TCR molecule and cell targeting KRAS G12 mutation and application of TCR molecule and cell

Abstract

The present invention provides a binding protein comprising a binding domain having antigen specificity for an antigen peptide: HLA complex, the binding domain comprising a T cell receptor (TCR) alpha chain variable region and a TCR beta chain variable region. Also provided herein are uses of the binding proteins and pharmaceutical compositions comprising the binding proteins in the treatment of cancers associated with the antigen peptides.

Description

TCR molecules and cells targeting KRAS G12 mutation and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application No. 202310484887.5 filed with the China Patent Office on April 28, 2023, the entire text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to TCR molecules and cells expressing the TCR molecules, in particular TCR molecules targeting target cells carrying KRAS G12 mutations and immune effector cells expressing the TCR molecules. The present invention also relates to therapeutic applications of the TCR molecules and cells. Background Art

[0004] The RAS gene family (HRAS, KRAS, NRAS) is the most common proto-oncogene that can cause tumorigenesis. The most common of these is the KRAS mutation (concentrated at codon 12), which is present in approximately 22-30% of tumors, and is particularly prevalent in pancreatic cancer (~60-90%), colorectal cancer (~40%), lung cancer (~10% in China, ~30% in Europe and the United States), bile duct cancer (~17%-37%), ovarian cancer, and endometrial cancer (~10-40%, related to pathological classification, with a high incidence of serous type). Taking pancreatic cancer as an example, in 2021, it is estimated that approximately 60,430 patients will be diagnosed with pancreatic cancer and approximately 48,220 patients will die from pancreatic cancer. Although advances have been made in the diagnosis and treatment of pancreatic cancer over the past decade, the 5-year survival rate of pancreatic cancer is approximately 9% due to the lack of effective treatments. Current treatment for unresectable pancreatic cancer mainly involves chemotherapy to shrink the tumor, but this is limited by the emergence of drug resistance. In addition, the success of targeted therapy is also limited. Furthermore, pancreatic cancer is also resistant to immunotherapy, possibly due in part to the low mutation burden of the disease, which leads to a lack of neoantigen-reactive tumor-infiltrating lymphocytes. Research targeting KRAS-G12 TCR-T cells could effectively improve the current predicament of having no effective treatment options for KRAS-mutant tumors.

[0005] Summary of the Invention

[0006] In one aspect, provided herein is a binding protein comprising a binding domain having antigenic specificity for an antigenic peptide:HLA complex, said binding domain comprising a T cell receptor (TCR) alpha chain variable region and a TCR beta chain variable region, wherein:

[0007] The antigenic peptide comprises the amino acid sequence VVVGAXGVGK (SEQ ID NO: 11), wherein X is any amino acid residue that is not G or R;

[0008] The HLA is HLA-A*11:01;

[0009] The CDR3 of the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO: 5; the CDR3 of the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0010] In some embodiments, the antigenic peptide comprises VVVGAVGVGK (SEQ ID NO: 12), VVVGADGVGK (SEQ ID NO: 13), VVVGASGVGK (SEQ ID NO: 14), VVVGAAGVGK (SEQ ID NO: 15), or VVVGACGVGK (SEQ ID NO: 16).

[0011] In some embodiments, in the α chain variable region, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO:5; in the β chain variable region, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO:10.

[0012] On the other hand, the present invention provides a binding protein, which includes a binding domain, wherein the binding domain includes a T cell receptor (TCR) α chain variable region and a TCR β chain variable region, wherein CDR1 in the α chain variable region includes the amino acid sequence shown in SEQ ID NO: 3, CDR2 includes the amino acid sequence shown in SEQ ID NO: 4, and CDR3 includes the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 5; and CDR1 in the β chain variable region includes the amino acid sequence shown in SEQ ID NO: 8, CDR2 includes the amino acid sequence shown in SEQ ID NO: 9, and CDR3 includes the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 10.

[0013] In some embodiments, the α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 2; the β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 7.

[0014] In some embodiments, the α chain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 1; the β chain comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 6.

[0015] In some embodiments, the binding protein is a protein in the following form:

[0016] 1) TCR molecules;

[0017] 2) antibody molecules; or

[0018] 3)CAR molecule.

[0019] In some embodiments, when T cells expressing the above-mentioned binding protein are incubated with HLA-A*11:01 cells in the presence of the antigenic peptide, or when T cells expressing the above-mentioned binding protein are incubated with HLA-A*11:01 cells expressing the antigenic peptide, the T cells specifically kill the HLA-A*11:01 cells.

[0020] In some embodiments, when T cells expressing the above-mentioned binding protein are incubated with HLA-A*11:01 cells in the presence of the antigenic peptide, or when T cells expressing the above-mentioned binding protein are incubated with HLA-A*11:01 cells expressing the antigenic peptide, the T cells express CD69, or the amount of CD69 expressed is increased.

[0021] In some embodiments, the binding protein further comprises a conjugate covalently or non-covalently linked to the binding domain; preferably, the conjugate is a detectable label, a radioactive isotope or a therapeutic agent.

[0022] In another aspect, provided herein are isolated nucleic acid molecules encoding the above-described binding proteins, α chain variable regions, β chain variable regions, α chains, or β chains.

[0023] In another aspect, provided herein is a vector comprising the above-described nucleic acid molecule.

[0024] In another aspect, provided herein is a host cell that expresses the above-mentioned binding protein, nucleic acid molecule or vector.

[0025] In some embodiments, the host cell is a mammalian cell, preferably a human cell.

[0026] In some embodiments, the host cell is a T cell or a NK cell.

[0027] In some embodiments, the host cell has killing activity against HLA-A*11:01 cells expressing the antigenic peptide VVVGAXGVGK (SEQ ID NO: 11), wherein X is any amino acid residue that is not G or R.

[0028] In some embodiments, the antigenic peptide comprises VVVGAVGVGK (SEQ ID NO: 12), VVVGADGVGK (SEQ ID NO: 13), VVVGASGVGK (SEQ ID NO: 14), VVVGAAGVGK (SEQ ID NO: 15), or VVVGACGVGK (SEQ ID NO: 16).

[0029] In another aspect, provided herein is a pharmaceutical composition comprising: 1) the binding protein, nucleic acid molecule, vector or host cell described above; and 2) a pharmaceutically acceptable carrier.

[0030] On the other hand, the present invention provides the use of the above-mentioned binding protein, nucleic acid molecule, vector or host cell in the preparation of a medicament for treating a tumor, wherein the tumor expresses the above-mentioned antigenic peptide.

[0031] In another aspect, provided herein is a method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the binding protein, nucleic acid molecule, vector, host cell, or pharmaceutical composition described above.

[0032] In some embodiments, the tumor is selected from pancreatic cancer, colorectal cancer, lung cancer, bile duct cancer, endometrial cancer, and ovarian cancer.

[0033] In another aspect, provided herein is a detection kit comprising the above-mentioned binding protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows a flow cytometry diagram of the enrichment and sorting of HLA-A*11:01 / KRAS G12V-specific T cells by tetramers after stimulation.

[0035] Figure 2A shows CD8 T cells that were not transduced and transduced with HLA-A*11:01 / KRAS G12V-specific TCR1. + The results of T cells killing K562-A*11:01 target cells loaded with different antigenic peptides. The loaded peptides in the figure are:

[0036] KRAS-G12V peptide: VVVGAVGVGK (SEQ ID NO: 12)

[0037] KRAS-G12D peptide: VVVGADGVGK (SEQ ID NO: 13)

[0038] KRAS-G12S peptide: VVVGASGVGK (SEQ ID NO: 14)

[0039] KRAS-G12A peptide: VVVGAAGVGK (SEQ ID NO: 15)

[0040] KRAS-G12C peptide: VVVGACGVGK (SEQ ID NO: 16)

[0041] WT-10 peptide: VVVGAGGVGK (SEQ ID NO: 17).

[0042] Figure 2B shows the CD8 T cells that were not transduced and transduced with HLA-A*11:01 / KRAS G12V-specific TCR1. + T cell killing results against BxPC3 cells overexpressing HLA-A*11:01 and / or KRAS G12V or G12D. BxPC3-A1101 is a BxPC3 cell transduced with HLA-A*11:01, BxPC3-A1101-G12V is a BxPC3 cell transduced with both HLA-A*11:01 and KRAS G12V, and BxPC3-A1101-G12D is a BxPC3 cell transduced with both HLA-A*11:01 and KRAS G12D.

[0043] Figure 3 shows the TCR1 transduced Jurkat cells and loaded with different concentrations of peptide (10 -11 -10 -6 M) target cells (K562-A*11:01) were co-incubated and the expression of CD69 was detected by flow cytometry. DETAILED DESCRIPTION

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0045] The term "or" refers to a single element of the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0046] The term "about" generally refers to a variation within a range of 10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0047] The terms "comprising" or "including" refer to including the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. As used herein, when the terms "comprising" or "including" are used, unless otherwise indicated, they also encompass the context of consisting of the recited elements, integers, or steps. For example, reference to a variable region "comprising" a specific sequence is intended to encompass a variable region consisting of that specific sequence.

[0048] The term "major histocompatibility complex (MHC)" is a general term for a group of genes that encode the major histocompatibility antigens of animals. At present, the structures of MHC class I and MHC class II molecules have been completely resolved and belong to the immunoglobulin superfamily. The human MHC is called HLA (human leukocyte antigen, HLA), that is, human leukocyte antigen. HLA class I molecules mainly include the protein products (heavy chains or α chains) of three functional genes HLA-A, HLA-B, and HLA-C. Each class I HLA heavy chain pairs with β2 microglobulin (β2m) to form a complete MHC class I molecule in the form of a heterodimer. Due to the polymorphism of the genes encoding HLA molecules, the HLA Nomenclature Committee has established naming guidelines for specific alleles. Key points include: for a specific allele, the locus name of the gene is written after the three letters HLA, separated by a hyphen, such as HLA-A; an asterisk (*) is added to the locus name, followed by the gene family number of the allele, corresponding to the corresponding serological typing as much as possible, followed by a colon (:) and the specific numeric number of the allele, such as HLA-A*02:01. One of the primary functions of MHC molecules is antigen presentation. MHC molecules bind to antigenic peptides through their peptide-binding grooves to form peptide-MHC complexes (pMHCs), which are then presented on the cell surface for T cell recognition (via the TCR) and the initiation of an immune response.

[0049] The term "antigenic peptide" refers to a short peptide capable of being bound by the peptide binding groove of an MHC molecule (or HLA molecule). The antigen binding groove of an HLA class I molecule is composed of the α1 and α2 domains of the heavy chain (whose extracellular region includes three structures, α1, α2, and α3). Each domain folds into an α helix and four β sheets. Two α helices form the walls of the groove, while eight β sheets form the bottom of the groove. Antigenic peptides suitable for accommodation in this binding groove are typically 8-11 amino acids in length. In one embodiment, the antigenic peptide is a mutant KRAS protein fragment, such as VVVGAVGVGK (SEQ ID NO: 12).

[0050] The term "antigenic peptide:MHC complex" or "antigenic peptide:HLA complex" refers to a complex (pMHC) consisting of an MHC molecule and an antigenic peptide, with the antigenic peptide located in the peptide-binding groove. TCR molecules recognize this complex and are expected to bind specifically to it via the CDR sequences within their binding domains. Binding of TCR molecules to the pMHC complex is MHC-dependent, meaning that the TCR molecule recognizes the pMHC complex simultaneously with the antigenic peptide.

[0051] The term "T cell receptor (TCR)" refers to the functional unit that recognizes antigens (pMHC) on T lymphocytes and belongs to the immunoglobulin superfamily. TCRs expressed on T cells are glycoproteins on the cell membrane surface, consisting of either α / β chains or γ / δ chains, present as heterodimers. In humans, the vast majority (90%-95%) of TCRs in peripheral blood are heterodimers composed of two polypeptide chains, α and β. Both α and β chains are divided into variable regions (Vα and Vβ), constant regions (Cα and Cβ), transmembrane regions, and cytoplasmic regions. The cytoplasmic regions of both α and β chains are very short, consisting of only a few amino acids. The sequence of the constant regions (Cα and Cβ) varies relatively little, and their specific amino acid sequences can be found, for example, in the public database of the International Immunogenetics Information System (IMGT). The transmembrane region contains positively charged amino acids that non-covalently bind to the negatively charged amino acids in the transmembrane region of the CD3 molecule, stabilizing the structure of the TCR-CD3 complex. Amino acid sequence analysis of the TCR variable region reveals that Vα and Vβ each have three hypervariable regions, termed complementarity-determining regions (CDRs), namely CDR1, CDR2, and CDR3. CDR3 exhibits the greatest variation and largely determines the antigen specificity of the TCR. In addition to the hypervariable regions, similar to antibodies, the variable region also includes four framework regions (FR1-FR4). The positions of CDR1-CDR3 and FR1-FR4 within the full-length TCR sequence are defined according to the IMGT nomenclature, which is well known and available in the IMGT public database. The variable regions of the α and β chains, specifically the three CDRs in the α chain and the three CDRs in the β chain, collectively form the portion of the TCR that specifically recognizes and binds to pMHC complexes (herein referred to as the "binding domain"). In the immune system, binding of the antigen-specific TCR to the pMHC complex initiates direct physical contact between a T cell and an antigen-presenting cell (APC) or target cell, leading to T cell activation or cytotoxicity. Unless the context indicates or implies otherwise, when referring to a TCR the term may also encompass functional fragments thereof that have antigen specificity, such as the Vα and Vβ variable regions connected by a short peptide.

[0052] The term "antibody" is used in its broadest sense herein, including immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, which are proteins or polypeptides that exhibit binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on the conservative differences in the amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind to the antigen, and the constant region (e.g., Fc fragment) is used to initiate downstream effects, such as antibody-dependent cellular cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions with a higher degree of variation in amino acid composition and arrangement order, which are key positions for antibody-antigen binding and are therefore also referred to as complementary determining regions (CDRs). The three complementarity determining regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3, respectively, and the three complementarity determining regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, respectively. Each heavy chain variable region (VH) and light chain variable region (VL) can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Herein, the antibody molecule can also include a fusion protein composed of the variable region of a TCR molecule and the constant region of an antibody molecule, which has both the antigen specificity of the TCR molecule and the effector function of the constant region of the antibody molecule.

[0053] The term "chimeric antigen receptor (CAR)" refers to an engineered membrane protein receptor molecule that can confer desired specificity to immune effector cells, such as the ability to bind to specific tumor antigens. Chimeric antigen receptors are generally composed of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen binding domain is a scFv sequence or a single domain antibody fragment that is responsible for recognizing and binding to a specific antigen. The intracellular signaling domain generally includes an immunoreceptor tyrosine activation motif (ITAM), such as a signaling domain derived from a CD3ζ molecule, which is responsible for activating immune effector cells and producing a killing effect. In addition, the chimeric antigen receptor may also include a signal peptide at the amino terminus that is responsible for the localization of the nascent protein in the cell, and a hinge region between the antigen binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also include a costimulatory domain derived from, for example, a 4-1BB or CD28 molecule.

[0054] The terms "antigen specificity", "targeting" or "specific binding" refer to the property of a TCR (or other fusion proteins including its binding domain, such as CAR) that can specifically recognize and bind to the corresponding pMHC complex with high affinity. TCR (or other fusion proteins including its binding domain, such as CAR) can have a higher binding affinity to another molecule (such as a pMHC complex) for which it has antigen specificity relative to other molecules present in the environment. The binding affinity of TCR to pMHC complex can be measured by a number of parameters, such as EC 50 value or KD value.

[0055] The term "binding protein" refers to a protein that has specific binding ability to a specific ligand (such as an antigen). In this article, binding proteins can include TCR molecules (including single-chain TCR and water-soluble TCR molecules) that have specific binding ability to pMHC complexes, fusion proteins formed by the TCR molecule with other proteins (such as antibody molecules or chimeric antigen receptors), and immunoconjugates (connected to detectable labels, therapeutic agents, or radioisotopes).

[0056] The term "fusion protein" refers to a protein molecule artificially generated (e.g., through genetic engineering) that is composed of at least two different peptide segments. These peptide segments do not exist in nature or do not exist in the same protein molecule. Common examples of fusion proteins including antibody fragments include multispecific antibodies, antibody-cytokine fusion proteins, enzyme-labeled antibodies for immunoassays, and chimeric antigen receptors (CARs).

[0057] When referring to a TCR (or antigen-specific fragment thereof), the term "variant" as used herein refers to a protein obtained by introducing one or more amino acid insertions, deletions, or substitutions into a parent TCR (or antigen-specific fragment thereof), which still retains at least some of the functions of the parent TCR (or antigen-specific fragment thereof) (particularly functions of interest, such as the ability to bind to a corresponding pMHC complex). For example, a variant of a TCR (or antigen-specific fragment thereof) may retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the binding ability of its parent TCR (or antigen-specific fragment thereof) to a pMHC complex, or even have a higher binding ability than the parent TCR (or antigen-specific fragment thereof). In some embodiments, a variant of a TCR (or antigen-specific fragment thereof) may retain at least 80%, 85%, 90%, 95%, or even 100% or more of the antigen-binding affinity of its parent TCR (or antigen-specific fragment thereof). Variants of TCRs (or antigen-specific fragments thereof) typically include amino acid changes in the variable region framework sequence and / or constant region, but this does not exclude the possibility of altering one or a few amino acids in the CDR region sequence. Therefore, those skilled in the art will also understand that, based on the specific TCR (or antigen-specific fragment thereof) amino acid sequence provided herein (CDR sequence, variable region sequence, or α / β chain sequence), a small number of amino acids can be replaced, deleted, or added, and the resulting products can be verified or screened for their binding ability to the corresponding pMHC complex or biological activity, thereby obtaining corresponding variants of the TCR (or antigen-specific fragment thereof) provided herein, and these variants are also intended to be included within the scope of the present invention.

[0058] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably herein to refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0059] The term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (e.g., a nucleic acid, a plasmid, or a virus). A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric analysis, such as β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0060] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include (but are not limited to) CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. In some embodiments, the host cell is used to produce a protein of interest, for example, the TCR α chain and / or β chain provided herein can be secreted and produced by a mammalian cell. In other embodiments, the host cell has a specific function after expressing the TCR molecule provided herein, for example, after the T cell expresses the TCR molecule provided herein, it has the ability to recognize and kill target cells expressing a specific antigenic peptide. The host cell can be an isolated cell or cell line, and also includes cells transfected with the nucleic acid molecule or expression vector provided herein in vivo. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.

[0061] "Subject" includes animals, such as mammals, including but not limited to primates, rodents, monkeys, felines, canines, equines, bovines, porcines, sheep, goats, mammalian experimental animals, mammalian farm animals, mammalian sports animals, and mammalian pets. The subject can be male or female and can be any age-appropriate subject, including infants, young children, young people, adults, and elderly subjects. In some instances, the subject refers to an individual who needs to diagnose or treat a disease or condition. In some instances, the subject receiving diagnosis or treatment can be a patient who suffers from a condition associated with the diagnosis or treatment, or is at risk of developing the condition. In a specific instance, the subject is a human, such as a human patient. The term is generally used interchangeably with "patient," "test subject," "treatment subject," etc.

[0062] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered, is suitable for administration to humans and / or animals without excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent contained therein.

[0063] When referring to disease treatment, "effective amount" refers to the amount of active molecules (such as antibodies) sufficient to induce the biological or medical response desired by the clinician in the subject. The "effective amount" at the time of administration can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, for protein drugs, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight. For immune effector cells (such as T cells) expressing the TCR provided herein, the number of cells administered per infusion can be, for example, from about 1×10 6 to about 1×10 12 In some embodiments, the number of cells that can be expressed is less than 1×10 6 Methods for administering active molecules (such as TCRs, antibodies, fusion proteins) or immune effector cells provided herein include, but are not limited to, injection, for example, intravenously, intramuscularly, intraarterially, subcutaneously, intraperitoneally, and the like.

[0064] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of consistency between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using the default settings. In some embodiments, "at least 80% sequence identity" as described herein includes, but is not limited to, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 even 100% sequence identity.

[0065] Provided herein are isolated or purified binding proteins, e.g., T cell receptors (TCRs), antibodies, or CARs, that have antigenic specificity for an antigenic peptide (having amino acid mutation sites relative to the wild-type sequence)-MHC complex, wherein the antigenic peptide is derived from a mutant KRAS protein.

[0066] This article also provides polypeptides and proteins related to the TCR or its binding domain, as well as related nucleic acid molecules, vectors (such as recombinant expression vectors), host cells (such as T cells) and pharmaceutical compositions.

[0067] Also provided herein are methods and kits for detecting the presence of a tumor in a subject, as well as methods of treating or preventing a tumor in a subject.

[0068] T cell receptor (TCR)

[0069] Provided herein are isolated or purified T cell receptors (TCRs) that are antigenically specific for an antigenic peptide (having amino acid mutations relative to the wild-type sequence)-MHC complex, wherein the antigenic peptide is derived from a mutant KRAS protein and comprises or consists of VVVGAXGVGK (SEQ ID NO: 11), wherein X is any amino acid residue that is not G or R. Preferably, the antigenic peptide is selected from: VVVGAVGVGK (SEQ ID NO: 12), VVVGADGVGK (SEQ ID NO: 13), VVVGASGVGK (SEQ ID NO: 14), VVVGAAGVGK (SEQ ID NO: 15), or VVVGACGVGK (SEQ ID NO: 16). Considering that MHC class I molecules do not strictly limit the length of antigenic peptides, the present inventors anticipate that one or two amino acids can be added or deleted (especially at the termini) on the basis of the above-mentioned antigenic peptide sequences provided herein, and that the binding proteins or TCR molecules provided herein also have the ability to bind to these antigenic peptide variant-MHC complexes.

[0070] The TCR provided herein can be an αβ heterodimer or a single-chain form (scTCR). Examples of single-chain forms can be αβTCR polypeptides in the form of Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ or Vα-L-Vβ-Cβ (wherein Vα and Vβ refer to TCRα chain and TCRβ chain variable regions, respectively, Cα and Cβ are TCRα chain constant regions and TCRβ chain constant regions, respectively, and L is a linker sequence, typically a short peptide). The linker sequence can be conventional in the art and is often used in antibody engineering and TCR engineering. The TCR provided herein can also be a water-soluble TCR molecule that does not contain the transmembrane region of α and β chains.

[0071] In some embodiments, provided herein are TCR molecules that have antigenic specificity for the antigenic peptide-MHC complex. The CDR3 of the α chain variable region of the TCR molecule comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence of SEQ ID NO: 5; the CDR3 of the β chain variable region of the TCR molecule comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence of SEQ ID NO: 10. Preferably, the MHC molecule is HLA-A*11:01.

[0072] Furthermore, in the α chain variable region, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO: 5; in the β chain variable region, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0073] Furthermore, in the TCR molecule, the α chain variable region includes the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 2; the β chain variable region includes the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 7.

[0074] In some specific embodiments, in the TCR molecule, the α chain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 1; the β chain comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 6.

[0075] It will be appreciated by those skilled in the art that, based on the specific sequences provided herein, variants of the TCR molecules provided herein can be obtained by replacing, deleting, or adding a small number of amino acids and then verifying or screening the binding ability or biological activity of the resulting products to the corresponding pMHC complex, and such variants are also intended to be included within the scope of the present invention. For example, the TCR molecules provided herein may have at least one and no more than ten, such as no more than five, four, three, two, or one, amino acid changes in their full-length or variable region sequences or CDR sequences. For example, there may be at least one and no more than ten, such as no more than five, four, three, two, or one, amino acid changes in the α or β variable region sequences, and there may also be no more than five, four, three, two, or one amino acid changes in the CDR sequences of the α or β variable regions.

[0076] It is contemplated that the TCRs described herein (or antigen-specific fragments thereof) may comprise conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as substitutions of one amino acid residue by another amino acid residue of similar chemical structure with little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions may, for example, be substitutions of one amino acid from the following groups (a)-(e) by another amino acid from the same group: (a) small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar positively charged residues: His, Arg, and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0077] In some embodiments, the TCR (or its antigen-specific fragment) provided herein may further comprise post-translational modifications. Examples of post-translational protein modifications include: phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination-like, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Therefore, the modified TCR (or its antigen-specific fragment) may comprise non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. One form of glycosylation is, for example, sialylation modification, which binds one or more sialic acid groups to a polypeptide. Sialic acid groups improve the solubility and serum half-life of proteins, while also reducing the possible immunogenetic properties of proteins.

[0078] When the N-terminus of the amino acid sequence of the TCR provided herein is a methionine residue (M), as is well known to those skilled in the art, the methionine may be removed during the production process of the recombinant protein.

[0079] Fusion proteins and immunoconjugates

[0080] Also provided herein are fusion proteins comprising the variable regions of the α chain and / or β chain of the TCR molecules provided herein, in particular, their CDR sequences.

[0081] In some embodiments, the fusion protein is in the form of a single-chain TCR. For example, the α chain variable region and the β chain variable region are connected by a linker sequence to form a complete binding domain with antigen specificity for the corresponding pMHC complex.

[0082] In some embodiments, the variable regions of the α chain and / or β chain of the TCR molecules provided herein, particularly their CDR sequences, can be used to form TCR molecules with multispecificity. In some embodiments, the fusion protein includes at least two functional parts, the first functional part having antigen specificity for the first pMHC complex, and the second functional part having the same or different antigen specificity as the first functional part.

[0083] In some embodiments, the variable regions of the α chain and / or β chain of the TCR molecules provided herein, in particular their CDR sequences, can be used to form a fusion protein with multispecificity. In some embodiments, the fusion protein includes at least two functional parts, the first functional part has antigen specificity for the pMHC complex, and the second functional part has an antigen specificity or targeting different from the first functional part. In some embodiments, the second functional part can be a targeting part that can specifically recognize and bind to tumor-specific antigens, tumor-associated antigens, or other target cell surface molecules, such as antibodies or antigen-binding fragments thereof (e.g., scFv), hormones, growth factors, cytokines, and any other natural or non-natural ligands that can bind to cell surface receptors (e.g., epidermal growth factor receptor (EGFR), CD28, platelet-derived growth factor receptor (PDGFR), nicotinic acetylcholine receptor (nAChR), etc.).

[0084] In some embodiments, the binding domain of an antibody molecule (such as a classical tetrameric antibody molecule) can be replaced with a TCR molecule (or an α chain and β chain variable region or a binding domain formed thereof) provided herein to form a recombinant antibody molecule with the antigen specificity of the TCR molecule. In some embodiments, the two heavy chain variable regions of the antibody molecule are replaced with the α chain variable region of the TCR molecule and connected to the heavy chain constant region, while the two light chain variable regions of the antibody molecule are replaced with the β chain variable region and connected to the light chain constant region. In some embodiments, the two heavy chain variable regions of the antibody molecule are replaced with the β chain variable region of the TCR molecule and connected to the heavy chain constant region, while the two light chain variable regions of the antibody molecule are replaced with the α chain variable region and connected to the light chain constant region. In some embodiments, the α chain variable region and the β chain variable region of the TCR molecule are used to replace one heavy chain variable region and one light chain variable region of the antibody molecule, respectively, keeping the original antigen binding domain of the antibody molecule unchanged, to form a recombinant antibody molecule with bispecificity, which targets the target antigen of the pMHC complex and the original antibody molecule, respectively. The recombinant antibody molecule thus formed can realize the effector function of the antibody molecule through its constant region (such as Fc fragment), such as mediating complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, etc. In addition, fusion with the Fc fragment can increase the half-life of the TCR molecule (or the α chain and β chain variable regions or the binding domain formed therefrom) in vivo, so as to increase the dosing interval when the TCR molecule (or the α chain and β chain variable regions or the binding domain formed therefrom) is used as a therapeutic drug. In some embodiments, the Fc fragment can be derived from the constant region of an immunoglobulin, such as IgG1, IgG2 or IgG4.

[0085] In some embodiments, the TCR molecules provided herein (or the α chain and β chain variable regions or the binding domains formed thereof) can be used as extracellular binding domains to construct chimeric antigen receptors (CARs). For example, the α chain and β chain variable regions can be connected by a linker sequence to form a binding molecule similar to scFv, replacing the extracellular binding domain in the conventional CAR molecule with the transmembrane region and the intracellular signal transduction domain. The CAR construct targets the pMHC complex corresponding to the original TCR molecule and activates the T cell expressing the CAR through the intracellular transduction domain of the CAR molecule to achieve the desired immunological effect (e.g., target cell killing function).

[0086] In some embodiments, the TCR molecule (or the α chain and β chain variable regions or the binding domain formed therefrom) can be linked to a conjugate, such as a detectable label, therapeutic agent, tracer, etc., to form an immunoconjugate.

[0087] In some embodiments, the TCR molecule (or the α chain and β chain variable regions or the binding domain formed thereof) can be connected to a protein tag to form a fusion protein. The protein tag may include a purification tag and a detectable tag. Purification tags include but are not limited to His6 tags, Flag tags, MBP tags, GST tags, SUMO tags, etc. Detectable tags can be used to indicate the presence or content of TCR molecules (or the α chain and β chain variable regions or the binding domain formed thereof) in a sample, or for tracking the location information of TCR molecules (or the α chain and β chain variable regions or the binding domain formed thereof) in a subject or cell. Examples of detectable tags include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.; fluorescent proteins, such as GFP. Due to the specific binding ability of TCR molecules (or α-chain and β-chain variable regions, or binding domains formed therefrom) to corresponding pMHC complexes, the amount of TCR molecules (or α-chain and β-chain variable regions, or binding domains formed therefrom) can be determined by the amount of detectable label attached to the TCR molecules (or α-chain and β-chain variable regions, or binding domains formed therefrom), and thus the content of the corresponding pMHC complex in the sample. When the corresponding pMHC complex can serve as a tumor marker, the fusion protein can be used for tumor detection and diagnosis.

[0088] In other embodiments, the TCR molecules provided herein (or the α chain and β chain variable regions or the binding domain formed therefrom) can be linked to cytokines or therapeutic proteins to form fusion proteins. In this case, the specific binding ability of the TCR molecules (or the α chain and β chain variable regions or the binding domain formed therefrom) to the corresponding pMHC complex can be utilized to purposefully deliver the cytokines or therapeutic proteins to specific tissues or cells, thereby achieving the therapeutic effects of the cytokines or therapeutic proteins.

[0089] In some embodiments, the TCR molecule (or the α chain and β chain variable regions or the binding domain formed therefrom) can be linked to a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc., so as to deliver the chemotherapeutic agent to specific cells or tissues through the antigen specificity of the TCR molecule.

[0090] In some embodiments, the TCR molecule (or the α and β chain variable regions or the binding domain formed thereof) can be combined with a radioactive isotope (e.g. 3 H. 14 C. 35 S) connection for tracking or therapeutic purposes.

[0091] Host cells expressing TCR molecules

[0092] Also provided herein are host cells that express the TCR molecules (or the α chain and β chain variable regions or the binding domains formed therefrom) provided herein.

[0093] The host cell used refers to any type of cell that can express the TCR molecules provided herein (or the α chain and β chain variable regions or the binding domains formed thereof). Preferably, the host cell expresses a complete TCR molecule, i.e., including the α chain and β chain variable regions, the α chain and β chain constant regions, the transmembrane region, and the intracellular region. The host cell can be a eukaryotic cell or a prokaryotic cell. Preferably, the host cell is a mammalian cell. Most preferably, the host cell is a human cell. Although the host cell can be a cell of any cell type, can be derived from any type of tissue, and can be a cell of any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. For the purposes of this article, 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 fluids. T cells can also be enriched or purified. T cells can be of any type and can be T cells of any developmental stage, 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 (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc.

[0094] Also provided herein is a host cell expressing the CAR provided herein. Preferably, the host cell is a human cell, especially a human T cell or NK cell.

[0095] The utility function of host cells (such as T cells) expressing TCR or CAR can be detected in a variety of ways. In some embodiments, the host cells (such as T cells) expressing TCR or CAR are incubated with target cells and the number or activity of target cells is detected to reflect the host cell's ability to kill target cells (such as by detecting the release of LDH). In some embodiments, the immune effect of the host cell is assessed by incubating host cells (such as T cells) expressing TCR or CAR with target cells and detecting the host cell's IFN-γ secretion or CD69 expression.

[0096] Pharmaceutical compositions and methods of treatment

[0097] The TCR molecules (or the α chain and β chain variable regions or the binding domains formed therefrom) and fusion proteins or immunoconjugates comprising the TCR molecules (or the α chain and β chain variable regions or the binding domains formed therefrom) provided herein can be formulated as pharmaceutical compositions together with a pharmaceutically acceptable carrier and administered to a subject for tumor prevention or treatment.

[0098] In some embodiments, the TCR molecules (or the α chain and β chain variable regions or the binding domains formed therefrom) and fusion proteins or immunoconjugates comprising the TCR molecules (or the α chain and β chain variable regions or the binding domains formed therefrom) provided herein can be administered in combination with one or more other drugs (e.g., anti-tumor agents).

[0099] In some embodiments, the disease or condition treated is a tumor, in particular a tumor expressing an antigenic peptide comprising a KRAS G12 mutation. These tumors include, but are not limited to, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, uterine cancer, Cancers of the cervix, gastrointestinal carcinoid tumors, gliomas, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and bladder cancer are preferred. Preferred cancers are pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, prostate cancer and the like.

[0100] Nucleic acid molecules encoding TCR molecules (or α chain and β chain variable regions or binding domains formed therefrom) provided herein and fusion proteins or immunoconjugates including TCR molecules (or α chain and β chain variable regions or binding domains formed therefrom), expression vectors including the nucleic acid molecules, and host cells (T cells, CAR cells) transfected with the nucleic acid molecules or expression vectors can also be used for the above-mentioned therapeutic purposes in various ways. For example, for expression vectors, gene therapy means known in the art can be introduced into the subject to express the target protein or polypeptide (such as TCR molecules (or α chain and β chain variable regions or binding domains formed therefrom) and fusion proteins or immunoconjugates including TCR molecules (or α chain and β chain variable regions or binding domains formed therefrom) provided herein), so as to achieve therapeutic purposes.

[0101] The effective amount of this purpose can depend on the severity of the disease and the overall state of the patient's own immune system.Dosage regimen will also change with disease state and subject state, and general scope will be from single large amount (bolus) administration or continuous infusion to multiple administration every day (for example, every 4-6 hours). Clinicians skilled in the art can, for example, by utilizing clinical trials, physical examination and subject's family history, easily determine whether a certain subject is a candidate for this treatment.

[0102] Detection or treatment kits

[0103] The TCR molecules (or the α chain and β chain variable regions or the binding domains formed therefrom) provided herein, as well as fusion proteins or immunoconjugates comprising TCR molecules (or the α chain and β chain variable regions or the binding domains formed therefrom), can specifically bind to corresponding pMHC complexes in a sample. By detecting the amount of the formed ternary complex (i.e., TCR-antigen peptide, MHC molecule), the amount (or presence) of the corresponding pMHC complex in the sample can be conveniently determined.

[0104] As described above, for this purpose, the TCR molecules provided herein (or the α and β chain variable regions or the binding domains formed therefrom) can be linked to various detection labels to facilitate detection by various means, including but not limited to bioluminescence, fluorescence, radiolabeling, and enzymatic reaction product production. After detecting the level of the corresponding pMHC complex in a sample and comparing it with the normal level of the corresponding pMHC complex in a healthy population, it can be used to determine the disease status or severity of the subject providing the sample. By repeatedly detecting changes in the level of the corresponding pMHC complex over time during the subject's treatment, it can also be used to determine whether the treatment is effective, thereby providing a basis for modifying the treatment plan.

[0105] The TCR molecules (or α chain and β chain variable regions or the binding domains formed thereof) provided herein and fusion proteins or immunoconjugates or host cells comprising TCR molecules (or α chain and β chain variable regions or the binding domains formed thereof) can be placed in containers to form detection or treatment kits. These containers can be boxes, ampoules, bottles, tubes, bags or suitable container forms known in the art. These containers can be made of plastic, glass, laminated paper, metal foil or other materials suitable for preserving medicines. If necessary, instructions for use are also provided with the container. The instructions may generally include information on how to use TCR molecules (or α chain and β chain variable regions or binding domains formed therefrom), compositions comprising TCR molecules (or α chain and β chain variable regions or binding domains formed therefrom), and host cells for treating or preventing tumors. For example, the instructions may include a description of the therapeutic agent (such as a TCR molecule (or α chain and β chain variable regions or binding domains formed therefrom), a nucleic acid molecule, a host cell, etc.); a dosage regimen for treating or preventing neoplasia; precautions; warnings; indications; contraindications; adverse reactions; animal pharmacology; clinical studies; and / or reference materials. The instructions may be printed directly on the container (if present), or as a label affixed to the container, or as a separate paper, booklet, card, or folded printed matter provided in or with the container.

[0106] Experimental materials and reagents:

[0107] 1. Healthy human PBMC

[0108] 2. Targeted peptides:

[0109] A*11:01KRAS-G12V peptide: VVVGAVGVGK (SEQ ID NO: 12)

[0110] A*11:01KRAS-G12D peptide: VVVGADGVGK (SEQ ID NO: 13)

[0111] A*11:01KRAS-G12S peptide: VVVGASGVGK (SEQ ID NO: 14)

[0112] A*11:01KRAS-G12A peptide: VVVGAAGVGK (SEQ ID NO: 15)

[0113] A*11:01KRAS-G12C peptide: VVVGACGVGK (SEQ ID NO: 16)

[0114] 3.Brilliant Violet 510 TManti-human CD3 Antibody:Biolegend 300448

[0115] 4.APC anti-human CD8a Antibody:Biolegend 301014

[0116] 5.Flex-T TM HLA-A*11:01Monomer UVX:Biolegend 280007

[0117] 6.Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1

[0118] 7. Lentiviral vectors

[0119] 8. Polybrene Viral Enhancer: Merck Millipore TR-1003-G

[0120] 9.anti-CD3 / CD28 magnetic beads: novoprotein GMP-B038

[0121] 10.X-VIVO 15:lonza 04418q

[0122] 11.OKM-100:CansBio sj18

[0123] 12.RPMI 1640:gibco 11875-093

[0124] 13.LDH kit: dojindo

[0125] 14.Human IFN-γ kit: Excell Bio EH008-96

[0126] 15.IFN-γELISPOT:Mabtech 3420-4AST-10

[0127] 16.APC-anti-human CD69 Antibody:biolegend 310910

[0128] Instruments and Equipment

[0129] Flow cytometer: BD LSRFortessa

[0130] Flow sorter: SONY SH800S

[0131] Microplate reader: Molecular Devices SpectraMax i3x

[0132] ELISPOT imager: Bio-sys Bioreader BIO-SYS Bioreader 6000-Eβ

[0133] Graphics and analysis software

[0134] FlowJo

[0135] Graphpad Prism 8.0.1

[0136] Example

[0137] T cells from a healthy donor were stimulated with dendritic cells loaded with the KRAS G12V 10 peptide (VVVGAVGVGK) (SEQ ID NO: 12). The proportion of A*11:01-G12V-specific T cells was determined by tetramer staining and flow cytometry using a BD LSRFortessa. Specific T cells were then enriched and sorted using a Sony SH800S flow cytometer and analyzed with FlowJo software. The flow cytometric profile is shown in Figure 1. The enriched cells were subjected to 10× single-cell sequencing to obtain paired TCRα and TCRβ chains (see TCR1 amino acid sequence below).

[0138] A lentiviral vector containing the TCR gene of the present invention is constructed and transduced into T cells. TCR-transduced T cells were incubated with (1) K562 cells expressing HLA-A*11:01 loaded with different peptides (WT peptide: VVVGAGGVGK (SEQ ID NO: 17), G12D-10 peptide: VVVGADGVGK (SEQ ID NO: 13), G12V-10 peptide: VVVGAVGVGK (SEQ ID NO: 12), G12S-10 peptide: VVVGASGVGK (SEQ ID NO: 14), G12A-10 peptide: VVVGAAGVGK (SEQ ID NO: 15), G12C-10 peptide: VVVGACGVGK (SEQ ID NO: 16)) and (2) K562 cells expressing HLA-A*11:01, co-expressing HLA-A*11:01 and KRAS G12V, co-expressing HLA-A*11:01 and KRAS After incubation with BxPC3 cells expressing G12D (effector cells: target cells = 5:1) for 16 h, the cytotoxicity of T cells against each target cell was assessed by detecting the release of LDH. The statistical data are shown in Figures 2A and 2B (Graphpad Prism 8.0.1 software). T cells transduced with the TCR of the present invention have effective and specific cytotoxicity against target cells expressing A*11:01-loaded KRAS G12D-10 peptide, KRAS G12V-10, KRAS G12S-10, KRAS G12A-10, and KRAS G12C-10 peptides, as well as target cells overexpressing KRAS G12V and KRAS G12D.

[0139] The functional affinity of the TCR was evaluated by measuring the expression of CD69 after T cells transduced by the TCR of the present invention were co-incubated with target cells loaded with different concentrations of KRAS G12V-10 peptide (VVVGAVGVGK) (SEQ ID NO: 12) and KRAS G12D-10 peptide (VVVGADGVGK) (SEQ ID NO: 13). As shown in Figure 3, the EC50 concentration of TCR recognition of G12V-10 peptide was approximately 45 nM, and the EC50 concentration of TCR recognition of G12D-10 peptide was approximately 77 nM.

[0140] Some of the amino acid sequences mentioned herein are as follows.

[0141] TCR1α chain amino acid sequence

[0142] TCR1α chain V region amino acid sequence

[0143] TCR1α chain CDR1 amino acid sequence

[0144] TCR1α chain CDR2 amino acid sequence

[0145] TCR1α chain CDR3 amino acid sequence

[0146] TCR1β chain amino acid sequence

[0147] TCR1β chain V region amino acid sequence

[0148] TCR1β chain CDR1 amino acid sequence

[0149] TCR1β chain CDR2 amino acid sequence

[0150] TCR1β chain CDR3 amino acid sequence

Claims

1. A binding protein comprising a binding domain having antigen specificity for an antigen peptide:HLA complex, wherein the binding domain comprises a T cell receptor (TCR) α chain variable region and a TCR β chain variable region, wherein: The antigenic peptide comprises an amino acid sequence VVVGAXGVGK (SEQ ID NO: 11), wherein X is any amino acid residue that is not G or R; The HLA is HLA-A*11:01; The CDR3 of the α chain variable region includes the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 5; the CDR3 of the β chain variable region includes the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO:

10.

2. The binding protein of claim 1, wherein the antigenic peptide comprises VVVGAVGVGK (SEQ ID NO: 12), VVVGADGVGK (SEQ ID NO: 13), VVVGASGVGK (SEQ ID NO: 14), VVVGAAGVGK (SEQ ID NO: 15), or VVVGACGVGK (SEQ ID NO: 16).

3. The binding protein of claim 1, wherein in the α chain variable region, CDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 5; in the β chain variable region, CDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO:

10.

4. A binding protein, comprising a binding domain, wherein the binding domain comprises a T cell receptor (TCR) α chain variable region and a TCR β chain variable region, wherein CDR1 in the α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 5; and CDR1 in the β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, and more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO:

10.

5. The binding protein according to any one of claims 1 to 4, wherein the α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 2; and the β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO:

7.

6. The binding protein according to any one of claims 1 to 5, wherein the α chain comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO: 1; and the β chain comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably 90% or 95% identical to the amino acid sequence shown in SEQ ID NO:

6.

7. The binding protein according to any one of claims 1 to 6, which is a protein in the form of: 1) TCR molecules; 2) Antibody molecules; or 3) CAR molecule.

8. The binding protein of any one of claims 1 to 7, wherein when T cells expressing the binding protein are incubated with HLA-A*11:01 cells in the presence of the antigenic peptide, or when T cells expressing the binding protein are incubated with HLA-A*11:01 cells expressing the antigenic peptide, the T cells specifically kill the HLA-A*11:01 cells.

9. The binding protein of any one of claims 1 to 8, wherein when T cells expressing the binding protein are incubated with HLA-A*11:01 cells in the presence of the antigenic peptide, or when T cells expressing the binding protein are incubated with HLA-A*11:01 cells expressing the antigenic peptide, the T cells express CD69, or the amount of CD69 expressed is increased.

10. The binding protein according to any one of claims 1 to 9, further comprising a conjugate covalently or non-covalently linked to the binding domain; preferably, the conjugate is a detectable marker, a radioactive isotope or a therapeutic agent.

11. An isolated nucleic acid molecule encoding the binding protein, α chain variable region, β chain variable region, α chain or β chain according to any one of claims 1 to 10.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A host cell expressing the binding protein according to any one of claims 1 to 10 or comprising the nucleic acid molecule according to claim 11 or the vector according to claim 12. The host cell according to claim 13 , which is a mammalian cell, preferably a human cell. The host cell according to claim 13 or 14, which is a T cell or a NK cell.

16. The host cell according to any one of claims 13 to 15, which has killing activity against HLA-A*11:01 cells expressing the antigenic peptide VVVGAXGVGK (SEQ ID NO: 11), wherein X is any amino acid residue that is not G or R.

17. The host cell of any one of claims 13-16, wherein the antigenic peptide comprises VVVGAVGVGK (SEQ ID NO: 12), VVVGADGVGK (SEQ ID NO: 13), VVVGASGVGK (SEQ ID NO: 14), VVVGAAGVGK (SEQ ID NO: 15), or VVVGACGVGK (SEQ ID NO: 16).

18. A pharmaceutical composition comprising: 1) the binding protein according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, or the host cell according to any one of claims 13 to 17; and 2) Pharmaceutically acceptable carrier.

19. Use of the binding protein according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12 or the host cell according to any one of claims 13 to 17 in the preparation of a medicament for treating a tumor, wherein the tumor expresses the antigenic peptide.

20. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the binding protein of any one of claims 1-10, the nucleic acid molecule of claim 11, the vector of claim 12, the cell of any one of claims 13-17, or the pharmaceutical composition of claim 18.

21. The use of claim 19 or the method of claim 20, wherein the tumor is selected from pancreatic cancer, colorectal cancer, lung cancer, bile duct cancer, endometrial cancer and ovarian cancer.

22. A detection kit comprising the binding protein according to any one of claims 1 to 10.