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

Abstract

The invention relates to the field of immunity, in particular to a modified T cell receptor targeting KRAS G12V or a fragment thereof and application. The modified T cell receptor targeting KRAS G12V or a fragment thereof provided by the invention comprises a TCR alpha chain variable region and a TCR beta chain variable region, wherein the TCR alpha chain variable region comprises CDR1, CDR2 and CDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and the TCR beta chain variable region comprises beta chains CDR1, CDR2 and CDR3, wherein the amino acid sequences of the beta chains CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The TCR can bind to a 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 includes three genes: NRAS, HRAS and KRAS, which encode a small GTPase membrane-bound protein that plays a major role in signal transduction for cell growth regulation. RAS plays a core role in the cell signal transduction process: it can regulate transcription, translation and other processes at the molecular level, and regulate cell proliferation, differentiation, aging, apoptosis and so on 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. In the activated state, 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 on the 12th codon of exon 2, and the most common types are G12D, G12V and G12C.

[0004] Although there is a broad consensus on the important role of KRAS mutations in tumors, no drugs targeting KRAS have been approved for marketing. In recent years, new drugs specifically targeting KRAS G12C mutations have made some progress: small molecule drugs targeting KRAS can directly inhibit KRAS protein activity. KRAS protein is widely expressed, and arbitrarily inhibiting wild-type and mutant KRAS proteins may produce unacceptable toxicity. Another way to directly inhibit KRAS activity is to interfere with the binding of KRAS to the lipid membrane periphery, which is also unlikely to distinguish between mutant proteins and wild-type proteins, leading to potential toxicity problems. KRAS protein can regulate many downstream effector molecules, mainly RAF / MEK / ERK and PI3K / AKT / mTOR pathways. However, due to the existence of a large number of feedback mechanisms, clinical trials have shown that the efficacy of multiple downstream molecule inhibitors is limited.

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

[0006] The present invention uses a human KRAS G12V-specific wild-type TCR sequence as a template, uses an in vitro mutagenesis method to mutate the complementary 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 a 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 having amino acid sequences as 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 having amino acid sequences as 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 the present invention has the ability to bind target antigen peptides with high affinity, significantly improves the target cell recognition ability, and exhibits good expression stability and membrane stability, ensuring its long-lasting and high efficiency in complex in vivo environments. By mediating the specific killing effect of effector cells on antigen-positive target cells, it can be used to treat a variety of cancers positive for KRAS G12V, providing an important tool for cell therapy, showing 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 It is shown that the affinity-optimized TCR provided by the present invention has good membrane expression stability.

[0016] Figure 3 It is shown that the affinity-optimized TCR provided by the present invention has highly efficient specific killing ability.

[0017] Figure 4 It is shown 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 reaction 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 those generally 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 "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 references unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" may be used interchangeably. Similarly, the terms "comprising," "including," and "having" may be used interchangeably.

[0021] When the term "comprising" is used herein and in the appended claims, it does not exclude other elements. For the purpose 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 of only these embodiments.

[0022] The present disclosure provides a modified T cell receptor or a fragment thereof targeting KRAS G12V, which comprises 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 having amino acid sequences as shown in 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 having amino acid sequences as shown in 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 in their research 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 affinity" 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 affinity respond to very low antigen doses in in vitro tests, while such cells with lower binding affinity require higher amounts of antigen before reaching an immune response similar to that of cells expressing TCRs with high affinity. Therefore, binding affinity can be considered as a quantitative determinant of the activation threshold of cells expressing TCRs. This is determined by exposing such cells to different amounts of cognate antigens in vitro. Cells with TCRs with high binding affinity 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 present 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 recognized by a binding domain. The term "binding domain" refers to an "antigen binding site" in its broadest sense, i.e., a domain that characterizes a molecule that binds / interacts with a specific epitope on an antigen target. An antigen target may contain a single epitope, but typically contains at least two epitopes, and depending on the size, conformation, and type of the antigen, the antigen target may include any number of epitopes. The term "epitope" generally includes linear epitopes and conformational epitopes. A linear epitope is a continuous epitope contained in an amino acid primary sequence, 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 a target antigen, and particularly a target (poly)peptide.

[0028] As used herein, the term "T cell receptor" or "TCR" can be used interchangeably to refer to a natural TCR or its variants, fragments and constructs. The term thus includes heterodimers, 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 a 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 complementary determining regions (CDR1α / β, CDR2α / β, and CDR3α / β) surrounded by framework (FR) regions. CDR3 is the main determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 mainly interact with MHC molecules presenting antigenic peptides.

[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 with 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 TCR binds to its specific epitope-MHC complex, the T cell is activated and exerts biological effector functions.

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

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

[0034] In some embodiments of the invention, the TCR α 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] The term "sequence identity" as used herein 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 usually 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 some 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] Therefore, the amino acid sequence of SEQ ID NO: 7 or 8 can be used as a "subject sequence" or "reference sequence", while the amino acid sequence of the variable region of the TCR α chain or β chain different therefrom can be used as a "query sequence".

[0038] In some embodiments, the TCR α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the TCR β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the TCR α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the TCR β chain variable region comprises the amino acid sequence shown 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 different second 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 a soluble TCR. Soluble TCR is generally a fragment or construct comprising a TCR α chain and / or β chain or its variable region or CDR, and optionally it is stabilized by a disulfide bond or covalently linked by a suitable linker. Typically, a soluble TCR does 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" may be a human constant region or derived from another species, thereby creating a "chimeric" TCR. For example, the human α and / or β chains may be replaced ("murinization") with their murine counterparts, 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 is reported that adding a disulfide bond 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 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 contains 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 the position of the cysteine ​​mutation, the position of the amino acid sequence of the wild-type TCR constant region in the present invention is numbered according to the naming rules of the International Immunogenetics Information System (IMGT). For example, an amino acid in the TCR α chain constant region (TRAC) is numbered 48 in the position listed in IMGT, and it is described herein as the 48th amino acid in the TCR α chain constant region (TRAC); an amino acid in the TCR β chain constant region (TRBC) is numbered 57 in the position listed in IMGT, and it is described herein as the 57th amino acid in the TCR β chain constant region (TRBC), and so on. Herein, the position numbering of the amino acid sequence of the variable region TRAV and TRBV follows the position numbering listed in IMGT. For example, an amino acid in TRAV is numbered 46 in the position listed in IMGT, and it is described herein as the 46th amino acid in TRAV, and so on. In the present invention, if the sequence position numbering of other amino acids is specially specified, it is in accordance with the special instructions.

[0051] In some embodiments, the TCR α chain constant region further comprises an LVL mutation or a 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 mouse constant region, the mouse 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 α 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 β 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 α chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 9, and the TCR β chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 10.

[0053] In some embodiments, the fragment of the TCR constant region is an 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 may include a leader sequence. For example, the leader sequence of the TCR α chain may have an amino acid sequence (MASAPISMLAMLFTLSGLR) as shown in SEQ ID NO: 18. The leader sequence of the TCR β chain may have an amino acid sequence (MDSWTFCCVSLCILVAKHT) as 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 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% identity to any one selected from SEQ ID NO: 11; in some embodiments, the TCR comprises a TCR beta chain comprising 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 selected from SEQ ID NO: 12. In some embodiments, the TCR comprises a TCR alpha chain as described in SEQ ID NO: 11 and a TCR beta chain as described in SEQ ID NO: 12.

[0057] In some embodiments, the TCR further comprises an intracellular signaling region. In some embodiments, the TCR further comprises one or more antigen binding regions 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 it 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 The present disclosure provides nucleic acids encoding the TCRs of the present disclosure.

[0061] 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 α 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 β chain variable region.

[0062] 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 with 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 with SEQ ID NO: 21.

[0063] 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.

[0064] The present disclosure provides vectors comprising a nucleic acid of the present disclosure.

[0065] The term "vector" as used herein is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule as a vector can, for example, be replicated and / or expressed. The term "vector" encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral 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 of autonomous replication in the host cell (if stable expression of polynucleotides is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may 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 number 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 Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is incorporated herein by reference in its entirety.

[0066] 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.

[0067] The present disclosure provides a cell comprising a TCR, a nucleic acid, or a vector according to the present disclosure.

[0068] The term "cell" as used herein 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, etc. 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.

[0069] 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 may be a lymphoid progenitor cell, an induced pluripotent stem cell (iPSC), or a hematopoietic stem cell (HSC). In some embodiments, stem cells do not include embryonic stem cells obtained by destroying human embryos, and / or do not include omnipotent stem cells for development and formation of animal individuals. 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.

[0070] In some embodiments, the stem cell is a lymphoid progenitor cell or an induced pluripotent stem cell (iPSC).

[0071] In some embodiments, the cell is a T cell. The T cell can be any T cell, such as a cultured T cell, such as 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 a person. 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.

[0072] 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.

[0073] As used herein, the term "transfection" is a process in which nucleic acid molecules or polynucleotides (including vectors) are intentionally introduced into target cells. An example is RNA transfection, which is the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into host cells. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of viral-mediated nucleic acid molecules or polynucleotides. The transfection of animal cells generally involves opening a transient hole or "hole" in the cell membrane to allow the uptake of materials. 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 into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation.

[0074] In some embodiments, the method further comprises the step of expanding and / or activating the cells either before or after the transduction or transfection.

[0075] 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.

[0076] In some embodiments, the product is a drug, which comprises the T cell receptor or a fragment thereof disclosed herein, the nucleic acid disclosed herein, or the vector disclosed herein.

[0077] In some embodiments, the drug is in the form of a conjugate, which includes a TCR of the present disclosure and an active agent coupled or conjugated to the TCR.

[0078] 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 having 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, rachlombycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, pyroximate sodium photofrin II, 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, which can be used to promote the binding of these radionuclides to molecules or their polymers; or heterologous protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0079] In some embodiments, the drug is in the form of a composition, which comprises the TCR, nucleic acid, vector, or cell of the present disclosure. Preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0080] The term "composition" refers in particular 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., active agent and optional carrier or excipient) are preferably pharmaceutically acceptable, i.e., capable of inducing the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutically acceptable composition of the present invention may be, for example, 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.

[0081] The term "excipient" includes fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavoring agents, 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 to select suitable excipients to prepare the compositions of the present invention. Exemplary carriers for the compositions of the present invention include saline, buffered saline, glucose and water. Typically, the selection of suitable excipients depends particularly on the desired dosage form of the active agent used, the disease to be treated and the compositions.

[0082] Depending on the active agent used (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. The processes known to the invention for producing drugs 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.

[0083] 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.

[0084] 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.

[0085] 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 sample to be tested, wherein the epitope comprises a KRAS G12V mutation epitope.

[0086] 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.

[0087] In some embodiments, the conjugate comprises a detectable label. Examples of detectable labels include, but are not limited to, 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 an enzyme or a catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent or chemiluminescent molecule. In some embodiments, the kit may further comprise instructions for how to use the kit.

[0088] In an embodiment 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.

[0089] The present invention is further described by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are carried out according to the conventional conditions in the art, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0090] Example Example 1 Screening of modified TCRs with enhanced affinity The inventors used the human KVA11-N02 sequence (202310065643.3) as a template, substituted the complementary determining region (CDR) of KVA11-N02 by in vitro substitution, and obtained high-affinity substitution clones by in vitro positive screening of the substituted TCR. Through this method, the preferred TCR sequence screened had amino acid mutations at three sites in CDR1α, thereby obtaining a modified TCR named KVA11-M02-321. The specific TCR information is shown in Table 1 below.

[0091] In order to increase the correct pairing and expression of exogenous TCR on the surface of human T cells, the α chain and β chain variable regions of TCR were fused to the mouse TCR constant region, and the classic α chain C region "LIV" substitution was introduced into the mouse TCR constant region (i.e., amino acid substitutions were introduced into the mouse 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 mouse TCR constant region.

[0092] Through the 2A self-cleavage peptide (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 22), the α chain and β chain of TCR (the N-termini of the α chain and β chain 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.

[0093] Table 1

[0094]

[0095] In order to determine the degree of improvement in affinity of the TCR provided by the present invention compared to the parent TCR, the parent TCR and the modified TCR sequences of the present invention were cloned into TCR tandem expression lentiviral vectors, packaged with lentivirus, transduced into reporter cells JKR9, and then sorted for TCR expression-positive cells. 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 JKR9 reporter cells transduced with parent TCR and TCR of the present invention for activation. After 16 hours of activation, the reporter gene was stained for flow cytometry analysis. The activation results of the high-affinity TCR of the present invention on reporter cells are shown in Figure 1. Figure 1 As shown. Figure 1 The EC50 value was calculated based on the results, the EC50 value of KVA11-N02 was set to 1, and the EC50 enhancement factor of the replaced TCR relative to KVA11-N02 was calculated. The results are shown in Table 2.

[0096] Table 2

[0097] As can be seen from Table 2, the affinity of KVA11-M02-321 molecule to KRAS G12V7-16 peptide is 13.85 times higher than that of its parent KVA11-N02. Compared with the parent TCR, the affinity of the TCR provided by the present invention is significantly improved.

[0098] Example 2 Evaluation of cell membrane expression stability of affinity-optimized TCR 293T cells were used to package lentiviruses that produced KVA11-N02 and affinity-optimized TCR, respectively. After transduction of PBMC (MOI=5), the cells were amplified and cultured until harvest on day 11. At the time of harvest, the cells proliferated well, with a proliferation multiple of 70-150 times and a cell viability of >90%. Human CD3-PE antibody was used to confirm T cells; anti-mouse TCR β-APC antibody was used to detect the expression rate of TCR-T and prove that TCR transduction was successful; CD8-FITC and CD4-PE-Cy7 antibodies were used to detect the ratio of CD4 / CD8.

[0099] The results of flow cytometry analysis showed that under the same transduction and culture conditions, the ratio of CD8 / CD4 expressed by each cell after affinity-optimized TCR transduction was close to 3:1, and the positive rate of KVA11-M02-321 TCR 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.

[0100] Example 3. Efficient and specific killing of antigen-positive tumor cells by affinity-optimized TCR-T cells

[0101] T cells expressing affinity-optimized TCR or KVA11-N02 were used as effector cells, and PBMCs without TCR transduction 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 was used as the positive target cell matched with HLA-antigen peptide. All target cells stably expressed the luciferase gene. The effector cells were incubated with different target cells at a 1:1 effector-target ratio (E:T) for 16-20 hours, and the luciferase substrate was added to detect the surviving target cells. The proportion of target cells killed was calculated based on the remaining target cells. The results are as follows: Figure 3 shown.

[0102] Depend on Figure 3It can be seen that affinity-optimized TCR-T cells have significant killing activity against HELA-A1101-G12V-EL, PANC1-A1101-G12V-EL, and T2KO-A1101 positive target cells loaded with KRAS G12V7-16 peptide, and their killing activity is significantly higher than that of KVA11-N02 TCR-T cells. These results show that affinity-optimized TCR-T cells have the ability to efficiently and specifically kill HLA-antigen peptide-matched target cells, and their killing activity increases with the increase in affinity.

[0103] Example 4. Specific INF-γ secretion by affinity-optimized TCR-T cells against antigen-positive tumor cells T cells expressing affinity-optimized TCR or KVA11-N02 were used as effector cells, and PBMCs without TCR transduction 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 a 1:1 effector-target ratio (E:T) for 16-20 h, and the IFN-γ secretion in the supernatant was detected using an IFN-ELISA kit (ExCell, Cat# EH008-96). The results are shown in Figure 4 shown.

[0104] The results showed that affinity-optimized TCR-T cells and positive target cells HELA-A1101-G12V-EL, PANC1-A1101-G12V-EL, T2KO-A1101 loaded with KRAS G12V 7-16 After co-incubation with peptides, significant IFN-γ secretion can be detected, and the amount of IFN-γ secretion is significantly higher than that of KVA11-N02 TCR-T cells. These results show that affinity-optimized TCR-T cells have good specific killing effects on HLA-antigen peptide-matched target cells, and this specific killing effect is enhanced with the increase of affinity.

[0105] Example 5 Activation of affinity-optimized TCR-T cells after co-incubation with non-target cells 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 cells, 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 the protein expression of different tissues, and to cover the human protein expression spectrum as much as possible through the combination of cells derived from multiple tissue types. All cell lines were transduced with the HLA-A*11:01 gene as nonspecific antigen presenting cells to detect whether the CRTKVA11-M02-321 molecule could respond to nonspecific antigens.

[0106] 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 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.

[0107] 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 from different tissues, showing excellent antigen specificity.

[0108] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications. Including the combination of various specific technical features in any suitable manner. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. However, these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A modified T cell receptor or fragment thereof targeting KRAS G12V, comprising 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 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 or fragment thereof according to claim 1, wherein The T cell receptor or a fragment thereof binds to an epitope comprising the amino acid sequence shown in SEQ ID NO: 17 or a complex of the epitope and an MHC molecule.

3. The T cell receptor or fragment thereof 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 or fragment thereof according to claim 1 or 2, wherein The TCR further comprises a TCR constant region or a fragment thereof, wherein the TCR constant region is a mouse constant region or a human constant region.

5. The T cell receptor or fragment thereof 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 or fragment thereof 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 a T cell receptor or a fragment thereof 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 a T cell receptor or a fragment thereof according to any one of claims 1 to 6, a nucleic acid according to claim 7 or 8, or a vector according to claim 9.

11. Use of a T cell receptor or a fragment thereof according to any one of claims 1 to 6, a nucleic acid according to claim 7 or 8, or a vector according to claim 9 in the preparation of a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12V-positive disease or condition.

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