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WO2025208068A2 - Constructions de récepteurs des lymphocytes t et leurs utilisations - Google Patents

Constructions de récepteurs des lymphocytes t et leurs utilisations

Info

Publication number
WO2025208068A2
WO2025208068A2 PCT/US2025/022094 US2025022094W WO2025208068A2 WO 2025208068 A2 WO2025208068 A2 WO 2025208068A2 US 2025022094 W US2025022094 W US 2025022094W WO 2025208068 A2 WO2025208068 A2 WO 2025208068A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
tcr
amino acid
set forth
acid sequence
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.)
Pending
Application number
PCT/US2025/022094
Other languages
English (en)
Other versions
WO2025208068A3 (fr
Inventor
Christina M. ARIETA
Özcan ÇINAR
Vikram JUNEJA
Ashley GAINES
Jared DIETZE
Kendra FOLEY
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.)
Biontech US Inc
Original Assignee
Biontech US Inc
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 Biontech US Inc filed Critical Biontech US Inc
Publication of WO2025208068A2 publication Critical patent/WO2025208068A2/fr
Publication of WO2025208068A3 publication Critical patent/WO2025208068A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • 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]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds

Definitions

  • T cell receptors are members of the immunoglobulin superfamily and usually consist - -subunits.
  • TCRs are thought to recognize the MHC.
  • the constant domain of the TCR domain consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which forms a link between the two chains.
  • the affinity of TCRs for a specific antigen makes them valuable for several therapeutic approaches. For example, cancer patients, such as melanoma patients, can be effectively treated by using adoptive immunotherapy as TCRs areakily sensitive for their antigen and can direct immune responses at tumor cells expressing their cognate antigen. Accordingly, there is a need for TCRs against peptide- MHC complexes for development of new and effective therapeutics.
  • TCR T cell receptor
  • CDR3 complementarity determining region 3
  • the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1) and a WSGR Docket No. 50401-775.601 complementarity determining region 2 (CDR2), wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 19; and the CDR2 has an amino acid sequence set forth in SEQ ID NO: 20.
  • the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 27.
  • the TCR beta chain construct comprises a variable region having at least 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 27.
  • the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity to an a mino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 27.
  • the recombinant nucleic acid encoding a T cell receptor (TCR) further comprising a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 16; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 17; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the TCR comprises (a) a sequence having at least 80% sequence identity with SEQ ID NOs: 25 or 26, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 21; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 22 or 23, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 18.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24.
  • the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24.
  • the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 24.
  • the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 110, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 110; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 109, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 109.
  • the recombinant nucleic acid encodes WSGR Docket No. 50401-775.601 a TCR that binds to an epitope from human GATA3 comprising a sequence set forth in SEQ ID NO: 30.
  • a recombinant nucleic acid encoding a T cell receptor comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having an amino acid sequence CASQDREGGNQPQHF (SEQ ID NO: 66).
  • the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1) and a complementarity determining region 2 (CDR2), wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 64; and the CDR2 has an amino acid sequence set forth in SEQ ID NO: 65.
  • the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 72.
  • the TCR further comprising a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 61; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 62; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 63.
  • the recombinant nucleic acid encoding the TCR comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NOs: 70 or 71, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 66; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 67 or 68, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 63.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 69.
  • the recombinant nucleic acid encoding the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 74, or SEQ ID NO: 138, or SEQ ID NO: 139, or SEQ ID NO: 140; or an amino acid sequence that is at least 80% identical to SEQ ID NO: 74 or SEQ ID NO: 138, or SEQ ID NO: 139, or SEQ ID NO: 140; (b) WSGR Docket No.
  • the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 116, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 116; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 115, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 115.
  • the TCR binds to an epitope from human GATA3 comprising a sequence set forth in SEQ ID NO: 75.
  • a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having an amino acid sequence CASSSDIFYEQYF (SEQ ID NO: 6).
  • TCR beta chain construct further comprises a complementarity determining region 1 (CDR1) and a complementarity determining region 2 (CDR2), wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 4; and the CDR2 has an amino acid sequence set forth in SEQ ID NO: 5.
  • TCR beta chain construct comprises a variable region having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 12.
  • TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 12.
  • TCR further comprises a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3.
  • the recombinant nucleic acid described above comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NOs: 10 or 11, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 6; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 7 or 8, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 3.
  • the recombinant nucleic acid encoding a T cell receptor (TCR) having the TCR alpha chain construct comprises a variable region having at least 80% , 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.
  • the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 9.
  • the TCR comprises:(a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 14, or SEQ ID NO: 122, or SEQ ID NO: 123 or SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14, SEQ ID NO: 122, SEQ ID NO: 123 or SEQ ID NO: 124; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or SEQ ID NO: 121 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13, or SEQ ID WSGR Docket No.
  • the TCR comprises:(a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 108, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 108; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 107, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 107.
  • the recombinant nucleic acid encodes the TCR as described above, wherein the TCR binds to an epitope from human GATA3 comprising a sequence set forth in SEQ ID NO: 15.
  • the TCR comprises a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 91; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 92; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 93.
  • the recombinant nucleic acid comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 100 or 101, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 96; and (b) a sequence having at least 80% sequence identity with SEQ ID NOs: 97 or 98, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 93.
  • the recombinant nucleic acid encodes a T cell receptor (TCR) wherein the TCR alpha chain construct comprises a variable region having at least 80% , 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 99.
  • TCR T cell receptor
  • the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 99.
  • the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 104, or SEQ ID NO: 146, or SEQ ID NO: 147, or SEQ ID NO: 148; or an amino acid sequence that is at least 80% identical to SEQ ID NO: 104, SEQ ID NO: 146, or SEQ ID NO: 147, or SEQ ID NO: 148; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 103, or SEQ ID NO: 145; or an amino acid sequence that is at least 80% identical to SEQ ID WSGR Docket No.
  • the recombinant nucleic acid encodes a TCR that further comprises a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein: the CDR1 has an amino acid sequence set forth in SEQ ID NO: 31; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 32; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 33.
  • the recombinant nucleic acid encodes the TCR, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 44, or SEQ ID NO: 130, or SEQ ID NO: 131, or SEQ ID NO: 132, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 44; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 43, or SEQ ID NO: 129, or an amino WSGR Docket No. 50401-775.601 acid sequence that is at least 80% identical to SEQ ID NO: 43 or SEQ ID NO: 129.
  • a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having an amino acid sequence CASSLSETQYF (SEQ ID NO: 51).
  • the recombinant nucleic acid encodes a T cell receptor (TCR) wherein the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1) and a complementarity determining region 2 (CDR2), wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 49; and the CDR2 has an amino acid sequence set forth in SEQ ID NO: 50.
  • the recombinant nucleic acid described herein comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 85 or 86, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 81; and (b) a sequence having at least 80% sequence identity with SEQ ID NOs: 82 or 83, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 78.
  • the recombinant nucleic acid encodes the TCR wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 89, or SEQ ID NO: 142, or SEQ ID NO: 143, or SEQ ID NO: 144; or an amino acid sequence that is at least 80% identical to SEQ ID NO: 89, or SEQ ID NO: 142, or SEQ ID NO: 143, or SEQ ID NO: 144; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 88, or SEQ ID NO: 141; or an amino acid sequence that is at least 80% identical to SEQ ID NO: 88 or SEQ ID NO: 141.
  • the recombinant nucleic acid encodes the TCR comprising: (a) a beta chain having an amino acid sequence set f orth in SEQ ID NO: 118, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 118; (b) an WSGR Docket No. 50401-775.601 alpha chain having an amino acid sequence set forth in SEQ ID NO: 117, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 117.
  • the recombinant nucleic acid encodes the TCR as described in this paragraph, wherein the TCR binds to an epitope from human GATA3 comprising a sequence set forth in SEQ ID NO: 90.
  • a vector comprising the recombinant nucleic acid of any one of the TCR constructs described in the embodiments above.
  • a cell comprising the recombinant nucleic acid of any one of the embodiments described above, or the vector as described.
  • a recombinant nucleic acid encoding a TCR construct comprising: (a) a TCR beta chain construct, and (b) a TCR alpha chain construct; wherein the TCR recognizes and binds to an epitope from human GATA3 comprising a frameshift mutation, the epitope being in a human MHC-protein complex, wherein the human MHC-protein is an HLA antigen encoded by the HLA-A*02:01 allele.
  • the epitope binds to the human MHC with a KD or an IC50 less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM or less than 10 nM.
  • the mutation is not present in non-cancer cells of a subject.
  • the TCR binds to a MHC-peptide complex with a KD or an IC50 of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM or less than 10 nM.
  • the vector is a lentiviral vector.
  • the cell is a CD4+ T cell.
  • the cell is a CD8+ T cell.
  • the cell is isolated from a subject having a GATA3 mutation.
  • the TCR binds to a complex comprising (i) an epitope from human GATA3 and (ii) an MHC protein, wherein the MHC protein is encoded by HLA-A*02:01 allele.
  • the TCR comprises a sequence provided in Table 2.
  • the TCR may be a TCR described in Table 3.
  • the vector is a lentiviral vector.
  • the cell is a CD4+ T cell.
  • the cell is a CD8+ T cell.
  • the cell is isolated from a subject having a GATA3 mutation.
  • a pharmaceutical composition comprising: (a) the nucleic acid encoding a TCR are described above; or, the vector described herein; or, the cell as described herein; and (b) a pharmaceutically acceptable excipient or diluent.
  • the pharmaceutical composition is for use in treating an immune disease or cancer.
  • provided herein is use of the pharmaceutical composition, for treating an immune disease or cancer.
  • provided herein is a method of treating a subject having a disease or condition, comprising administering to the subject in need thereof the pharmaceutical composition described herein.
  • a method of identifying a subject with cancer as a candidate for a therapeutic comprising determining the subject as a subject that expresses a protein encoded by an HLA-A*02:01 allele wherein therapeutic is the pharmaceutical composition described herein.
  • a soluble TCR comprising at least a TCR variable domain, comprising a sequence selected from the group : CATSDRGDSQETQYF (SEQ ID NO: 21); CASQDREGGNQPQHF (SEQ ID NO: 66); CASSSDIFYEQYF (SEQ ID NO: 6); CASSLISLGAGEDTEAFF (SEQ ID NO: 96); CASQGPYSLDTQYF (SEQ ID NO: 36); CASSLSETQYF (SEQ ID NO: 51); and CASSTLTISTDTQYF (SEQ ID NO: 81).
  • a recombinant nucleic acid encoding a TCR comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having an amino acid sequence CASSQGVGESPEAFF (SEQ ID NO: 206).
  • the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1) and a complementarity determining region 2 (CDR2), wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 204; and the CDR2 has an amino acid sequence set forth in SEQ ID NO: 205.
  • the TCR beta chain construct comprises a variable region having about 80%, 85%, WSGR Docket No.
  • the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 210.
  • the recombinant nucleic acid comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 209, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 206; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 207, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 203.
  • the TCR alpha chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 208.
  • the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 208.
  • a TCR having a TCR alpha chain and a TCR beta chain wherein the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 203; and the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 206, and where in the TCR binds to a complex comprising (i) an epitope MLTGPPARV (SEQ ID NO: 15), or an epitope SMLTGPPARV (SEQ ID NO: 200) and (ii) an MHC protein.
  • the recombinant nucleic acid encoding a TCR further comprises a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 291; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 292; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 293.
  • the recombinant nucleic acid comprise: (a) a WSGR Docket No.
  • a TCR having a TCR alpha chain and a TCR beta chain
  • the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 293
  • the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 296, and where in the TCR binds to a complex comprising (i) an epitope MLTGPPARV (SEQ ID NO: 15), or an epitope SMLTGPPARV (SEQ ID NO: 200) and (ii) an MHC protein.
  • the TCR beta chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 330. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 330.
  • the recombinant nucleic acid further comprising a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 321; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 322; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 323.
  • the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 338.
  • a TCR having a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 333; and the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 336, and where in the TCR binds to a complex comprising (i) an epitope MLTGPPARV (SEQ ID NO: WSGR Docket No.
  • the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1) and a complementarity determining region 2 (CDR2), wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 344; and the CDR2 has an amino acid sequence set forth in SEQ ID NO: 345.
  • the TCR beta chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 350.
  • the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 350.
  • the recombinant nucleic acid encoding a TCR further comprises a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 341; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 342; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 343.
  • the recombinant nucleic acid comprising: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 389, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 386; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 387, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 383.
  • the TCR alpha chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 388.
  • the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 388.
  • a TCR having a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 383; and the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 386, and where in the TCR binds to a complex comprising (i) an epitope MLTGPPARV (SEQ ID NO: 15), or an epitope SMLTGPPARV (SEQ ID NO: 200), and (ii) an MHC protein.
  • TCR beta chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 400. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 400. [0041] In some embodiments, the TCR further comprising a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 391; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 392; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 393.
  • the recombinant nucleic acid encoding the TCR comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 399, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 396; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 397, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 393.
  • the TCR alpha chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 398.
  • TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 398.
  • a TCR having a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 393; and the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 396, and where in the TCR binds to a complex comprising (i) an epitope MLTGPPARV (SEQ ID NO: 15), or an epitope SMLTGPPARV (SEQ ID NO: 200), and (ii) an MHC protein.
  • the TCR comprises: (a) a beta chain comprising a variable region having at least 80% sequence identity to SEQ ID NO: 220; and (b) an alpha chain, comprising a variable region having at least 80% sequence identity to SEQ ID NO: 218.
  • the TCR comprises: (a) a beta chain comprising a variable region having at least 80% sequence identity to SEQ ID NO: 230; and (b) an alpha chain, comprising a variable region having at least 80% sequence identity to SEQ ID NO: 228.
  • the TCR comprises: (a) a beta chain, comprising a CDR1 having a sequence of SEQ ID NO: 234, a CDR2 having a sequence of SEQ ID NO: 235, and a CDR3 having a sequence of SEQ ID WSGR Docket No.
  • the TCR comprises: (a) a beta chain comprising a variable region having at least 80% sequence identity to SEQ ID NO: 240; and (b) an alpha chain, comprising a variable region having at least 80% sequence identity to SEQ ID NO: 238.
  • the TCR comprises: (a) a beta chain comprising a variable region having at least 80% sequence identity to SEQ ID NO: 250; and (b) an alpha chain, comprising a variable region having at least 80% sequence identity to SEQ ID NO: 248.
  • the TCR comprises: (a) a beta chain, comprising a CDR1 having a sequence of SEQ ID NO: 274, a CDR2 having a sequence of SEQ ID NO: 275, and a CDR3 having a sequence of SEQ ID NO: 276; and (b) an alpha chain, comprising a CDR1 having a sequence of SEQ ID NO: 271, a CDR2 having a sequence of SEQ ID NO: 272 and a CDR3 having a sequence of SEQ ID NO: 273.
  • the TCR comprises: (a) a beta chain comprising a variable region having at least 80% sequence identity to SEQ ID NO: 310; and (b) an alpha chain, comprising a variable region having at least 80% sequence identity to SEQ ID NO: 308.
  • the TCR comprises: (a) a beta chain, comprising a CDR1 having a sequence of SEQ ID NO: 354, a CDR2 having a sequence of SEQ ID NO: 355, and a CDR3 having a sequence of SEQ ID NO: 356; and (b) an alpha chain, comprising a CDR1 having a sequence of SEQ ID NO: 351, a CDR2 having a sequence of SEQ ID NO: 352 and a CDR3 having a sequence of SEQ ID NO: 353.
  • the TCR comprises: (a) a beta chain comprising a variable region having at least 80% sequence identity to SEQ ID NO: 360; and (b) an alpha chain, comprising a variable region having at least 80% sequence identity to SEQ ID NO: 358.
  • a TCR having a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 423; and the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: WSGR Docket No. 50401-775.601 426, and where in the TCR binds to a complex comprising (i) an epitope KPKRDGYMF, or an epitope KPKRDGYMFL, and (ii) an MHC protein.
  • the MHC is encoded by HLA-B*07:02 allele.
  • a recombinant nucleic acid encoding a TCR comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having an amino acid sequence CASSPTSGISYEQYF (SEQ ID NO: 516).
  • the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1) and a complementarity determining region 2 (CDR2), wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 514; and the CDR2 has an amino acid sequence set forth in SEQ ID NO: 515.
  • the TCR beta chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 520. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 520. In some embodiments, the TCR further comprising a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 511; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 512; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 513.
  • the recombinant nucleic acid comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 519, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 516; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 517, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 513.
  • the TCR alpha chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 518.
  • the TCR beta chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 530. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 530. In some embodiments, the TCR comprises a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 521; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 522; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 523.
  • the TCR comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 529, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 526; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 527, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 523.
  • the TCR alpha chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 528.
  • the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 528.
  • a TCR having a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 523; and the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 526, and where in the TCR binds to a complex comprising (i) an epitope KPKRDGYMF, or an epitope KPKRDGYMFL, and (ii) an MHC protein.
  • the recombinant nucleic acid comprises: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 599, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 596; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 597, wherein the sequence comprises a sequence encoding at least SEQ ID NO: 593.
  • the TCR alpha chain construct comprises a variable region having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 598. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 598.
  • a TCR having a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 593; and the TCR beta chain comprises a CDR3 has an amino acid sequence set forth in SEQ ID NO: 596, and where in the TCR binds to a complex comprising (i) an epitope KPKRDGYMF, or an epitope KPKRDGYMFL, and (ii) an MHC protein.
  • the TCR further comprising a TCR alpha chain construct having a CDR1, a CDR2, and a CDR3, wherein, the CDR1 has an amino acid sequence set forth in SEQ ID NO: 681; the CDR2 has an amino acid sequence set forth in SEQ ID NO: 682; and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 683.
  • FIG.3 depicts experimental EC50 results of a TCR functional avidity assay.
  • TCR2 and TCR5 showed the lowest EC50 values.
  • GATA3neoORF specific TCR expressing T cells successfully recognize target cells with the GATA3 neoORF mutation.
  • T cells were transduced with nucleic acid encoding sample mutation-specific TCRs (TCRs-B7-2, B74, B7-11, B7-12, and B7-19 respectively) (Table 5A-5B). These T cells can recognize naturally processed/presented peptide-MHC on K562 cells expressing full-length GATA3neoORF and HLA-B*07:02, without external peptide pulsing.
  • FIG.6 depicts exemplary data demonstrating that multiple TCRs generated and presented in Tables 6A and 6B are functional. The data show that T cells transduced with mutation-specific TCRs generated herein can recognize naturally processed/presented peptide-MHC on K562 cells expressing full-length GATA3neoORF and HLA-B*08:01, without external peptide pulsing.
  • the TCR may be an intact or full-length TCR.
  • the TCR may be an antigen-binding portion that may be less than a full-length TCR but that binds to a specific antigenic peptide bound to (i.e., in the context of) an MHC molecule, i.e., an MHC-peptide complex.
  • an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet may be able to bind the epitope (e.g., MHC-peptide complex) to which the full TCR binds.
  • FR-H1, FR-H2, FR-H3, and FR-H4 there are four FRs in each full-length alpha chain WSGR Docket No. 50401-775.601 variable region
  • FR-L1, FR-L2, FR-L3, and FR-L4 four FRs in each full-length beta chain variable region
  • gamma or delta chain that may be involved in binding the TCR to antigen-MHC complexes.
  • the ures with each -MHC complex.
  • TCR fragments including antigen-binding fragments.
  • the TCR may be a single-chain TCR fragment comprising an alpha chain variable region linked to a beta chain variable region, such as a scTv.
  • a scTv may be also referred to as a scFv.
  • a single-chain Tv or scTv refers in some aspects to polypeptide chain.
  • the Tv polypeptide further comprises a polypeptide linker between the antigen binding.
  • a diabody refers in some aspects to TCR fragments with two antigen-binding sites, which frag - - pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • a linear TCR refers in some aspects to a pair of tandem Fd segments (e.g. - 1- - 1) which form a pair of antigen binding regions.
  • Linear TCRs can be bispecific or monospecific.
  • An antigen-binding domain refers in some aspects to one or more fragments of a TCR that retain the ability to specifically bind to a peptide-MHC complex.
  • TCRs are substituted with corresponding residues from a non-human TCR (e.g., the TCR from which may be a TCR with an amino acid sequence corresponding to that of a TCR produced by a human or a human cell, or non-human source that utilizes human TCR repertoires or other human TCR-encoding sequences, including human TCR libraries.
  • the term excludes humanized forms of non-human TCRs comprising non-human peptide-MHC complex binding regions, such as those in which all or substantially all CDRs are non-human.
  • Human TCRs may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human TCRs or intact TCRs with human variable regions in response to antigenic challenge.
  • a neoantigen represents either oncogenic viral proteins or abnormal proteins that arise as a consequence of somatic mutations.
  • a neoantigen can arise by the disruption of cellular mechanisms through the activity of viral proteins.
  • Another example can be an exposure of a carcinogenic compound, which in some cases can lead to a somatic mutation. This somatic mutation can ultimately lead to the formation of a tumor/cancer.
  • a neoantigen can be a class of tumor antigens which arise from tumor-specific changes in proteins.
  • Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, a substitution in a protein sequence, a frame shift mutation, a fusion polypeptide, an in-frame deletion, an insertion, and expression of an endogenous retroviral polypeptide.
  • a neoepitope can be an epitope that is not present in a reference, such as a non- diseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell.
  • an epitope refers to a portion of a peptide-MHC complex capable of forming a binding interaction with the variable region binding pocket of a TCR.
  • covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions under physiological conditions and includes interactions such as salt bridges and water bridges, as well as any other conventional means of binding.
  • reference to a TCR with specific binding refers to a situation in which a TCR will not show any significant binding to molecules other than the peptide-MHC complex containing the epitope recognized by the TCR.
  • an antigen binding domain may be specific for a particular epitope which is carried by a number of peptide-MHC complexes, in which case the selected TCR or peptide-MHC complex binding fragment thereof carrying the peptide-MHC complex binding domain will be able to bind to the various peptide- MHC complexes carrying the epitope. that the TCRs or fragments thereof bind to an epitope with greater affinity than it binds unrelated amino acid sequences, and if cross-reactive to other polypeptides containing the epitope, are not toxic WSGR Docket No. 50401-775.601 at the levels at which they are formulated for administration to human use.
  • binding pair e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or II HLA, or a peptide-HLA complex and a T cell receptor (TCR)
  • HLA human leukocyte antigen
  • TCR T cell receptor
  • Affinity can be expressed as an equilibrium constant of the reversible binding of two agents and can be expressed as KD, KA, Koff or Kon.
  • KD refers to the dissociation constant between two members of a binding pair and has units of molarity.
  • K A refers to the affinity constant between two members of a binding pair is the inverse of the dissociation constant. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units.
  • SPR surface plasmon resonance
  • Affinity of a binding protein to a ligand such as affinity of a TCR for an epitope can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 resistance of a complex of two or more agents to dissociation after dilution.
  • 50 Affinity may also be expressed as the inhibitory concentration 50 (IC 50 ), or the concentration at which 50% of a first member of a binding pair (e.g., a peptide) is displaced.
  • IC 50 inhibitory concentration
  • ln(IC 50 ) refers to the natural log of the IC 50 .
  • binding can be expressed relative to binding by a reference WSGR Docket No. 50401-775.601 standard peptide. Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol.2:443 (1990); Hill et al., J. Immunol.147:189 (1991); del Guercio et al., J. Immunol.
  • MHC class III region can encode for other immune components, such as complement components and cytokines.
  • the MHC can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).
  • HLA alleles found on one chromosome and the proteins encoded thereby. Haplotype may also refer to the allele present at any one locus within the MHC.
  • Each class of MHC may be represented by several loci: e.g., HLA-A (Human Leukocyte Antigen-A).
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • Polynucleotides can include nonstandard nucleotides, such as nucleotide analogs or modified nucleotides.
  • nonstandard nucleotides can stabilize hybrid formation.
  • nonstandard nucleotides can destabilize hybrid formation.
  • nonstandard nucleotides can enhance hybridization specificity.
  • nonstandard nucleotides can reduce hybridization specificity.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • WSGR Docket No. 50401-775.601 [0095] another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity can indicate the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • Sequence identity such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally with default settings), the BLAST algorithm (see e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm. Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.
  • Needleman-Wunsch algorithm e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally with default settings
  • the BLAST algorithm see e
  • polypeptide can comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 peptides or amino acids.
  • polypeptides include, but are not limited to, amino acid chains, proteins, peptides, hormones, polypeptide saccharides, lipids, glycolipids, phospholipids, antibodies, enzymes, kinases, receptors, transcription factors, and ligands.
  • Polypeptides including the provided TCRs and TCR chains and other peptides, e.g., linkers and binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • Examples of unconventional amino acids include: 4 - - - -N,N,N-trimethyll -N-acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5- -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left hand direction is the amino terminal direction
  • the right hand direction is the carboxy- terminal direction, in accordance with standard usage and convention.
  • Percent (%) sequence identity with respect to a reference polypeptide sequence (or nucleic acid sequence) may be the percentage of amino acid residues (or nucleotides in case of nucleic acid sequence) in a candidate sequence that are identical with the amino acid residues (or nucleotides) in the reference polypeptide sequence (or nucleic acid sequence), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B.
  • treatment of cancer includes, but is not limited to, stasis, partial or total elimination of a cancerous growth or tumor.
  • Treatment or partial elimination includes, for example, a fold reduction in growth or tumor size and/or volume such as about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or any fold reduction in between.
  • subjects and hosts include, but are not limited to, horses, cows, camels, sheep, p igs, goats, dogs, cats, rabbits, guinea pigs, rats, mice (e.g., humanized mice), gerbils, non-human primates (e.g., macaques), humans and the like, non-mammals, including, e.g., non-mammalian vertebrates, such as birds (e.g., chickens or ducks) fish (e.g., sharks) or frogs (e.g., Xenopus), and non-mammalian invertebrates, as well as transgenic species thereof.
  • non-mammalian vertebrates such as birds (e.g., chickens or ducks) fish (e.g., sharks) or frogs (e.g., Xenopus), and non-mammalian invertebrates, as well as transgenic species thereof.
  • a subject refers to a single organism (e.g., human).
  • a subject from whom samples are obtained can either be inflicted with a disease and/or disorder (e.g., one or more allergies, infections, cancers or WSGR Docket No. 50401-775.601 autoimmune disorders or the like) and can be compared against a negative control subject which is not affected by the disease. [00101] ing out a method disclosed herein.
  • a disease and/or disorder e.g., one or more allergies, infections, cancers or WSGR Docket No. 50401-775.601 autoimmune disorders or the like
  • the packaging material can maintain the components sterilely and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc.).
  • the label or packaging insert can include appropriate written instructions. Kits, therefore, can additionally include labels or instructions for using the kit components in any method of the invention.
  • a kit can include a compound in a pack, or dispenser together with instructions for administering the compound in a method described herein.
  • gene or the host cell containing the gene to become resistant to treatment with a drug for example, BTK C481S mutation is a resistance mutation which can confer ibrutinib resistance.
  • TCRs T cell receptors
  • isolated nucleic acid molecules encoding TCRs against neoantigens
  • T cells expressing said TCRs T cells expressing said TCRs
  • the TCR alpha chain construct comprises a variable region having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least WSGR Docket No. 50401-775.601 about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9, 24, 39, 54, 69, 84, and 99.
  • the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs: 12, 27, 42, 57, 72, 87, 102. In some embodiments, the TCR beta chain construct comprises a variable region have at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 12, 27, 42, 57, 72, 87, 102.
  • the TCR alpha chain construct comprises a complementarity determining region 1 (CDR1) having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 16, 31, 46, 61, 76, and 91.
  • CDR1 complementarity determining region 1
  • the TCR beta chain construct comprises a complement arity determining region 1 (CDR1) having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 19, 34, 49, 64, 79, and 94.
  • CDR1 complement arity determining region 1
  • the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 19, 34, 49, 64, 79, and 94.
  • CDR1 complementarity determining region 1
  • the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2, 17, 32, 47, 62, 77, and 92.
  • CDR2 complementarity determining region 2
  • the TCR alpha chain construct WSGR Docket No.
  • the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 5, 20, 35, 50, 65, 80, and 95.
  • the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NOs: 3, 18, 33, 48, 63, 78, or 93.
  • the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NOs: 6, 21, 36, 51, 66, 81, or 96.
  • CDR3 complementarity determining region 3
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 24; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 27.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 17, and a CDR3 of SEQ ID NO: 18; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 19, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 21.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 69; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 72.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 99; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 102.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 91, a CDR2 of SEQ ID NO: 92, and a CDR3 of SEQ ID NO: 93; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 94, a CDR2 of SEQ ID NO: 95, and a CDR3 of SEQ ID NO: 96.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 39; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 42.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 31, a CDR2 of SEQ ID NO: 32, and a CDR3 of SEQ ID NO: 33; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of SEQ ID NO: 36.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 54; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 57.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 46, a CDR2 of SEQ ID NO: 47, and a CDR3 of SEQ ID NO: 48; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 49, a CDR2 of SEQ ID NO: 50, and a CDR3 of SEQ ID NO: 51.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 84; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 87.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 76, a CDR2 of SEQ ID NO: 77, and a CDR3 of SEQ ID NO: 78; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 79, a CDR2 of SEQ ID NO: 80, and a CDR3 of SEQ ID NO: 81.
  • the present disclosure provides a host cell comprising the nucleic acid encoding a TCR against a neoantigen provided herein, a vector containing the nucleic acid sequence, or a protein encoded by the nucleic acid provided herein.
  • the host cell is a CD4 + T cell. In some embodiments, the host cell is a CD8+ T cell.
  • the host cell may be a natural killer (NK) cell or a B cell.
  • the host cell may be an immortalized cell line.
  • the present disclosure provides pharmaceutical compositions comprising the nucleic acid encoding a TCR against a neoantigen provided herein, a host cell comprising the nucleic acid encoding a TCR against a neoantigen provided herein, a vector containing the nucleic acid WSGR Docket No. 50401-775.601 sequence, or a protein encoded by the nucleic acid provided herein.
  • TCRs provided herein target may be engineered TCRs, for example, chimeric antigen receptors (CARs).
  • CARs can be composed of three regions: an ectodomain, a transmembrane domain and an endodomain.
  • Zinc finger nucleases are heterodimeric arrays that co-localize at a target DNA site.
  • ZFNs include individual finger subunits that bind DNA and are tethered to the Fokl nuclease domain that cleaves DNA.
  • Transcription activator-like effector nucleases include repeating units that bind DNA by virtue of a hypervariable two amino acid sequence (repeat variable diresidue; RVD) that governs DNA base recognition. Similar WSGR Docket No. 50401-775.601 to ZFNS, TALENs function as dimeric proteins that are fused to the Fokl endonuclease domain for DSB generation.
  • introducing the genome-editing nuclease into the T cell includes introducing into the T cell a polynucleotide that encodes the genome-editing nuclease.
  • introducing the genome-editing nuclease into the T cell includes introducing into the T cell a Cas9 polypeptide.
  • the genome-editing nuclease includes a TALEN nuclease, a CRISPR/Cas9 nuclease, or a megaTAL nuclease.
  • the CRISPR/Cas9 nuclease is derived from either Streptococcus pyogenes or Staphylococcus aureus.
  • the CRISPR/Cas9 nuclease includes a nuclease-resistant gRNA such as, for example, at least one 2'-OMe-phosphorothioate modified base, at least one 2'-O-methyl modified base, or at least one 2'-O-methyl 3' thioPACE modified base.
  • the TALEN nuclease or the megaTAL nuclease is encoded by an RNA that has an exogenous polyadenylation signal.
  • the method described herein may further include culturing the T cell under conditions effective for expanding the population of genome-modified T cells.
  • disrupting expression of further disrupts .
  • disrupting expre involves fur .
  • a genetically-modified T cell comprises a disrupted TCR alpha chain and/or beta chain and an inactivated gene encoding immune checkpoint protein such as PD1 and CTLA-4. This may be made possible by gene inactivation using specific TALE-nucleases directed against TCR alpha or TCR beta coupled with inactivation of genes encoding immune checkpoint protein such as PD1 and CTLA-4.
  • the genetic modification relies on the inactivation of one gene, or two genes selected from the group consisting of PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha and TCR beta. In some embodiments, the genetic modification relies on the inactivation of two genes selected from the group consisting of PD1 and TCR alpha, PD1 WSGR Docket No.
  • the genetic modification relies on the inactivation of more than two genes.
  • the genetic modification may be operated ex-vivo.
  • TCRs Specific to GATA3 peptide-MHC complexes [00145] GATA3 is frequently mutated in breast cancer and appears to regulate b reast cancer via numerous molecular mechanisms. GATA3 distal deletion mutations also cause HDR syndrome (hypoparathyroidism, sensorineural deafness, and renal dysplasia) and DiGeorge syndrome.
  • GATA3 gene is on chromosome 10p15. It consists of six exons and encodes a transcription factor and two transactivating domains and two zinc finger domains on exons 2-6.
  • GATA3 Tumor suppressor functions of GATA3 have been demonstrated primarily in basal-like breast cancers and luminal breast cancers.
  • ER- (ER negative) basal-like breast cancer (BLBC) GATA3 function is largely attributed to negative regulation of genes associated with invasion and dedifferentiation, and loss of GATA3 expression is involved in aggressiveness of BLBC.
  • luminal breast cancer GATA3 directly upregulated proto- oncogenes.
  • neoORFs Several frameshift mutations in GATA3 gene in various cancers result in neoORFs, which can translate to generate mutated GATA3 protein containing segments encoded by the neoORFs, and which in turn may comprise neoantigens.
  • a therapeutic composition e.g., a vaccine may comprise a GATA3 polypeptide, or a polynucleic acid encoding the GATA3 polypeptide.
  • the WSGR Docket No. 50401-775.601 GATA3 polypeptide may comprise a sequence, PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGP PARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH.
  • antigen presenting cells e.g., via electroporation
  • the APCs of the APC cell therapeutic express the polynucleotide and present the neoepitopes in combination with an MHC molecule encoded by an HLA.
  • antigen presenting cells expressing the polynucleic acid (e.g. mRNA) having a sequence encoding the polypeptide as provided above may be used to stimulate T cells ex vivo.
  • TCRs may be generated that are capable of binding to a neoantigen, e.g.
  • TCRs as is known for all neoantigen-specific TCRs is that T cells comprising the TCR will selectively attack and destroy cells that express the mutated GATA3 gene expressing the neoantigens.
  • the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 19, 34, 49, 64, 79, and 94.
  • CDR1 complementarity determining region 1
  • the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence of SEQ ID NOs: 1, 16, 31, 46, 61, 76, or 91, or a variant thereof comprising 1-3 amino acid modifications.
  • the TCR alpha chain construct comprises a complementarity determining region 1 (CDR1) having 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 16, 31, 46, 61, 76, and 91.
  • the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2, 17, 32, 47, 62, 77, and 92.
  • the recombinant nucleic acid encoding a T cell receptor comprises a TCR beta chain construct; wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR beta chain construct comprises a variable region having at least 70%, 75%, 80%, or 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 12, 27, 42, 57, 72, 87, 102.
  • the TCR beta chain construct comprises a variable region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 12, 27, 42, 57, 72, 87, 102.
  • the nucleic acid encoding at least one T cell receptor (TCR) comprises a TCR alpha chain construct; wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the recombinant nucleic acid encoding a T cell receptor comprises a TCR beta chain construct; wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR beta chain construct comprises a variable region having at least 70%, 75%, 80%, or 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 108, 110, 112, 114, 116, 118, and 120.
  • the TCR beta chain construct comprises a variable region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 108, 110, 112, 114, 116, 118, and 120.
  • the nucleic acid encoding at least one T cell receptor (TCR) comprises a TCR alpha chain construct; wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR alpha chain construct comprises a variable region having at least 70%, 75%, 80%, or 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 107, 109, 111, 113, 115, 117, and 119.
  • the TCR alpha chain construct comprises a WSGR Docket No. 50401-775.601 variable region having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 107, 109, 111, 113, 115, 117, and 119.
  • the TCR comprises a beta chain construct having at least 70%, 75%, 80%, or 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 14, 29, 44, 59, 74, 89, and 104. In some embodiments, the TCR comprises a beta chain construct having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 14, 29, 44, 59, 74, 89, and 104. In some embodiments, the TCR comprises an alpha chain construct having at least 70%, 75%, 80%, or 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 13, 28, 43, 58, 73, 88, and 103.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs: 24 and 69, wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR alpha chain construct as described above comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 16 and 61.
  • the TCR beta chain construct as described above comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 19 and 64.
  • the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence selected from SEQ ID NOs: 21 and 66.
  • CDR3 complementarity determining region 3
  • a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct, wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs: 12 and 102 wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9 and 99, wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR alpha chain construct as described above comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1 and 91.
  • the TCR beta chain construct as described above comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4 and 94.
  • the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2 and 92.
  • a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct, wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs: 42 and 57 wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid WSGR Docket No. 50401-775.601 sequence selected from SEQ ID NOs: 39 and 54, wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • the TCR alpha chain construct as described above comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 31 and 46.
  • the TCR beta chain construct as described above comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 34 and 49.
  • the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 32 and 47.
  • TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 35 and 50.
  • the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence selected from SEQ ID NOs: 33 and 48.
  • the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence selected from SEQ ID NOs: 36 and 51.
  • CDR3 complementarity determining region 3
  • a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct, wherein the TCR specifically binds to an epitope from GATA3 in complex with a human MHC encoded by an HLA-A02:01 allele.
  • TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 80.
  • CDR2 complementarity determining region 2
  • the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence selected from SEQ ID NOs: 78.
  • the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence selected from SEQ ID NOs: 81.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 17, and a CDR3 of SEQ ID NO: 18; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 19, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 21.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 24; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 27.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 61, a CDR2 of SEQ ID NO: 62, and a CDR3 of SEQ ID NO: 63; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 64, a CDR2 of SEQ ID NO: 65, and a CDR3 of SEQ ID NO: 66.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 69; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 72.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 9; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 12.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 91, a CDR2 of SEQ ID NO: 92, and a CDR3 of SEQ ID NO: 93; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 94, a CDR2 of SEQ ID NO: 95, and a CDR3 of SEQ ID NO: 96.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 99; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 102.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 31, a CDR2 of SEQ ID NO: 32, and a CDR3 of SEQ ID NO: 33; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of SEQ ID NO: 36.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 39; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 42.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 46, a CDR2 of SEQ ID NO: 47, and a CDR3 of SEQ ID NO: 48; and the TCR WSGR Docket No. 50401-775.601 beta chain construct comprises a CDR1 of SEQ ID NO: 49, a CDR2 of SEQ ID NO: 50, and a CDR3 of SEQ ID NO: 51.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 54; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 57.
  • the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 76, a CDR2 of SEQ ID NO: 77, and a CDR3 of SEQ ID NO: 78; and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 79, a CDR2 of SEQ ID NO: 80, and a CDR3 of SEQ ID NO: 81.
  • the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 84; and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 87.
  • a soluble T cell receptor comprising at least a TCR variable domain, comprising a CDR3 having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 6, 21, 36, 51, 66, 81, and 96.
  • the nucleic acid sequence encoding a TCR is codon optimized. Codon optimization is a technique that may maximize protein expression by increasing the translational efficiency of the encoding gene. Codon optimization may enhance translational efficiency through modification of the nucleic acid sequence. In some embodiments, codon optimization may further enhance vector yield.
  • a TCR alpha chain variable domain may be codon optimized.
  • the TCR alpha chain construct comprises a variable region having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to a codon optimized sequence as set forth in SEQ ID NOs: 8, 23, 38, 53, 68, 83, 98.
  • a TCR alpha chain variable domain may not be t.
  • the TCR alpha chain construct comprises a variable region having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NOs: 7, 22, 37, 67, 82, 97.
  • a TCR beta chain variable domain may be codon optimized.
  • the TCR beta chain construct comprises a variable region having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least WSGR Docket No. 50401-775.601 about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to a codon optimized sequence as set forth in SEQ ID NOs: 11, 26, 41, 56, 71, 86, 101.
  • a TCR beta chain variable domain may not be codon optimized.
  • the TCR beta chain construct comprises a variable region having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to a nucleotide sequence as set forth in SEQ ID NOs: 10, 25, 40, 55, 70, 85, 100.
  • T cells can combine through V(D)J recombination to generate diverse and functional TCRS capable of proper expression and MHC recognition.
  • a TCR is composed of an alpha and beta chain which can be generated through V(D)J recombination.
  • the TCR alpha chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 18.
  • the TCR alpha chain comprises a CDR3 generated from TRAV9-2*02 and/or TRAJ48*01.
  • the TCR beta chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 21.
  • the TCR alpha chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 93.
  • the TCR alpha chain comprises a CDR3 generated from TRAV8-1*01 and/or TRAJ6*01.
  • the TCR beta chain WSGR Docket No. 50401-775.601 comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 96.
  • the TCR beta chain comprises a CDR3 generated from TRBV28*01, TRBJ1-1*01, TRBD1*01.
  • the TCR alpha chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 33.
  • the TCR alpha chain comprises a CDR3 generated from TRAV41*01 and/or TRAJ45*01.
  • the TCR beta chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 36.
  • the TCR beta chain comprises a CDR3 generated from TRBV28*01, TRBJ2-3*01, and/or TRBD1*01.
  • the TCR alpha chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 48.
  • the TCR alpha chain comprises a CDR3 generated from TRAV8-1*01 and/or TRAJ45*01. In some embodiments, the TCR beta chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 51. In some embodiments, the TCR beta chain comprises a CDR3 generated from TRBV7-8*01 and/or TRBJ2- 5*01. [00183] In some embodiments, the TCR alpha chain comprises a complementarity determining region 3 (CDR3) as set forth in SEQ ID NO: 78. In some embodiments, the TCR alpha chain comprises a CDR3 generated from TRAV13-1*01 and/or TRAJ37*02.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least one amino acid WSGR Docket No. 50401-775.601 encoded by a GATA3 neoORF sequence and at least one amino acid encoded by a GATA3 wild type sequence.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids encoded by a GATA3 neoORF sequence and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids encoded by a GATA3 wild type sequence.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least one amino acid encoded by a GATA3 neoORF sequence and at least one amino acid not encoded by a GATA3 neoORF sequence.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids encoded by a GATA3 neoORF sequence and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids not encoded by a GATA3 neoORF sequence.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein each amino acid of the GATA peptide is an amino acid encoded by a GATA3 neoORF sequence.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the GATA3 peptide comprises a sequence of MLTGPPARV (SEQ ID NO: 15).
  • the human MHC is encoded by an HLA-A02:01, HLA-B07:02 or HLA-B08:01 allele.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the human MHC is encoded by an HLA-A02:01 allele.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the human MHC is encoded by an HLA-B07:02 allele.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the human MHC is encoded by an HLA-B08:01 allele.
  • the TCR binds to an MHC:GATA3 peptide complex, wherein the human MHC is encoded by an HLA-B08:01 allele and wherein the GATA3 peptide comprises a sequence of MLTGPPARV (SEQ ID NO: 15).
  • Delivery of Nucleic Acid or Vector Nucleic acids encoding TCRs or vectors containing such nucleic acids can be delivered to host cells for expression and processing.
  • HIV human immunodeficiency virus: including HIV type 1, and HIV type 2
  • equine infectious anemia virus feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • FMV feline immunodeficiency virus
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and maedi/visna virus. Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian target cells with viral particles containing TCRs transgenes are well known in the art and have been previous WSGR Docket No.
  • retroviral and lentiviral viral vectors and packaging cells for transducing mammalian target cells with viral particles containing TCRs transgenes are well known in the art and have been previous WSGR Docket No.
  • Retroviral and lentiviral vector constructs and expression systems are also commercially available.
  • a viral vector is used to introduce the non-endogenous nucleic acid sequence encoding hematopoietic progenitor cells.
  • the viral vector may be a retroviral vector or a lentiviral vector.
  • the viral vector may also include a nucleic acid sequence encoding a marker for transduction.
  • Transduction markers for viral vectors are known in the art and include selection markers, which may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry.
  • the viral vector genome comprises more than one nucleic acid sequence to be expressed in the host cell as separate transcripts, the viral vector may also comprise additional sequence between the two (or more) transcripts allowing bicistronic or multicistronic expression.
  • viral vectors examples include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide.
  • Other vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., 1998, Gene Ther. 5: 1517-30).
  • HSVs herpes simplex viruses
  • Other vectors include those derived from baculoviruses and alpha-viruses. (Jolly D J.1999. Emerging viral vectors.
  • a vector may include nucleic acid sequences that permit the nucleic acid to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art.
  • a vector preferably is an expression vector that includes a nucleic acid according to the present invention operably linked to sequences allowing for the expression of said nucleic acid.
  • a vector comprising a nucleic acid encoding a TCR disclosed herein.
  • the vector is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion.
  • the vector is a viral vector.
  • the vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, pox virus, alpha virus, vaccina virus, hepatitis B virus, human papillomavirus or a pseudotype thereof.
  • the vector is a non-viral vector.
  • non-viral vector is a nanoparticle, a cationic lipid, a cationic polymer, a metallic nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cell-penetrating peptide, or a liposphere.
  • constructs for example, nucleic acid constructs that encode an alpha chain and a beta chain of a TCR for expression in a cell.
  • the constructs comprise one or more polynucleotides encoding a TCR alpha chain and a TCR beta chain.
  • the polynucleotides are incorporated in a suitable vector.
  • a cell may be transduced or transfected with a nucleic acid encoding a TCR, wherein the cell is capable of expressing the TCR and the cell is used as a therapeutic.
  • the cell is derived from a subject or a host, wherein the subject or the host is a human.
  • the subject or the host comprises a cell having a mutation in an epitope, and the TCR expressed in the cell is capable of binding specifically to the epitope having the mutation.
  • the cell is a lymphocyte cell.
  • the T-lymphocyte In some embodiments, the cell is a lymphocytic precursor cell. In some embodiments, the cell is a T lymphocyte precursor cell. In some embodiments, the cell is a T lymphocyte progenitor cell. In some embodiments the cell is a thymocyte.
  • the T cells are immature T cells. In some embodiments, the T cells are antigen naive T cells.
  • the host cell may be cultured ex vivo for 1, 2, 3, 4, 5 or more days for monitoring and recover after transfectionor transduction with the polynucleotide(s) encoding the TCR.
  • Neoantigens [00197]
  • the TCRs disclosed herein are specific to immunogenic neoantigens.
  • the neoantigen peptide is from RAS.
  • the neoantigen peptide is from GATA3.
  • the neoantigen peptide is from BTK.
  • the neoantigen peptide is from TMPRSS2:ERG.
  • one or more neoantigen peptides are loaded on to APCs, wherein the peptide loaded APCs are then used to stimulate T cells to produce antigen specific T cells.
  • the APCs used for peptide loading are dendritic cells.
  • Immunogenic neoantigen sequences can be identified by any suitable method known in the art.
  • the neoantigen comprises an epitope. In both animals and humans, mutated epitopes can be potentially effective in inducing an immune response or activating T cells.
  • the epitope comprises a mutation. In some embodiments, the mutation is selected WSGR Docket No.
  • the neoantigenic peptide can be at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or less amino acid residues in length.
  • the neoantigenic peptide has a total length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.
  • the neoantigenic peptide has a total length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.
  • the neoantigenic peptide can have a pI value of about 0.5 and about 12, about 2 and about 10, or about 4 and about 8.
  • the neoantigenic peptide can WSGR Docket No. 50401-775.601 have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more.
  • th e neoantigenic peptide can have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.
  • the neoantigenic peptide can have an HLA binding affinity of between about 1pM and about 1mM, about 100pM and about 500 ⁇ M, about 500pM and abou t 10 ⁇ M, about 1nM and about 1 ⁇ M, or about 10nM and about 1 ⁇ M.
  • the neoantigenic peptide can have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 ⁇ M, or more.
  • the neoantigenic peptide can have an HLA binding affinity of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 ⁇ M.
  • a neoantigenic peptide described herein can comprise carriers such as those well known in the art, e.g., thyroglobulin, albumins such as human serum albumin , tetanus toxoid, polyamino acid residues such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.
  • carriers such as those well known in the art, e.g., thyroglobulin, albumins such as human serum albumin , tetanus toxoid, polyamino acid residues such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.
  • a neoantigenic peptide described herein can be modified by terminal- NH2 acylation, e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for linking to a support or other molecule.
  • a neoantigenic peptide described herein can contain modifications such as but not limited to glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of a surface active material, e.g.
  • Half-life of the peptides described herein is conveniently determined using a 25% human serum (v/v) assay.
  • the protocol is as follows: pooled human serum (Type AB, non-heat inactivated) is dilapidated by centrifugation before use. The serum is then diluted to 25% with RPMI-1640 or another suitable tissue culture medium. At predetermined time intervals, a small amount of reaction solution is removed and added to either 6% aqueous trichloroacetic acid (TCA) or ethanol.
  • TCA aqueous trichloroacetic acid
  • a neoantigenic peptide described herein can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or can be isolated from natural sources such as native tumors or pathogenic organisms. Epitopes can be synthesized individually or joined directly or indirectly in a peptide. Although a neoantigenic peptide described herein will be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments, the peptide can be synthetically conjugated to be joined to native fragments or particles.
  • a neoantigenic peptide described herein can be prepared in a wide variety of ways.
  • the peptides can be synthesized in solution or on a solid support according to conventional techniques.
  • Various automatic synthesizers are commercially available and can be used according to known protocols. (See, for example, Stewart & Young, SOLID PHASE WSGR Docket No. 50401-775.601 PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co., 1984).
  • individual peptides can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.
  • compositions can be formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active agents into preparations which can be used pharmaceutically. Proper formulation can be dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients can be used as suitable and as understood in the art.
  • a pharmaceutical composition is formulated as cell based therapeutic, e.g., a T cell therapeutics.
  • a pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and/or a cell-based therapy.
  • a pharmaceutical composition comprises a peptide-based therapeutic, or nucleic acid based therapeutic in which the nucleic acid encodes the polypeptides.
  • a composition can comprise T cells specific for two or more immunogenic antigen or neoantigen peptides.
  • the T cell specific therapeutic may be supplemented by one or more additional therapies.
  • a pharmaceutical composition comprising: a nucleic acid encoding a TCR targeting a neoantigen disclosed herein, a vector containing the nucleic acid, the protein encoded by the nucleic acid, or a host cell comprising the nucleic acid, the protein or the vector; and a pharmaceutically acceptable excipient or diluent.
  • the pharmaceutical composition further comprises an immunomodulatory agent or an adjuvant.
  • the immunomodulatory agent is a cytokine.
  • the adjuvant is poly I:C.
  • Pharmaceutical compositions can include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. WSGR Docket No. 50401-775.601 Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.
  • Acceptable carriers, excipients, or stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • compositions including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration.
  • Pharmaceutical compositions or pharmaceutical formulations therefore refer to a composition suitable for pharmaceutical use in a subject.
  • Compositions can be formulated to be compatible with a particular route of administration (i.e., systemic or local).
  • routes of administration i.e., systemic or local.
  • compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • a pharmaceutical composition can further comprise an acceptable additive in order to improve the stability of the composition.
  • Acceptable additives may not alter the WSGR Docket No. 50401-775.601 specific activity of the active agent, e.g. immune cells.
  • acceptable additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof.
  • Acceptable additives can be combined with acceptable carriers and/or excipients such as dextrose.
  • examples of acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase stability of the peptide and decrease gelling of the solution.
  • the pharmaceutical composition comprises a therapeutic which is a T cell expressing one or more polynucleotides, encoding a T cell receptor.
  • the pharmaceutical composition comprises physiologically acceptable carrier suitable for a cell suspension.
  • the pharmaceutical composition can be administered, for example, by injection.
  • Pharmaceutical compositions for injection include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.
  • Isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride can be included in the composition.
  • the resulting solutions can be packaged for use as is, or lyophilized; the lyophilized preparation can later be combined with a sterile solution prior to administration.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as needed.
  • Sterile injectable solutions can be prepared by incorporating an active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the WSGR Docket No. 50401-775.601 active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation can be vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof .
  • compositions can be conventionally administered intravenously, such as by injection of a unit dose, for example.
  • an active ingredient can be in the form of a parenterally acceptable aqueous solution which is substantially pyrogen-free and has suitable pH, isotonicity and stability.
  • suitable solutions using, for example, isotonic vehicles such stabilizers, buffers, antioxidants and/or other additives can be included, as required.
  • compositions can be administered via aerosolization.
  • the compositions can be substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human patient.
  • Acceptable carriers can contain a compound that stabilizes, increases or delays absorption, or increases or delays clearance.
  • Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buff ers.
  • Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers.
  • the compound can be complexed with a composition to render it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately resistant carrier such as a liposome.
  • Means of protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res.13:17601764; Samanen (1996) J. Pharm. Pharmacol.48:119135; and U.S. Pat. No.5,391,377).
  • the pharmaceutical compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the degree of binding capacity desired. Precise amounts of active ingredient requ ired to be administered WSGR Docket No.
  • the present disclosure is directed to an immunogenic composition, e.g., a pharmaceutical composition capable of raising a neoantigen-specific response (e.g., a humoral or cell-mediated immune response).
  • a neoantigen-specific response e.g., a humoral or cell-mediated immune response
  • the immunogenic composition comprises neoantigen therapeutics (e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.) described herein corresponding to a tumor specific antigen or neoantigen.
  • neoantigen therapeutics e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.
  • a pharmaceutical composition described herein is capable of raising a specific cytotoxic T cells response, specific helper T cell response, or a B cell response.
  • antigen polypeptides or polynucleotides can be provided as antigen presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides.
  • antigen presenting cells are used to stimulate T cells for use in patien ts.
  • the antigen presenting cells are dendritic cells.
  • the dendritic cells are autologous dendritic cells that are pulsed with the neoantigen peptide or nucleic acid.
  • the neoantigen peptide can be any suitable peptide that gives rise to an appropriate T cell response.
  • the T cell is a CTL.
  • the T cell is an HTL.
  • an immunogenic composition containing at least one antigen presenting cell (e.g., a dendritic cell) that is pulsed or loaded with one or more neoantigen polypeptides or polynucleotides described herein.
  • APCs are autologous (e.g., autologous dendritic cells).
  • PBMCs peripheral blood mononuclear cells isolated from a patient can be loaded with neoantigen peptides or polynucleotides ex vivo.
  • such APCs or PBMCs are injected back into the patient.
  • the polynucleotide can be any suitable polynucleotide that is capable of transducing the dendritic cell, thus resulting in the presentation of a neoantigen peptide and induction of immunity.
  • APCs antigen presenting cells
  • PBMCs peripheral blood mononuclear cells
  • T cell e.g., an autologous T cell, or an allogeneic T cell.
  • the T cell is a CTL.
  • the T cell is an HTL.
  • the T cells are CD8 + T cells.
  • the T cells are CD4 + T cells.
  • T cells are then injected into the patient.
  • CTL is injected into the patient.
  • HTL is injected into WSGR Docket No. 50401-775.601 the patient.
  • both CTL and HTL are injected into the patient.
  • Administration of either therapeutic can be performed simultaneously or sequentially and in any order.
  • pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment can be formulated for parenteral, topical, nasal, oral or local administration.
  • the pharmaceutical compositions described herein are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • an increase in humoral immunity can be manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T cell activity can be manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion.
  • An adjuvant can also alter an immune response, for example, by changing a primarily humoral or T helper 2 response in to a primarily cellular, or T helper 1 response.
  • Suitable adjuvants include, but are not limited to poly(I:C), poly-ICLC, STING agonist, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF- - WSGR Dock
  • lymphoid tissues e.g., TNF- -presenting cells for T- lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL- -4, IL-6 and CD40L) (U.S. Pat. No. 5,849,589 incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL- 12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol.1996 (6):414-418).
  • TNF- -presenting cells for T- lymphocytes e.g., GM-CSF, PGE1, PGE2, IL-1, IL- -4, IL-6 and CD40L
  • immunoadjuvants e.g., IL- 12
  • CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a therapeutic setting.
  • CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.
  • TLR Toll-like receptors
  • CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cellular immunogenic pharmaceutical compositions and polysaccharide conjugates in both prophylactic and therapeutic immunogenic pharmaceutical compositions.
  • CTL cytotoxic T-lymphocyte
  • U.S. Pat. No.6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response.
  • a commercially available CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, DE), which is a component of the pharmaceutical composition described herein.
  • TLR binding molecules such as RNA binding TLR7, TLR8 and/or TLR9 can also be u sed.
  • WSGR Docket No. 50401-775.601 Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g.
  • an immunogenic composition according to the present disclosure can comprise more than one different adjuvant.
  • the invention encompasses a pharmaceutical composition comprising any adjuvant substance including any of the above or combinations thereof.
  • the immunogenic composition comprises neoantigen therapeutics (e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.) and the adjuvant can be administered separately in any appropriate sequence.
  • neoantigen therapeutics e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.
  • the adjuvant can be administered separately in any appropriate sequence.
  • Lipidation can be classified into several different types, such as N-myristoylation, palmitoylation, GPI-anchor addition, prenylation, and several additional types of modifications.
  • N- myristoylation is the covalent attachment of myristate, a C14 saturated acid,
  • Palmitoylation is thioester linkage of long-chain fatty acids (C16) to cysteine residues.
  • GPI-anchor addition is glycosyl-phosphatidylinositol (GPI) linkage via amide bond.
  • Prenylation is the thioether linkage of an isoprenoid lipid (e.g. farnesyl (C-15), geranylgeranyl (C-20)) to cysteine residues.
  • Additional types of modifications can include attachment of S-diacylglycerol by a sulfur atom of cysteines, O-octanoyl conjugation via serine or threonine residues, S-archaeol conjugation to cysteine residues, and cholesterol attachment.
  • Fatty acids for generating a lipidated peptides can include C2 to C30 saturated, monounsaturated, or polyunsaturated fatty acyl groups.
  • Exemplary fatty acids can include palmitoyl, myristoyl, stearoyl and decanoyl groups.
  • a lipid moiety that has adjuvant property is attached to a polypeptide of interest to elicit or enhance immunogenicity in the absence of an extrinsic adjuvant.
  • a lipidated peptide or lipopeptide can be referred to as a self -adjuvant lipopeptide. Any of the fatty acids described above and elsewhere herein can elicit or enhance immunogenicity of a polypeptide of interest.
  • the nucleic acid of interest comprises one or more sequences encoding a T cell receptor.
  • Liposomes may be used to deliver a DNA or an RNA. Liposomes may be used to deliver a nucleic acid incorporated in a vector.
  • the nucleic acid may be 50-200,000 nucleotides long, or may be 100-500,000 nucleotides long, or may be 20-500,000 nucleotides long.
  • Exemplary liposomes suitable for incorporation in the formulations include, and are not limited to, multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MVV), single or oligolamellar vesicles made by reverse-phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), ve
  • liposomes can be unilamellar or multilamellar, and can adsorb many types of cells and then release an incorporated agent (e.g., a peptide described herein). In some cases, the liposomes fuse with the target cell, whereby the contents of the liposome then empty into the target cell.
  • a liposome can be endocytosed by cells that are phagocytic. Endocytosis can be followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents.
  • the liposomes provided herein can also comprise carrier lipids. In some embodiments, the carrier lipids are phospholipids.
  • Carrier lipids capable of forming liposomes include, but are not limited to dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS).
  • DPPC dipalmitoylphosphatidylcholine
  • PC phosphatidylcholine
  • lecithin phosphatidic acid
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • Suitable phospholipids further include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidyglycerol (DPPG), distearoylphosphatidyglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidyethanolamine (DPPE), WSGR Docket No.
  • DSPC distearoylphosphatidylcholine
  • DMPC dimyristoylphosphatidylcholine
  • DPPG dipalmitoyl
  • the liposomes further comprise a sterol (e.g., cholesterol) which modulates liposome formation.
  • the carrier lipids can be any known non-phosphate polar lipids.
  • a pharmaceutical composition can be encapsulated within liposomes using well-known technology. Biodegradable microspheres can also be employed as carriers for the pharmaceutical compositions of this invention.
  • the pharmaceutical composition can be administered in liposomes or microspheres (or microparticles).
  • Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. Essentially, material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
  • Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months.
  • Cell-based immunogenic pharmaceutical compositions can also be administered to a subject.
  • an antigen presenting cell (APC) based immunogenic pharmaceutical composition can be formulated using any of the well-known techniques, carriers, and excipients as suitable and as understood in the art.
  • APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells.
  • an APC based immunogenic pharmaceutical composition can be a dendritic cell- based immunogenic pharmaceutical composition.
  • a dendritic cell-based immunogenic pharmaceutical composition can be prepared by any methods well known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared through an ex vivo or in vivo method.
  • the ex vivo method can comprise the use of autologous DCs pulsed ex vivo with the polypeptides described herein, to activate or load the DCs prior to administration into the patient.
  • the in vivo method can comprise targeting specific DC receptors using antibodies coupled with the polypeptides described herein.
  • the DC-based immunogenic pharmaceutical composition can further comprise DC activators such as TLR3, TLR-7- 8, and CD40 agonists.
  • the DC-based immunogenic pharmaceutical composition can further comprise adjuvants, and a pharmaceutically acceptable carrier.
  • An adjuvant can be used to enhance the immune response (humoral and/or cellular) elicited in a patient receiving the immunogenic pharmaceutical composition. Sometimes, adjuvants can elicit WSGR Docket No.
  • Th1-type response can be characterized by the production of cytokines such as IFN- -type response which can be characterized by the production of cytokines such as IL-4, IL-5 and IL-10.
  • lipid-based adjuvants such as MPLA and MDP, can be used with the immunogenic pharmaceutical compositions disclosed herein.
  • MPLA Monophosphoryl lipid A
  • MPLA is an adjuvant that causes increased presentation of liposomal antigen to specific T Lymphocytes.
  • Adjuvant can also comprise stimulatory molecules such as cytokines.
  • cytokines in - - - -CSF, epidermal growth factor (EGF), cutaneous T cell- attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-la, MIP-1-, IL-8, L- selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50
  • Additional adjuvants include: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, -1,
  • an adjuvant can be a modulator of a toll like recepto r.
  • modulators of toll-like receptors include TLR9 agonists and are not limited to small molecule modulators of toll-like receptors such as Imiquimod.
  • an adjuvant is selected from bacteria toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or a combination thereof.
  • an adjuvant is an oil-in-water emulsion.
  • an immunogenic pharmaceutical composition can include carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, and other acceptable additives, adjuvants, or binders, other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like.
  • carriers and excipients including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents
  • excipients examples include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the pharmaceutical preparation is substantially free of preservatives.
  • the pharmaceutical preparation can contain at least one preservative. It will be recognized that, while any suitable carrier known to those of ordinary skill in the art can be employed to administer the pharmaceutical compositions described herein, the type of carrier will vary depending on the mode of administration.
  • An immunogenic pharmaceutical composition can include preservatives such as thiomersal or 2-phenoxyethanol.
  • the immunogenic pharmaceutical composition is substantially - -Tocopherol succinate may be used as an alternative to mercurial compounds.
  • a physiological salt such as sodium salt can be included in the immunogenic pharmaceutical composition.
  • Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, or the like.
  • An immunogenic pharmaceutical composition can have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, between 240-360 mOsm/kg, or within the range of 290-310 mOsm/kg.
  • An immunogenic pharmaceutical composition can comprise one or more buffers, such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included in the 5 -20 or 10-50 mM range.
  • the pH of the immunogenic pharmaceutical composition can be between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8.
  • An immunogenic pharmaceutical composition can be sterile.
  • the immunogenic pharmaceutical composition can be non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and can be ⁇ 0.1 EU per dose.
  • the composition can be gluten free.
  • An immunogenic pharmaceutical composition can include detergent e.g. a polyoxyethylene -9 (Triton X-100) or t-octylphenoxypolyethoxyethanol). The detergent can be present only at trace amounts.
  • the immunogenic pharmaceutical composition can include less than 1 mg/mL of each of octoxynol-10 and polysorbate 80. Other residual components in trace amounts can be an tibiotics (e.g. neomycin, kanamycin, polymyxin B).
  • An immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension, in suitable vehicles, well known in the art. The pharmaceutical compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions comprising, for example, an active agent such as immune cells disclosed herein, in combination with one or more adjuvants can be formulated to comprise certain molar ratios.
  • an active agent such as immune cells disclosed herein
  • one or more adjuvants can be formulated to comprise certain molar ratios.
  • molar ratios of about 99:1 to about 1:99 of an active a gent such as an immune cell described herein, in combination with one or more adjuvants can be used.
  • the range of molar ratios of an active agent such as an immune cell described herein, in combination with one or more adjuvants can be selected from about 80:20 to about 20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90.
  • the molar ratio of an active agent such as an immune cell described herein, in combination with one or more adjuvants can be about 1:9, and in some cases can be about 1:1.
  • an immunogenic pharmaceutical composition can be administered with an additional agent.
  • the choice of the additional agent can depend, at least in part, on the condition being treated.
  • the additional agent can include, for example, a checkpoint inhibitor agent such as an anti- PD1, anti-CTLA4, anti-PD-L1, anti CD40, or anti-TIM3 agent (e.g., an anti-PD1, anti-CTLA4, anti- PD-L1, anti CD40, or anti-TIM3 antibody); or any agents having a therapeutic effect for a pathogen infection (e.g. viral infection), including, e.g., drugs used to treat inflammatory conditions such as an NSAID, e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin.
  • an NSAID e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin.
  • the checkpoint inhibitor can be a PD-1/PD- L1 antagonist selected from the group consisting of: nivolumab (ONO- 4538/BMS-936558, MDX1106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA (ROCHE).
  • formulations can additionally contain one or more supplements, such as vitamin C, E or other anti-oxidants.
  • a pharmaceutical composition comprising an active agent such as an immune cell described herein, in combination with one or more adjuvants can be formulated in conventional manner using one or more physiologically acceptable carriers, comprising excipients, diluents, and/or auxiliaries, e.g., which facilitate processing of the active agents into preparations that can be administered. Proper formulation can depend at least in part upon the route of administration chosen.
  • the agent(s) described herein can be delivered to a patient using a number of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular applications, as well as by inhalation.
  • the vehicle can be chosen from those known in art to be suitable, including aqueous solutions or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.
  • the formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly(lactic-co-glycolic)acid. These materials can be made into WSGR Docket No. 50401-775.601 micro or nanospheres, loaded with drug and further coated or derivatized to provide superior sustained release performance.
  • Vehicles suitable for periocular or intraocular injection include, for example, suspensions of therapeutic agent in injection grade water, liposomes and vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are well known in the art.
  • pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the pharmaceutical composition does not comprise an adjuvant or any other substance added to enhance the immune response.
  • the active agents can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection or use of a transdermal patch.
  • the agents can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the TCRs, recombinant nucleic acids encoding the TCRs, or cells comprising the TCRs or recombinant nucleic acids encoding the TCRs described herein can be administered (concurrently, prior to, or subsequent to) with a cancer vaccine into a subject in need thereof.
  • the cancer vaccine can comprise a GATA3 polypeptide or a nucleic acid encoding the GATA3 polypeptide.
  • the GATA3 peptide can comprise a full-length GATA3 protein.
  • the GATA3 polypeptide can comprise one or more epitopes described herein from GATA3 protein.
  • the present invention is directed to a vaccine formulation comprising the GATA3 polypeptide, e.g., a pharmaceutical composition capable of raising a neoantigen-specific response (e.g., a humoral or cell-mediated immune response).
  • the vaccine formulation comprises a GATA3 polypeptide or recombinant nucleic acid encoding a GATA3 polypeptide.
  • the vaccine formulation comprises an immune cell comprising the recombinant nucleic acid encoding the GATA3 polypeptide.
  • a vaccine formulation described herein is capable of raising a specific cytotoxic T cells response, specific helper T cell response, or a B cell response.
  • antigen polypeptides or polynucleotides can be provided as antigen presenting cells (e.g., dendritic cells) containing, for example, a GATA3 polypeptide or recombinant nucleic acid encoding a GATA3 polypeptide.
  • antigen presenting cells e.g., dendritic cells
  • such antigen presenting cells are used to stimulate T cells for use in patients.
  • the antigen presenting cells are dendritic cells.
  • the dendritic cells are autologous dendritic cells that are pulsed with the neoantigen peptide or nucleic acid.
  • the neoantigen peptide can be any suitable peptide that gives rise to an appropriate T cell response.
  • the T cell is a CTL.
  • the T WSGR Docket No. 50401-775.601 cell is an HTL.
  • one embodiment of the present disclosure is a vaccine formulation containing at least one antigen presenting cell (e.g., a dendritic cell) that is pulsed or loaded with one or more neoantigen polypeptides or polynucleotides described herein.
  • APCs are autologous (e.g., autologous dendritic cells).
  • PBMCs peripheral blood mononuclear c ells isolated from a patient can be loaded with neoantigen peptides or polynucleotides ex vivo.
  • such APCs or PBMCs are injected back into the patient.
  • the polynucleotide can be any suitable polynucleotide that is capable of transducing the dendritic cell, thus resulting in the presentation of a neoantigen peptide and induction of immunity.
  • such antigen presenting cells e.g., dendritic cells
  • PBMCs peripheral blood mononuclear cells
  • APCs antigen presenting cells
  • PBMCs peripheral blood mononuclear cells
  • T cell e.g., an autologous T cell
  • the T cell is a CTL.
  • the T cell is an HTL.
  • the T cells are CD8 + T cells.
  • One exemplary GATA3 polypeptide may comprise a sequence encoded by a neoORF, for example, PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGP PARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH or a fragment thereof; or a polynucleic acid encoding the same.
  • a neoORF for example, PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGP PARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH or a fragment thereof; or a polynucleic acid encoding the same.
  • an exemplary GATA3 polypeptide may comprise a sequence encoded by a neoORF, for example PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHEA or a fragment thereof; or a polynucleic acid encoding the same.
  • an exemplary GATA3 polypeptide may comprise a sequence encoded by a neoORF, for example AQAKAVCSQESRDVLCELSDHHNHTLEEECQWGPCLQCLWALLQASQY or a fragment thereof; or a polynucleic acid encoding the same.
  • the GATA3 polypeptide or recombinant nucleic acid may comprise one or more neoepitopes selected from the GATA3 polypeptide comprising a sequence encoded by a GATA3 neoORF.
  • the vaccine may comprise one or more neoepitopes provided herein, but not limited to: HVLPEPHLAL (B07.02), RPLQTHVLPE (B07.02), VLWTTPPLQH (A03.01), APSESPCSPF (B07.02), CPLDHTTPPA (B07.02), FLQEQYHEA (A02.01, B08.01), RLAFLQEQYH (A03.01), SPCSPFRLAF (B07.02), SPPWVRALL (B07.02), YPACPLDHTT (B07.02), AIQPVLWTT (A02.01), ALQPLQPHA WSGR Docket No.
  • a vaccine formulation described herein for therapeutic treatment can be formulated for parenteral, topical, nasal, oral or local administration.
  • the vaccine formulation described herein are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • the vaccine formulation can be administered intratumorally.
  • compositions for parenteral administration which comprise a solution of the neoantigen peptides and vaccine formulations are dissolved or suspended in an acceptable carrier, for example, an aqueous carrier.
  • an aqueous carrier e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.
  • compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • an increase in humoral immunity can be manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T cell activity can be manifested in increased WSGR Docket No. 50401-775.601 cell proliferation, or cellular cytotoxicity, or cytokine secretion.
  • An adjuvant can also alter an immune response, for example, by changing a primarily humoral or T helper 2 response into a primarily cellular, or T helper 1 response.
  • Suitable adjuvants include, but are not limited to poly(I:C), poly-ICLC, STING agonist, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel ® vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF- - stimulon (
  • cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF- lerating the maturation of dendritic cells into efficient antigen-presenting cells for T- lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL- -4, IL-6 and CD40L) (U.S. Pat. No. 5,849,589 incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL- 12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol.1996 (6):414-418).
  • TNF- lerating the maturation of dendritic cells into efficient antigen-presenting cells for T- lymphocytes e.g., GM-CSF, PGE1, PGE2, IL-1, IL- -4, IL-6 and CD40L
  • immunoadjuvants e.g.,
  • CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a therapeutic setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccine formulations, autologous cellular vaccine formulations and polysaccharide conjugates in both prophylactic and therapeutic vaccine formulations.
  • TLR Toll-like receptors
  • CTL cytotoxic T-lymphocyte
  • U.S. Pat. No.6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response.
  • a commercially available CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, DE), which is a component of the pharmaceutical composition described herein.
  • Other TLR binding molecules such as RNA binding TLR7, TLR8 and/or TLR9 can also be used.
  • CpGs e.g. CpR, Idera
  • non-CpG bacterial DNA or RNA e.g., ssRNA40 for TLR8, as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP- 547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and/or as an adjuvant.
  • CpGs e.g. CpR, Idera
  • a vaccine formulation according to the present disclosure can comprise more than one different adjuvant.
  • the invention encompasses a pharmaceutical composition comprising any adjuvant substance including any of the above or combinations thereof.
  • the vaccine formulation comprises a GATA3 polypeptide or recombinant nucleic acid encoding a GATA3 polypeptide.
  • the vaccine formulation comprises an immune cell comprising the recombinant nucleic acid encoding the GATA3 polypeptide.
  • the adjuvant can be administered separately in any appropriate sequence.
  • Lipidation can be classified into several different types, such as N-myristoylation, palmitoylation, GPI-anchor addition, prenylation, and several additional types of modifications.
  • N- myristoylation is the covalent attachment of myristate, a C14 saturated acid, to a glycine residue. Palmitoylation is thioester linkage of long-chain fatty acids (C16) to cysteine residues.
  • GPI-anchor addition is glycosyl-phosphatidylinositol (GPI) linkage via amide bond.
  • Prenylation is the thioether linkage of an isoprenoid lipid (e.g., farnesyl (C-15), geranylgeranyl (C-20)) to cysteine residues.
  • Additional types of modifications can include attachment of S-diacylglycerol by a sulfur atom of WSGR Docket No. 50401-775.601 cysteines, O-octanoyl conjugation via serine or threonine residues, S-archaeol conjugation to cysteine residues, and cholesterol attachment.
  • Fatty acids for generating lipidated peptides can include C2 to C30 saturated, monounsaturated, or polyunsaturated fatty acyl groups.
  • Exemplary fatty acids can included e palmitoyl, myristoyl, stearoyl and decanoyl groups.
  • a lipid moiety that has adjuvant property is attached to a polypeptide of interest to elicit or enhance immunogenicity in the absence of an extrinsic adjuvant.
  • a lipidated peptide or lipopeptide can be referred to as a self-adjuvant lipopeptide. Any of the fatty acids described above and elsewhere herein can elicit or enhance immunogenicity of a polypeptide of interest.
  • a fatty acid that can elicit or enhance immunogenicity can include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl groups.
  • Polypeptides such as naked peptides or lipidated peptides can be incorporated into a liposome. Sometimes, lipidated peptides can be incorporated into a liposome. For example, the lip id portion of the lipidated peptide can spontaneously integrate into the lipid bilayer of a liposome.
  • a incorporation in the formulations include, and are not limited to, multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium- sized unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MVV), single or oligolamellar vesicles made by reverse-phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), vesicles prepared
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the DEC205 antigen, or with other therapeutic or vaccine formulations.
  • a desired peptide or polynucleotide described herein can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic polypeptide/polynucleotide compositions.
  • Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged WSGR Docket No.
  • the GATA3 polypeptide and recombinant nucleic acids are target to dendritic cells using the markers DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, TSLP receptor, or CD1a.
  • conventional or nanoparticle nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10 -95% of active ingredient, that is, one or more GATA3 polypeptides and recombinant nucleic acids described herein at a concentration of 25%-75%.
  • the GATA3 polypeptide and recombinant nucleic acids can be supplied in finely divided form along with a surfactant and propellant.
  • the recombinant nucleic acids encoding the GATA3 polypeptide may be part of a synthetic lipid nanoparticle formulation.
  • the recombinant nucleic acid is DNA.
  • the recombinant nucleic acid is mRNA.
  • the recombinant nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids.
  • nucleic acids can be encapsulated in lipid nanoparticles (e.g., comprising cationic lipid, non-cationic lipids (e.g., phospholipids and/or sterol), and/or PEG-lipids).
  • liposomes can be unilamellar or multilamellar, and can adsorb many types of cells and then release an incorporated agent (e.g., a peptide described herein).
  • the liposomes fuse with the target cell, whereby the contents of the liposome then empty into the target cell.
  • a liposome can be endocytosed by cells that are phagocytic. Endocytosis can be followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents.
  • the liposomes provided herein can also comprise carrier lipids.
  • the carrier lipids are phospholipids.
  • Carrier lipids capable of forming liposomes include, but are not limited to dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS).
  • DPPC dipalmitoylphosphatidylcholine
  • PC phosphatidylcholine
  • lecithin phosphatidic acid
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • Suitable phospholipids further include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidyglycerol (DPPG), distearoylphosphatidyglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidyethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE) and the like, or combinations thereof.
  • DSPC distearoylphosphatid
  • An adjuvant can be used to enhance the immune response (humoral and/or cellular) elicited in a patient receiving the vaccine formulation. Sometimes, adjuvants can elicit a Th1-type response. Other times, adjuvants can elicit a Th2-type response.
  • a Th1-type response can be characterized by the production of cytokines such as IFN- -type response which can be characterized by the production of cytokines such as IL-4, IL-5 and IL-10.
  • lipid-based adjuvants such as MPLA and MDP, can be used with the vaccine formulations disclosed herein.
  • IL-7 nerve growth factor
  • vascular endothelial growth factor Fas
  • TNF receptor Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap- ctive NIK, SAP K, SAP- TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP2.
  • an adjuvant can be a modulator of a toll like receptor.
  • modulators of toll-like receptors include TLR9 agonists and are not limited to small molecule modulators of toll-like receptors such as Imiquimod.
  • an adjuvant is selected from bacteria toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or a combination thereof.
  • an adjuvant is an oil-in-water emulsion.
  • 50401-775.601 approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like.
  • excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the pharmaceutical preparation is substantially free of preservatives.
  • the pharmaceutical preparation can contain at least one preservative.
  • a vaccine formulation can include preservatives such as thiomersal or 2-phenoxyethanol. In e.g., thiomersal- -Tocopherol succinate may be used as an alternative to mercurial compounds.
  • a physiological salt such as sodium salt can be included in the vaccine formulation.
  • Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, or the like.
  • the pH of the vaccine formulation can be between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8.
  • a vaccine formulation can be sterile.
  • the vaccine formulation can be non-pyrogenic e.g., containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and can be ⁇ 0.1 EU per dose.
  • the composition can be gluten free.
  • a vaccine formulation can include detergent e.g., a polyoxyethylene sorbitan ester surfactant -9 (Triton X-100) or t- octylphenoxypolyethoxyethanol). The detergent can be present only at trace amounts.
  • the particles can be frozen and thawed without formation of aggregates, while maintaining the original particle size profile, and maintaining the biological activity.
  • the particles can be lyophilized and reconstituted with water without formation of aggregates, while WSGR Docket No. 50401-775.601 maintaining the original particle size profile and maintaining the biological activity.
  • the particles can be manufactured by different protocols which are scalable and which can be performed under controlled conditions. With such properties the lipoplex formulations of the present disclosure can fulfill important requirements for pharmaceutical formulations for application to patients, in terms of particle size distribution profile and stability.
  • the RNA nanoparticles described herein can be less toxic and to display less undesired serum interactions.
  • the formulations can be suitable for parenteral administration, including intravenous and subcutaneous administration.
  • the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, in some cases 8:2 to 3:7, and in some cases of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, in some cases 1.6:2 to 1:2, in some cases 1.4:2 to 1.1:2 and in some cases about 1.2:2.
  • the nanoparticles are lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 10:0 to 1 :9, in some cases 8:2 to 3:7, and in some cases of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, in some cases 1.6:2 to 1:2, in some cases 1.4:2 to 1.1:2 and in some cases about 1.2:2.
  • the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, in some cases 8:2 to 3:7, and in some cases of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, in some cases 1.6:2 to 1:2, in some cases 1.4:2 to 1.1:2 and in some cases about 1.2:2.
  • the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 2:1 to 1:2, in some cases 2:1 to 1:1, and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4:1 or less.
  • the nanoparticles are lipoplexes comprising DOTMA and cholesterol in a molar ratio of 2:1 to 1:2, in some cases 2:1 to 1:1, and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4:1 or less.
  • the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 2:1 to 1:2, in some cases 2: to 1:1, and wherein the charge ratio of positive charges in DOTAP to negative charges in the RNA is 1.4:1 or less.
  • the nanoparticles have an average diameter in the range of from about 50 nm to about 1000 nm, in some cases from about 50 nm to about 400 nm, in some cases about 100 nm to about 300 nm such as about 150 nm to about 200 nm. In some embodiments, the nanoparticles have a diameter in the range of about 200 to about 400 nm.
  • the polydispersity index of the nanoparticles described herein as measured by dynamic light scattering is 0.5 or less, in some cases 0.4 or less or in some cases 0.3 or less.
  • the nanoparticles described herein are obtainable by one or more of the following: (i) incubation of liposomes in an aqueous phase with the RNA in an aqueous phase, (ii) incubation of the lipid dissolved in an organic, water miscible solvent, such as ethanol, with the RNA in aqueous solution, (iii) reverse phase evaporation technique, (iv) freezing and thawing of the product, (v) dehydration and rehydration of the product, (vi) lyophilization and rehydration of the of the product, or (vii) spray drying and rehydration of the product.
  • the lipid solutions, liposomes and RNA lipoplex particles described herein include a cationic lipid.
  • a "cationic lipid” refers to a lipid having a net positive charge. Cationic lipids bind negatively charged RNA by electrostatic interaction to the lipid matrix. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and the head group of the lipid typically carries the positive charge.
  • cationic lipids include, but are not limited to l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3 -trimethylammonium propane (DOTAP); l,2- dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammonium propanes; l,2-dialkyloxy-3- dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl)-dimethylazanium (DMRIE), 1 ,2-dimyristoyl-sn- glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethyl
  • the at least one cationic lipid is DOTMA and/or DOTAP. In some embodiments, the at least one cationic lipid is DOTMA, in particular (R)-DOTMA.
  • An additional lipid may be incorporated to adjust the overall positive to negative charge ratio and physical stability of the RNA lipoplex particles. In certain embodiments, the additional lipid is a neutral lipid. As used herein, a "neutral lipid" refers to a lipid having a net charge of zero.
  • neutral lipids include, but are not limited to, l,2-di-(9Z-octadecenoyl)-sn- glycero-3- phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), diacylphosphatidyl choline, diacylphosphatidyl ethanol amine, ceramide, sphingoemyelin, cephalin, cholesterol, and cerebroside.
  • the second lipid is DOPE, cholesterol and/or DOPC.
  • the RNA lipoplex particles include both a cationic lipid and an additional lipid.
  • the cationic lipid is DOTMA and the additional lipid is DOPE.
  • the amount of the at least one cationic lipid compared to the WSGR Docket No. 50401-775.601 amount of the at least one additional lipid may affect important RNA lipoplex particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1 :9, about 4:1 to about 1 :2, o r about 3 : 1 to about 1 : 1.
  • the stabilizer is at a concentration from about 5% (w/v) to about 35% (w/v), or from about 10% (w/v) to about 25% (w/v). In specific embodiments, the stabilizer is at a concentration of about 10% (w/v), about 11% (w/v), about 12% (w/v), about 13% (w/v), about 14% (w/v), about 15% (w/v), about 16% (w/v), about 17% (w/v), about 18% (w/v), about 19% (w/v), about 20% (w/v), about 21% (w/v), about 22% (w/v), about 23% (w/v), about 24% (w/v), or about 25% (w/v).
  • the heavy chain diseases such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous sys tem cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, WSGR Docket No.
  • the heavy chain diseases such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung
  • the cancer whose phenotype is determined by the method of the present disclosure is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma.
  • the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, brenner, or undifferentiated.
  • the present disclosure is used in the treatment, diagnosis, and/or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.
  • the subject has a breast cancer that is resistant to anti-estrogen therapy, is an MSI breast cancer, is a metastatic breast cancer, is a Her2 negative breast cancer, is a Her2 WSGR Docket No.
  • At least one or more chemotherapeutic agents may be administered in addition to the pharmaceutical composition comprising an immunogenic therapy.
  • the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents.
  • therapeutically -effective amounts of the pharmaceutical compositions can be administered to a subject having a disease or condition.
  • a therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.
  • Subjects can be, for example, mammal, humans, pregnant women, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, newborn, or neonates.
  • a subject can be a patient.
  • a subject can be a human.
  • a subjec t can be a child (i.e. a young human being below the age of puberty).
  • a subject can be an infant.
  • the subject can be a formula-fed infant.
  • a subject can be an individual enrolled in a clinical study.
  • a subject can be a laboratory animal, for example, a mammal, or a rodent.
  • the subject can be a mouse. In some cases, the subject can be an obese or overweight subject. [00365] In some embodiments, the subject has previously been treated with one or more different cancer treatment modalities. In some embodiments, the subject has previously been treated with one or more of radiotherapy, chemotherapy, or immunotherapy. In some embodiments, the subject has been treated with one, two, three, four, or five lines of prior therapy. In some embodiments, the prior therapy is a cytotoxic therapy. [00366] In some embodiments, the disease or condition that can be treated with the methods disclosed herein is abnormal growth of cells. In some embodiments, the disease or condition that can be treated with the methods disclosed herein is cancer. In some embodiments, the cancer is a malignant cancer.
  • the cancer is a benign cancer. In some embodiments, the cancer is an invasive cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid cancer. [00367] The methods of the disclosure can be used to treat any type of cancer known in the art.
  • a cancer to be treated by the methods of the present disclosure include, for example, carcinoma, squamous carcinoma (for example, cervical canal, eyelid, tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder, tongue, larynx, and gullet), and adenocarcinoma (for example, prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, gullet, rectum, uterus, stomach, mammary gland and ovary).
  • carcinoma for example, cervical canal, eyelid, tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder, tongue, larynx, and gullet
  • adenocarcinoma for example, prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, gullet, rectum, uterus, stomach, mammary gland and ovary.
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.
  • Example 1 Workflow for antigen-specific T cell identification and validation
  • mutant target epitopes e.g., mutant GATA3 peptides
  • the synthesized peptides were used to load APCs to stimulate T cells from a sample of healthy donor peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • PBMCs from patients harboring neoantigens of interest can also be used to obtain neoantigen-specific T cells.
  • T cell populations were analyzed by flow cytometry. Antigen-specific T cells were isolated using flow cytometry.
  • TCRs were assessed for percentage of CD69+ cells across increasing concentrations of peptide.
  • TCR-2 and TCR-5 showed the greatest percentage of CD69+ cells compared to other TCR constructs (FIG. 2).
  • TCR-2 and TCR-5 also showed superior EC50 values compared to those of the other TCR constructs (FIG.3).
  • TCR-2 had a EC50 of 8.8 and TCR-5 had a EC50 of 2.3.
  • FIGs.4A-4B show TCRs were able to recognize target antigen bound to the MHC encoded by HLA A*02:01 allele.
  • Example 2 Summary of recombinant TCRs developed [00374] This example provides the details of the TCRs developed.
  • the TCRs are generated using a mouse T cell receptor beta constant region (TRBC1), having the following sequence for the mouse TRBC1 region: X 1 DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVX 2 TDP QAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEA WGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS; (SEQ ID NO: 150); wherein, X 1 is K, E, or absent; X 2 is S or C.
  • TRBC1 mouse T cell receptor beta constant region
  • a TCR thus generated comprises a sequence of a mouse T cell receptor beta constant region (TRBC1), having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the mouse TRBC1 sequence: X 1 DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVX 2 TDP QAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEA WSGR Docket No.
  • TRBC1 mouse T cell receptor beta constant region
  • a TCR thus generated comprises a sequence of a mouse T cell receptor beta constant region (TRBC2), having at least 80% sequence identity to the mouse TRBC2 sequence: XDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQ AYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW GRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS; (SEQ ID NO: 151); wherein, X is K, E, or absent.
  • TRBC2 mouse T cell receptor beta constant region
  • the TCRs are generated using a human TCR alpha chain constant region (TRAC), having the following sequence for the human TRAC sequence: XIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS; (SEQ ID NO: 152); wherein, X is D, N, or absent.
  • TRAC human TCR alpha chain constant region
  • a TCR thus generated comprises a sequence of a human TCR alpha chain constant region (TRAC), having at least 80% sequence identity to the human TRAC sequence: XIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS; (SEQ ID NO: 152); wherein, X is D, N, or absent.
  • a TCR thus generated comprises a sequence of a human TCR alpha chain constant region (TRAC), having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the human TRAC sequence: XIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS; (SEQ ID NO: 152); wherein, X is D, N, or absent.
  • TRAC alpha chain constant region
  • the TCRs are generated using a human TCR beta chain constant region (TRBC1), having the following sequence for the human TRBC1 sequence: XDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF; (SEQ ID NO: 153); wherein, X is K, E, or absent.
  • TRBC1 human TCR beta chain constant region
  • a TCR thus generated comprises a sequence of a human TCR beta chain constant region (TRBC1), having at least 80% sequence identity to the human TRBC1 sequence: XDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF; (SEQ ID NO: 153); wherein, X is K, E, or absent.
  • TRBC1 human TCR beta chain constant region
  • a TCR thus generated comprises a sequence of a human TCR beta chain constant region (TRBC1), having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the human TRBC1 sequence: XDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF; (SEQ ID NO: 153); wherein, X is K, E, or absent.
  • TRBC1 human TCR beta chain constant region
  • the TCRs are generated using a human TCR beta chain constant region (TRBC2), having the following sequence for the human TRBC2 sequence: XDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG; (SEQ ID NO: 154); wherein, X is K, E, or absent.
  • TRBC2 human TCR beta chain constant region
  • a TCR thus generated comprises a sequence of a human TCR beta chain constant region (TRBC2), having at least 80% sequence identity to the human TRBC2 sequence: WSGR Docket No. 50401-775.601 XDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG; (SEQ ID NO: 154); wherein, X is K, E, or absent.
  • TRBC2 human TCR beta chain constant region
  • the GATA3 TCR comprises TCR-1 beta chain full length sequence, including a human TCR beta Constant domain (TRBC2) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 122.
  • TRBC2 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-1 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC1) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 123.
  • TRBC1 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-1 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC2) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 124.
  • TRBC2 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-2 alpha chain full length sequence including a human TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 28.
  • TCR human TCR alpha Constant domain
  • the GATA3 TCR comprises TCR-2 beta chain full length sequence, including a human TCR beta Constant domain (TRBC1) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 29.
  • TRBC1 Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 29.
  • the GATA3 TCR comprises TCR-2 beta chain full length sequence, including a human TCR beta Constant domain (TRBC2) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 126.
  • TRBC2 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-2 alpha chain full length sequence including a mouse TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 125.
  • TCR alpha Constant domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 125.
  • the GATA3 TCR comprises TCR-2 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC2) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 128.
  • TRBC2 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-3 beta chain full length sequence, including a human TCR beta Constant domain (TRBC1) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 44.
  • TRBC1 Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 44.
  • the GATA3 TCR comprises TCR-3 beta chain full length sequence, including a human TCR beta Constant domain (TRBC2) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 130.
  • TRBC2 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-3 alpha chain full length sequence including a mouse TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 129.
  • TCR alpha Constant domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 129.
  • the GATA3 TCR comprises TCR-3 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC1) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 131.
  • TRBC1 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-3 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC2) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 132.
  • TRBC2 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-4 alpha chain full length sequence including a human TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 58.
  • TCR human TCR alpha Constant domain
  • the GATA3 TCR comprises TCR-4 beta chain full length sequence, including a human TCR beta Constant domain (TRBC1) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 59.
  • TRBC1 Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 59.
  • the GATA3 TCR comprises TCR-4 beta chain full length sequence, including a human TCR beta Constant domain (TRBC2) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 134.
  • TRBC2 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-4 alpha chain full length sequence including a mouse TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 133.
  • TCR alpha Constant domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 133.
  • the GATA3 TCR comprises TCR-4 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC1) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 135.
  • TRBC1 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-4 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC2) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 136.
  • TRBC2 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-5 alpha chain full length sequence including a human TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 73.
  • TCR human TCR alpha Constant domain
  • the GATA3 TCR comprises TCR-5 beta chain full length sequence, including a human TCR beta Constant domain (TRBC1) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 74.
  • TRBC1 Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 74.
  • the GATA3 TCR comprises TCR-5 beta chain full length sequence, including a human TCR beta Constant domain (TRBC2) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 138.
  • TRBC2 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-5 alpha chain full length sequence including a mouse TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 137.
  • TCR alpha Constant domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 137.
  • the GATA3 TCR comprises TCR-5 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC1) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 139.
  • TRBC1 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-5 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC2) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 140.
  • TRBC2 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-6 alpha chain full length sequence including a human TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 88.
  • TCR human TCR alpha Constant domain
  • the GATA3 TCR comprises TCR-6 beta chain full length sequence, including a human TCR beta Constant domain (TRBC1) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 89.
  • TRBC1 Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 89.
  • the GATA3 TCR comprises TCR-6 beta chain full length sequence, including a human TCR beta Constant domain (TRBC2) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 142.
  • TRBC2 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-6 alpha chain full length sequence including a mouse TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 141.
  • TCR alpha Constant domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 141.
  • the GATA3 TCR comprises TCR-6 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC1) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 143.
  • TRBC1 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-6 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC2) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 144.
  • TRBC2 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-7 alpha chain full length sequence including a human TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 103.
  • TCR alpha Constant domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 103.
  • the GATA3 TCR comprises TCR-7 beta chain full length sequence, including a human TCR beta Constant domain (TRBC1) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 104.
  • TRBC1 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-7 beta chain full length sequence, including a human TCR beta Constant domain (TRBC2) Constant Domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 146.
  • TRBC2 human TCR beta Constant domain
  • the GATA3 TCR comprises TCR-7 alpha chain full length sequence including a mouse TCR alpha Constant domain (TRAC) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 145.
  • TCR alpha Constant domain comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 145.
  • the GATA3 TCR comprises TCR-7 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC1) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 147.
  • TRBC1 mouse TCR beta Constant domain
  • the GATA3 TCR comprises TCR-7 alpha chain full length sequence including a mouse TCR beta Constant domain (TRBC2) comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 148.
  • TRBC2 mouse TCR beta Constant domain
  • GATA3 binding TCR sequences specific to epitope MLTGPPARV (9mer) (SEQ ID NO: 15) in complex with HLA-A*02:01, or to SMLTGPPARV (10mer) (SEQ ID NO: 200) in complex with HLA-A*02:01.
  • GATA3 binding TCR sequences wherein the TCRs bind to a complex comprising (i) an epitope having a sequence, MLTGPPARV (9mer) (SEQ ID NO: 15); or a sequence SMLTGPPARV (10mer) (SEQ ID NO: 200); and (ii) an MHC protein, encoded by HLA-A*02:01.
  • TCR sequences or parts thereof comprising any one of the TCR alpha chain variable regions (TRAV), beta chain variable regions (TRBV), and corresponding constant domains (TRAC, TRBC1, TRBC2 etc) depicted in Table 4A, or sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the TCR alpha chain variable domains, regions, beta chain variable regions, corresponding constant domains depicted in Table 4A.
  • Complete TCR sequences were constructed as shown in Table 4B, each row corresponding to the components to construct a full TCR.
  • Each TCR sequence is specific for a complex comprising WSGR Docket No.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA-A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCTGCAGGCTCCTCTGCTGTGTGGTCTTCTGCCTCCTCCAAGCAGGTCCCTTGGAC ACAGCTGTTTCCCAGACTCCAAAATACCTGGTCACACAGATGGGAAACGACAAGTCCAT TAAATGTGAACAAAATCTGGGCCATGATACTATGTATTGG
  • TCR A2-1 Table 4A
  • the TCR A2-1 (Table 4A) is further described in Table 4B, comprising a TCR beta chain WSGR Docket No. 50401-775.601 with a CDR3 sequence, CASSQGVGESPEAFF (SEQ ID NO: 206); joined with the TCR beta V chain of TRBV3-1; the J chain TRBJ1-1; and the constant domain TRBC1; and a TCR alpha chain CDR3 sequence CVVSDLSGGGYQKVTF (SEQ ID NO: 203) joined with V chain sequence of TRAV8-2, J chain sequence of TRAJ13 and TRAC constant domain.
  • Table 4B Table 4B, comprising a TCR beta chain WSGR Docket No. 50401-775.601 with a CDR3 sequence, CASSQGVGESPEAFF (SEQ ID NO: 206); joined with the TCR beta V chain of TRBV3-1; the J chain TRBJ1-1; and the constant domain TRBC1; and a TCR alpha chain CDR3 sequence CVVSDL
  • TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCTGCAGGCTCCTCTGCTGTGTGGTCTTCTGCCTCCTCCAAGCAGGTCCCTTGGAC ACAGCTGTTTCCCAGACTCCAAAATACCTGGTCACACAGATGGGAAACGACAAGTCCAT TAAATGTGAACAAAATCTGGGCCATGATACTATGTATTGGT
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGAT TCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGC TGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGAC CAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAAAACA TTCTTGAACGATTCTCCGCACA
  • AGCCCTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAG comprises a sequence of the TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR alpha chain, encoded by the sequence, ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATT GCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGA CTCTGGACTGCACATATGACACCAGTGATCCAAGTTATGGTCTATTCTGGTACAAGCAG CCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACCAGCAAAATGC AACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAA
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAA GCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAG TGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGG CTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGT TCCTGAAGGGTACAAAGTCTCTCTCT
  • GCCCATATGAGCGACGCGGCTGAGTACTTCTGTGCTGTGAGTCGCATGAAAACCTCCTA CGACAAGGTGATATTTGGGCCAGGGACAAGCTTATCAGTCATTCCAA or comprises a sequence of the TCR alpha chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRAV nucleic acid sequence above.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAA GCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAG TGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGG CTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGT TCCTGAAGGGTACAAAGTCTCTCTCT
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, WSGR Docket No.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC TGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGT CTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAAAAAGGAGATAT TCCTGAGGGGTACAGTGTC
  • TCR alpha chain encoded by the sequence, ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTAAGTGGAGAGCT GAAAGTGGAACAAAACCCTCTGTTCCTGAGCATGCAGGAGGGAAAAAACTATACCATC TACTGCAATTATTCAACCACTTCAGACAGACTGTATTGGTACAGGCAGGATCCTGGGAA AAGTCTGGAATCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGACGAT TAATGGCCTCACTTGATACCAAAGCCCGTCTCAGCACCCTCCACATCACAGCTGCCGTG CATGACCTCTCTGCCACCTACTTCTGTGCCGTGGACATCCATTCAGGAGGAGGTGCTGAC GGACTCACCTTTGGCAAAGGGACTCATCTAATCATCCAGCCCT, or comprises a sequence of the TCR alpha chain that is at least 80%, or at least 85%, or at least
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC TGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGT CTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATAT TCCTGAGGGGTACAGTGTC
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC TGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAG
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC WSGR Docket No.
  • TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR alpha chain, encoded by the sequence, ATGCTACTCATCACATCAATGTTG
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC TGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGT CTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAAAAAGGAGATAT TCCTGAGGGGTACAGTGTCTCTC
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC TGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGT CTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATAT TCCTGAGGGGTACAGTGTC
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC TGGAATGTGTCCAGGATA
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGACTCCTGGACCCTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGAT GCTGGAGTTATCCAGTCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTC TGAGATGTAAACCAATTTCAGGACACGACTACCTTTTCTGGTACAGACAGACCATGATG WSGR Docket No.
  • TCR beta chain comprises a sequence of the TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR alpha chain, encoded by the sequence, ATGAGGCAAGTGGCGAGAGTGATCGTGTTCCTGACCCTGAGTACTTTGAGCCTTGCTAA GACCACCCAGCCCATCTCCATGGACTCATATGAAGGACA
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCGGGCTCCTCTGCTGGGCACTGCTTTGTCTCCTGGGAGCAGGCTTAGTGGA CGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACT CTGAGATGCTCTCCTAAGTCTGGGCATGACACTGTGTCCTGGTACCAACAGGCCCTGGG TCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGGAGGAAGAGAGACAGAGAGGCAACT TCCCTGATCGATTCTCAGGTC
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCGGGCTCCTCTGCTGGGCACTGCTTTGTCTCCTGGGAGCAGGCTTAGTGGA CGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACT CTGAGATGCTCTCCTAAGTCTGGGCATGACACTGTGTCCTGGTACCAACAGGCCCTGGG TCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGGAGGAAGAGAGACAGAGAGGCAACT TCCCTGATCGATTCTCAGGTC
  • WSGR Docket No. 50401-775.601 Provided herein is an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACATAGAA GCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCT GAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGACGTGTTATGGGAA AAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGATGAGTCCGG
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACATAGAA GCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCT GAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGACGTGTTATGGGAA AAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGATGAGTCCGGTATG WSGR Docket No.
  • TCR beta chain comprises a sequence of the TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR alpha chain, encoded by the sequence, ATGACACGTGTTAGCTTGCTGTGGGCAGTCGTGGTCCACCTGTCTTGAATCCGGCATG GCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGAC CCTGAGTTGCACATATG
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, MLTGPPARV (9mer) and (ii) an MHC protein, encoded by HLA- A*02:01; or (i) an epitope having the sequence SMLTGPPARV (10mer) in and (ii) an MHC protein, encoded by HLA-A*02:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCCTGGGAGCGGTCCCCATGGAA ACGGGAGTTACGCAGACACCAAGACACCTGGTCATGGGAATGACAAATAAGAAGTCTT TGAAATGTGAACAACATCTGGGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAG AAGCCACTGGAGCTCATGTTTGTCTACAGTCTTGAAGAACGGGTTGAAAACAACAGTGT GCCAAGTCGCTTCACCTGA
  • an exemplary TCR A2-1 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 203, a TCR alpha variable region of SEQ ID NO: 208, a TCR alpha chain comprising TRAV8-2; joined with TRAJ13, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 206, a TCR beta variable domain of SEQ ID NO: 210, a TCR beta chain comprising TRBV3-1; joined with TRBJ1-1, and a constant domain having a sequence of human TRBC1 domain of SEQ ID NO: 153.
  • an exemplary TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 203, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 208.
  • TRAV and TRAC domains for example, a TCR comprising a sequence having at least 80% sequence identity to TRAV domain of SEQ ID NO: 208, a constant domain comprising mouse TRAC sequence of SEQ ID NO: 149.
  • an exemplary TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 323, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 328.
  • GATA3 binding TCR sequences that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and an MHC protein, encoded by HLA-B*07:02, or to an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGA TACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACT TTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGG GCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGC TGCTCAGTGATCGGTTCTGCAGA
  • the TCR described herein comprises the sequence of TCR B7-1 (Table 5A), e.g., a sequence comprising a TRBV sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% sequence identity to SEQ ID NO: 420, and a sequence comprising a TRAV sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% sequence identity to SEQ ID NO: 418.
  • the TCR B7-1 (Table 5A) is further described in Table 5B, comprising a TCR beta chain with a CDR3 sequence, CASSSEIVGPSQYF (SEQ ID NO: 416); joined with the TCR beta V chain of TRBV7-9; the J chain TRBJ2-7; and the constant domain TRBC2; and a TCR alpha chain CDR3 sequence CALRLNSGNTPLVF (SEQ ID NO: 413); joined with TRAV19 V chain sequence, TRAJ29 J chain sequence and TRAC constant domains.
  • Table 5B Table 5B
  • Table 5B comprising a TCR beta chain with a CDR3 sequence, CASSSEIVGPSQYF (SEQ ID NO: 416); joined with the TCR beta V chain of TRBV7-9; the J chain TRBJ2-7; and the constant domain TRBC2; and a TCR alpha chain CDR3 sequence CALRLNSGNTPLVF (SEQ ID NO: 413); joined with TRAV19 V chain sequence, TRA
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA-B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGAT GTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTC TGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGT CTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATAT TCCTGAGGGGTACAGT
  • ACGGGCAGGAGAGCACTTACTTTTGGGAGTGGAACAAGACTCCAAGTGCAACCAA or comprises a sequence of the TCR alpha chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRAV nucleic acid sequence above.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGGGCAGGTCCAGTGAAT GCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACT GCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCA TGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTC CCCAATGGCTACAATGTCT
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCCCAGTGGA WSGR Docket No.
  • TCR beta chain comprises a sequence of the TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAA GCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAG TGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGG CTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGT TCCTGAAGGGTACAAAGTCTCTCTCT
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCAGAACTCACAGA AGCTGGAGTTGCCCAGTCTCCCAGATATAAGATTATAGAGAAAAGGCAGAGTGTGGCTT TTTGGTGCAATCCTATATCTGGCCATGCTACCCTTTACTGGTACCAGCAGATCCTGGGAC WSGR Docket No.
  • TCR beta chain comprises a sequence of the TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR alpha chain, encoded by the sequence, ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATT GCCCAGAAGATAACTCAA
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCCCAGTGGA CGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAAACGAGAGGACAGCACGTGACT CTGAGATGCTCTCCTATCTCTGGGCACAAGAGTGTGTCCTGGTACCAACAGGTCCTGGG TCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGAAAGAAGAGAGAGAGGAAGGAAAC TTCCCTGATCGATTCT
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACATAGAA GCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCT GAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGACGTGTTATGGGAA AAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGATCGATTCTTAGCTGA
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAA GCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAG TGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGG CTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGT TCCTGAAGGGTACAAAGTCTCTCTCT
  • TTAACCTTTGGGACTGGAACAAGACTCACCATCATACCCA or comprises a sequence of the TCR alpha chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRAV nucleic acid sequence above.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAA GCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAG TGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGG CTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGT TCCTGAAGGGTACAAAGTCTCTCTCT
  • TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- WSGR Docket No.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGA TACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACT TTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGG GCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGC TGCTCAGTGATCGGTTCTGCA
  • AATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCCT or comprises a sequence of the TCR alpha chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRAV nucleic acid sequence above.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, KPKRDGYMF (9mer) and (ii) an MHC protein, encoded by HLA- B*07:02; or (i) an epitope having the sequence KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGA TACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACT TTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGG GCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGC TGCTCAGTGATCGGTTCTGCA
  • an exemplary TCR B7-2 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 423, a TCR alpha variable region of SEQ ID NO: 428, a TCR alpha chain comprising TRAV24; joined with TRAJ5, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 426, a TCR beta variable domain of SEQ ID NO: 430, a TCR beta chain comprising TRBV28; TRBD1; joined with WSGR Docket No.
  • TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 423, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 428.
  • TCR beta chain having a CDR3 sequence of SEQ ID NO: 426, a TCR beta variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 430.
  • an exemplary TCR B7-4 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 443, a TCR alpha variable region of SEQ ID NO: 448, a TCR alpha chain comprising TRAV38-1; joined with TRAJ53, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 446, a TCR beta variable domain of SEQ ID NO: 450, a TCR beta chain comprising TRBV5-5; joined with TRBJ1-2, and a constant domain having a sequence of human TRBC1 domain of SEQ ID NO: 153.
  • an exemplary TCR B7-11 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 513, a TCR alpha variable region of SEQ ID NO: 518, a TCR alpha chain comprising TRAV19; joined with TRAJ34, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 516, a TCR beta variable domain of SEQ ID NO: 520, a TCR beta chain comprising TRBV27; joined with TRBJ2-7, and a constant domain having a sequence of human TRBC2 domain of SEQ ID NO: 154.
  • an exemplary TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 513, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 518.
  • a TCR beta chain having a CDR3 sequence of SEQ ID NO: 516, a TCR beta variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 520.
  • an exemplary TCR B7-12 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 523, a TCR alpha variable region of SEQ ID NO: 528, a TCR alpha chain comprising TRAV8-3; joined with TRAJ47, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 526, a TCR beta variable domain of SEQ ID NO: 530, a TCR beta chain comprising TRBV28; joined with WSGR Docket No.
  • TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 523, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 528.
  • TCR beta chain having a CDR3 sequence of SEQ ID NO: 526, a TCR beta variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 530.
  • an exemplary TCR B7-19 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 593, a TCR alpha variable region of SEQ ID NO: 598, a TCR alpha chain comprising TRAV19; joined with TRAJ37, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 596, a TCR beta variable domain of SEQ ID NO: 600, a TCR beta chain comprising TRBV9; joined with TRBJ1-4, and a constant domain having a sequence of human TRBC1 domain of SEQ ID NO: 153.
  • an exemplary TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 593, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 598.
  • a TCR beta chain having a CDR3 sequence of SEQ ID NO: 596, a TCR beta variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 600.
  • a mature protein translated from a nucleic acid sequence does not contain a signal peptide sequence.
  • a transmembrane protein as expressed on the membrane of a cell does not contain the signal peptide.
  • the instant disclosure and the sequences described herein, indicating the sequence of a signal peptide invariably describes the complete protein in its mature form, read without the signal peptide sequence.
  • CDRs, partial and full length sequences of TCRs that bind to a complex comprising (i) KPKRDGYMF (9mer) or KPKRDGYMFL (10mer) and (ii) an MHC protein, encoded by HLA-B*07:02. Italicized sequences denote signal peptide regions. Underlined denote CDR sequences.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, ESKIMFATL and (ii) an MHC protein, encoded by HLA-B*08:01; the TCR having a TCR beta chain, encoded by the sequence, ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTCTCCTGGGAGCAAACACCGTGGA TGGTGGAATCACTCAGTCCCCAAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCC TGAGTTGTGAACAGAATTTGAACCACGATGCCATGTACTGGTACCGACAGGACCCAGGG CAAGGGCTGAGATTGATCTACTACTCACAGATAGTAAATGACTTTCAGAAAGGAGATAT AGCTGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCTCACTGTGACAT CGGCCCAAAAGAACCCGACAGCTTTCTATCTGTGCCAGTAG
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, ESKIMFATL. and (ii) an MHC protein, encoded by HLA-B*08:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCAGTTCCCATAGAC ACTGAAGTTACCCAGACACCAAAACACCTGGTCATGGGAATGACAAATAAGAAGTCTTT GAAATGTGAACAACATATGGGGCACAGGGCTATGTATTGGTACAAGCAGAAAGCTAAG AAGCCACCGGAGCTCATGTTTGTCTACAGCTATGAGAAACTCTCTATAAATGAAAGTGT WSGR Docket No.
  • TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR alpha chain, encoded by the sequence, ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACTACAGGACAAAA CATTGACCAGCCCACTGAGATGACAGCTACGGAAGGTGCCATTGTCCAGATCAACTGCA CGTACCAGACATCTGGGTTCAACGGGCT
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, ESKIMFATL. and (ii) an MHC protein, encoded by HLA-B*08:01; the TCR having a TCR beta chain, encoded by the sequence, ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAA GCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAG TGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGG CTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGT TCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGT CGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTAAGTCGG
  • ACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACC TAGTGATGTAGGCATCTACTTCTGTGCTGGGGGAGGAAGCCAAGGAAATCTCATCTTTG GAAAAGGCACTAAACTCTCTGTTAAACCAA or comprises a sequence of the TCR alpha chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRAV nucleic acid sequence above.
  • an exemplary TCR that specifically binds to a complex comprising (i) an epitope having the sequence, ESKIMFATL. and (ii) an MHC protein, encoded by HLA-B*08:01; the TCR having a TCR beta chain, encoded by the sequence, ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGAAT WSGR Docket No.
  • TCR beta chain comprises a sequence of the TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR beta chain that is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 96%, or at least 97%, or at least 98%, or at least 99% or at least 100% identical to a sequence encoded by the TRBV nucleic acid sequence above; and a TCR beta chain that is at least 80%, or at least 85%, or at
  • exemplary GATA3 binding TCR sequences specific to epitope ESKIMFATL in complex with HLA-B*08:01.
  • an exemplary TCR B8-5 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 673, a TCR alpha variable region of SEQ ID NO: 678, a TCR alpha chain comprising TRAV13-2; joined with TRAJ8, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 676, a TCR beta variable domain of SEQ ID NO: 680, a TCR beta chain comprising TRBV6-2; joined with TRBJ2-7, and a constant domain having a sequence of human TRBC2 domain of SEQ ID NO: 154.
  • an exemplary TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 673, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 678.
  • an exemplary TCR B8-6 having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 683, a TCR alpha variable region of SEQ ID NO: 688, a TCR alpha chain comprising TRAV10; joined with TRAJ36, and a constant domain having a sequence of human TRAC domain of SEQ ID NO: 152; and a TCR beta chain having a CDR3 of SEQ ID NO: 686, a TCR beta variable domain of SEQ ID NO: 690, a TCR beta chain comprising TRBV18; joined with TRBJ1-2, and a constant domain having a sequence of human TRBC2 domain of SEQ ID NO: 154.
  • an exemplary TCR having a TCR alpha chain having a CDR3 sequence of SEQ ID NO: 683, a TCR alpha variable region having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 688.
  • Table 6B Summary of TCRs from Table 6A that bind to a complex comprising (i) an epitope having the sequence, ESKIMFATL and (ii) HLA-B*08:01. WSGR Docket No. 50401-775.601 [00511]
  • FIG.5 shows T cells transduced with mutation-specific TCRs can recognize naturally processed/presented peptide-MHC on K562 cells expressing full-length GATA3neoORF and HLA-B*07:02, without external peptide pulsing.
  • FIG.5 shows T cells transduced with mutation-specific TCRs can recognize naturally processed/presented peptide-MHC on K562 cells expressing full-length GATA3neoORF and HLA-B*07:02, without external peptide pulsing.
  • FIG.5 shows T cells transduced with mutation-specific TCRs can recognize naturally processed/presented peptide-MHC on K562 cells expressing full-length GATA3
  • T cells transduced with mutation-specific TCRs can recognize naturally processed/presented peptide-MHC on K562 cells expressing full-length GATA3neoORF and HLA-B*08:01, without external peptide pulsing.
  • 4-1BB is a marker of epitope recognition and activation by T cells, and is known as T cell WSGR Docket No. 50401-775.601 exhaustion marker, which results from the TCR-specific T cell activation.
  • FIG. 7 are avidity data for TCRs that bind to a complex containing the epitope having the sequence, ESKIMFATL and HLA-B*08-01. The avidity values are shown in Table 7. Each of the tested TCRs show efficient binding.
  • TCR B8-5 shows highly efficient binding avidity score.
  • Table 7. EC50 values of TCRs shown in FIG. 7.
  • FIG. 8 shows binding avidity data for representative TCR samples generated that bind to a peptide-MHC complex comprising (i) an epitope KPKRDGYMF, or an epitope KPKRDGYMFL, and (ii) an MHC protein encoded by HLA-B*07:02 allele.
  • Table 8 shows binding avidity data for representative TCR samples generated that bind to a peptide-MHC complex comprising (i) an epitope KPKRDGYMF, or an epitope KPKRDGYMFL, and (ii) an MHC protein encoded by HLA-B*07:02 allele.
  • Table 8 shows binding avidity data for representative TCR samples generated that bind to a peptide-MHC complex comprising (i) an epitope KPKRDGYMF, or an epitope KPKRDGYMFL, and (ii) an
  • FIG. 9 demonstrates T cells expressing a TCR of the TCRs generated herein MCF7 cells with natural expression of HLA-A*02-01. T cells transduced with mutation-specific TCRs can recognize naturally expressing HLA-A*02:01 positive MCF-7 breast cancer cells engineered with full- length WT GATA3 or GATA3neoORF, without external peptide pulsing.

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Abstract

La présente divulgation concerne des récepteurs des lymphocytes T (TCR) qui se lient à des complexes peptide-CMH; des molécules d'acide nucléique isolées codant pour des TCR contre des complexes peptide-CMH, des lymphocytes T exprimant des TCR contre des complexes peptide-CMH et des compositions pharmaceutiques destinées à être utilisées dans le traitement de maladies.
PCT/US2025/022094 2024-03-29 2025-03-28 Constructions de récepteurs des lymphocytes t et leurs utilisations Pending WO2025208068A2 (fr)

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