[go: up one dir, main page]

WO2024148181A2 - Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*11:01 dans le cancer du poumon - Google Patents

Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*11:01 dans le cancer du poumon Download PDF

Info

Publication number
WO2024148181A2
WO2024148181A2 PCT/US2024/010342 US2024010342W WO2024148181A2 WO 2024148181 A2 WO2024148181 A2 WO 2024148181A2 US 2024010342 W US2024010342 W US 2024010342W WO 2024148181 A2 WO2024148181 A2 WO 2024148181A2
Authority
WO
WIPO (PCT)
Prior art keywords
tcr
cell
seq
aspects
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/010342
Other languages
English (en)
Other versions
WO2024148181A3 (fr
Inventor
Alexandre REUBEN
Minying ZHANG
John Victor HEYMACH
Gregory A. LIZEE
Peixin JIANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
University of Texas at Austin
Original Assignee
University of Texas System
University of Texas at Austin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Texas System, University of Texas at Austin filed Critical University of Texas System
Publication of WO2024148181A2 publication Critical patent/WO2024148181A2/fr
Publication of WO2024148181A3 publication Critical patent/WO2024148181A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

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.
  • ACT adoptive cell transfer
  • HLA human leukocyte antigen
  • 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 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, wherein the TCR binds an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homo
  • the TCR-a comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9, 17, or 33; and the TCR-P comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, 19, or 35.
  • the T cell receptor (TCR), or antigen-binding fragment thereof, of the disclosure comprises (a) an alpha chain (TCR-a) comprising an amino acid sequence of SEQ ID NO: 9, 17, or 33, and (b) a beta chain (TCR-P) comprising an amino acid sequence of SEQ ID NO: 10, 19, or 35.
  • TCR T cell receptor
  • TCR-P beta chain
  • the T cell receptor (TCR), or antigen-binding fragment thereof, of the disclosure is humanized or chimeric.
  • the disclosure is directed to a modified T cell comprising the T cell receptor (TCR), or antigen-binding fragment thereof, the polynucleotide, or the expression vector, disclosed herein.
  • T cell receptor TCR
  • the T cell receptor (TCR) is expressed on the surface of the modified T cell.
  • the modified T cell is capable of recognition of a cancer cell expressing a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation, which comprises an epitope presented on the human leucocyte antigen- A (HLA-A)* 11:01.
  • the epitope comprises the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the modified cell is capable of recognition of a cell expressing a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation.
  • the modified cell is capable of recognition of a cell 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 disclosure is directed to a bi-specific T-cell engager (BiTE) comprising a T cell receptor (TCR) comprising: (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 (CDRip) of SEQ ID NO: 10, 19, or 35, (ii) a CDR2P of SEQ ID NO: 10, 19, or 35, and (iii) a CDR3P of SEQ ID NO: 10, 19, or 35, wherein the TCR binds an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein
  • 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 modified T cell or the BiTE of the disclosure comprise a TCR alpha chain 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; and a TCR beta chain 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: 10, 19, or 35.
  • the TCR alpha chain comprises an amino acid sequence of SEQ ID NO: 9, 17, or 33
  • the TCR beta chain comprises an amino acid sequence of SEQ ID NO: 10, 19, or 35.
  • the TCR-a comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9, 17, or 33; and the TCR-P comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, 19, or 35.
  • the TCR alpha chain comprises an amino acid sequence of SEQ ID NO: 9, 17, or 33
  • the TCR beta chain comprises an amino acid sequence of SEQ ID NO: 10, 19, or 35.
  • the disclosure is directed to a method for prophylaxis and/or therapy of a subject diagnosed with, suspected of having or at risk for developing or recurrence of a cancer (e.g., lung cancer), comprising administering to the subject the recombinant TCR, the polynucleotide, the vector, the host cell, the T cell, the modified T cell, or the BiTE of the disclosure.
  • 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)* 11 :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).
  • reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), P-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.
  • a sequence alignment performed for determining percent amino acid sequence identity can be carried out according to procedures known in the art, as described for example in EP 1 241 179 Bl, which is incorporated herewith by reference, including in particular page 9, line 35 to page 10, line 40 with the definitions used therein and Table 1 regarding possible conservative substitutions.
  • a skilled person can use publicly available computer software.
  • Computer program methods for determining sequence identity include, but are not limited to BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • the software alignment program used can be BLAST.
  • a skilled person can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences subjected to comparison.
  • allogeneic refers to any material derived from a different animal or a different patient of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, two or more individuals are considered to be allogeneic to each other. In some aspects, allogeneic materials from individuals of the same species may be genetically different enough for antigenic interaction to occur.
  • 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
  • crystallization can be accomplished using any of the known methods in the art (e.g., Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269- 1274; McPherson A (1976) J Biol Chem 251 : 6300-6303).
  • direct binding refers to an antigen binding molecule that recognizes and binds a protein of a binding partner (such as a tumor antigen).
  • KRAS Krsten Rat Sarcoma Viral Oncogene Homolog
  • chromosome 12pl2.1 refers to member of the RAS canonical family of genes that also includes HRAS (chromosome 1 Ip 15.5) and NRAS (chromosome Ip 13.1).
  • HRAS chromosome 1 Ip 15.5
  • NRAS chromosome Ip 13.1
  • the three genes are highly conserved across different species and encode monomeric GTPases that cycle between active (GTPbound) and inactive (GDPbound) states in response to extracellular cues.
  • GTPbound active
  • GDPbound inactive
  • TCRs Recombinant T Cell Receptors
  • Certain aspects of the disclosure are directed to recombinant T cell receptors
  • TCRs TCRs
  • KRAS Oncogene Homolog
  • the recombinant T cell receptors (TCRs), or antigen-binding fragments thereof are 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).
  • HLA-A human leucocyte antigen-A
  • the recombinant T cell receptors 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)* 11 :01.
  • the (HLA- A)* 11 :01 allele presents the epitope comprising the amino acid sequence of VVGACGVGK (SEQ ID NO: 1).
  • the (HLA-A)* 11 :01 allele presents the epitope comprising 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 TCRs of the present disclosure include the variable chain portions of the TCR or any functional fragment thereof, such as an antigen-binding portion of a TCR (e.g., antigen-binding fragments) that binds to a specific antigenic peptide bound in an MHC molecule, i.e. MHC-peptide complex.
  • an antigen-binding portion of a TCR e.g., antigen-binding fragments
  • MHC-peptide complex e.g. MHC-peptide complex
  • the antigen-binding portion or antigen-binding fragment of a TCR refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC-peptide complex) to which the full TCR binds.
  • an antigen-binding portion contains the variable domains of a TCR, such as a variable alpha (a) chain and variable beta (P) chain, or a variable gamma (y) chain and a variable delta (5) chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as generally where each chain contains three complementarity determining regions (CDRs).
  • a TCR variable alpha (a) chain and variable beta (P) chain, or a variable gamma (y) chain and a variable delta (5) chain of a TCR
  • 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 TCR1.
  • the recombinant TCR of the present disclosure, or antigen-binding fragment thereof comprises a chain gamma (y) and a chain delta (5), or antigen-binding fragments thereof.
  • the recombinant TCR of the present disclosure, or antigen-binding fragment thereof comprises a variable chain gamma (y) and a variable chain delta (5), or antigen-binding fragments thereof.
  • a recombinant TCR of the present disclosure comprises a full length chain gamma (y) and a full length chain delta (5).
  • 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).
  • a recombinant TCR of the present disclosure comprises a TCR a and a TCR P chain (or antigen-binding fragments thereof), or a TCR y and a TCR 5 chain (or antigen-binding fragments thereof), wherein the two chains (or antigen-binding fragments thereof), are present in a physical association with one another (e.g., in a complex) and are non-covalently joined to one another, or wherein the two chains (or antigen-binding fragments thereof) are distinct polypeptides but are covalently joined to one another, such as by a disulfide or other covalent linkage that is not a peptide bond.
  • linkages can comprise, for example, substituted or unsubstituted polyalkylene glycol, and combinations of ethylene glycol and propylene glycol in the form of, for example, copolymers.
  • two polypeptides that constitute a TCR aand a TCR P chain or antigen-binding fragments thereof, or two polypeptides that constitute a TCR y and a TCR 5 chain or antigenbinding fragments thereof can both be included in a single polypeptide, such as a fusion protein.
  • two polypeptides that constitute a TCR aand a TCR P chain or antigen-binding fragments thereof, or two polypeptides that constitute a TCR y and a TCR 5 chain or antigen-binding fragments thereof can be included as separate polypeptides expressed on a T cell.
  • variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity.
  • CDRs complementarity determining regions
  • TCRs that exist in aP and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions.
  • the vast majority of T lymphocytes (over 90%) carry TCR2.
  • Few T-lymphocytes with TCR1 are located beneath the mucous membranes and skin.
  • a TCR can be found anchored to the surface of a cell or in soluble form.
  • a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • a TCR can contain a variable domain, a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al, Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997).
  • each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end.
  • a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.
  • the recombinant TCR of the present disclosure binds an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation. In some aspect, the recombinant TCR of the present disclosure binds 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 recombinant TCR of the present disclosure binds 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)* 11 :01.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the recombinant TCR of the present disclosure comprises a target-specific binding element, which is also referred to as an antigen recognition portion.
  • the antigen recognition portion can be selected to bind the target antigen as a cell surface marker associated with a specific disease state on the target cell (e.g., a neoantigen).
  • the antigen recognition portion binds an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the antigen recognition portion binds 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).
  • the antigen recognition portion binds 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)* 11 :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). In some aspects, the epitope consists of the amino acid sequence of VVGACGVGK (SEQ ID NO: 1). In some aspect, the epitope consists of the amino acid sequence of VVVGACGVGK (SEQ ID NO: 2). In some aspects, the recombinant TCR of the present disclosure specifically binds the epitope.
  • the recombinant TCRs of the disclosure, or the antigen-binding fragments thereof can be derived from natural sources or from recombinant sources. In some aspects, the recombinant TCR, or the antigen-binding fragment thereof can be derived from any protein, especially a membrane-bound or transmembrane protein. In some aspects, the recombinant TCR of the disclosure, or the antigen-binding fragment thereof, is derived from a tumor antigen-specific T cell. In some aspects, the antigenbinding domain can associate with the transmembrane domain. In some aspects, antigenbinding fragments can particularly useful in the present invention.
  • 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.
  • recombinant TCRs and antigenbinding 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* 11 :01) and comprise the a and P CDRs of a TCR sequence disclosed herein, e.g., shown in Table 1, determined by IMGT method, for example, as described in Lefranc M-P (1999) supra and Lefranc M-P et aL, (1999) supra).
  • 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 CDRs of the recombinant TCRs, or of 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)* 11 :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 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 CDRs of the recombinant TCRs, or of the antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).
  • 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 CDRs of the recombinant TCRs, or of the antigen-binding domain 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 (HL A- A) (e.g., a HLA-A* 11 :01), provided herein, are determined by the IMGT numbering scheme.
  • 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 recombinant T-cell Receptor (TCR), or the antigen-binding domain thereof 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 TCR, or the antigen-binding domain thereof binds an epitope comprising the amino acid sequence of VVGACGVGK (SEQ ID NO: 1) of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)* 11 :01.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • 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)* 11 :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)* 11:01 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 9
  • 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 (CDR1 a) comprising the amino acid sequence of SEQ ID NO: 3, (ii) a variable TCR alpha chain complementarity determining region 2 (CDR2 a) comprising the amino acid sequence of SEQ ID NO: 4, (iii) a variable TCR alpha chain complementarity determining region 3 (CDR3 a) comprising the amino acid sequence of SEQ ID NO: 5; and (b) a beta chain (TCR-P comprising (i) a variable TCR beta chain complementarity determining region 1 (CDR1 P) comprising the amino acid sequence of SEQ ID NO: 6, (ii) a variable TCR beta chain complementarity determining region 2 (CDR2 P) comprising the amino acid sequence of SEQ ID
  • 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 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 TCR-P comprises 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: 10, 19, or 35.
  • 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 (TCR), or the antigen-binding fragment thereof 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 CDR2 which is encoded by a nucleic acid sequence of SEQ ID NO: 38, 20, or 36, and (i
  • the recombinant TCR binds an epitope comprising the amino acid sequence of VVGACGVGK (SEQ ID NO: 1) of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation presented on a human leucocyte antigen-A (HLA-A)* 11 :01.
  • the recombinant TCR 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)* 11 :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) 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)* 11 :01 comprises: (a) a TCR-alpha chain comprising (i) a variable TCR alpha chain complementarity determining region 1 (CDRloc) 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 90%, at least 91%, 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 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 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- 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-P comprising an amino acid sequence which is encoded by a nucleic acid sequence of SEQ ID NO: 38, 20, or 36.
  • TCRs T cell receptors
  • the recombinant TCR, or the antigen-binding fragment thereof, of the disclosure is humanized or chimeric.
  • chimeric TCR refers to TCRs wherein the amino acid sequence is derived from two or more species.
  • the variable regions of the TCR correspond to the variable region of TCRs derived from one species of mammals (e.g. mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in TCRs derived from another (usually human) to avoid eliciting an immune response in that species.
  • humanized TCR refers to forms of non-human (e.g. murine) TCRs that contain minimal non-human (e.g., murine) sequences.
  • humanized TCRs are human TCRs in which residues from the complementarity determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g. mouse, rat, rabbit, and hamster) that have the desired specificity, affinity, and capability ("CDR grafted". Examples of methods used to generate humanized TCRs are described in (Chen et al., A humanized TCR retaining authentic specificity and affinity conferred potent anti-tumour cytotoxicity, Immunology, 2018, 155(1): 123-136).
  • the disclosure is directed to polynucleotides encoding one or more of the recombinant TCRs of the disclosure, or one or more of the antigen-binding fragments thereof.
  • the polynucleotides encode the alpha chain (TCR- 00 of the recombinant TCR, or of the antigen-binding fragment thereof, of the disclosure.
  • the polynucleotides encode a CDRloc, a CDR2a, and a CDR3a.
  • the polynucleotides encode a variable alpha chain (TCR-00.
  • the polynucleotides encode a full length alpha chain (TCR-00.
  • 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-P).
  • the polynucleotides encode the alpha chain (TCR- 00 and the beta chain (TCR- ) of the recombinant TCR, or of the antigen-binding fragment thereof, of the disclosure.
  • the disclosure is directed to vectors (e.g., expression vectors) comprising the polynucleotides encoding the recombinant TCR, or the antigen-binding fragment thereof, of the disclosure.
  • the polynucleotides comprised in such vectors may encode the alpha chain (TCR-00, or the beta chain (TCR-P), or both 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 and the vectors of the disclosure may be obtained by any of techniques well-known in the art for the manipulation of nucleic acid sequences outside an organism.
  • the nucleotide sequences encoding the desired T cell receptors (TCRs), or antigen-binding fragments thereof, derived from tumor antigen-specific T cells of interest may be isolated and cloned in a vector (e.g., expression vector) by any of the techniques well-known in the art (see for example Green & Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-3, 4 th edition).
  • 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 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.
  • the disclosure is directed to a Bi-specific T-cell engager (BiTE) comprising the recombinant TCRs of the disclosure or antigen-binding fragment 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. In some aspects, 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).
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • HLA-A human leucocyte antigen-A
  • 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 tumour cell. This leads to activation of the T-cells and triggers the T cells to exert their cytotoxic effects on tumour 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)* 11:01.
  • 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)* 11:01.
  • 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 is specific for at least a surface antigen on a T cell of interest.
  • T cell surface antigens include but are not limited to: CD3, CD2, VLA-1, CD8, CD4, CCR6, CXCR5, CD25, CD31, CD45RO, CD197, CD127, CD38, CD27, CD196, CD277 and CXCR3, particularly CD2, CD3, CD31 and CD277.
  • 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)* 11 :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 (K
  • 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 BiTEs disclosed herein bind to an epitope of a Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) protein comprising a G12C mutation. In some aspects, the BiTEs disclosed herein 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).
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • 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)* 11 :01 on a cancer cell.
  • 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 * 11 :01) on a lung cancer cell.
  • 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 * 11 :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).
  • 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, 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.
  • the cancer cell is selected from the group consisting of: a lung cancer cell, a non-small cell lung cancer (NSCLC) cell, a colorectal cancer cell, a pancreatic cancer cell, or any combination thereof.
  • the BiTEs disclosed herein are expressed in a cell.
  • the BiTEs disclosed herein are expressed in a host cell.
  • the host cell is a T cell.
  • the recombinant TCR of the disclosure, or antigenbinding domains thereof are expressed on the surface of the host cell (e.g., T cell).
  • the BiTEs disclosed herein 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.
  • KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog
  • the cancer cell expresses 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 cell expresses 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)* 11:01.
  • 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). 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).
  • a method of treating cancer or a tumor in an subject comprising administering to the individual a therapeutically effective amount of any of the of the BiTEs 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
  • 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.
  • the cell is a modified cell.
  • the term "modified cell” can refer to any type of cell (e.g., a primary cell, a cell in culture, or a cell from a cell line).
  • a modified cell can be a cell which is manipulated in vitro, by any technique known in the art, to achieve expression of a specific protein (e.g., a cell exposed to an APC presenting a specific antigen to achieve expression of a specific TCR).
  • the modified cell can be a cell which is transformed, transfected, or transduced with a nucleic acid molecule (e.g., and expression vector), and the progeny or potential progeny of such a cell.
  • 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 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 presented on a human leucocyte antigen-A (HLA- A).
  • 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 presented on a human leucocyte antigen-A (HLA-A)* 11:01.
  • the recombinant T cell receptor (TCR), or antigen-binding fragment thereof is expressed on the surface of the host cell (e.g., engineered T cell).
  • 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 TCR is a recombinant TCR.
  • 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 TCR is a recombinant TCR.
  • the T cell of the present disclosure can be expanded by contacting with a surface to which an agent that stimulates the CD3/TCR complex-related signal and a ligand that stimulates a costimulatory molecule on the surface of the T cell are attached.
  • the T cell population can be stimulated, for example by contacting with an anti-CD3 antibody or antigen-binding fragments thereof or an anti- CD2 antibody immobilized on a surface, or by contacting a protein kinase C activator (for example, bryostatin) and calcium ionophore.
  • CD3 Cluster of Differentiation 3
  • T cell co-receptor is a protein complex composed of four different chains. In mammals, the complex contains one CD3 gamma chain, CD3 delta chain, and two CD3 epsilon chains. These chains have a molecule of accessory T cell receptor (TCR) and zeta-chain to generate activation signals for T lymphocytes.
  • TCR accessory T cell receptor
  • the TCR, zeta, chain and CD3 molecule together constitute a T cell receptor complex.
  • the CD3 molecule is connected to the T cell receptor (TCR) through a salt bridge to form a TCR-CD3 complex, which participates in the signaling of T cells, and is mainly used to label thymocytes, T lymphocytes and T cell lymphomas.
  • 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.
  • T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
  • PBMCs peripheral blood mononuclear cells
  • a specific subpopulation of T cells such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
  • enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method for use herein is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • PBMCs can be used directly for genetic modification with the various methods as disclosed herein.
  • T lymphocytes after isolation of PBMC, T lymphocytes are further isolated and in some aspects, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.
  • CD8+ cells can be obtained by using standard methods.
  • CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those types of CD8+ cells.
  • memory T cells are present in both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes.
  • PBMC are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies.
  • the expression of phenotypic markers of central memory TCM include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B.
  • central memory T cells are CD45RO+, CD62L+, CD8+ T cells.
  • effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.
  • naive CD8+T lymphocytes are characterized by the expression of phenotypic markers of naive T cells including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.
  • CD4+ T cells are further sorted into subpopulations.
  • CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
  • CD4+ lymphocytes can be obtained by standard methods.
  • 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.
  • T cells can be expanded in vitro or in vivo.
  • 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.
  • T cell populations can be stimulated in vitro such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that can contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL- 10, IL-2, IL- 15, TGFp, and TNF-a or any other additives for the growth of cells known to the skilled artisan.
  • Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • 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 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.
  • 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 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)* 11:01.
  • 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 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).
  • a method for prophylaxis and/or therapy of an individual diagnosed with, suspected of having or at risk for developing or recurrence of a cancer comprising administering to the individual any of the recombinant molecules or modified cells disclosed herein (e.g., the recombinant T cell receptors (TCRs), or antigenbinding fragment thereof, the polynucleotides, the vectors, or the host cells (e.g., TCR-T cell)).
  • TCRs T cell receptors
  • the host cells e.g., TCR-T cell
  • the subject is a human.
  • 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 is resistant to sotorasib and/or adagrasib.
  • TNF-a Tumor Necrosis Factor alpha
  • IL-6 and IL-lb the matured DC were pulsed with G12C peptide.
  • the peptide pulsed DC together with IL-21 were subsequently co-cultured with the PBMC from the same donor, at a ratio 1 :35 of DC to PBMC, for one week to stimulate antigen specific T cell growth. The same procedure was then repeated twice.
  • T cells were collected and stained with PE-labeled G12C tetramer and a CD8 antibody. The Tetramer/CD8 double positive T cells were then sorted by flowcytometry after a first stimulation (FIG. 1).
  • 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- H1792 (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 HLAA1 101 and used as a target for cytotoxicity killing assay assessment.
  • the 1792 parental cell, HLAA1101 expressing 1792 cells (1792A11) were then appropriately labeled with chromium Cr51 for two hours, and co-cultured with G12C specific TCR-T cells at different effectors to target ratios for a total duration of 4 hours.
  • the released Cr51 was then measured by a gamma counter and calculated for killing efficacy as shown in FIG. 5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des récepteurs de lymphocytes T recombinants (TCR) qui se lient à une protéine homologue d'oncogène viral de sarcome de rat de Kirsten (KRAS) comprenant une mutation G12C présentée sur un antigène leucocytaire humain-A (HLA-A)*11:01, des polynucléotides codant pour de tels TCR, des lymphocytes T modifiés comprenant de tels TCR, et des procédés d'utilisation de ceux-ci.
PCT/US2024/010342 2023-01-04 2024-01-04 Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*11:01 dans le cancer du poumon Ceased WO2024148181A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363478489P 2023-01-04 2023-01-04
US63/478,489 2023-01-04

Publications (2)

Publication Number Publication Date
WO2024148181A2 true WO2024148181A2 (fr) 2024-07-11
WO2024148181A3 WO2024148181A3 (fr) 2024-08-15

Family

ID=91804275

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/010342 Ceased WO2024148181A2 (fr) 2023-01-04 2024-01-04 Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*11:01 dans le cancer du poumon

Country Status (1)

Country Link
WO (1) WO2024148181A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2618835B1 (fr) * 2010-09-20 2017-07-05 BioNTech Cell & Gene Therapies GmbH Récepteur des cellules t spécifiques des antigènes et épitopes des cellules t
RS67172B1 (sr) * 2016-03-31 2025-09-30 Biontech Us Inc Neoantigeni i postupci za njihovu upotrebu
US12331097B2 (en) * 2018-08-16 2025-06-17 Biontech Us Inc. T cell receptor constructs and uses thereof
GB2596461B (en) * 2019-02-20 2023-09-27 Fred Hutchinson Cancer Center Binding proteins specific for RAS neoantigens and uses thereof

Also Published As

Publication number Publication date
WO2024148181A3 (fr) 2024-08-15

Similar Documents

Publication Publication Date Title
KR102809909B1 (ko) P53 암-특이적 돌연변이에 대한 항원 특이성을 갖는 t 세포를 단리하는 방법
JP7730765B2 (ja) Ｍａｇｅ－ａ４ ｔ細胞受容体およびその使用方法
TWI789348B (zh) 表現免疫機能控制因子之免疫活性細胞及表現載體
JP2023503163A (ja) キメラ抗原受容体及びその使用
BR112021003305A2 (pt) métodos para produzir células que expressam receptor de antígeno quimérico
CA3173394A1 (fr) Methodes de production de cellules exprimant un recepteur antigenique chimerique
JP2023515211A (ja) キメラ抗原受容体発現細胞を作製する方法
AU2021326504B2 (en) Improving immune cell function
RU2680588C2 (ru) Гены рецепторов антигенспецифичных хелперных т-клеток
TW201934575A (zh) 一種分離的嵌合抗原受體以及包含其的修飾ｔ細胞及用途
TWI840766B (zh) 改善免疫細胞功能
CA3165827A1 (fr) Antigene exprime de preference dans des recepteurs de lymphocytes t de melanome (prame) et leurs procedes d'utilisation
JP2023524873A (ja) 方法
JP2023516538A (ja) Ｕｃａｒｔ細胞を精製する方法及び応用
CA3202725A1 (fr) Peptide epitope mutant ras et recepteur de lymphocyte t reconnaissant un mutant ras
TWI875737B (zh) Ｔ細胞受體及其使用方法
TWI877144B (zh) Ｔ細胞受體及其使用方法
EP3935174A1 (fr) Récepteurs de lymphocytes t et leurs procédés d'utilisation
WO2024148181A2 (fr) Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*11:01 dans le cancer du poumon
WO2024148185A2 (fr) Récepteurs de lymphocytes t ciblant la mutation kras g12v hautement prévalente sur hla-a*11:01 dans le cancer du poumon
WO2024148183A2 (fr) Récepteurs de lymphocytes t ciblant la mutation kras g12v hautement prévalente sur hla-a*03:01 dans le cancer du poumon
WO2024148178A2 (fr) Récepteurs de lymphocytes t ciblant la mutation kras g12c hautement prévalente sur hla-a*03:01 dans le cancer du poumon
CN118055944A (zh) 调节Bcl-2增强嵌合抗原受体癌症免疫疗法功效
WO2024054863A1 (fr) Récepteur de lymphocytes t ciblant une mutation egfr et ses procédés d'utilisation
CN117736298A (zh) T细胞抗原受体及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24738925

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE