Binding protein, isolated nucleic acid molecule, vector, host cell, pharmaceutical composition, use of binding protein, method for treating tumor in an individual, and detection kit.

Abstract

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. The use of the binding protein and a pharmaceutical composition comprising the binding protein in the treatment of cancers associated with the antigen peptide.

Description

1 / 44 “Binding protein, isolated nucleic acid molecule, vector, host cell, pharmaceutical composition, use of binding protein, method for treating tumor in an individual and detection kit” Cross-reference to related requests

[001] This application requests priority from a Chinese patent application, application number 202310484887.5, filed with the China Patent Office on April 28, 2023, which is incorporated herein by reference in its entirety. Technical Field

[002] This document concerns TCR molecules and cells expressing such TCR molecules, in particular TCR molecules targeting target cells carrying the KRAS G12 mutation, as well as immune effector cells expressing these TCR molecules. This document also concerns the therapeutic application of said TCR molecules and the cells expressing them. State of the Art

[003] The RAS gene family (HRAS, KRAS, NRAS) constitutes the group of proto-oncogenes most frequently associated with tumor genesis. Among them, mutations in the KRAS gene—concentrated mainly in codon 12—are the most prevalent, being present in approximately 22% to 30% of tumors. The frequency is particularly high in pancreatic adenocarcinoma (~60-90%), colorectal cancer (~40%), lung cancer (~10% in China; ~30% in Europe and the USA), cholangiocarcinoma (~17%-37%), as well as in ovarian and endometrial carcinomas (~10%-40%, depending on the histopathological subtype, with a higher incidence in Petition 870250073464, dated 08 / 20 / 2025, p. 18 / 73 2 / 44 serous variant). As an example, in the case of pancreatic adenocarcinoma, it was estimated that in 2021, approximately 60,430 people would be diagnosed with the disease, with approximately 48,220 deaths attributed to pancreatic cancer. Despite advances in the diagnosis and treatment of pancreatic cancer in the last decade, the five-year survival rate remains around 9%, due to the scarcity of effective therapeutic options. Currently, the treatment of unresectable pancreatic cancer is mainly based on chemotherapy with the aim of reducing tumor volume, but its effectiveness is limited by the development of resistance. In addition, the success of targeted therapies has been modest to date. Furthermore, pancreatic cancer also exhibits resistance to immunotherapy, which may be partly due to the low mutational load of the disease, resulting in a scarcity of tumor-infiltrating lymphocytes reactive to neoantigens.Research with TCR-T cells targeting KRAS-G12 may effectively improve the current therapeutic limitations for tumors with mutations in the KRAS gene, for which there are still no effective treatment options. Disclosure of the Invention

[004] In one aspect, the present document provides a fusion protein that includes a binding domain with antigenic specificity for the antigenic peptide:HLA complex. Said binding domain comprises the variable region of the α chain of the T cell receptor (TCR) and the variable region of the β chain of the TCR, where: - the aforementioned antigenic peptide includes the sequence of Petition 870250073464, dated 08 / 20 / 2025, p. 19 / 73 3 / 44 amino acids VVVGAXGVGK (SEQ ID NO: 11), where X represents any amino acid residue except G or R; The HLA in question is HLA-A*11:01; - The CDR3 region of the α variable chain includes the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence with at least 80% identity to SEQ ID NO: 5, preferably at least 85%, more preferably 90% or 95% identity; the CDR3 region of the β variable chain includes the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence with at least 80% identity to SEQ ID NO: 10, preferably at least 85%, more preferably 90% or 95% identity.

[005] In some embodiments, the said antigenic peptide includes 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).

[006] In some embodiments, in the variable chain α, CDR1 includes the amino acid sequence shown in SEQ ID NO: 3, CDR2 includes the sequence shown in SEQ ID NO: 4 and CDR3 includes the sequence shown in SEQ ID NO: 5, or an amino acid sequence with at least 80% identity with SEQ ID NO: 5, preferably at least 85%, more preferably 90% or 95% identity; in the variable chain β, CDR1 includes the amino acid sequence shown in SEQ ID NO: 8, CDR2 includes the sequence shown in SEQ ID NO: 9 and CDR3 includes the sequence shown in SEQ ID NO: 10, or an amino acid sequence with at least 80% identity with SEQ ID NO: 10, preferably at least 85%, more Petition 870250073464, dated 08 / 20 / 2025, p. 20 / 73 4 / 44 preferably 90% or 95% identity.

[007] In another aspect, the present document provides a binding protein that includes a binding domain, comprising the variable region of the α chain and the variable region of the β chain of the T cell receptor (TCR), wherein, in the variable chain α, CDR1 includes the amino acid sequence shown in SEQ ID NO: 3, CDR2 includes the sequence shown in SEQ ID NO: 4 and CDR3 includes the sequence shown in SEQ ID NO: 5, or an amino acid sequence with at least less than 80% identity with SEQ ID NO: 5, preferably at least 85%, more preferably 90% or 95% identity; and where, in the variable β chain, CDR1 includes the amino acid sequence shown in SEQ ID NO: 8, CDR2 includes the sequence shown in SEQ ID NO: 9 and CDR3 includes the sequence shown in SEQ ID NO: 10, or an amino acid sequence with at least 80% identity with SEQ ID NO: 10, preferably at least 85%, more preferably 90% or 95% identity.

[008] In some embodiments, the variable α chain region above includes the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2. The amino acid sequence shown has an amino acid sequence consistent in at least 80%, preferably consistent in at least 85%, more preferably consistent in 90% or 95%; The variable β chain region above includes the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7. The amino acid sequence shown has an amino acid sequence consistent in at least 80%, preferably consistent in at least 85%, more preferably consistent in 90% or 95%. Petition 870250073464, dated 08 / 20 / 2025, p. 21 / 73 5 / 44

[009] In some embodiments, the aforementioned α chain includes the amino acid sequence presented in SEQ ID NO: 1, or an amino acid sequence with at least 80% identity with SEQ ID NO: 1, preferably at least 85%, more preferably 90% or 95% identity; and the aforementioned β chain includes the amino acid sequence presented in SEQ ID NO: 6, or an amino acid sequence with at least 80% identity with SEQ ID NO: 6, preferably at least 85%, more preferably 90% or 95% identity.

[0010] In some embodiments, the said binding protein is a protein with the following form: 1) TCR molecule; 2) antibody molecule; or 3) CAR molecule.

[0011] In some embodiments, when T lymphocytes expressing said binding protein are incubated with HLA-A*11:01 cells in the presence of said antigenic peptide, or when they are incubated with HLA-A*11:01 cells expressing said antigenic peptide, said T lymphocytes specifically eliminate HLA-A*11:01 cells.

[0012] In some embodiments, when T lymphocytes expressing the said binding protein are incubated with HLA-A11:01 cells in the presence of the said antigenic peptide, or when they are incubated with HLA-A11:01 cells expressing the said antigenic peptide, the said T lymphocytes express CD69, or show an increase in CD69 expression.

[0013] In some embodiments, the aforementioned Petition 870250073464, dated 08 / 20 / 2025, page 22 / 73 6 / 44 The binding protein also includes a conjugate linked, covalently or non-covalently, to the binding domain; preferably, said conjugate is a detectable marker, a radioactive isotope, or a therapeutic agent.

[0014] In another aspect, the present document provides an isolated nucleic acid molecule encoding said binding protein, the variable region of the α chain, the variable region of the β chain, the α chain, or the β chain.

[0015] In another aspect, the present document provides a vector that includes said nucleic acid molecule.

[0016] In another aspect, the present document provides a host cell expressing said binding protein, nucleic acid molecule, or vector.

[0017] In some embodiments, the host cell is a mammalian cell, preferably a human cell.

[0018] In some embodiments, the host cell is a T lymphocyte or an NK cell.

[0019] In some embodiments, the host cell exhibits cytotoxic activity against HLA-A*11:01 cells that express the antigenic peptide VVVGAXGVGK (SEQ ID NO: 11), where X represents any amino acid residue except G or R.

[0020] In some embodiments, the said antigenic peptide includes 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).

[0021] In another aspect, this document provides a pharmaceutical composition that includes: 1) the aforementioned binding protein, nucleic acid molecule, vector or Petition 870250073464, dated 08 / 20 / 2025, p. 23 / 73 7 / 44 host cell; and 2) a pharmacologically acceptable vehicle.

[0022] In another aspect, the present document provides the use of said binding protein, nucleic acid molecule, vector or host cell in the preparation of a medicament for the treatment of tumors expressing said antigenic peptide.

[0023] In another aspect, the present document provides a method for treating tumors in an individual, comprising administering to said individual a therapeutically effective amount of said binding protein, nucleic acid molecule, vector, host cell or pharmaceutical composition.

[0024] In some embodiments, the tumor in question is selected from pancreatic cancer, colorectal cancer, lung cancer, cholangiocarcinoma, endometrial carcinoma, and ovarian carcinoma.

[0025] In another aspect, the present document provides a detection kit that includes the aforementioned binding protein. Description of the Figures

[0026] Figure 1 shows a flow cytometry diagram illustrating the selection of HLA-A*11:01 / KRAS G12V specific T lymphocytes, enriched by tetramer after stimulation.

[0027] Figure 2A shows the results of the cytotoxic activity of non-transduced and transduced CD8+ T cells with the HLA-A*11:01 / KRAS G12V specific TCR1 against K562-A*11:01 target cells loaded with different antigenic peptides. The loaded peptides Petition 870250073464, dated 08 / 20 / 2025, page 24 / 73 8 / 44 of the cells in the figure are respectively: KRAS-G12V peptide: VVVGAVGVGK (SEQ ID NO: 12) KRAS-G12D peptide: VVVGADGVGK (SEQ ID NO: 13) KRAS-G12S peptide: VVVGASGVGK (SEQ ID NO: 14) KRAS-G12A peptide: VVVGAAGVGK (SEQ ID NO: 15) KRAS-G12C peptide: VVVGACGVGK (SEQ ID NO: 16) WT-10 peptide: VVVGAGGVGK (SEQ ID NO: 17).

[0028] Figure 2B shows the results of the cytotoxic activity of non-transduced and transduced CD8+ T cells with the TCR1 specific for HLA-A*11:01 / KRAS G12V against BxPC3 cells that overexpress HLA-A11:01 and / or KRAS G12V or G12D. BxPC3-A1101 refers to BxPC3 cells transfected with HLA-A*11:01; BxPC3-A1101-G12V refers to BxPC3 cells transfected simultaneously with HLA-A*11:01 and KRAS G12V; and BxPC3-A1101-G12D refers to BxPC3 cells transfected simultaneously with HLA-A*11:01 and KRAS G12D.

[0029] Figure 3 shows the expression of CD69, evaluated by flow cytometry, in Jurkat cells transduced with TCR1 after co-incubation with K562-A*11:01 target cells loaded with different peptide concentrations (10-11 to 106M). Specific Modes of Implementation

[0030] Unless otherwise indicated, all technical and scientific terms used in this document have the meaning normally understood by a specialist in the subject matter.

[0031] The term or refers to a single element among the elements listed as alternatives, unless the context expressly indicates otherwise. The term and / or Petition 870250073464, dated 08 / 20 / 2025, p. 25 / 73 9 / 44 refers to any one, any two, any three, any more, or all of the elements listed as alternatives.

[0032] The term “about” generally refers to a variation of up to 10% above or below the specified value, for example, a variation of ±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%.

[0033] The terms “comprises” or “includes” mean that the elements, wholes or steps mentioned are present, but do not exclude the presence of other additional elements, wholes or steps. In this document, unless otherwise indicated, the terms “comprises” or “includes” also cover situations where the invention consists only of the elements, wholes or steps mentioned. For example, when reference is made to a variable region that “includes” a particular sequence, such reference also aims to cover a variable region that consists of that specific sequence.

[0034] The term “major histocompatibility complex (MHC)” refers to a set of genes that encode the major histocompatibility antigens in animals. Currently, the structures of MHC class I and class II molecules have been fully elucidated, both being members of the immunoglobulin superfamily. The human MHC is called HLA (human leukocyte antigen). HLA class I molecules mainly include the protein products (heavy chain or α chain) of three functional genes: HLA-A, HLA-B, and HLA-C. Petition 870250073464, dated 08 / 20 / 2025, p. 26 / 73 10 / 44 Each heavy chain of HLA class I associates with β2-microglobulin (p2m), forming a heterodimer that constitutes the complete MHC class I molecule. Due to the polymorphism of the genes encoding HLA molecules, the HLA Nomenclature Committee has established specific principles for allele designation. The main points include: for a given allele, the genetic locus name is written after the three-letter HLA prefix, separated by a hyphen, for example, HLA-A; after the locus name, an asterisk (*) is added, followed by the genetic family number corresponding to the allele, which reflects, whenever possible, the associated serological typing; then, a colon (:) is added and the specific allele number. For example: HLA-A*02:01.One of the main functions of MHC molecules is to participate in antigen presentation; that is, MHC molecules bind to antigenic peptides through their peptide-binding groove, forming peptide-MHC complexes (pMHCs), which are presented on the cell surface to be recognized by T lymphocytes (through the TCR), thus triggering an immune response.

[0035] The term antigenic peptide refers to a short peptide capable of binding to the peptide-binding groove of an MHC molecule (or an HLA molecule). The antigen-binding groove of HLA class I molecules is formed by the α1 and α2 domains of the heavy chain, whose extracellular region comprises three structural domains: α1, α2, and α3. Each of the α1 and α2 domains adopts a conformation composed of one α-helix and four β-sheets. The two α-helices form the walls of the groove, while the eight Petition 870250073464, dated 08 / 20 / 2025, page 27 / 73 11 / 44 β-sheets form the bottom of the same. The length of antigenic peptides suitable for lodging in said binding groove is typically 8 to 11 amino acids. In a specific example, the antigenic peptide is a mutated fragment of the KRAS protein, such as VVVGAVGVGK (SEQ ID NO: 12).

[0036] The term “peptide antigenic complex: MHC” or “peptide antigenic complex: HLA” refers to the complex (pMHC) formed by an MHC molecule and an antigenic peptide, in which the peptide is located in the peptide-binding groove of the MHC molecule. The TCR molecule recognizes this complex through the CDR sequences present in its binding domain, predicting a specific binding to it. The binding of the TCR molecule to the pMHC complex is MHC-dependent, that is, the TCR molecule simultaneously recognizes the antigenic peptide and the pMHC complex.

[0037] The term “T cell receptor (TCR)” refers to the functional unit responsible for the recognition of antigens (pMHC) by T lymphocytes, belonging to the immunoglobulin superfamily. The TCR expressed on T cells is a membrane glycoprotein that exists in the form of a heterodimer composed of an α chain and a β chain, or a γ chain and a δ chain. In the human body, the vast majority (90%-95%) of TCRs present in peripheral blood are heterodimers consisting of two polypeptide chains: an α chain and a β chain. Petition 870250073464, dated 08 / 20 / 2025, p. 28 / 73 12 / 44 cytoplasmic α and β chains are very short, containing only a few amino acids. The constant regions (Ca, Oβ) exhibit relatively little sequence variation, and specific amino acid sequences can be found, for example, in the public database of the International Immunogenetics Information System (IMGT). The transmembrane region includes positively charged amino acid residues, allowing non-covalent interaction with negatively charged amino acid residues present in the transmembrane region of CD3 molecules, thus contributing to the stabilization of the TCR-CD3 complex structure. Analysis of the amino acid sequences of the variable regions of the TCR reveals that both Va and νβ possess three hypervariable regions, designated complementarity-determining regions (CDRs), namely CDR1, CDR2, and CDR3. Among these, CDR3 is the most variable and plays a determining role in the antigenic specificity of the TCR.In addition to the hypervariable regions, the variable regions also include four support structure regions (FR1 to FR4), similar to what occurs in antibody molecules. The determination of the positions of the CDR1 to CDR3 and FR1 to FR4 regions in the complete TCR sequence is based on the nomenclature defined by the IMGT, which is widely recognized and can be consulted in the IMGT public database. The variable regions of the α and β chains—namely, the three CDRs of the α chain and the three CDRs of the β chain—together form the part of the TCR responsible for specific recognition and binding to the pMHC complex (referred to in this document as the “binding domain”). In the immune system, binding... Petition 870250073464, dated 08 / 20 / 2025, p. 29 / 73 13 / 44 The interaction between the antigen-specific TCR and the pMHC complex triggers direct physical contact between T lymphocytes and antigen-presenting cells (APCs) or target cells, leading to T lymphocyte activation or target cell elimination. Unless otherwise indicated or implied by the context, the term TCR also encompasses functional fragments with antigen specificity, such as the Va and νβ variable regions linked by a short peptide.

[0038] The term antibody is used in this document in its broadest sense, encompassing immunoglobulins or other molecules that include one or more antigen-binding domains with binding specificity, being proteins or polypeptides that demonstrate binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain molecules), single-chain antibodies, single-domain antibodies, and multispecific antibodies, among others. Classic antibody molecules are generally tetramers composed of two identical heavy chains and two identical light chains, linked together by disulfide bonds. Based on the conservation differences in amino acid sequences, the heavy and light chains are divided into a variable region (V), located at the N-terminal end, and a constant region (C), located at the C-terminal end.The variable region is responsible for antigen recognition and binding, while the constant region (such as the Fc fragment) participates in the activation of downstream effects, such as antibody-mediated cellular cytotoxicity (ADCC). Petition 870250073464, dated 08 / 20 / 2025, page 30 / 73 14 / 44 Variable Regions of the Heavy and Light Chains: There are three local regions whose composition and amino acid sequence exhibit the greatest degree of variability. These are crucial for antibody-antigen binding and are therefore designated complementarity-determining regions (CDRs). The three complementarity-determining regions of the heavy chain are designated HCD1, HCD2, and HCD3, while those of the light chain are designated LCDR1, LCDR2, and LCDR3. Each variable region of the heavy chain (HC) and light chain (LC) is composed of three CDRs and four support structure regions (SRs), organized, from the N-terminal end to the C-terminal end, in the following order: SR1, HCD1, SR2, HCD2, SR3, HCD3, and SR4.In this document, the antibody molecule may also include a fusion protein composed of the recombination of the variable regions of a TCR with the constant region of an antibody, simultaneously conferring the antigenic specificity of the TCR and the effector functions of the antibody's constant region.

[0039] The term “chimeric antigen receptor (CAR)” refers to a genetically modified membrane receptor molecule that can confer a desired specificity to effector cells of the immune system, such as the ability to recognize and bind to specific tumor antigens. A chimeric antigen receptor is generally composed of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen-binding domain consists of an scFv sequence or a single-domain antibody fragment, being responsible for recognition and binding to an antigen. Petition 870250073464, dated 08 / 20 / 2025, page 31 / 73 15 / 44 specific antigen. The intracellular signaling domain generally includes tyrosine-based activation motifs of immune receptors (ITAMs), such as the signal transduction domain derived from the CD3Z molecule, responsible for activating effector cells of the immune system and inducing cytotoxic activity. Additionally, the chimeric antigen receptor may include, at the N-terminal end, a signal peptide responsible for intracellular targeting of the newly synthesized protein, as well as a hinge region between the antigen-binding domain and the transmembrane domain. Beyond the signal transduction domain, the intracellular signaling domain may also include a co-stimulatory domain, derived, for example, from the 4-1BB or CD28 molecules.

[0040] The terms antigenic specificity, “targeting,” or “specific binding” refer to the ability of the TCR (or other fusion proteins that include its binding domain, such as CARs) to specifically recognize and bind with high affinity to the corresponding pMHC complex. Compared to other molecules present in the environment, the TCR (or other fusion proteins that include its binding domain, such as CARs) exhibits a higher binding affinity for a specific molecule with which it possesses antigenic specificity, such as a pMHC complex. The binding affinity of the TCR to the pMHC complex can be assessed through certain measurement parameters, such as the EC50 value or the KD value.

[0041] The term “binding protein” refers to a Petition 870250073464, dated 08 / 20 / 2025, page 32 / 73 16 / 44 protein with the ability to bind specifically to a specific ligand, such as an antigen. In this document, the binding protein may include TCR molecules with specific binding capacity to the pMHC complex (including single-chain TCRs and soluble TCRs), fusion proteins formed between the TCR and other proteins (such as antibody molecules or chimeric antigen receptors), as well as immunoconjugates linked to detectable markers, therapeutic agents, or radioactive isotopes.

[0042] The term “fusion protein” refers to an artificially generated protein molecule (e.g., by genetic engineering techniques) composed of at least two distinct peptide segments. These peptide segments do not exist naturally or are not found in nature in the same protein molecule.Common examples of fusion proteins containing antibody fragments include multispecific antibodies, antibody-cytokine fusion proteins, enzyme-labeled antibodies for immunological detection, and chimeric antigen receptors (CARs).

[0043] When referring to the TCR (or its antigenically specific fragment), the term “variant” used in this document designates a protein obtained from the original TCR (or its antigenically specific fragment) by introducing one or more amino acid insertions, deletions, or substitutions, while maintaining at least some of the functions of the original TCR—in particular, the relevant function, such as the ability to bind to the corresponding pMHC complex. For example, a variant of the TCR (or its antigenically specific fragment) Petition 870250073464, dated 08 / 20 / 2025, page 33 / 73 17 / 44 antigenic specificity) may retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the binding capacity to the pMHC complex of the original TCR (or its antigenically specific fragment), or even exhibit a binding capacity superior to that of the original TCR. In some embodiments, the TCR variant (or its antigenically specific fragment) may retain at least 80%, 85%, 90%, 95%, or even 100% or more of the antigen-binding affinity of the original TCR (or its antigenically specific fragment). TCR variants (or its antigenically specific fragment) generally include amino acid alterations in the structural support sequence of the variable region and / or the constant region, without excluding, however, the possibility of one or a few modifications in the sequence of the CDR regions.Thus, it is also understood by those skilled in the art that, based on the specific amino acid sequences of the TCR (or its antigenically specific fragment) provided in this document—including the sequences of the CDR regions, the variable regions, or the α / β chains—it is possible to obtain corresponding variants by substitution, deletion, or addition of a limited number of amino acids, followed by validation or screening of the ability to bind to the corresponding pMHC complex or biological activity. These variants should also be considered to be included within the scope of the present invention.

[0044] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably in this document and refer to a polymer of nucleotides. These nucleotide polymers can Petition 870250073464, dated 08 / 20 / 2025, p. 34 / 73 18 / 44 contain natural and / or non-natural nucleotides and include (but are not limited to) DNA, RNA, and PNA. Nucleic acid sequence refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0045] The term vector refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (e.g., the coding sequence of a polypeptide of interest) or a nucleic acid molecule capable of replicating in a host cell (e.g., nucleic acid, plasmid, or virus). The vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that control the expression of the polynucleotide of interest (such as promoters and / or enhancers), and / or one or more selection marker genes (such as antibiotic resistance genes and genes usable in colorimetric analyses, such as β-galactosidase). The term expression vector refers to a vector used to express a polypeptide of interest in a host cell.

[0046] Host cell refers to a cell that can receive or has received a vector or an isolated polynucleotide. The host cell can be a prokaryotic or eukaryotic cell. Examples of eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting examples of mammalian cells include (but are not limited to) CHO cells, HEK-293 cells, Petition 870250073464, dated 08 / 20 / 2025, page 35 / 73 19 / 44 BHK cells or PER-C6 cells, as well as cells derived from them, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. In some embodiments, the host cell is used for the production of the protein of interest; for example, the α and / or β chain of the TCR provided in this document can be secreted by mammalian cells. In other embodiments, the host cell acquires a specific function after expressing the TCR molecule provided in this document; for example, after expressing said TCR molecule in T cells, these cells acquire the ability to recognize and eliminate target cells expressing a specific antigenic peptide. The host cell can be a single cell or cell line, including cells that have been transfected in vivo with the nucleic acid molecule or expression vector provided in this document.The host cell includes the descendants of a single host cell, and these descendants may not be entirely identical to the original progenitor cell (in terms of morphology or genomic DNA complementarity) due to natural, accidental, or induced mutations.

[0047] The term individual includes animals, such as mammals, including (but not limited to) primates, rodents, apes, felines, canines, equines, bovines, swine, ovines, caprines, laboratory mammalian animals, livestock mammalian animals, mammalian animals used in sporting activities, and mammalian pets. Individuals may be male or female and may belong to any suitable age range, including Petition 870250073464, dated 08 / 20 / 2025, page 36 / 73 20 / 44 individuals of infant, juvenile, young adult, adult, or senior age. In some examples, the term individual refers to an individual who needs diagnosis or treatment for a disease or pathological condition. In some cases, the individual receiving the diagnosis or treatment may be a patient suffering from a condition associated with that diagnosis or treatment, or who is at risk of developing such a condition. In specific instances, the individual is a human being, such as a human patient. This term can generally be used interchangeably with "patient," "individual under evaluation," or "individual under treatment."

[0048] When referring to a pharmaceutical composition, the term “pharmaceutically acceptable vehicle” designates substances such as solid or liquid diluents, excipients, antioxidants, stabilizers, among others, that can be safely administered, are suitable for administration to humans and / or animals without excessive adverse effects, and are also appropriate for maintaining the activity of the drug or active agent contained therein.

[0049] When referring to the treatment of a disease, the term "effective dose" designates the amount of an active molecule (such as an antibody) sufficient to elicit, in the individual's organism, a biological or medical response desired by the clinical professional. The "effective dose" to be administered can be determined by a technical professional in the field, taking into account factors such as route of administration, body weight, age, clinical condition of the individual, among others. For example, in the case of Petition 870250073464, dated 08 / 20 / 2025, page 37 / 73 21 / 44 protein-based drugs, the typical daily dose may vary between 0.01 mg and 100 mg of the active ingredient per kilogram of body weight. For immune effector cells expressing the TCR provided in this document (such as T lymphocytes), the number of cells administered per infusion may vary, for example, from about 1x10⁶ to about 1x10¹² cells or even more. In certain embodiments, administration may be done with fewer than 1x10⁶ T lymphocytes. Methods of administration of the active molecules provided in this document (such as TCRs, antibodies or fusion proteins) or of immune effector cells include, but are not limited to, injection, for example intravenously, intramuscularly, intra-arterially, subcutaneously or intraperitoneally.

[0050] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also called "sequence homology") refers to the degree of correspondence between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), usually expressed as a percentage. Typically, before calculating the percentage of identity between two amino acid or nucleotide sequences, the sequences are first aligned and, if necessary, gaps are introduced. If, at a given position in the alignment, the amino acid residues or nucleotides of the two sequences are identical, there is considered to be correspondence or coincidence at that position; if they are different, there is considered to be a discrepancy or incompatibility at that position. In certain algorithms, sequence identity is Petition 870250073464, dated 08 / 20 / 2025, p. 38 / 73 22 / 44 is obtained by dividing the number of coincident positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and / or the length of the gaps are also considered in the sequence identity calculation. For the purposes of the present invention, the public alignment software BLAST (available at ncbi.nlm.nih.gov) can be used, employing the predefined settings to obtain the best sequence alignment and calculate the identity between two amino acid or nucleotide sequences. In certain embodiments, the expression "at least 80% sequence identity as described in this document" includes, but is not limited to, cases of 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.

[0051] This document provides isolated or purified binding proteins with antigenic specificity for peptide-MHC antigen complexes (in which the antigenic peptide has amino acid mutations relative to the wild-type sequence), such as T cell receptors (TCRs), antibodies or CARs, the antigenic peptide being derived from a mutated KRAS protein.

[0052] This document also provides polypeptides and proteins associated with the aforementioned TCR or its binding domain, as well as corresponding nucleic acid molecules, vectors (such as recombinant expression vectors), host cells (such as Petition 870250073464, dated 08 / 20 / 2025, page 39 / 73 23 / 44 T lymphocytes) and associated pharmaceutical compositions.

[0053] This document also provides methods and kits for detecting the presence of tumors in an individual, as well as methods for treating or preventing tumors in that individual. T-cell receptor (TCR)

[0054] This document provides isolated or purified T cell receptors (TCRs) with antigenic specificity for antigen-MHC peptide complexes (in which the antigenic peptide has amino acid mutations relative to the wild-type sequence), the antigenic peptide being derived from a mutated KRAS protein and including, or consisting of, the sequence VVVGAXGVGK (SEQ ID NO:11), where X represents any amino acid residue that is neither G (glycine) nor R (arginine). 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).Given that MHC class I molecules do not impose a strict limitation on the length of the antigenic peptide, the inventors of this document foresee that, based on the antigenic peptide sequences provided herein, one or two amino acid residues can be added or removed (particularly at the ends), while maintaining the binding capacity of the binding proteins or TCR molecules described in this document to the MHC complexes formed with these antigenic peptide variants.

[0055] The TCRs provided in this document may Petition 870250073464, dated 08 / 20 / 2025, page 40 / 73 24 / 44 can be in the form of αβ heterodimers or in single-chain form (scTCR). Examples of the single-chain form may include αβ TCR polypeptides with the structures Va-L-νβ, νβ-L-Va, Va-Ca-L-νβ, or Va-L-νβce, where Va and Ve represent, respectively, the variable regions of the α and β chains of the TCR, Ca and Ce are the constant regions of the α and β chains of the TCR, and L is a linker sequence, usually consisting of a short peptide. The linker sequence may be a conventional sequence in the field, frequently used in antibody engineering and TCR engineering. The TCRs provided in this document may also take the form of water-soluble TCR molecules, which do not contain the transmembrane regions of the α and β chains.

[0056] In certain embodiments, the present document provides TCR molecules with antigenic specificity for the referenced antigenic peptide-MHC complex. The CDR3 region of the variable α chain of the TCR molecule may include the amino acid sequence shown as SEQ ID NO: 5, or an amino acid sequence with at least 80% identity to SEQ ID NO: 5, preferably at least 85%, more preferably 90% or 95% identity. The CDR3 region of the variable β chain may include the amino acid sequence shown as SEQ ID NO: 10, or a sequence with at least 80% identity to SEQ ID NO: 10, preferably at least 85%, more preferably 90% or 95% identity. Preferably, the MHC molecule is of the HLA-A*11:01 type.

[0057] Additionally, in the variable region of the α chain, CDR1 includes the amino acid sequence presented as SEQ Petition 870250073464, dated 08 / 20 / 2025, p. 41 / 73 25 / 44 ID NO: 3, CDR2 includes the sequence shown as SEQ ID NO: 4, and CDR3 includes the sequence shown as SEQ ID NO: 5, or an amino acid sequence with at least 80% identity with SEQ ID NO: 5, preferably at least 85%, more preferably 90% or 95% identity. In the variable region of the β chain, CDR1 includes the amino acid sequence shown as SEQ ID NO: 8, CDR2 includes the sequence shown as SEQ ID NO: 9, and CDR3 includes the sequence shown as SEQ ID NO: 10, or a sequence with at least 80% identity with SEQ ID NO: 10, preferably at least 85%, more preferably 90% or 95% identity.

[0058] Furthermore, in the TCR molecule, the variable region of the α chain includes the amino acid sequence shown as SEQ ID NO: 2, or a sequence with at least 80% identity with SEQ ID NO: 2, preferably at least 85%, more preferably 90% or 95% identity; and the variable region of the β chain includes the sequence shown as SEQ ID NO: 7, or a sequence with at least 80% identity with SEQ ID NO: 7, preferably at least 85%, more preferably 90% or 95% identity.

[0059] In certain specific embodiments, in the TCR molecule, the α chain includes the amino acid sequence shown as SEQ ID NO: 1, or a sequence with at least 80% identity with SEQ ID NO: 1, preferably at least 85%, more preferably 90% or 95% identity; and the β chain includes the sequence shown as SEQ ID NO: 6, or a sequence with at least 80% identity with SEQ ID NO: 6, preferably Petition 870250073464, dated 08 / 20 / 2025, p. 42 / 73 26 / 44 at least 85%, more preferably 90% or 95% identity.

[0060] It will be understood by a professional in the field that, based on the specific sequences provided in this document, it is possible to obtain variants of the TCR molecules described herein by means of substitutions, deletions or additions of a limited number of amino acid residues, followed by validation or screening of the ability to bind to the corresponding pMHC complex or its biological activity. These variants should also be considered included within the scope of this document. For example, the TCR molecules provided in this document may present, in their total length sequence, in the variable region or in the CDR sequences, at least 1 and at most 10 amino acid alterations, and in certain cases the number of alterations does not exceed 5, 4, 3, 2 or even only 1 amino acid residue.For example, at least 1 and at most 10 amino acid changes may be introduced into the variable sequence of the α or β chain, and in certain cases the number of changes does not exceed 5, 4, 3, 2, or 1 residue. Additionally, the CDR sequences of the variable regions of the α or β chains may contain, in total, no more than 5, 4, 3, 2, or 1 amino acid change.

[0061] It is expected that the TCRs (or their antigenically specific fragments) described in this document may include conservative amino acid substitutions. Conservative amino acid substitutions are generally described as the replacement of one amino acid residue with another of similar chemical structure. Petition 870250073464, dated 08 / 20 / 2025, p. 43 / 73 27 / 44 resulting in little or no impact, or minimal impact, on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the technical field. Conservative substitutions may, for example, involve the substitution of one amino acid for another belonging to the same of the following groups (a) to (e): (a) small, aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) negatively charged polar residues and their respective amides (uncharged): Asp, Asn, Glu, and Gln; (c) positively charged polar residues: His, Arg, and Lys; (d) large, nonpolar aliphatic residues: Met, Leu, Ile, Val, and Cys; (e) aromatic residues: Phe, Tyr, and Trp.

[0062] In certain embodiments, the TCRs provided in this document (or their antigen-specific fragments) may additionally include post-translational modifications. Examples of post-translational modifications of proteins include: phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitin-type modifications, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Thus, modified TCRs (or their antigen-specific fragments) may include non-peptidic components such as lipids, polysaccharides, or monosaccharides, as well as phosphate groups. One form of glycosylation is, for example, sialylation modification, in which one or more sialic acid groups are conjugated to the polypeptide. Sialic acid groups improve the solubility and serum half-life of proteins, while reducing their Petition 870250073464, dated 08 / 20 / 2025, page 44 / 73 28 / 44 potential immunogenicity.

[0063] When the N-terminal end of the TCR amino acid sequence provided in this document is a methionine residue (M), it is known to professionals in the field that, during the recombinant protein production process, this methionine can be removed. Fusion proteins and immunoconjugates

[0064] This document also provides fusion proteins that include the variable region of the α and / or β chain of the TCR molecule provided here, especially their CDR sequences.

[0065] In certain embodiments, the aforementioned fusion protein is in the form of a single-chain TCR (scTCR). For example, by linking the variable region of the α chain to the variable region of the β chain via a linker sequence, a complete binding domain with antigenic specificity for the corresponding pMHC complex can be formed.

[0066] In certain embodiments, the variable regions of the α and / or β chain of the TCR molecule provided in this document, especially their CDR sequences, can be used to form multispecific TCR molecules. In certain embodiments, said fusion protein includes at least two functional parts: a first functional part with antigenic specificity for a first pMHC complex, and a second functional part that has identical or different antigenic specificity from the first.

[0067] In certain embodiments, the variable regions of the α and / or β chain of the supplied TCR molecule Petition 870250073464, dated 08 / 20 / 2025, page 45 / 73 29 / 44 in this document, especially their CDR sequences, can be used to form multispecific fusion proteins. In certain embodiments, said fusion protein includes at least two functional parts: a first functional part with antigenic specificity for a pMHC complex, and a second functional part with antigenic specificity or targeting capability different from the first part. In certain embodiments, the second functional part may be a targeting domain, capable of specifically recognizing and binding to specific tumor antigens, tumor-associated antigens, or other molecules present on the surface of target cells.Examples include antibodies or their antigen-binding fragments (such as scFv), hormones, growth factors, cytokines, as well as any other natural or non-natural ligands that bind to cell surface receptors, such as the epidermal growth factor receptor (EGFR), CD28, the platelet-derived growth factor receptor (PDGFR), the nicotinic acetylcholine receptor (nAChR), among others.

[0068] In certain embodiments, the binding domain of an antibody molecule (e.g., a classic tetrameric antibody) can be replaced by the TCR molecule provided in this document (or by the variable regions of the α and β chains, or even by the binding domain formed by these), in order to form a recombinant antibody molecule with the antigenic specificity of that TCR. In certain embodiments, the variable region of the α chain of the TCR molecule is used. Petition 870250073464, dated 08 / 20 / 2025, p. 46 / 73 30 / 44 to replace the two variable regions of the heavy chains of an antibody, binding to the respective constant regions of the heavy chains; simultaneously, the variable region of the β chain replaces the two variable regions of the light chains of the antibody, binding to the respective constant regions of the light chains. In certain embodiments, the variable region of the β chain of the TCR molecule is used to replace the two variable regions of the heavy chains of an antibody, binding to the respective constant regions of the heavy chains; simultaneously, the variable region of the α chain replaces the two variable regions of the light chains of the antibody, binding to the respective constant regions of the light chains.In certain embodiments, the variable region of the α chain and the variable region of the β chain of the TCR molecule are used to replace, respectively, a variable region of the heavy chain and a variable region of the light chain of an antibody, while leaving one of the antibody's original antigen-binding domains unchanged. In this way, a bispecific recombinant antibody molecule is obtained, capable of recognizing both the pMHC complex and the antibody's original target antigen. The recombinant antibody molecule thus formed can exert effector functions typical of antibodies through its constant region (such as the Fc fragment), including, for example, mediating complement-dependent cellular cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and facilitating phagocytosis. Additionally, fusion to the Fc fragment can increase the half-life of TCR molecules (or the variable regions of the TCR). Petition 870250073464, dated 08 / 20 / 2025, p. 47 / 73 31 / 44 α and β chains, or the binding domain formed by them) in the body, allowing for longer intervals between administrations when these molecules are used as therapeutic agents. In certain embodiments, the aforementioned Fc fragment may be derived from the constant region of an immunoglobulin, such as IgG1, IgG2, or IgG4.

[0069] In certain embodiments, the TCR molecules provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them) can be used as an extracellular binding domain in the construction of chimeric antigen receptors (CARs). For example, the variable regions of the α and β chains can be linked via a linker sequence, forming a binding molecule similar to an scFv, which replaces the conventional extracellular binding domain of a CAR, and is then linked to the transmembrane region and intracellular signaling domains. This CAR construct is directed to the pMHC complex recognized by the original TCR molecule and, through the CAR's intracellular signaling domains, activates the T cell that expresses it, triggering the desired immunological effects, such as the destruction of target cells.

[0070] In certain embodiments, the TCR molecule (or the variable regions of the α and β chains, or the binding domain formed by them) can be linked to a conjugate to form an immunoconjugate, said conjugate being, for example, a detection tag, a therapeutic agent, a screening marker, among others.

[0071] In certain embodiments, the TCR molecule (or the variable regions of the α and β chains, or the domain of Petition 870250073464, dated 08 / 20 / 2025, p. 48 / 73 The 32 / 44 linkage formed by them) can be linked to a protein tag, forming a fusion protein. Protein tags can include purification tags and detection tags. Purification tags include, but are not limited to: His6 tag, Flag tag, MBP tag, GST tag, SUMO tag, among others. Detection tags can be used to indicate the presence or quantity of the TCR molecule (or the variable regions of the α and β chains, or the binding domain formed by them) in a sample, or even to track the location of this molecule within the organism or the individual's cells. Examples of detection tags include various enzymes used in immunochemical assays, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), as well as fluorescent proteins such as GFP (green fluorescent protein).Due to the specific binding capacity of the TCR molecule (or the variable regions of the α and β chains, or the binding domain formed by them) to the corresponding pMHC complex, the quantity of the TCR molecule (or the variable regions of the α and β chains, or the binding domain formed by them) can be determined by measuring the amount of the detection tag bound to the TCR. Consequently, it is possible to infer the quantity of the corresponding pMHC complex present in the sample. When the corresponding pMHC complex is used as a tumor marker, the aforementioned fusion protein can be employed for tumor detection and diagnosis.

[0072] In other embodiments, the TCR molecule provided in this document (or the variable regions) Petition 870250073464, dated 08 / 20 / 2025, p. 49 / 73 33 / 44 of the α and β chains, or the binding domain formed by them) can be linked to cytokines or therapeutic proteins, forming fusion proteins. In this case, the specific binding capacity of the TCR molecule (or of the variable regions of the α and β chains, or the binding domain formed by them) to the corresponding pMHC complex can be used to target the cytokine or therapeutic protein specifically to certain tissues or cells, thus enabling the desired therapeutic effect of the cytokine or therapeutic protein.

[0073] In certain embodiments, the TCR molecule (or the variable regions of the α and β chains, or the binding domain formed by them) can be conjugated to chemotherapeutic agents, such as asparaginase, bleomycin, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, vinorelbine, among others, in order to allow the targeted delivery of these agents to specific cells or tissues through the antigenic specificity of the TCR.

[0074] In certain embodiments, the TCR molecule (or the variable regions of the α and β chains, or the binding domain formed by them) can be conjugated to radioactive isotopes, such as 3H, 14C or 35S, for screening or therapeutic purposes. Host cells for expression of TCR molecules

[0075] This document also provides host cells that express the TCR molecule provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them). Petition 870250073464, dated 08 / 20 / 2025, pp. 50 / 73 34 / 44

[0076] The host cells used refer to any type of cell capable of expressing the TCR molecule provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them). Preferably, the host cell expresses the complete TCR molecule, including the variable and constant regions of the α and β chains, as well as the transmembrane and intracellular regions. The host cell can be a eukaryotic or 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 of any cell type, originate from any tissue type, and belong to any developmental stage, it is preferred that the host cell be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).Even more preferably, the host cell is a T lymphocyte. For the purposes of this document, T cells may be any T cells, such as cultured T cells, for example, primary T cells or T cells from cultured cell lines such as Jurkat, SupTl, among others, or T cells obtained from mammals. When obtained from mammals, T cells may be isolated from various sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. T cells may be of any type and at any stage of development, including but not limited to: double-positive T cells. Petition 870250073464, dated 08 / 20 / 2025, pp. 51 / 73 35 / 44 CD4+ / CD8+, 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, among others.

[0077] This document also provides host cells that express the CAR provided in this document. Preferably, the host cell is a human cell, especially a human T cell or a human NK cell.

[0078] The functionality of host cells expressing TCR or CAR (such as T cells) can be assessed by various methods. In certain embodiments, the cytotoxic capacity of host cells expressing TCR or CAR (such as T cells) can be assessed by incubating these cells with target cells and measuring the quantity or activity of the target cells, for example, by detecting LDH release. In certain embodiments, the immune effect of host cells expressing TCR or CAR (such as T cells) can be assessed by incubating these cells with target cells and measuring IFN-γ secretion or CD69 expression by the host cells. Pharmaceutical compositions and therapeutic methods

[0079] The TCR molecules provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them), as well as the fusion proteins or immunoconjugates that include them, may be formulated with acceptable pharmaceutical vehicles to compose a Petition 870250073464, dated 08 / 20 / 2025, pp. 52 / 73 36 / 44 pharmaceutical composition intended for administration to an individual for the prevention or treatment of tumors.

[0080] In certain embodiments, the TCR molecules provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them), as well as the fusion proteins or immunoconjugates that include them, can be administered in combination with one or more other drugs, such as antitumor agents.

[0081] In certain embodiments, the disease or condition treated is a tumor, especially tumors that express antigenic peptides containing KRAS G12 mutations. These tumors include, but are not limited to: acute lymphoblastic leukemia, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, cervical cancer, gallbladder cancer or pleural mesothelioma, nasal cancer, nose cancer, or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer,Non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, cancer of, Petition 870250073464, dated 08 / 20 / 2025, pp. 53 / 73 37 / 44 skin cancer, small bowel cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureter cancer, and bladder cancer. Preferred cancers include pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, prostate cancer, among others.

[0082] Nucleic acid molecules encoding the TCR molecules provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them), as well as fusion proteins or immunoconjugates that include them, including expression vectors containing such nucleic acid molecules, and host cells (such as T cells or CAR cells) transfected with such nucleic acid molecules or expression vectors, can also be used in various ways for the aforementioned therapeutic purposes. For example, expression vectors can be introduced into the individual's body by known gene therapy methods, promoting the expression of the desired protein or polypeptide (such as the TCR molecules provided in this document, or the variable regions of the α and β chains, or the binding domain formed by them, as well as the fusion proteins or immunoconjugates that include them), in order to achieve the intended therapeutic effect.

[0083] The effective dose for this application may depend on the severity of the disease and the overall state of the patient's immune system. The administration regimen may also vary depending on the disease state and the individual's condition, generally ranging from one administration in Petition 870250073464, dated 08 / 20 / 2025, pp. 54 / 73 38 / 44 single bolus dose or continuous infusion up to multiple daily administrations (e.g., every 4 to 6 hours). An experienced clinician can, for example, easily determine if an individual is a candidate for this treatment using clinical trials, physical examinations, and the individual's family history. Diagnostic or treatment kits

[0084] The TCR molecules provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them), as well as the fusion proteins or immunoconjugates that include them, can specifically bind to the corresponding pMHC complex present in a sample. By detecting the amount of the ternary complex formed (i.e., TCR-antigenic peptide-MHC molecule), it is possible to conveniently determine the amount (or presence) of the corresponding pMHC complex in the sample.

[0085] To this end, as described above, the TCR molecules provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them) can be conjugated to various detection tags, facilitating their identification by several methods, including, but not limited to, bioluminescence, fluorescence, radioactive labeling, or the production of enzymatic reaction products. After determining the amount of the corresponding pMHC complex in the sample, comparison with normal levels found in healthy individuals can be used to assess the state or severity of the disease in the individual who provided the sample. Repeated monitoring over time of Petition 870250073464, dated 08 / 20 / 2025, pp. 55 / 73 39 / 44 variations in the amount of the corresponding pMHC complex during the individual's treatment can also be used to assess the effectiveness of the therapy, thus providing a basis for adjusting the therapeutic regimen as needed.

[0086] The TCR molecules provided in this document (or the variable regions of the α and β chains, or the binding domain formed by them), as well as the fusion proteins or immunoconjugates that include them, or the host cells that express them, can be packaged in containers to form diagnostic or treatment kits. These containers can take the form of boxes, vials, small bottles, tubes, bags or other suitable forms known in the technical field. These containers can be made of plastic, glass, foil, metal foil or other materials suitable for drug storage. When necessary, the container can be supplied together with an instruction leaflet.The instruction leaflet may include information on the use of TCR molecules (or the variable regions of the α and β chains, or the binding domain they form), the compositions containing them, and the host cells for the treatment or prevention of tumors. This information may include, for example: descriptions of the therapeutic agents (such as TCR molecules, nucleic acid molecules, host cells, etc.); dosage regimens for the treatment or prevention of neoplasms; precautions; warnings; indications; contraindications; adverse effects; pharmacological data in animals; clinical trials; and / or bibliographic references. The instruction leaflet may... Petition 870250073464, dated 08 / 20 / 2025, pp. 56 / 73 40 / 44 can be printed directly on the container (if present), or presented as a label affixed to the container, or provided as a separate document on paper, booklet, card or foldable material included inside the container or delivered together with it. Experimental materials and reagents: 1. PBMC of healthy individuals 2. Target peptide: A*11:01 KRAS-G12V peptide: VVVGAVGVGK (SEQ ID NO:12) A*11:01 KRAS-G12D peptide: VVVGADGVGK (SEQ ID NO:13) A*11:01 KRAS-G12S peptide: VVVGASGVGK (SEQ ID NO:14) A*11:01 KRAS-G12A peptide: VVVGAAGVGK (SEQ ID NO:15) A*11:01 KRAS-G12C peptide: VVVGACGVGK (SEQ ID NO:16) 3. Brilliant Violet 510™ Anti-Human CD3 Antibody: Biolegend 300448 4. APC Anti-Human CD8a Antibody: Biolegend 301014 5. Flex-T™ HLA-A*11:01 UVX Monomer: Biolegend 280007 6. Chromium Next GEM Single Cell V(D)J v1.1 Reagent Kits 7. Lentiviral vector 8. Polybrene Viral Enhancer: Merck Millipore TR1003-G 9. Anti-CD3 / CD28 magnetic beads: novoprotein GMPB038 10. X-VIVO 15: lonza 04418q 11. OKM-100: CansBio sj18 12. RPMI 1640: gibco 11875-093 13. LDH reagent kit: dojindo 14. Human IFN-γ Reagent Kit: Excell Bio EH008-96 15. ELISPOT for IFN-γ: Mabtech 3420-4AST-10 Petition 870250073464, dated 08 / 20 / 2025, pp. 57 / 73 41 / 44 16. Human anti-CD69 APC antibody: BioLegend 310910 Equipment and Instruments • Flow cytometer: BD LSRFortessa • Flow cytometry sorter: SONY SH800S • Microplate reader (spectrophotometer): Molecular Devices SpectraMax i3x • Imaging system for ELISPOT: Bio-sys Bioreader 6000-Ep • Charting and analysis software • FlowJo • Graphpad Prism 8.0.1 Examples of Implementation

[0087] T lymphocytes from a healthy donor were stimulated with dendritic cells loaded with the 10-amino acid KRAS G12V peptide (VVVGAVGVGK) (SEQ ID NO: 12). The ratio of A*11:01-G12V specific T lymphocytes was assessed by tetramer staining and flow cytometry analysis using the BD LSRFortessa. Subsequently, the specific T cells were enriched and selected using the SONY SH800S flow cytometry sorter. The data obtained were analyzed using FlowJo software. The resulting flow cytometry graph is shown in Figure 1. The enriched cells were subjected to 10-cell sequencing, obtaining the paired chains of TCRa and TCRp (see the amino acid sequence of TCR1 below).

[0088] A lentiviral vector containing the TCR gene of the present invention was constructed and translated into T lymphocytes. The T cells transduced with the TCR were Petition 870250073464, dated 08 / 20 / 2025, pp. 58 / 73 42 / 44 co-incubated for 16 hours 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) BxPC3 cells expressing HLA-A*11:01, co-expressing HLA-A*11:01 and KRAS G12V, or co-express HLA-A*11:01 and KRAS G12D, in a ratio of effector cells to target cells of 5:1. The cytotoxic capacity of T lymphocytes against the different target cells was evaluated by LDH release. Statistical analysis of the data was performed using GraphPad Prism 8.0.1 software, as shown in Figures 2A and 2B.The results demonstrate that T cells transduced with the TCR of the present invention exhibit specific and effective cytotoxicity against target cells expressing HLAA*11:01 loaded with the peptides KRAS G12D-10, KRAS G12V-10, KRAS G12S-10, KRAS G12A-10 and KRAS G12C-10, as well as against cells overexpressing KRAS G12V or KRAS G12D.

[0089] The functional affinity of the TCR was evaluated based on CD69 expression after co-incubation of T cells transduced with the TCR of the present invention and target cells loaded with different concentrations of the peptides KRAS G12V-10 (VVVGAVGVGK) (SEQ ID NO: 12) and KRAS G12D-10 (VVVGADGVGK) (SEQ ID NO: 13). As illustrated in Figure 3, the EC50 concentration for recognition of the G12V-10 peptide by the TCR was approximately 45 nM, while the EC50 concentration for recognition of the peptide Petition 870250073464, dated 08 / 20 / 2025, pp. 59 / 73 43 / 44 G12D-10 was approximately 77 nM.

[0090] Some of the amino acid sequences mentioned in this document are presented below. Amino acid sequence of the α chain of TCR1 METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDP GKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVNSYSGAG SYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVY ITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 1) Amino acid sequence of the variable (V) region of the α chain of TCR1 KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTS GRLNASLDKSSGRSTLYIAASQPGDSATYLCAVNSYSGAGSYQLTFGKGTKLSVIP (SEQ ID NO: 2) Amino acid sequence of CDR1 of the α chain of TCR1 DSAIYN (SEQ ID NO: 3) Amino acid sequence of CDR2 of the α chain of TCR1 IQSSQRE (SEQ ID NO: 4) Amino acid sequence of CDR3 of the α chain of TCR1 AVNSYSGAGSYQLT (SEQ ID NO: 5) Amino acid sequence of the β chain of TCR1 MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILG QKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASRGGGE QYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG (SEQ ID NO: 6) Amino acid sequence of the variable (V) region of the β chain of TCR1 EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSE Petition 870250073464, dated 08 / 20 / 2025, pages 60 / 73 44 / 44 IFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASRGGGEQYFGPGTRLTVT ID NO: 7) Amino acid sequence of CDR1 of the β chain of TCR1 SNHLY (SEQ ID NO: 8) Amino acid sequence of CDR2 of the β chain of TCR1 FYNNEI (SEQ ID NO: 9) Amino acid sequence of CDR3 of the β chain of TCR1 ASRGGGEQY (SEQ ID NO: 10) (SEQ Petition 870250073464, dated 08 / 20 / 2025, pp. 61 / 73

Claims

1 / 4 CLAIMS (modified) 1. A binding protein, characterized in that it comprises a binding domain specific for an antigenic peptide: HLA complex, said binding domain being constituted by the variable region of the α chain of the T cell receptor (TCR) and the variable region of the β chain of the TCR, wherein: said antigenic peptide includes the amino acid sequence VVVGAXGVGK (SEQ ID NO: 11), wherein X represents any amino acid residue except G or R; said HLA is HLA-A*11:01; The variable region CDR3 of the α chain includes the amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence with at least 80% or 90% identity with the sequence represented by SEQ ID NO: 5; The variable region CDR3 of the β chain includes the amino acid sequence represented by SEQ ID NO: 10, or an amino acid sequence with at least 80% or 90% identity to the sequence represented by SEQ ID NO:

10. 2.Binding protein, according to claim 1, characterized in that it comprises the antigenic peptide including 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. Binding protein, according to claim 1, characterized in that it comprises in the variable region of the α chain, CDR1 including the amino acid sequence represented by SEQ ID NO: 3, CDR2 including the amino acid sequence represented by SEQ ID NO: 4, and CDR3 including the amino acid sequence represented by SEQ Petition 870250075335, dated 08 / 26 / 2025, page.7 / 10 2 / 4 ID NO: 5, or an amino acid sequence with at least 80% or 90% identity to the sequence represented by SEQ ID NO: 5; in the variable region of the β chain, CDR1 including the amino acid sequence represented by SEQ ID NO: 8, CDR2 including the amino acid sequence represented by SEQ ID NO: 9, and CDR3 including the amino acid sequence represented by SEQ ID NO: 10, or an amino acid sequence with at least 80% or 90% identity to the sequence represented by SEQ ID NO:

10. 4.A binding protein according to claim 1, characterized in that the variable region of the α chain includes the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence with at least 85% or 90% identity to the sequence represented by SEQ ID NO: 2; the variable region of the β chain includes the amino acid sequence represented by SEQ ID NO: 7 or an amino acid sequence with at least 85% or 90% identity to the sequence represented by SEQ ID NO: 7; preferably, the α chain comprises the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence with at least 85% or 90% identity to the sequence represented by SEQ ID NO: 1; the β chain comprises the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence with at least 85% or 90% identity to the sequence represented by SEQ ID NO:

6. 5.Binding protein, according to claim 1, characterized in that the binding protein is a protein in the form of: Petition 870250075335, dated 08 / 26 / 2025, page 8 / 10 3 / 4 1) TCR molecule; 2) antibody molecule; or 3) CAR molecule.

6. Binding protein, according to claim 1, characterized in that it further comprises a conjugate covalently or non-covalently linked to the binding domain; preferably, said conjugate being a detectable marker, a radioactive isotope or a therapeutic agent.

7. Isolated nucleic acid molecule, characterized in that it encodes the binding protein, the variable region of the α chain, the variable region of the β chain, the α chain or the β chain as described in claim 1.

8. Vector, characterized in that it comprises the nucleic acid molecule according to claim 7.

9. Host cell, characterized in that it expresses the binding protein as defined in claim 1, preferably the host cell being a mammalian cell, more preferably a human cell; even more preferably, it being a T cell or an NK cell. 10.Host cell, according to claim 9, characterized in that it exhibits cytotoxic activity against HLA-A*11:01 cells expressing the antigenic peptide VVVGAXGVGK (SEQ ID NO: 11), wherein X represents any amino acid residue except G or R; preferably, the antigenic peptides include 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). Petition 870250075335, dated 08 / 26 / 2025, p. 9 / 10 4 / 4 11. Pharmaceutical composition, characterized in that it comprises: 1) The binding protein as defined in claim 1 or the host cell expressing said binding protein; and 2) An acceptable pharmaceutical vehicle. 12.Use of the binding protein, as defined in claim 1, or of the host cell expressing said binding protein, characterized in that it is directed to the preparation of a medicament for the treatment of tumors, wherein the tumor expresses the antigenic peptide; wherein said 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).

13. Use according to claim 12, characterized in that the tumor is selected from pancreatic cancer, colorectal cancer, lung cancer, cholangiocarcinoma, endometrial cancer and ovarian cancer. Petition 870250075335, dated 08 / 26 / 2025, page 10 / 10.

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