WO2024148178A2 - Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*03:01 dans le cancer du poumon - Google Patents

Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*03:01 dans le cancer du poumon Download PDF

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WO2024148178A2
WO2024148178A2 PCT/US2024/010339 US2024010339W WO2024148178A2 WO 2024148178 A2 WO2024148178 A2 WO 2024148178A2 US 2024010339 W US2024010339 W US 2024010339W WO 2024148178 A2 WO2024148178 A2 WO 2024148178A2
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cell
tcr
seq
aspects
cancer
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WO2024148178A3 (fr
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Alexandre REUBEN
Minying ZHANG
John Victor HEYMACH
Gregory A. LIZEE
Peixin JIANG
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A61K40/4253—GTPases, e.g. Ras or Rho

Definitions

  • Lung cancer is a common form of cancer in men and women, and it is responsible for the highest number of cancer-related deaths globally. Because of the high rate (>50%) of late diagnosis, the 5 year overall survival rate of patients with lung cancer has improved very little over the past 3 decades, hovering around the 13%- 16% range.
  • Neoantigens a class of tumor-specific antigens
  • CTLs Cytolytic T lymphocytes
  • HLA class I molecules represent antitumor immune cells that are capable of causing regression of large tumors in cancer patients.
  • T-cell receptors (TCRs) derived from such tumor antigenspecific T cells can be cloned and isolated to create engineered TCR-T cells.
  • the T cell receptor (TCR), or antigen-binding fragment thereof, of the disclosure comprises (a) an alpha chain (TCR-a) comprising a CDR1 a having the sequence of SEQ ID NO: 3, a CDR2a having the sequence of SEQ ID NO: 4, a CDR3a having the sequence of SEQ ID NO: 5; and (b) a beta chain (TCR-P) comprising a CDRip having the sequence of SEQ ID NO: 6, a CDR2[3 having the sequence of SEQ ID NO: 7, and a CDR3[3 having the sequence of SEQ ID NO: 8.
  • the disclosure is directed to a polynucleotide encoding the TCR, or antigen-binding fragment thereof, of the disclosure.
  • the polynucleotide encodes the alpha chain (TCR-a) of the recombinant TCR, or antigenbinding fragment thereof, of the disclosure.
  • the polynucleotide encodes the beta chain (TCR-P) of the recombinant TCR, or an antigen-binding fragment thereof, of the disclosure.
  • the polynucleotide encodes the alpha chain (TCR- 00 and the beta chain (TCR-P) of the recombinant TCR, or antigen-binding fragments thereof, of the disclosure.
  • the vector comprises a polynucleotide encoding the alpha chain (TCR-00 and the beta chain (TCR-P) of the recombinant TCR, or antigen-binding fragment thereof, of the disclosure.
  • the expression vector comprises a promoter.
  • the disclosure is directed to a Bi-specific T-cell engager (BiTE) comprising a TCR, or antigen-binding fragment thereof, of the disclosure.
  • BiTE Bi-specific T-cell engager
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the TCR is expressed on the surface of the modified T cell.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the cancer is selected from the group consisting of: a lung cancer (e.g., a non-small cell lung cancer (NSCLC)), a colorectal cancer, a pancreatic cancer, an appendiceal cancer, a small bowel adenocarcinoma, an hepatobiliary cancer, a gynecological malignancy, a hematopoietic cancer, a breast cancer, a bladder cancer, a prostate cancer, a skin cancer, and any combination thereof.
  • a lung cancer e.g., a non-small cell lung cancer (NSCLC)
  • NSCLC non-small cell lung cancer
  • a colorectal cancer e.g., a colorectal cancer
  • pancreatic cancer e.g.
  • FIG. 2 shows flow cytometry analysis indicating the proportion of Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01 antigen-specific T cells from healthy donor blood following the first rapid expansion protocol.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • FIG. 3 shows flow cytometry analysis indicating the proportion of Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01 antigen-specific T cells from healthy donor blood following the second rapid expansion protocol, and used in killing and TCR a/p determination.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • FIG. 5 shows the ability of TCR transduced T cell population to kill targeted tumor cells expressing the antigen of interest endogenously by chromium-51 release assay.
  • vector includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors.
  • polypeptides dipeptides, tripeptides, oligopeptides, "protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
  • a % sequence identity value can be determined by dividing (a) the number of matching identical amino acid residues between a particular amino acid sequence as set forth herein which is subjected to comparison (e.g. a particular polypeptide sequence characterized by a sequence identifier in the sequence listings) and the candidate amino acid sequence of interest to be compared, for example the number of matching identical amino acid residues as determined by WU-BLAST-2, by (b) the total number of amino acid residues of the polypeptide sequence as set forth herein which is subjected to comparison (e.g. a particular polypeptide sequence characterized by a SEQ. ID. NO. in the sequence listings).
  • the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides in length (Or 20, 50, 200 or more amino acids).
  • a sequence serves as a reference sequence against which the test sequence is compared.
  • a sequence comparison algorithm is used, a test sequence and a reference sequence are input into a computer, and the sub-sequence coordinates and the sequence algorithm program parameters are specified, if necessary. Default program parameters can be used, or alternative parameters can be specified.
  • the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Methods of sequence alignment for comparison are well known in the art.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode polypeptides eliciting a desired immune response.
  • the antigen does not need to be encoded by a "gene” at all.
  • the antigen can be produced synthetically, or it can be derived from a biological sample, or it can be a macromolecule other than a polypeptide.
  • biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.
  • antigen recognition unit or "antigen-binding fragment” as used herein can include any molecular structure containing a polypeptide chain that has a specific shape that matches the epitope and recognizes the epitope, in which one or more non- covalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • the term "cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells (e.g., malignant cells) in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues through local spread and can also metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • the methods of the present disclosure can be used to reduce the size of a primary tumor or a metastatic tumor, or treat a primary tumor or a metastatic tumor.
  • the conditions that can be treated or prevented by the method of the present invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like.
  • the method of the present disclosure can achieve the inhibition and/or reversal of the undesirable hyperproliferative cell growth involved in such conditions.
  • the cancer can be lung cancer, e.g., non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • tumor refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.
  • primary tumor refers to the original, or first, tumor formed in the subject’s body.
  • metalastasis refers to cancer (e.g., a tumor) formed by cancer cells derived from a primary cancer (e.g., tumor) that spread to further locations or areas of the body.
  • a primary cancer e.g., tumor
  • refractory refers to a disease, such as cancer, which does not respond to treatment.
  • a refractory cancer may be resistant to treatment before or at the beginning of the treatment.
  • a refractory cancer may become resistant during treatment.
  • Refractory cancers are also called resistant cancers.
  • refractory or recurrent malignant tumors can use the treatment methods disclosed herein.
  • relapsed refers to the return of the signs and symptoms of a disease (e.g. cancer) or the return of a disease such as cancer during a period of improvement, for example, after a therapy, such as a previous treatment of cancer therapy.
  • a disease e.g. cancer
  • a therapy such as a previous treatment of cancer therapy.
  • administration refers to the administration of a composition of the present disclosure to a subject or system. Administration to an animal subject (e.g., to a human) can be by any appropriate route.
  • Administration refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the terms "effective amount,” “therapeutically effective amount,” and a “sufficient amount” refer to an amount of an agent that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system, and, as such, an "effective amount” depends on the context in which it is being applied.
  • an "effective amount”, or synonym thereto, of an agent is an amount that results in a beneficial or desired result in a subject as compared to a control, e.g., a beneficial result can take the form of an improvement over baseline (i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method).
  • a beneficial result can take the form of an improvement over baseline (i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method).
  • the precise number of immune effector cells and therapeutic agents of the present invention to be administered can be determined by a physician in consideration of the individual's age, weight, tumor size, degree of infection or metastasis, and the condition of a patient (subject).
  • An effective amount of immune effector cells refers to, but is not limited to, the number of immune effector cells which can increase, enhance or prolong the anti-tumor activity of immune effector cells; increase the number of anti-tumor immune effector cells or activated immune effector cells; promote tumor regression, tumor shrinkage and/or tumor necrosis.
  • prophylactically effective amount includes the amount of an agent, (e.g., a modified T cell or composition thereof disclosed herein) that, when administered to a subject having or predisposed to have a disease or disorder (e.g., cancer) is sufficient to prevent, reduce the symptoms of, or ameliorate the disease or disorder or one or more symptoms of the disease or disorder. Ameliorating the disease or disorder includes slowing the course of the disease or disorder or reducing the severity of later-developing disease or disorder.
  • an agent e.g., a modified T cell or composition thereof disclosed herein
  • the terms “treat,” “treated,” and “treating” mean both therapeutic and prophylactic treatment or preventative measures wherein the object is to reverse, alleviate, ameliorate, lessen, inhibit, slow down progression, development, severity or recurrence of an undesired symptom, complication, condition, biochemical indicia of a disorder, or disease, or obtain beneficial or desired clinical results.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease.
  • treatment includes eliciting a clinically significant response without excessive levels of side effects.
  • treatment includes prolonging survival as compared to expected survival if not receiving treatment.
  • the terms “individual” and “subject” have the same meaning herein, and can be a human and animal from other species.
  • the terms “subject” and “patient” are used interchangeably.
  • the subject can be an animal.
  • the subject is a mammal such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.).
  • the subject is a human.
  • the patient is a subject who has a disease, disorder, or condition, or is at risk of suffering from a disease, disorder, or condition, or is otherwise in need of the compositions and methods provided herein.
  • immune response refers to a biological response within an organism against a foreign agent or abnormal cell (e.g., a tumor cell), wherein the response protects the organism against such agents/cells and diseases caused by them.
  • a foreign agent or abnormal cell e.g., a tumor cell
  • An immune response is mediated by the action of a cell of the immune system (e.g., a T lymphocyte (T cell), B lymphocyte (B cell), natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the organism's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • T cell T lymphocyte
  • B cell B lymphocyte
  • NK natural killer
  • an immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+ T cell, or the inhibition of a regulatory T cell (Treg cell).
  • a T cell e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+ T cell, or the inhibition of a regulatory T cell (Treg cell).
  • the term "immune effector function” includes any function mediated by the composition of the immune system, which can lead to inhibition of tumor growth and/or inhibition of tumorigenesis, including inhibition the spread and metastasis of a tumor.
  • the immune effector function kills tumor cells.
  • the term "antigen presenting cell” or “APC” refers to a cell in the immune system that display a complex of foreign antigens and major histocompatibility complex (MHC) on the surface, such as helper cells (such as B cells, dendritic cells, etc.). T cells can recognize these complexes using a T cell receptor (TCR) thereof. An APC can process the antigen and present it to T cells.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • An APC can process the antigen and present it to T cells.
  • immunotherapy refers to the treatment of a disease by inducing, enhancing, suppressing or otherwise modifying an immune response.
  • autologous refers to any material derived from an individual that will later be reintroduced into that same individual.
  • anti-tumor effect refers to a biological effect that can be manifested in various ways, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in the number of metastases, increase in life expectancy, reduction in tumor cell proliferation, and reduction in tumor cell survival rate, or improvement in various physiological symptoms related to cancerous conditions.
  • the "anti-tumor effect” can also be expressed by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure to prevent or reduce the frequency of tumorigenesis.
  • T cell receptor also referred to herein as a "TCR protein", “TCR portion” or “TCR unit” is a protein complex found on the surface of all T cells.
  • a TCR can bind to CD3 by a non-covalent bond to form a TCR-CD3 complex.
  • TCRs can recognize antigens bound to major histocompatibility complex molecules.
  • TCRs are heterodimers including two different peptide chains. There are two categories of TCRs: TCR1 and TCR2.
  • TCR1 is composed of peptide chains gamma (y) and delta (5); and TCR2 is composed of peptide chains alpha (a) and beta (P).
  • Each peptide chain can be divided into several parts, such as variable region (V region), constant region (C region), transmembrane region and cytoplasmic region, characterized in that the cytoplasmic region is typically short in length.
  • TCR molecules are immunoglobulins, and their antigen specificity resides in the V regions (e.g., Va and VP), each of which has three hypervariable regions CDR1, CDR2, and CDR3, in which CDR3 has the largest variation and determines the binding specificity of the TCR to an antigen.
  • TCR When a TCR recognizes a MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the side wall of the antigen binding groove of the MHC molecule, and CDR3 binds to the antigen peptide.
  • a partial genetic modification can be used to improve the "affinity" and effectiveness of TCRs to target, thereby providing a high- affinity TCR.
  • TCR should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full- length TCRs, including TCRs in the aP form or y5 form.
  • a "TCR T cell” or “engineered TCR T cell” is a T cell that has been transduced with (for example, according to the methods disclosed herein) and expresses a recombinant TCR, or antigen-binding fragment thereof.
  • the T cell is a CD4+ T cell, CD8+ T cell, or CD4+/CD8+ T cell.
  • the T cell is a natural killer (NK) cell.
  • variable region typically refers to a portion of an immunoglobulin molecule, generally, a portion of a light or heavy chain, which differ in sequence among immunoglobulin molecule and are used in the binding and specificity of a particular immunoglobulin molecule for its particular antigen.
  • the variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the hypervariable regions in each chain are held together in close proximity by FRs, and with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of immunoglobulin molecule (see Kabat et al, Sequences of Proteins of Immunological Interest, 1992).
  • the hypervariable region generally comprises amino acid residues from a "complementary determining region” or "CDR", the latter being of highest sequence variability and/or involved in antigen recognition.
  • CDR complementary determining region
  • a number of CDR definitions are in use and are encompassed herein.
  • the Kabat definition is based on sequence variability and is the most commonly used (Kabat EA et al., supra).
  • alternate CDR sequences are provided for the same framework amino acid sequences of a variable region.
  • the alternate CDR sequences are generated using software programs.
  • different software can be used to generate alternate CDR sequences for the framework sequences of a variable region with different CDR sequences resulting from the use of the different software programs.
  • the use of alternate CDR sequences can improve binding affinities of a bispecific antibody molecule to at least one antigen.
  • alternate CDR sequences are used for affinity optimization of one or both antigen binding sites of a bispecific antibody molecule according to the present invention.
  • the alternate CDRs are defined according to Kabat, Chothia, Paratome, AbM, Contact and/or IMGT annotations. In some aspects, the CDRs are defined according to more than one annotation.
  • the CDRs of a recombinant TCRs, or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al, (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontane A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226).
  • Chothia numbering scheme refers to the location of immunoglobulin structural loops see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al, (1997) J Mol Biol 273: 927-9
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the terms "constant region” and “constant domain” are interchangeable and have their meaning common in the art.
  • the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • a recombinant TCR of the present disclosure comprises a variable region (V region), a constant region (C region), a transmembrane region, a cytoplasmic region, or any combination thereof.
  • a recombinant TCR of the present disclosure comprises a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region.
  • the recombinant TCR of the present disclosure, or antigenbinding fragment thereof is a TCR2.
  • the recombinant TCR of the present disclosure, or antigen-binding fragment thereof comprises a chain gamma (a) and a chain delta (P), or antigen-binding fragments thereof.
  • the recombinant TCR of the present disclosure, or antigen-binding fragment thereof comprises a variable chain gamma (a) and a variable chain delta (P), or antigen-binding fragments thereof.
  • the recombinant TCR of the present disclosure comprises a full length chain gamma (a) and a full length chain delta (P).
  • the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the recombinant TCRs of the present disclosure specifically bind the epitope.
  • the CDRs of a recombinant TCR, or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212.
  • CDR1B is at positions 26 to 35
  • CDR2B is at positions 51 to 57
  • CDR3B is at positions 93 to 102
  • CDR1 A is at positions 27 to 32
  • CDR2A is at positions 50 to 52
  • CDR3A is at positions 89 to 97.
  • the CDRs of recombinant TCRs, or the antigen-binding fragment thereof can be determined according to MacCallum RM et aL, (1996) J Mol Biol 262: 732-745. See also, e.g., Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).
  • recombinant TCRs and antigen-binding fragments thereof that bind to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A) (e.g., a HLA- A*03:01) and comprise the a and P CDRs of a TCR sequence disclosed herein, e.g., shown in Table 1, determined by the numbering method of MacCallum RM et al.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the recombinant T-cell Receptor comprises: (a) a TCR alpha chain comprising (i) a variable TCR alpha chain complementary determining region 1 (CDRla) of SEQ ID NO: 9, 17, or 33, (ii) a CDR2a of SEQ ID NO: 9, 17, or 33, and (iii) a CDR3a of SEQ ID NO: 9, 17, or 33; and (b) a TCR beta chain comprising (i) a variable TCR beta chain complementary determining region 1 (CDR1 ) of SEQ ID NO: 10, 19, or 35, (ii) a CDR20 of SEQ ID NO: 10, 19, or 35, and (iii) a CDR30 of SEQ ID NO: 10, 19, or 35.
  • the recombinant T cell receptor (TCR), or the antigen-binding domain thereof, capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01 comprises: (a) a TCR-alpha chain comprising (i) a variable TCR alpha chain complementarity determining region 1 (CDRloc) having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, sequence identity to SEQ ID NO: 3, (ii) a variable TCR alpha chain complementarity determining region 2 (CDR2a) having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
  • the recombinant T cell receptors (TCRs), or the antigen-binding fragment thereof, of the disclosure comprise a TCR-a comprising an amino acid sequence of SEQ ID NO: 9, 17, or 33, and a TCR-P comprising an amino acid sequence of SEQ ID NO: 10, 19, or 35.
  • the recombinant T-cell Receptor comprises: (a) a TCR alpha chain comprising (i) a variable TCR alpha chain complementary determining region 1 (CDRla) which is encoded by a nucleic acid sequence of SEQ ID NO: 37, 18, or 34, (ii) a CDR2a which is encoded by a nucleic acid sequence of SEQ ID NO: 37, 18, or 34, and (iii) a CDR3a which is encoded by a nucleic acid sequence of SEQ ID NO: 37, 18, or 34; and (b) a TCR beta chain comprising (i) a variable TCR beta chain complementary determining region 1 (CDRip) which is encoded by a nucleic acid sequence of SEQ ID NO: 38, 20, or 36, (ii) a CDR2P which is encoded by a nucleic acid sequence of SEQ ID NO: 38, 20, or 36, and
  • the recombinant TCR, or the antigen-binding domain thereof binds an epitope comprising the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2) of a KRAS protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the recombinant T cell receptor (TCR), or the antigen-binding domain thereof, capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01 comprises: (a) a TCR-alpha chain comprising (i) a variable TCR alpha chain complementarity determining region 1 (CDRla) which is encoded by a nucleic acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, sequence identity to SEQ ID NO: 11, (ii) a variable TCR alpha chain complementarity determining region 2 (CDR2a) which is encoded by a nucleic acid sequence having at least 85%, at least
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the recombinant TCR is capable of direct binding to the epitope.
  • the recombinant T cell receptors (TCRs), or the antigen-binding fragment thereof, of the disclosure comprises (a) an alpha chain (TCR-a) comprising (i) a variable TCR alpha chain complementarity determining region 1 (CDRloc) which is encoded by a nucleic acid sequence comprising SEQ ID NO: 11, (ii) a variable TCR alpha chain complementarity determining region 2 (CDR2a) which is encoded by a nucleic acid sequence comprising SEQ ID NO: 12, (iii) a variable TCR alpha chain complementarity determining region 3 (CDR3a) which is encoded by a nucleic acid sequence comprising SEQ ID NO: 13; and (b) a beta chain (TCR-P) comprising (i) a variable TCR beta chain complementarity determining region 1 (CDR1P) which is encoded by a nucleic acid sequence comprising SEQ ID NO: 14, (ii) a variable TCR alpha chain complementarity
  • the recombinant T cell receptors (TCRs), or the antigen-binding fragment thereof, of the disclosure comprise an alpha chain (TCR-a) and a beta chain (TCR-P).
  • TCR-a comprises an amino acid sequence which is encoded by a nucleic acid sequence having at least 85%, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 18, or 34
  • the TCR-P comprises an amino acid sequence which is encoded by a nucleic acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38, 20, or 36.
  • the recombinant T cell receptors (TCRs), or the antigen-binding fragment thereof, of the disclosure comprise a TCR-a comprising an amino acid sequence which is encoded by a nucleic acid sequence of SEQ ID NO: 37, 18, or 34, and a TCR- comprising an amino acid sequence which is encoded by a nucleic acid sequence of SEQ ID NO: 38, 20, or 36.
  • the cytoplasmic domain of the engineered TCR can include intracellular signaling domains; and TCR alpha and TCR beta subunits usually lack signaling domains.
  • the intracellular signaling domain is generally responsible for activating at least one normal effector function of immune cells into which the TCR has been introduced.
  • the polynucleotides encode the beta chain (TCR-P) of the recombinant TCR, or of the antigen-binding fragment thereof, of the disclosure.
  • the polynucleotides encode a CDRip, a CDR2[3, and a CDR3[3.
  • the polynucleotides encode a variable beta chain (TCR-P).
  • the polynucleotides encode a full length beta chain (TCR- ).
  • the polynucleotides encode the alpha chain (TCR- 00 and the beta chain (TCR-P) of the recombinant TCR, or of the antigen-binding fragment thereof, of the disclosure.
  • the polynucleotides encode a CDRloc, a CDR2a, a CDR3a, a CDRip, a CDR2P, and a CDR3p. In some aspects, the polynucleotides encode a variable alpha chain (TCR-00 and a variable beta chain (TCR- P). In some aspects, the polynucleotides encode a full length alpha chain (TCR-00 and a full length beta chain (TCR-P).
  • the epitope is presented on a human leucocyte antigen-A (HLA-A). In some embodiments, the epitope is presented on a human leucocyte antigen-A (HLA- A)*03:01. In some aspects, the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the polynucleotides of the disclosure encoding one or more of the recombinant TCRs, or of the antigen-binding fragments thereof, disclosed herein, are cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Viruses that can be used as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.
  • the present invention is not limited to the use of constitutive promoters, while inducible promoters are also considered.
  • the polynucleotides of the disclosure encode recombinant T cell receptors (TCRs), or antigen-binding fragments thereof, comprising an alpha chain (TCR- a) and/or a beta chain (TCR- ), or antigen-binding fragments thereof.
  • TCRs T cell receptors
  • the polynucleotides of the disclosure encode a TCR-a comprising an amino acid sequence having at least 85%, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9, 17, or 33.
  • the polynucleotides of the disclosure encode a beta chain (TCR- P) comprising (i) a variable TCR beta chain complementarity determining region 1 (CDR1P) of SEQ ID NO: 6, (ii) a variable TCR beta chain complementarity determining region 2 (CDR2P) of SEQ ID NO: 7, (iii) variable TCR beta chain complementarity determining region 3 (CDR3P) of SEQ ID NO: 8.
  • TCR- P beta chain comprising (i) a variable TCR beta chain complementarity determining region 1 (CDR1P) of SEQ ID NO: 6, (ii) a variable TCR beta chain complementarity determining region 2 (CDR2P) of SEQ ID NO: 7, (iii) variable TCR beta chain complementarity determining region 3 (CDR3P) of SEQ ID NO: 8.
  • the polynucleotides of the disclosure comprise (i) a nucleotide sequence of SEQ ID NO: 11, encoding a variable TCR alpha chain complementarity determining region 1 (CDRla), (ii) a nucleotide sequence of SEQ ID NO: 12, encoding a variable TCR alpha chain complementarity determining region 2 (CDR2a), (iii) a nucleotide sequence of SEQ ID NO: 13, encoding a variable TCR alpha chain complementarity determining region 3 (CDR3a), (iv) a nucleotide sequence of SEQ ID NO: 14, encoding a variable TCR beta chain complementarity determining region 1 (CDR1P), (v) a nucleotide sequence of SEQ ID NO: 15, encoding a variable TCR beta chain complementarity determining region 2 (CDR2[3), and (vi) a nucleotide sequence of SEQ ID NO: 16, encoding a variable TCR alpha chain
  • the cancer cell is derived from a cancer selected from the group consisting of: a lung cancer (e.g., a non-small cell lung cancer (NSCLC)), a colorectal cancer, a pancreatic cancer, an appendiceal cancer, a small bowel adenocarcinoma, an hepatobiliary cancer, a gynecological malignancy, a hematopoietic cancer, a breast cancer, a bladder cancer, a prostate cancer, a skin cancer, and any combination thereof.
  • a lung cancer e.g., a non-small cell lung cancer (NSCLC)
  • NSCLC non-small cell lung cancer
  • a colorectal cancer e.g., a colorectal cancer
  • pancreatic cancer e.g., an appendiceal cancer
  • a small bowel adenocarcinoma e.g., an hepatobiliary cancer
  • the recombinant TCR, or antigen-binding fragments thereof, of the disclosure are expressed in a cell.
  • the recombinant TCR, or antigenbinding fragments thereof, of the disclosure are expressed in a host cell.
  • the host cell is a T cell.
  • the recombinant TCR, or the antigen-binding fragments thereof, of the disclosure are expressed on the surface of the host cell (e.g., T cell).
  • the recombinant TCR, or antigen-binding fragments thereof of the disclosure direct the host cell (e.g., T cell) to a cancer cell.
  • the cancer cell expresses a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation. In some aspects, the cancer cell expresses a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation comprising an epitope presented on a human leucocyte antigen-A (HLA-A). In some aspects, the cancer cell expresses a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation comprising an epitope presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the cancer cell is selected from the group consisting of: a lung cancer cell (e.g., a non-small cell lung cancer (NSCLC) cell), a colorectal cancer cell, a pancreatic cancer cell, an appendiceal cancer cell, a small bowel adenocarcinoma cell, an hepatobiliary cancer cell, a gynecological malignancy cell, a hematopoietic cancer cell, a breast cancer cell, a bladder cancer cell, a prostate cancer cell, a skin cancer cell, or any combination thereof.
  • a lung cancer cell e.g., a non-small cell lung cancer (NSCLC) cell
  • NSCLC non-small cell lung cancer
  • a colorectal cancer cell e.g., a non-small cell lung cancer (NSCLC) cell
  • a pancreatic cancer cell e.g., an appendiceal cancer cell
  • a small bowel adenocarcinoma cell e.g
  • the cancer cell is selected from the group consisting of: a lung cancer cell (e.g., a non-small cell lung cancer (NSCLC) cell), a colorectal cancer cell, a pancreatic cancer cell, or any combination thereof.
  • a lung cancer cell e.g., a non-small cell lung cancer (NSCLC) cell
  • NSCLC non-small cell lung cancer
  • colorectal cancer cell e.g., a colorectal cancer cell
  • pancreatic cancer cell e.g., pancreatic cancer cell, or any combination thereof.
  • a method of treating cancer or a tumor in an subject comprising administering to the subject a therapeutically effective amount of any of the recombinant T cell receptors (TCRs), or antigen-binding fragment thereof, the polynucleotides, the vectors, or the host cells (e.g., T cell) disclosed herein.
  • the cancer comprises cancer cells expressing a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the cancer comprises cancer cells expressing a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation, which comprises an epitope presented on a human leucocyte antigen-A (HLA-A).
  • the cancer comprises cancer cells expressing a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation, which comprises an epitope presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the subject is a human.
  • the subject or cancer in the subject is resistant to small molecules inhibitors of Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the subject or cancer in the subject is resistant to sotorasib and/or adagrasib.
  • BiTEs Bispecific T cell engagers
  • the disclosure is directed to a Bi-specific T-cell engager (BiTE) comprising the recombinant TCRs of the disclosure or antigen-binding fragments thereof.
  • BiTE Bi-specific T-cell engager
  • the BiTE is capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • a bi-specific antibody molecule as employed herein refers to a molecule with two antigen binding domains, which may bind the same or different antigens.
  • a BiTE is a subclass of bispecific antibody molecules.
  • Non-IgG-like antibodies include antibodies that lack an Fc portion such as bispecific T cell engagers (BiTE), DART, tetravalent antiparallel structures (TandAbs) and VH-only bi-nanobodies.
  • the non-IgG-like antibodies due to their lack of an Fc portion are smaller and have shorter in vivo halflives.
  • Bi-specific T-cell engagers refer to a class of artificial bispecific monoclonal antibodies that are investigated for the use as anticancer drugs. They direct a host's immune system, more specifically the T cells' cytotoxic activity, against cancer cells. BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule.
  • scFvs single-chain variable fragments
  • BiTEs form a link between T cells and tumor cells. This causes T cells to exert cytotoxic activity on tumor cells by producing proteins like perforin and granzymes, independently of the presence of MHC I or co-stimulatory molecules. These proteins enter tumor cells and initiate the cell's apoptosis. This action mimics physiological processes observed during T cell attacks against tumor cells.
  • BiTE is a registered trademark of Micromet AG (fully owned subsidiary of Amgen Inc).
  • BiTEs are able to form a link between T cells and tumor cells by virtue of their specificities for an antigen on the T cell and an antigen on the tumor cell. This leads to activation of the T-cells and triggers the T cells to exert their cytotoxic effects on tumor cells, independently of MHC I or co-stimulatory molecules.
  • the present disclosure BiTE is capable of activation of antigen specific T cells, which can kill targeted cancer cells expressing a particular epitopes of interest (e.g., a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation, which comprises an epitope presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • a particular epitopes of interest e.g., a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation, which comprises an epitope presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the epitope consists of the amino acid sequence of VVGACGVGK
  • the BiTE comprises (i) a binding region specific to a surface antigen on a T cell of interest (e.g., selected from CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma), and (ii) an antigen binding region (e.g., specific to an antigen comprising a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation which comprises an epitope presented on a human leucocyte antigen-A (HLA-A)*03:01) comprising TCR-a chain and TCR-P chain disclosed herein.
  • a binding region specific to a surface antigen on a T cell of interest e.g., selected from CD3 (such as CD3 delta, CD3 epsilon or CD3 gamma)
  • an antigen binding region e.g., specific to an antigen comprising a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRA
  • the epitope of the Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation is presented on a human leucocyte antigen-A (HLA-A) (e.g., HLA-A *03:01) on a nonsmall cell lung cancer (NSCLC) cancer cell.
  • HLA-A human leucocyte antigen-A
  • NSCLC nonsmall cell lung cancer
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • Some aspects of the disclosure are directed to a polynucleotide encoding a BiTE disclosed herein.
  • the polynucleotide of the disclosure encodes a BiTE comprising a TCR of the disclosure.
  • Certain aspects of the disclosure are directed to engineered cells comprising a recombinant T cell receptor (TCR), or an antigen-binding fragment thereof, of the disclosure, a polynucleotide encoding such a TCR, and/or an expression vector comprising such a polynucleotide.
  • the cell is a T cell.
  • the T cell is a CD4+ cell, a CD8+ cell, or a CD4+/CD8+ cell.
  • the host cell is a T cell which is a CD4+ cell, a CD8+ cell, or a CD4+/CD8+ cell.
  • a modified cell transformed, transfected, or transduced with a nucleic acid molecule can be a host cell, a recombinant cell, or an engineered cell.
  • an engineered cell can include a cell which has been transformed, transfected or transduced with an expression vector comprising a nucleic acid sequence encoding a specific TCR.
  • the progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • the host cells (e.g., T cells) of the disclosure are modified to comprise a recombinant TCR, or antigen-binding fragment thereof, of the disclosure.
  • the recombinant T cell receptor (TCR), or antigen-binding fragment thereof is capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the exogenous receptor that can bind to the target antigen and trigger CD3 signal activation includes at least one CD3 binding site and at least one additional antigen binding site specific to bacterial substance, viral protein, autoimmune marker, or antigen present specific cells (e.g., cell surface proteins of B cells, T cells, natural killer (NK) cells, bone marrow cells, phagocytes, or tumor cells).
  • Such exogenous receptors can cross-link two kinds of cells and can be used to direct T cells to specific targets and trigger the cytotoxic activity of T cells on the target cells. Examples of such targets may be tumor cells or infectious agents, such as viral pathogens or bacterial pathogens.
  • the source of cells can be obtained from a subject.
  • the immune effector cells for use with the TCRs as disclosed herein comprise T cells.
  • the T cells are obtained from peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of an infection, ascites, pleural effusion, spleen tissue, or a tumor.
  • the T cells are obtained from peripheral blood mononuclear cells.
  • the T cells are obtained from tumor cells (e.g., NSCLC cells).
  • T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing.
  • the cells are washed with PBS.
  • the washed solution lacks calcium, and can lack magnesium or can lack many, if not all, divalent cations.
  • a washing step can be accomplished by methods known to those in the art, such as by using a semiautomated flowthrough centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In some aspects, the undesirable components of the apheresis sample can be removed in the cell directly resuspended culture media.
  • such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti- CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2.
  • a stimulatory agent and costimulatory agent such as anti- CD3 and anti-CD28 antibodies
  • cytokines such as IL-2.
  • Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a "surrogate" antigen presenting cell (APC).
  • the T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those disclosed in U.S. Pat. Nos. 6,040,177; 5,827,642; and WO2012129514.
  • the T cells of the disclosure are expanded by contact with an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T-cell.
  • an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T-cell.
  • chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T-cell.
  • Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% 02). T cells that have been exposed to varied stimulation times can exhibit different characteristics.
  • the cells can be expanded by co-culturing with tissue or cells.
  • the cells can also be expanded in vivo, for example in the subject's blood after administrating said cell into the subject.
  • the modified cells (e.g., engineered T cells) of the disclosure comprise a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation.
  • TCR recombinant T cell receptor
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the modified cells (e.g., engineered T cells) of the disclosure comprise a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A).
  • TCR recombinant T cell receptor
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • HLA-A human leucocyte antigen-A
  • the modified cells (e.g., engineered T cells) of the disclosure comprise a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the modified cells (e.g., engineered T cells) of the disclosure comprise a polynucleotide encoding a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A).
  • TCR recombinant T cell receptor
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • HLA-A human leucocyte antigen-A
  • the modified cells (e.g., engineered T cells) of the disclosure comprise a polynucleotide encoding a recombinant T cell receptor (TCR), or antigenbinding fragment thereof, capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • the modified cells comprise (a) a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, and/or (b) a polynucleotide encoding a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, and/or (c) a vector comprising a polynucleotide encoding a recombinant T cell receptor (TCR) or antigen-binding fragment thereof, wherein the recombinant T cell receptor is capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA- A).
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the modified cells comprise (a) a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, and/or (b) a polynucleotide encoding a recombinant T cell receptor (TCR), or antigen-binding fragment thereof, and/or (c) a vector comprising a polynucleotide encoding a recombinant T cell receptor (TCR) or antigen-binding fragment thereof, wherein the recombinant T cell receptor is capable of binding to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)*03:01.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • a method of treating cancer or a tumor in a subject comprising administering to the individual a therapeutically effective amount of any of the modified T cells disclosed herein.
  • the subject is a human.
  • the cancer comprises cancer cells expressing a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation.
  • the subject is a human.
  • the cancer comprises cancer cells expressing a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation which comprises an epitope presented on a human leucocyte antigen-A (HLA-A).
  • HLA-A human leucocyte antigen-A
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope comprises the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspects, the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2).
  • T cells from the first stimulation and sorting were collected and utilized in rapid T cell replication procedure by stimulation with anti-CD3 (OKT) antibody and IL-2 for two weeks (FIG. 2).
  • T cells were then stained and sorted again for second replication and used in killing and TCR a/p determination, as shown in FIG. 3.
  • the lung cancer cell line 1792 (NCI- 111792 (RRID:CVCL_1495) as described in Mitsudomi T., et al., Oncogene 7:171- 180(1992).), which exhibits the endogenous G12C mutation, was stably transduced with HLAA0301 and used as a target for cytotoxicity killing assay assessment.

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Abstract

La présente invention concerne des récepteurs de lymphocytes T (TCR) recombinants qui se lient à une protéine de l'homologue de l'oncogène viral du sarcome du rat de Kirsten (KRAS) comprenant une mutation G12C présentée sur un antigène leucocytaire humain A (HLA-A)*03:01, des polynucléotides codant pour de tels TCR, des lymphocytes T modifiés comprenant de tels TCR, et des procédés d'utilisation de ceux-ci.
PCT/US2024/010339 2023-01-04 2024-01-04 Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*03:01 dans le cancer du poumon Ceased WO2024148178A2 (fr)

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