVA11-N02 was calculated. The results are shown in Table 2.

[0100] Table 2

[0101]

[0102] As can be seen from Table 2, the affinity of KVA11-M02-321 molecule for KRAS G12V7-16 peptide is 13.85 times higher than that of its parent KVA11-N02. The TCR provided by the present invention has significantly improved affinity compared to the parent TCR.

[0103] Example 2 Evaluation of Cell Membrane Expression Stability of Affinity-Optimized TCR

[0104] Lentiviral vectors encoding KVA11-N02 and affinity-optimized TCRs were packaged in 293T cells and transduced into PBMCs (MOI = 5). Cells were then expanded and cultured until harvest on day 11. At harvest, cells showed good proliferation, ranging from 70 to 150 fold, with viability exceeding 90%. T cells were identified by staining with human CD3-PE antibody; anti-mouse TCR β-APC antibody was used to detect TCR-T cell expression, confirming successful TCR transduction; and CD8-FITC and CD4-PE-Cy7 antibodies were used to determine the CD4 / CD8 ratio.

[0105] The results of flow cytometry analysis showed that under the same transduction and culture conditions, the CD8 / CD4 ratio of each cell after affinity-optimized TCR transduction was close to 3:1, and the TCR positive rate of KVA11-M02-321 was 78.21%, which was significantly higher than that of its parent KVA11-N02 (positive rate was 53.55%) ( Figure 2 This indicates that the affinity-optimized TCR molecule has good membrane expression stability, suggesting that it has the potential to have good antigen reactivity and anti-tumor activity.

[0106] Example 3. Efficient and specific killing of antigen-positive tumor cells by affinity-optimized TCR-T cells

[0107] T cells expressing affinity-optimized TCR or KVA11-N02 were used as effector cells, and PBMCs not transduced with TCR were expanded and cultured in parallel as negative control of effector cells. HELA-A1101-G12V-EL (KRAS G12V positive, HLA-A*11:01 positive), PANC1-A1101-G12V-EL cells (KRAS G12V positive, HLA-A*11:01 positive), T2KO-A1101 loaded with KRAS G12V 7-16 The peptide is used as the positive target cell for HLA-antigen peptide matching. All target cells stably express the luciferase gene. Effector cells are incubated with different target cells at a 1:1 effector-to-target ratio (E:T) for 16-20 hours. Luciferase substrate is added to detect surviving target cells. The proportion of target cells killed is calculated based on the remaining target cells. The results are as follows Figure 3 shown.

[0108] Depend on Figure 3It can be seen that affinity-optimized TCR-T cells have significant cytotoxicity against HELA-A1101-G12V-EL, PANC1-A1101-G12V-EL, and T2KO-A1101 target cells loaded with KRAS G12V7-16 peptide, and their cytotoxicity is significantly higher than that of KVA11-N02 TCR-T cells. These results indicate that affinity-optimized TCR-T cells have the ability to specifically and efficiently kill HLA-antigen peptide-matched target cells, and their cytotoxicity increases with increasing affinity.

[0109] Example 4. Specific INF-γ secretion by affinity-optimized TCR-T cells against antigen-positive tumor cells

[0110] T cells expressing affinity-optimized TCR or KVA11-N02 were used as effector cells, and PBMCs not transduced with TCR were expanded and cultured in parallel as negative control of effector cells. HELA-A1101-G12V-EL (KRAS G12V positive, HLA-A*11:01 positive), PANC1-A1101-G12V-EL cells (KRAS G12V positive, HLA-A*11:01 positive), T2KO-A1101 loaded with KRAS G12V 7-16 The peptides were used as HLA-antigen peptide-matched positive target cells; effector cells were incubated with different target cells at an effector-target ratio (E:T) of 1:1 for 16-20 h, and IFN-γ secretion in the supernatant was detected using an IFN- ELISA kit (ExCell, Cat# EH008-96). The results are shown in Figure 2. Figure 4 shown.

[0111] The results showed that affinity-optimized TCR-T cells had strong affinity with positive target cells HELA-A1101-G12V-EL, PANC1-A1101-G12V-EL, T2KO-A1101 loaded with KRAS G12V 7-16 After co-incubation with the peptides, significant IFN-γ secretion was detected, and the amount of IFN-γ secreted was significantly higher than that of KVA11-N02 TCR-T cells. These results indicate that affinity-optimized TCR-T cells have a good specific killing effect on HLA-antigen peptide-matched target cells, and this specific killing effect is enhanced with the increase in affinity.

[0112] Example 5 Activation of affinity-optimized TCR-T cells after co-incubation with non-target cells

[0113] In order to further verify the specificity of the affinity-enhancing molecules, tumor cell lines derived from different human tissues were selected for activation testing. One or more cell lines derived from cervical cancer, human lung squamous cell carcinoma, liver cancer, prostate cancer, esophageal cancer, breast cancer, bladder transitional carcinoma, renal cell adenocarcinoma, colorectal adenocarcinoma, glioma, melanoma, tongue squamous cell carcinoma, and ovarian adenocarcinoma are used as target cells, specifically including NCI-H1703, HUH7-A1101, PC-3-A1101, TE-1-A1101, MDA-MB-231-A1101, J82-A1101, Siha-A1101, ACHN-A1101, HT29-A1101, GOS3-A1101, SK-MEL-5-A1101, CAL-27-A1101, and OVCAR-3-A1101, to simulate protein expression in different tissues. The combination of cells derived from multiple tissue types is used to cover the human protein expression spectrum as much as possible. All cell lines were transduced with the HLA-A*11:01 gene as nonspecific antigen-presenting cells to test whether the CRTKVA11-M02-321 molecule could respond to nonspecific antigens.

[0114] Reporter cells expressing CRTKVA11-M02-321 were used as effector cells, and Hela-A1101-G12V (overexpressing KRAS G12V and HLA-A*11:01) were used as target antigen-positive cells, with 1×10 cells of each type. 5 The cells were incubated with CRTKVA11-M02-321 reporter cells (target ratio 1:1) overnight to detect the activation of the reporter cells. Figure 5 and Figure 6 shown.

[0115] The results showed that CRTKVA11-M02-053 produced an activation response to all non-specific antigen-presenting target cells, indicating that CRTKVA11-M02-053 TCR did not have a non-specific activation response to HLA-A*11:01-positive tumor cell lines of different tissue origins, demonstrating excellent antigen specificity.

[0116] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made. This includes combining the specific technical features in any suitable manner. To avoid unnecessary repetition, the present invention will not further describe various possible combinations. However, these simple variations and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A modified T cell receptor targeting KRAS G12V, comprising a T cell receptor (TCR) α chain variable region and a TCR β chain variable region, wherein: The TCR α chain variable region comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and the TCR β chain variable region comprises β chain CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

2. The T cell receptor according to claim 1, wherein The T cell receptor binds to an epitope comprising the amino acid sequence VVVGAVGVGK (SEQ ID NO: 17) or a complex of the epitope with an MHC molecule.

3. The T cell receptor according to claim 1 or 2, wherein The TCR α chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and / or the TCR β chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

4. The T cell receptor according to claim 1 or 2, wherein The TCR further comprises a TCR constant region, which is a mouse constant region or a human constant region.

5. The T cell receptor according to claim 4, wherein The TCR constant region includes a TCR α chain constant region and a TCR β chain constant region; Wherein, the TCR α chain constant region comprises the amino acid sequence shown in SEQ ID NO: 9, and / or the TCR β chain constant region comprises the amino acid sequence shown in SEQ ID NO:

10.

6. The T cell receptor according to claim 4, wherein The T cell receptor comprises a TCR α chain having an amino acid sequence as shown in SEQ ID NO: 11; and / or a TCR β chain having an amino acid sequence as shown in SEQ ID NO:

12.

7. A nucleic acid encoding the T cell receptor according to any one of claims 1 to 6.

8. The nucleic acid according to claim 7, wherein The nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 13, and / or the nucleotide sequence shown in SEQ ID NO: 14; or The nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:15 and / or the nucleotide sequence shown in SEQ ID NO:

16.

9. A vector comprising the nucleic acid according to claim 7 or 8. 10 . A cell comprising the T cell receptor according to claim 1 , the nucleic acid according to claim 7 or 8 , or the vector according to claim 9 .

11. Use of the T cell receptor according to any one of claims 1 to 6, the nucleic acid according to claim 7 or 8, or the vector according to claim 9 in the preparation of a product for alleviating or treating KRAS G12V-positive and HLA-A1101-restricted pancreatic cancer.

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