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WO2024163371A1 - Protéines de liaison spécifiques pour p53 mutant et leurs utilisations - Google Patents

Protéines de liaison spécifiques pour p53 mutant et leurs utilisations Download PDF

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WO2024163371A1
WO2024163371A1 PCT/US2024/013407 US2024013407W WO2024163371A1 WO 2024163371 A1 WO2024163371 A1 WO 2024163371A1 US 2024013407 W US2024013407 W US 2024013407W WO 2024163371 A1 WO2024163371 A1 WO 2024163371A1
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amino acid
domain
cell
binding protein
variant
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Thomas M. Schmitt
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Fred Hutchinson Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4238—Regulators of development
    • A61K40/424—Apoptosis related proteins, e.g. survivin or livin
    • A61K40/4241—Apoptosis related proteins, e.g. survivin or livin p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • P53 (also called Tumor protein P53, tumor antigen p53, transformation-related protein 53, TP53, LFS1, Phosphoprotein P53, Antigen NY-CO-13, Mutant Tumor Protein 53, Tumor Suppressor P53, BMFS5, TRP53, and BCC7) is a regulatory protein that is often mutated in human cancers. Therapeutic modalities targeting P53 (also spelled “p53” herein) are needed.
  • FIG. 1 shows activation of T cells engineered to express the indicated T cell receptor (TCR) in the presence of various concentrations of (top) an HLA-A*02:01-restricted P53RI?5H mutant peptide (P53 amino acids 168-176 with a R175H mutation; SEQ ID NO:3) or (bottom) the corresponding wild-type P53 peptide (SEQ ID NO:2).
  • T cells expressed a reporter gene under the control of a promoter for the Nur77 T cell activation marker.
  • TCR discovery campaigns were performed (6 human donors in total), and several P53Ri75H-reactive TCRs were identified and tested.
  • mutant P53Ri75H-specific CD8 + T cell lines were generated from 6 healthy donors and T cells with high reactivity towards the mutant P53RI?5H peptide HMTEVVRHC (SEQ ID NO:3) were isolated by sorting the combined T cell lines for T cells that bound peptide/MHC tetramers at low tetramer concentration, or that bound peptide/MHC tetramers that were modified to eliminate associations with CD8 (CD8-independent peptide/MHC tetramers).
  • TCRa and TCRP chain genes were identified by single cell immune profiling (lOx Genomics).
  • Codon-optimized polynucleotides encoding TCRs (TCR beta and alpha chains with cysteine-modified constant domains, linked by a P2A sequence with a N- terminal GSG linker sequence) were produced to generate a single polycistronic construct expressing both TCR chains.
  • This cassette was introduced into a lentiviral expression vector, and the resulting lentivirus was used to transduce gene-modified Jurkat T cells that lack endogenous TCRa and TCRP and have a green fluorescent protein gene knocked into the Nur77 locus. These cells efficiently express transgenic TCR genes and produce GFP in proportion to the degree of TCR signaling.
  • the TCRs exhibited varying degrees of specificity for the cognate P53RI?5H mutant peptide (HMTEVVRHC; SEQ ID NO:3) versus the corresponding wild-type (wt) P53 peptide (HMTEVVRRC; SEQ ID NO:2). Curves were fit to the dose-response data by non-linear regression using Graphpad Prism®. The most promising TCRs exhibited a low logEC50 for the mutant P53 peptide but were much less responsive to the wt peptide. TCR4 (shown as R175H#4) fits both of these criteria, with a logEC50 of -7.81M for P53RI?5H but almost no response to the wt peptide.
  • the calculated peptide logEC50 for this TCR compares favorably with a p53Ri75H-specific TCR isolated from tumor infiltrating lymphocytes (TILs) (est. IFN-y logEC50: -7.62) reported by Lo, Rosenberg and colleagues (see Figure 3E of Lo et cd.. Cancer Immunol. Res. 7(4):534-543 (2019); doi: 10.1158/2326-6066.CIR-18-0686) and with a single-chain variable fragment (scFv) with specificity for HLA-A*02:01 p53Ri75Hthat was made into a bispecific diabody (est. IFN-y log EC50: -6.86), see Fig.
  • FIG. 2 shows response of TCR-transduced Jurkat cells to mutant P53-expressing tumors that naturally present the peptide antigen.
  • TCR-transduced Jurkat T cells were cultured for 12 hours with the indicated tumor cell lines (left panel), or with tumor cell lines pre-treated with IFN-y (right panel), which acts to increase HLA surface expression and enhances immunoproteasome expression and activity in the target cell.
  • FIG. 1 The response of Jurkat cells expressing TCR4 was directly compared to Jurkat cells expressing a high affinity WTl-specific TCR (shown as “P37”), which has been shown to have high level activity against WT1 -expressing tumor lines (Lahman et al., Sci Transl Med. 16(63 l):eabg8070 (2022); doi: 10.1126/scitranslmed.abg8070), as well as to untransduced Jurkat cells as a negative control.
  • Figure 3 shows HLA-A*02:01 expression by mutant P53-expressing tumor cell lines. All of the tumor lines assessed in Figure 2 have been reported to express P53RI?5H as well as HLA-A*02:01.
  • FIG. 4 shows activation of T cells engineered to express “TCR8” (shown as R175H#8, with TCR4 as comparator), specific for HLA-A*02:01-restricted P53RI?5H mutant peptide (SEQ ID NO:3).
  • TCR vector construction, expression, and activation assay were as described for Figure 1.
  • TCR8 has a similar reactivity profile as TCR4, but with a lower avidity by this assay.
  • Figure 5A provides a table showing CDR amino acid sequences of certain binding proteins of the present disclosure.
  • the CDR3 amino acid sequences in Figure 5A shown are according to the IMGT definition; it will be appreciated that CDR3 amino acid sequences may also be identified according to the IMGT junction definition, as provided herein and shown in Figure 5B.
  • Figure 6 shows expression of P53Ri75H-specific TCRs by peptide:HLA tetramer staining of primary CD8+ T cells.
  • Primary CD8+ T cells were (untransduced or) transduced as indicated, stained with P53RI?5H tetramer, and evaluated by FACS. Tetramer staining is specific and allows for surface TCR quantification.
  • Figure 7 shows increases in the percentage of CD 137-positive T cells and expression level of CD137 (MFI), which demonstrates T cell activation.
  • MFI CD137-positive T cells and expression level of CD137
  • FIGS 8-13 show amino acid sequences of certain binding protein polypeptides and expression constructs of the present disclosure. Each figure shows (top) a full-length TCRa chain, with signal peptide and wild-type constant domain, (middle) a full-length TCRP chain, with signal peptide and wild-type constant domain, and (bottom) amino acid sequence of: [TCRP chain with signal peptide and cysteine-modified constant domain - P2A self-cleaving peptide with N-terminal G-S-G linker (z.e., GSGATNFSLLKQAGDVEENPGP; SEQ ID NO: 103) - TCRa chain with signal peptide and cysteine-modified constant domain].
  • the shaded sequences are as follows, in N-terminal to C-terminal direction: signal peptide; CDR1; CDR2; CDR3.
  • CDR1 and CDR2 as shown in each TCR chain are according to the IMGT definition
  • CDR3 as shown in each TCR chain is according to the IMGT junction definition (z.e., including the N-terminal Cys and the C-terminal Trp or Phe). It will be understood that CDR3s according to the IMGT definition (z.e., not including the N-terminal Cys and the C-terminal Trp or Phe) are also contemplated.
  • the TCRp chain may be encoded upstream of the TCRa chain, or the TCRa chain may be encoded upstream of the TCRP chain.
  • self-cleaving peptides other than P2A are known (e.g., T2A, E2A, F2A) and one or more of these may be used.
  • an internal ribosome entry site (IRES) and/or a protease cleavage site may be used.
  • a signal peptide is typically removed, in whole or in part, from the expressed polypeptide (e.g., TCR chain) during or once localization or secretion is completed.
  • Signal peptides other than those illustrated in Figures 8-13 may be employed.
  • Figure 8 TCR P53R175H#2 aka FH TCR2 aka TCR R175H#2 aka TCR2.
  • Figure 9 TCR P53R175H#3 aka FH TCR3 aka TCR R175H#3 aka TCR3.
  • Figure 10 TCR P53R175H#4 aka FH_TCR4 aka TCR R175H#4 aka TCR4.
  • FIG. 11 TCR P53R175H#5 aka FH_TCR5 aka TCR R175H#5 aka TCR5.
  • Figure 12 TCR P53R175H#6 aka FH TCR6 aka TCR R175H#6 aka TCR6.
  • Figure 13 TCR P53R175H#8 aka FH TCR8 aka TCR R175H#8 aka TCR8.
  • Figures 14A-19B show polynucleotide sequences encoding the amino acid sequences shown in Figures 8-13.
  • Figures 14A, 15A, 16A, 17A, 18A, and 19A show polynucleotide sequences encoding TCRa and P chains (with signal peptide) for TCRs P53R175H#2, #3, #4, #5, #6, and #8, respectively.
  • Figures 14B, 15B, 16B, 17B, 18B, and 19B show codon-optimized polynucleotide sequences encoding [TCRP chain with signal peptide and cysteine-modified constant domain - P2A self-cleaving peptide with N-terminal G-S-G linker - TCRa chain with signal peptide and cysteine-modified constant domain] for TCRs P53R175H#2, #3, #4, #5, #6, and #8, respectively.
  • FIGS. 20A-20D show activation of T cells engineered to express the indicated TCR in the presence of various concentrations of HLA-A*02:01-restricted P53RI?5H mutant peptide (SEQ ID NO:3).
  • TCR vector construction, expression, and activation assay were as described for Figure 1.
  • TCRs F38 ( Figure 20A), F40 (Figure 20B), G46 (Figure 20C), and G41 ( Figure 20D) were tested, with TCR4 (shown as p53#4) included as a comparator.
  • Figure 20E shows functional avidity of the indicated TCRs, with the estimated logEC50 values calculated as described for Figure 1.
  • TCR4 is shown as #4 in Figure 20E.
  • FIGS 21-24 show amino acid sequences of certain binding protein polypeptides and expression constructs of the present disclosure. Each figure shows (top) a full-length TCRa chain, with signal peptide and wild-type constant domain, (middle) a full-length TCRP chain, with signal peptide and wild-type constant domain, and (bottom) amino acid sequence of: [TCRP chain with signal peptide and cysteine-modified constant domain - P2A self-cleaving peptide with N-terminal G-S-G linker (i.e. GSGATNFSLLKQAGDVEENPGP; SEQ ID NO: 103) - TCRa chain with signal peptide and cysteine-modified constant domain].
  • the underlined sequences are as follows, in N-terminal to C-terminal direction: signal peptide; CDR1; CDR2; CDR3.
  • CDR1 and 2 as shown in each TCR chain are according to the IMGT definition
  • CDR3 as shown in each TCR chain is according to the IMGT junction definition (zie., including the N-terminal Cys and the C-terminal Trp or Phe). It will be understood that CDR3s according to the IMGT definition (i.e., not including the N-terminal Cys and the C-terminal Trp or Phe) are also contemplated.
  • the TCRP chain may be encoded upstream of the TCRa chain, or the TCRa chain may be encoded upstream of the TCRP chain.
  • self-cleaving peptides other than P2A are known (e.g., T2A, E2A, F2A) and one or more of these may be used.
  • an internal ribosome entry site (IRES) and/or a protease cleavage site (e.g., furin cleavage site) may be used.
  • a signal peptide is typically removed, in whole or in part, from the expressed polypeptide (e.g.
  • FIG. 21 TCR P53R175H F38 aka P53R175H F38 aka FH TCR F38 aka TCR F38 aka F38.
  • Figure 22 TCR P53R175H F40 aka P53R175H F40 aka FH TCR F40 aka TCR F40 aka F40.
  • Figure 23 TCR P53R175H G41 aka
  • Figures 25A-28C show polynucleotide sequences encoding the amino acid sequences shown in Figures 21-24.
  • Figures 25 A, 26A, 27A, and 28 A show polynucleotide sequences encoding TCRa chains (with signal peptide) for TCRs P53R175H F38, _F40, _G41, and _G46, respectively.
  • Figures 25B, 26B, 27B, and 28B show polynucleotide sequences encoding TCRP chains (with signal peptide) for TCRs P53R175H F38, _F40, _G41, and _G46, respectively.
  • Figures 25C, 26C, 27C, and 28C show codon-optimized polynucleotide sequences encoding [TCRP chain with signal peptide and cysteine-modified constant domain - P2A self-cleaving peptide with N-terminal G-S-G linker - TCRa chain with signal peptide and cysteine-modified constant domain] for TCRs P53R175H F38, _F40, _G41, and _G46, respectively.
  • the present disclosure generally relates to binding proteins specific for P53 R175H mutant peptides (such as, for example, the peptide HMTEVVRHC; SEQ ID NO:3, also referred to as a P53 antigen or a P53RI?5H antigen) in complex with an HLA molecule, host cells (e.g., immune cells) expressing the same, polynucleotides that encode the binding proteins, vectors, host cells, compositions, and related uses.
  • disclosed binding proteins comprise a TCR Va domain and a TCR VP domain and are capable of binding to a HMTEVVRHC :HLA-A2 (e.g. HLA-*02:01) complex.
  • a binding protein can be or can comprise, for example, a T cell receptor (TCR), a single-chain T cell receptor variable fragment (scTv), a single-chain TCR, or a chimeric antigen receptor (CAR).
  • a binding protein is expressed by a host cell e.g., a T cell, optionally a Jurkat cell, expressing the binding protein at a cell surface) and is capable of binding to HMTEVVRHC:HLA-A*02:01 tetramers at low concentrations, and/or is capable of binding to HMTEVVRHC :HLA-A* 02:01 tetramers modified to eliminate associate with CD8 (i.e., the binding protein is capable of CD8-independent binding to the HMTEVVRHC: HL A-A *02:01 complex or tetramer).
  • a host cell e.g., an immune cell such as a T cell
  • a binding protein is activated e.g., as determined by expression of a reporter gene such as GFP knocked-in to the locus of the Nur77 activation marker) in the presence of HMTEVVRHC (SEQ ID NO.:3) peptide.
  • At least 50% of the host cells in a sample are activated e.g., produce a reporter signal for/Vwr77) at a concentration of less than 10' 6 M peptide, or of less than 10' 7 M peptide, or of beween 10' 6 M peptide and 1 O' 7 M peptide, or of between 10' 7 M peptide and 1 O' 8 M peptide.
  • a binding protein has a Nur77 logEC50 (HMTEVVRHC; SEQ ID NO.:3) of -6 to -7, or of -6 to -6.5, or of -6.5 to -7, or of -7 to -7.5, or of -7.5 to -8, or of -6.0, -6.1, -6.2, -6.3, -6.4, -6.5, -6.6, -6.7, -6.8, -6.9, -7.0, -7.1, -7.2, -7.3, -7.4, -7.5, -7.6, -7.7, or -7.8.
  • HMTEVVRHC Nur77 logEC50
  • a binding protein has Nur77 logEC50 (HMTEVVRHC; SEQ ID NO.:3) of about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, or about -7.8 or less.
  • a binding protein has a Nur77 logEC50 (HMTEVVRHC; SEQ ID N0.:3) of -7.8 or -7.81.
  • a binding protein does not substantially recognize a peptide having the amino acid sequence HMTEVVRRC (SEQ ID NO:2, a wild-type P53168-176 amino acid sequence).
  • the binding proteinexpressing host cells e.g., T cells
  • the binding proteinexpressing host cells e.g., T cells
  • certain embodiments provide a binding protein that is sensitive for the mutant P53 peptide HMTEVVRHC (SEQ ID NO.:3) and is less sensitive for, or displays no substantial or no detectable sensitivity for, the wild-type P53 peptide HMTEVVRRC (SEQ ID NO:2).
  • a binding protein of the present disclosure is capable of binding to a P53RI75H peptide (such as HMTEVVRHC (SEQ ID NO:3)) naturally processed and presented by one or more tumor cell line that expresses a mutated P53 protein.
  • the one or more tumor cell line comprises AU565, KLE, SKBR3, SKUT1, TYKNU, or any combination thereof.
  • the one or more tumor cell line comprises KLE, TYKNU, or both.
  • a binding protein comprises a CDR3a of any one of the binding proteins shown in Figure 5A or 5B, and a CDR3P of any one of the binding proteins shown in Figure 5A or 5B. In some embodiments, a binding protein comprises the CDR3a of a binding protein as shown in Figure 5 A and a CDR3P of the same binding protein as shown in Figure 5B.
  • the binding protein comprises a CDRla selected from any one of the CDRla sequences shown in Figure 5 A, a CDR2a selected from any one of the CDR2a sequences shown in Figure 5 A, a CDRip selected from any one of the CDRip sequences shown in Figure 5A, and a CDR2P selected from any one of the CDR2P sequences shown in Figure 5A.
  • a binding protein comprises the CDRla, the CDR2a, the CDRip, and the CDR2P of a binding protein as shown in Figure 5B.
  • a binding protein comprises a CDR3a and a CDR3P of binding protein FH TCR2, as shown in Figure 5 A or 5B.
  • the binding protein comprises a CDRla selected from any one of the CDRla sequences shown in Figure 5 A, a CDR2a selected from any one of the CDR2a sequences shown in Figure 5 A, a CDRip selected from any one of the CDRip sequences shown in Figure 5 A, and a CDR2P selected from any one of the CDR2P sequences shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of FH TCR2, as shown in Figure 5 A. In some embodiments, a binding protein comprises CDRla, CDR2a, CDRip, and CDR2P of FH TCR2, as shown in Figure 5A, and further comprises CDR3a and CDR3P of FH TCR2, as shown in Figure 5B.
  • a binding protein comprises a CDR3a and a CDR3P of FH TCR3, as shown in Figure 5A or 5B.
  • the binding protein comprises a CDRla selected from any one of the CDRla sequences shown in Figure 5 A, a CDR2a selected from any one of the CDR2a sequences shown in Figure 5 A, a CDRip selected from any one of the CDRip sequences shown in Figure 5 A, and a CDR2P selected from any one of the CDR2P sequences shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of FH TCR3, as shown in Figure 5 A. In some embodiments, a binding protein comprises CDRla, CDR2a, CDRip, and CDR2P of FH TCR3, as shown in Figure 5 A, and further comprises CDR3a and CDR3P of FH TCR3, as shown in Figure 5B.
  • a binding protein comprises a CDR3a and a CDR3P of FH TCR4, as shown in Figure 5A or 5B.
  • the binding protein comprises a CDRla selected from any one of the CDRla sequences shown in Figure 5 A, a CDR2a selected from any one of the CDR2a sequences shown in Figure 5 A, a CDRip selected from any one of the CDRip sequences shown in Figure 5 A, and a CDR2P selected from any one of the CDR2P sequences shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of FH TCR4, as shown in Figure 5 A. In some embodiments, a binding protein comprises CDRla, CDR2a, CDRip, and CDR2P of FH TCR4, as shown in Figure 5 A, and further comprises CDR3a and CDR3P of FH TCR4, as shown in Figure 5B.
  • a binding protein comprises a CDR3a and a CDR3P of FH TCR5, as shown in Figure 5A or 5B.
  • the binding protein comprises a CDRla selected from any one of the CDRla sequences shown in Figure 5 A, a CDR2a selected from any one of the CDR2a sequences shown in Figure 5 A, a CDRip selected from any one of the CDRip sequences shown in Figure 5 A, and a CDR2P selected from any one of the CDR2P sequences shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of FH TCR5, as shown in Figure 5A. In some embodiments, a binding protein comprises CDRla, CDR2a, CDRip, and CDR2P of FH TCR5, as shown in Figure 5 A, and further comprises CDR3a and CDR3P of FH TCR5, as shown in Figure 5B.
  • a binding protein comprises a CDR3a and a CDR3P of
  • the binding protein comprises a CDRla selected from any one of the CDRla sequences shown in Figure 5 A, a CDR2a selected from any one of the CDR2a sequences shown in Figure 5 A, a CDRip selected from any one of the CDRip sequences shown in Figure 5 A, and a CDR2P selected from any one of the CDR2P sequences shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of FH TCR6, as shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDRip, and CDR2P of FH TCR6, as shown in Figure 5 A, and further comprises CDR3a and CDR3P of FH TCR6, as shown in Figure 5B.
  • a binding protein comprises a CDR3a and a CDR3P of
  • the binding protein comprises a CDRla selected from any one of the CDRla sequences shown in Figure 5 A, a CDR2a selected from any one of the CDR2a sequences shown in Figure 5 A, a CDRip selected from any one of the CDRip sequences shown in Figure 5 A, and a CDR2P selected from any one of the CDR2P sequences shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of FH TCR8, as shown in Figure 5 A.
  • a binding protein comprises CDRla, CDR2a, CDRip, and CDR2P of FH TCR8, as shown in Figure 5 A, and further comprises CDR3a and CDR3P of FH TCR8, as shown in Figure 5B.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR2, as shown in Figure 8.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR2, as shown in Figure 8.
  • a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR2, as shown in Figure 8.
  • a binding protein is provided that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR2, as shown in Figure 8.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR3, as shown in Figure 9.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR3, as shown in Figure 9. In some embodiments, a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR3, as shown in Figure 9. In some embodiments, a binding protein is provided that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR3, as shown in Figure 9.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR4, as shown in Figure 10.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR4, as shown in Figure 10. In some embodiments, a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR4, as shown in Figure 10. In some embodiments, a binding protein is provided that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR4, as shown in Figure 10.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR5, as shown in Figure 11.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR5, as shown in Figure 11. In some embodiments, a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR5, as shown in Figure 11. In some embodiments, a binding protein is provided that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR5, as shown in Figure 11.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR6, as shown in Figure 12.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR6, as shown in Figure 12. In some embodiments, a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR6, as shown in Figure 12. In some embodiments, a binding protein is provided that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR6, as shown in Figure 12.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR8, as shown in Figure 13.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR8, as shown in Figure 13.
  • a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR8, as shown in Figure 13.
  • a binding protein is provided that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR8, as shown in Figure 13.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR F38, as shown in Figure 21.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR F38, as shown in Figure 21.
  • a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR F38, as shown in Figure 21.
  • a binding protein is provided that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR F38, as shown in Figure 21.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR F40, as shown in Figure 22.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR F40, as shown in Figure 22.
  • a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR F40, as shown in Figure
  • a binding protein comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR F40, as shown in Figure 22.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR G41, as shown in Figure 23.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR G41, as shown in Figure 23. In some embodiments, a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR G41, as shown in Figure
  • a binding protein comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR G41, as shown in Figure 23.
  • a binding protein comprises the Va domain amino acid sequence, the VP domain amino acid sequence, the TCRa chain amino acid sequence, and/or the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR G46, as shown in Figure 24.
  • a binding protein that comprises the Va CDRs 1-3 and the VP CDRs 1-3 of FH TCR G46, as shown in Figure 24. In some embodiments, a binding protein is provided that comprises the Va domain amino acid sequence and the VP domain amino acid sequence (with or without the signal peptide) of FH TCR G46, as shown in Figure
  • a binding protein that comprises the TCRa chain amino acid sequence and the TCRP chain amino acid sequence (with or without the signal peptide) of FH TCR G46, as shown in Figure 24.
  • an isolated polynucleotide or an expression construct is provided that encodes a TCRP-GSG-P2A-TCRa amino acid sequence, as shown in Figure 8 (TCR2), Figure 9 (TCR3), Figure 10 (TCR4), Figure 11 (TCR5), Figure 12 (TCR6), Figure 13 (TCR8), Figure 21 (TCR F38), Figure 22 (TCR F40), Figure 23 (TCR G41), or Figure 24 (TCR G46).
  • a GSG linker is absent or is replaced by an alternative linker (e.g., GPP or PGP or AAA or a glycine-serine linker other than GSG), and/or wherein an alternative 2A peptide is used, and/or wherein an IRES and/or a protease cleavage site is used.
  • an alternative linker e.g., GPP or PGP or AAA or a glycine-serine linker other than GSG
  • an alternative linker e.g., GPP or PGP or AAA or a glycine-serine linker other than GSG
  • an alternative linker e.g., GPP or PGP or AAA or a glycine-serine linker other than GSG
  • an alternative 2A peptide is used
  • an IRES and/or a protease cleavage site is used.
  • the order of the TCRP and TCRa chains may be different to what is shown in Figures 8, 9,
  • polynucleotides that encode a binding protein that encode a binding protein
  • vectors that comprise the polynucleotide that comprise the polynucleotide
  • host cells e.g, immune cells such as T cells or NK cells
  • pharmaceutical compositions comprising a binding protein, polynucleotide, vector, or host cell disclosed herein.
  • a disease or disorder associated with a P53 mutation e.g, a P53RI?5H mutation
  • a P53 mutation e.g, a P53RI?5H mutation
  • the disease or disorder is a cancer.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more" of the enumerated components.
  • a protein domain, region, or module e.g., a binding domain, hinge region, linker module
  • a protein which may have one or more domains, regions, or modules
  • protein or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers.
  • a "peptide” e.g., a peptide antigen refers to a polymer of about 8-10 amino acid residues in length.
  • hematopoietic progenitor cell is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cell types (e.g., immune system cells).
  • exemplary hematopoietic progenitor cells include those with a CD24 LO Lin CD117 + phenotype or those found in the thymus (referred to as progenitor thymocytes).
  • an "immune system cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
  • myeloid progenitor cell which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes
  • lymphoid progenitor cell which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells.
  • Exemplary immune system cells include a CD4 + T cell, a CD8 + T cell, a CD4' CD8' double negative T cell, a y5 T cell, a regulatory T cell, a natural killer cell, a natural killer T cell, and a dendritic cell.
  • Macrophages and dendritic cells can be referred to as "antigen presenting cells" or "APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
  • MHC major histocompatibility complex
  • T cell or "T lymphocyte” is an immune system cell that matures in the thymus and produces a T cell receptor (TCR).
  • T cells can be naive ("TN”; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased or no expression of CD45RO as compared to T CM (described herein)), memory T cells (T M ) (antigen experienced and long-lived), including stem cell memory T cells, and effector cells (antigen- experienced, cytotoxic).
  • T M can be further divided into subsets of central memory T cells (T CM expresses CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (T E M, express CD45RO, decreased expression of CD62L, CCR7, CD28, and CD45RA).
  • Effector T cells (T E ) refers to antigen-experienced CD8 + cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 as compared to T CM , and are positive for granzyme and perforin.
  • Helper T cells (TH) are CD4 + cells that influence the activity of other immune cells by releasing cytokines.
  • CD4 + T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals.
  • T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection.
  • Other exemplary T cells include regulatory T cells, such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8 + CD28‘, and Qa-1 restricted T cells.
  • T cell receptor refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e. g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor.
  • a TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and P chains (also known as TCR a and TCRP, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively).
  • a polynucleotide encoding a binding protein of this disclosure can be codon optimized to enhance expression in a particular host cell, such, for example, as a cell of the immune system, a hematopoietic stem cell, an embryonic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten et aL, Clin. Immunol. 119: 135, 2006).
  • Exemplary T cells that can express binding proteins and TCRs of this disclosure include CD4 + T cells, CD8 + T cells, and related subpopulations thereof (e.g., naive, central memory, stem cell memory, effector memory).
  • the extracellular portion of TCR chains can contain two immunoglobulin domains, a variable domain (e.g., a- chain variable domain or V a P-chain variable domain or Vp at the N-terminus, and one constant domain (e.g., a-chain constant domain or C a , P-chain constant domain or Cp) adjacent the cell membrane.
  • the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., lores et cd., Proc. Nat'l Acad. Sci.
  • the source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit, or other mammal.
  • variable region refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR a-chain or P-chain (or y chain and 5 chain for y5 TCRs)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g., TCR) to antigen.
  • immunoglobulin superfamily binding protein e.g., a TCR a-chain or P-chain (or y chain and 5 chain for y5 TCRs)
  • the variable domains of the a-chain and P-chain (Va and VP, respectively) of a native TCR generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs.
  • the Va domain is encoded by two separate DNA segments, the variable gene segment, and the joining gene segment (V-J); the VP domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J).
  • a single Va or VP domain may be sufficient to confer antigen-binding specificity.
  • TCRs that bind a particular antigen may be isolated using a Va or VP domain from a TCR that binds the antigen to screen a library of complementary Va or VP domains, respectively.
  • CDR complementarity determining region
  • HVR hypervariable region
  • CDRs confer antigen specificity and binding affinity and are separated from one another in primary amino acid sequence by framework regions.
  • aCDRl a-chain variable region
  • PCDR1, PCDR2, PCDR3 three CDRs in each TCR P-chain variable region
  • CDR3 is thought to be the main CDR responsible for recognizing processed antigen (see e.g., Rosati et al.
  • CDR1 and CDR2 interact mainly or exclusively with the MHC.
  • CDR1 and CDR2 are encoded within the variable gene segment of a TCR variable region-coding sequence
  • CDR3 is encoded by the region spanning the variable and joining segments for Va, or the region spanning variable, diversity, and joining segments for Vp.
  • the sequences of their corresponding CDR1 and CDR2 can be deduced; e.g., according to a numbering scheme as described herein.
  • CDR3, and in particular CDR3P is typically significantly more diverse due to the addition and loss of nucleotides during the recombination process.
  • TCR variable domain sequences can be aligned to a numbering scheme (e.g., IMGT, Kabat, Chothia, EU, Enhanced Chothia, or Aho, or a hybrid of two or more of these), allowing equivalent residue positions to be annotated and for different molecules to be compared using, for example, ANARCI software tool (2016, Bioinformatics 15:298-300).
  • a numbering scheme provides a standardized delineation of framework regions and CDRs in the TCR variable domains.
  • a CDR of the present disclosure is identified according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol.
  • a CDR3 is defined in accordance with the IMGT junction definition (including CYS 104 and PHE 118 or TRP 118). In some embodiments, a CDR3 is defined in accordance with the IMGT definition (rearranged CDR3 corresponding to IMGT positions 105-117 within the variable domain).
  • a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the IMGT numbering scheme.
  • a CDR3 (or both CDR3s of a binding protein) is identified or defined according to the IMGT-junction numbering scheme.
  • a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the Kabat numbering scheme.
  • a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the Chothia numbering scheme.
  • a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the EU numbering scheme. In some embodiments, a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the Enhanced Chothia numbering scheme. In some embodiments, a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined of the present disclosure is identified according to the Aho numbering scheme.
  • TCR constant domain sequences may be from, for example, human, mouse, marsupial (e.g., opossum, bandicoot, wallaby), shark, or non-human primate.
  • TCR constant domain sequences are human or comprise engineered variants of human sequences. TCR constant domains may be engineered to improve pairing, expression, stability, or any combination of these.
  • TCR Ca and CP examples include mutation of a native amino acid to a cysteine so that a disulfide bond forms between the introduced cysteine of one TCR constant domain and a native cysteine of the other TCR constant domain.
  • Such mutations can include T48C in Ca, T57C in CP, or both.
  • cognate TCR constant domains comprise mutations so that, for example, one TCR constant domain (e.g., one of Ca and CP) comprises an introduced "cavity" (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having smaller side chains) and the other (e.g., the other of Ca and CP) comprises a compensatory "protuberance” (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having larger side chains), similar to a "knob-into-hole” configuration used to promote preferential pairing of antibody heavy chains.
  • TCR constant domain amino acids are mutated to introduce charge properties that favor pairing of the mutated constant domains.
  • Mutations to improve stability can include a mutation in the Ca transmembrane domain from the sequence LSVIGF to the sequence LLVIVL ("L-V-L" mutation; see Haga-Friedman et aL, J Immunol 755:5538-5546 (2012), the TCR mutations and mutant TCR constant domain sequences of which are incorporated herein by reference).
  • CD8 co-receptor means the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer.
  • the CD8 co-receptor assists in the function of cytotoxic T cells (CD8 + ) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21 :630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1 :81-88, 2004).
  • CD4 is an immunoglobulin co-receptor glycoprotein that assists the TCR in communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002)). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and includes four immunoglobulin domains (DI to D4) that are expressed at the cell surface. During antigen presentation, CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII P2, while the TCR complex binds MHCII al/pi).
  • TCR complex close proximity to the TCR complex allows CD4-associated kinase molecules to phosphorylate the immunoreceptor tyrosine activation motifs (IT AMs) present on the cytoplasmic domains of CD3.
  • IT AMs immunoreceptor tyrosine activation motifs
  • a TCR is found on the surface of T cells (or T lymphocytes) and associates with a CD3 complex.
  • CD3 is a multi -protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999) that is associated with antigen signaling in T cells.
  • the complex comprises a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of CD3( ⁇ chains.
  • the CD3y, CD3P, and CD3s chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3y, CD3P, and CD3s chains are negatively charged, which is believed to allow these chains to associate with positively charged regions of T cell receptor chains.
  • the intracellular tails of the CD3y, CD3P, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3( ⁇ chain has three.
  • ITAMs are important for the signaling capacity of a TCR complex.
  • CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
  • TCR complex refers to a complex formed by the association of CD3 with TCR.
  • a TCR complex can be composed of a CD3y chain, a CD3P chain, two CD3s chains, a homodimer of CD3( ⁇ chains, a TCRa chain, and a TCRP chain.
  • a TCR complex can be composed of a CD3y chain, a CD3P chain, two CD3s chains, a homodimer of CD3( ⁇ chains, a TCRy chain, and a TCRP chain.
  • a “component of a TCR complex”, as used herein, refers to a TCR chain (z.e., TCRa, TCRP, TCRy or TCR5), a CD3 chain (z.e., CD3y, CD35, CD3s or CD3Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCRP, CD3y, CD35, and two CD3s chains).
  • CAR Chimeric antigen receptor
  • CARs can include an extracellular portion comprising an antigen-binding domain (e.g., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived or obtained from a TCR specific for a cancer antigen, a scFv derived or obtained from an antibody, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol.
  • an antigen-binding domain e.g., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived or obtained from a TCR specific for a cancer antigen, a sc
  • CARs of the present disclosure that specifically bind to a P53 antigen (e.g, in the context of a peptide:HLA complex) comprise a TCR Va domain and a VP domain.
  • Any polypeptide of this disclosure can, as encoded by a polynucleotide sequence, comprise a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide).
  • Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell. In some contexts, signal peptides are from about 15 to about 22 amino acids in length.
  • Non-limiting examples of signal peptides include: a signal peptide native to a mammalian (e.g., human) TCRa or TCRP chain; MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.:322; from GM-CSF); the signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO.:323; from CD8a); the signal peptide MRPRLWLLLAAQLTVLHGNSV (SEQ ID NO.:324; from CD8P); and the signal peptide METDTLLLWVLLLWVPGSTG (SEQ ID NO : 325, from murine IgG kappa light chain).
  • MLLLVTSLLLCELPHPAFLLIP SEQ ID NO.:322; from GM-CSF
  • MALPVTALLLPLALLLHAARP SEQ ID NO.:323; from CD8a
  • MRPRLWLLLAAQLTVLHGNSV SEQ ID NO.:3
  • any suitable naturally occurring or engineered signal peptide can be employed. Certain signal peptides and characteristics of these are decribed in Owji et al., European Journal of Cell Biology 97(6):422-441 (2016), and in Ling et al. Front. Immunol. (2020) doi.org /10.3389/fimmu.2020.604318; the signal peptides of which are incorporated herein by reference. A signal peptide may be removed from the polypeptide during or once localization or secretion is completed.
  • a binding protein or fusion protein comprises, or is, a mature protein, or is or comprises a pre-protein.
  • a “linker” refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTv, scTCR) to a target molecule or retains signaling activity (e.g., TCR complex).
  • a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.
  • a linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids.
  • Exemplary linkers include glycine-serine linkers.
  • Antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells (e.g., T cells), or both.
  • An antigen immunologically competent cells (e.g., T cells), or both.
  • An antigen immunologically competent cells
  • An antigen may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen, or that endogenously (e.g., without modification or genetic engineering by human intervention) express a mutation or polymorphism that is immunogenic.
  • a “neoantigen,” as used herein, refers to a host cellular product containing a structural change, alteration, or mutation that creates a new antigen or antigenic epitope that has not previously been observed in the subject’s genome (z.e., in a sample of healthy tissue from the subject) or been "seen” or recognized by the host's immune system, which: (a) is processed by the cell’s antigen-processing and transport mechanisms and presented on the cell surface in association with an MHC (e.g., HLA) molecule; and (b) elicits an immune response (e.g., a cellular (T cell) response).
  • MHC e.g., HLA
  • Neoantigens may originate, for example, from coding polynucleotides having alterations (substitution, addition, deletion) that result in an altered or mutated product, or from the insertion of an exogenous nucleic acid molecule or protein into a cell, or from exposure to environmental factors (e.g., chemical, radiological) resulting in a genetic change. Neoantigens may arise separately from a tumor antigen or may arise from or be associated with a tumor antigen. "Tumor neoantigen” (or “tumor-specific neoantigen”) refers to a protein comprising a neoantigenic determinant associated with, arising from, or arising within a tumor cell or plurality of cells within a tumor.
  • Tumor neoantigenic determinants are found on, for example, antigenic tumor proteins or peptides that contain one or more somatic mutations or chromosomal rearrangements encoded by the DNA of tumor cells (e.g., pancreas cancer, lung cancer, colorectal cancers), as well as proteins or peptides from viral open reading frames associated with virus-associated tumors (e.g., cervical cancers, some head and neck cancers).
  • tumor cells e.g., pancreas cancer, lung cancer, colorectal cancers
  • proteins or peptides from viral open reading frames associated with virus-associated tumors e.g., cervical cancers, some head and neck cancers.
  • epitope includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain, or protein.
  • a cognate binding molecule such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain, or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • P53 antigen or “P53R175H antigen” or “P53R175H peptide” (P53R175H is also written as P53RI?5H) refers to a naturally or synthetically produced peptide portion of a P53 protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, or about 10 amino acids, up to about 20 amino acids, and comprising an R175H mutation, which peptide can form a complex with a MHC (e.g., HLA) molecule, and a binding protein of this disclosure specific for a P53 antigen:MHC (e.g., HLA) complex can specifically bind to such as complex.
  • An exemplary P53 antigen comprises, consists essentially of, or consists of a peptide having the amino acid sequence of SEQ ID NO.:3. In some embodiments, a peptide consists of the amino acid sequence of SEQ ID NO.:3.
  • MHC Major histocompatibility complex
  • MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non-covalently associated P2 microglobulin.
  • MHC class II molecules are composed of two transmembrane glycoproteins, a and P, both of which span the cell membrane. Each chain comprises two domains.
  • MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8 + T cells.
  • HLAs corresponding to "class I" MHC present peptides from inside the cell and include, for example, HLA-A, HLA-B, and HLA-C. Alleles include, for example, HLA A*2, such as HLA-A*02:01. HLAs corresponding to "class II" MHC present peptides from outside the cell and include, for example, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.
  • APC antigen presenting cells
  • MHC major histocompatibility complex
  • processed antigen peptides originating in the cytosol are generally from about 7 amino acids to about 11 amino acids in length and will associate with class I MHC (HLA) molecules
  • peptides processed in the vesicular system e.g., bacterial, viral
  • HLA class I MHC
  • peptides processed in the vesicular system will vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC (HLA) molecules.
  • P53R175H-specific binding protein refers to a protein or polypeptide, such as, for example, a TCR, a scTv, a scTCR, or CAR, that binds to a P53R175H peptide antigen (or to a P53R175H peptide antigen:HLA complex, e.g., on a cell surface), and does not bind a peptide that does not contain the P53R175H peptide antigen and does not bind to an HLA complex containing such a peptide.
  • a P53R175H peptide antigen comprises or consists of the amino acid sequence of SEQ ID NO:3.
  • Binding proteins of this disclosure will contain a binding domain specific for a target.
  • a "binding domain” also referred to as a "binding region” or “binding moiety” refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target (e.g., P53R175H peptide or P53R175H peptide:MHC (e.g., HLA-A*02:01) complex).
  • a target e.g., P53R175H peptide or P53R175H peptide:MHC (e.g., HLA-A*02:01) complex.
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (z.e., complex comprising two or more biological molecules), or other target of interest.
  • binding domains include immunoglobulin variable regions or single chain constructs comprising the same (e.g., single chain TCR (scTCR)).
  • a P53R175H-specific binding protein binds to a P53R175H peptide (or a P53R175H:HLA (e.g., HLA-A*02:01) complex) with a Kd of less than about 10' 8 M, less than about 10' 9 M, less than about IO' 10 M, less than about 10' 11 M, less than about 10' 12 M, or less than about 10' 13 M, or with an affinity that is about the same as, at least about the same as, or is greater than at or about the affinity exhibited by an exemplary P53R175H-specific binding protein provided herein, such as any of the P53R175H-specific TCRs provided herein, for example, as measured by the same assay.
  • a P53R175H-specific binding protein comprises a P53R175H-specific immunoglobulin superfamily binding protein or binding portion thereof.
  • binding protein e.g., TCR receptor
  • binding domain or fusion protein thereof
  • K a z.e., an equilibrium association constant of a particular binding interaction with units of 1/M
  • 10 5 M' 1 which equals the ratio of the on- rate [k on ]to the off-rate [k o ff] for this association reaction
  • Binding proteins or binding domains may be classified as “high affinity” binding proteins or binding domains (or fusion proteins thereof) or as “low affinity” binding proteins or binding domains (or fusion proteins thereof).
  • "High affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a K a of at least 10 7 M’ 1 , at least 10 8 M’ 1 , at least 10 9 M’ 1 , at least IO 10 M’ 1 , at least 10 11 M’ 1 , at least 10 12 M’ 1 , or at least 10 13 M’ 1 .
  • Bind affinity binding proteins or binding domains refer to those binding proteins or binding domains having a K a of up to 10 7 M’ 1 , up to 10 6 M’ 1 , or up to 10 5 M' 1 .
  • affinity can be defined as an equilibrium dissociation constant (Ka) of a particular binding interaction with units of M (e.g., 10' 5 M to IO’ 13 M).
  • a receptor or binding domain may have "enhanced affinity," which refers to a selected or engineered receptors or binding domain with stronger binding to a target antigen than a wild type (or parent) binding domain.
  • enhanced affinity may be due to a K a (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a Ka (dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (koff) for the target antigen that is less than that of the wild type binding domain, or a combination thereof.
  • binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51 :660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
  • a P53R175H-specific binding domain alone i.e., without any other portion of a P53R175H-specific binding protein
  • a P53R175H -specific binding domain includes a P53R175H-specific scTv or scTCR (e.g., single chain aPTCR proteins such as Va-L- VP, VP-L-Va, Va-Ca-L-Va, or Va-L-VP-CP, wherein Va and VP are TCRa and P variable domains respectively, Ca and CP are TCRa and P constant domains, respectively, and L is a linker, such as a linker described herein).
  • a P53R175H-specific scTv or scTCR e.g., single chain aPTCR proteins such as Va-L- VP, VP-L-Va, Va-Ca-L-Va, or Va-L-VP-CP, wherein Va and VP are TCRa and P variable domains respectively, Ca and CP are TCRa and P constant domains, respectively, and L is a linker, such as a linker
  • the term "functional avidity”, as used herein, refers to a biological measure or activation threshold of an in vitro immune cell (e.g., T cell, NK cell, NK-T cell) response to a given concentration of a ligand, wherein the biological measure may include or be cytokine production (e.g., IFN-y production, IL-2 production, etc.), cytotoxic activity, activation markers (e.g., CD137, Nur77) or proliferation.
  • cytokine production e.g., IFN-y production, IL-2 production, etc.
  • cytotoxic activity e.g., CD137, Nur77
  • T cells that biologically (immunologically) respond in vitro to a low antigen dose by, for example, expressing an activation marker, producing cytokines, exhibiting cytotoxic activity, or proliferating are considered to have high or higher functional avidity, while T cells having low or lower functional avidity require higher amounts of antigen before an immune response, similar to the high-avidity T cells, is elicited.
  • functional avidity is different from affinity and avidity. Affinity refers to the strength of any given bond between a binding protein and its antigen/ligand. Some binding proteins are multivalent and bind to multiple antigens - in this case, the strength of the overall connection is the avidity.
  • T cell functions e.g., proliferation, cytokines production, etc.
  • Factors that affect functional avidity can include (a) the affinity of a TCR for the pMHC- complex, that is, the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol.
  • the concentration of antigen needed to induce a half-maximum response (e.g., response in the form of production of a cytokine or expression of an activation marker by a host cell; or in the form of fluorescence intensity when binding to a labeled peptide:HLA multimer) between the baseline and maximum response after a specified exposure time is referred to as the "half maximal effective concentration" or "EC50".
  • the EC50 value is generally presented as a molar (moles/liter) amount, but it is often converted into a logarithmic value as follows - logio(EC50). For example, if the EC50 equals 1 pM (10‘ 6 M), the logio(EC50) value is -6.
  • the functional avidity of a binding protein of this disclosure comprises a measure of an ability of the binding protein to promote activation and/or IFNy production by host T cells, which can be measured using assays known in the art and described herein.
  • functional avidity will comprise a measure of the ability of the binding protein, upon binding to antigen, to activate a host cell, such as a T cell.
  • fusion proteins comprising a scTCR or scTv of the present disclosure linked to a constant domain of an antibody (e.g., a heavy chain constant domain or Fc polypeptide of IgG (1, 2, 3, 4), IgE, IgD, IgA, IgM, and variants thereof) or a fragment thereof (e.g., a fragment that, in some embodiments, retains binding to one or more Fc receptors, to Clq, to Protein A, to Protein G, or any combination thereof), and including immunoglobulin heavy chain monomers and multimers, such as Fc dimers; see, e.g., Wong et al., J. Immunol. 198: 1 Supp. (2017).
  • Variant Fc polypeptides comprising mutations that enhance, reduce, or abrogate binding to or by, e.g., FcRn or other Fc receptors, are known and are contemplated within this disclosure.
  • a binding protein or fusion protein (e.g., TCR, scTv, scTCR, CAR) of the present disclosure is expressed by a host cell (e.g., by a T cell, NK cell, or NK-T cell heterologously expressing the binding protein or fusion protein).
  • Avidity of such a host cell for a P53 peptide antigen (e.g., P53R175H peptide, such as SEQ ID NO:3) or P53 peptide antigen:HLA complex can be determined by, for example, exposing the host cell to the peptide, or to a peptide:HLA complex (e.g., organized as a tetramer), or to an antigen-presenting cell (APC) that presents the peptide to the host cell, optionally in a peptide:HLA complex, and then measuring an activity of the host cell, such as, for example, production or secretion of cytokines (e.g., IFN-y; TNFa); increased expression of host cell signaling or activation components (e.g., CD137 (4-1BB), Nur77); proliferation of the host cell; or killing of the APC (e.g., using a labeled-chromium release assay).
  • cytokines e.g., IFN-y; T
  • nucleic acid or “nucleic acid molecule” or “polynucleotide” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, polynucleotides, fragments thereof generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and also to fragments generated by any of ligation, scission, endonuclease action, or exonuclease action.
  • the nucleic acids of the present disclosure are produced by PCR.
  • Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • Analogs of phosphodiester linkages include phosphonothioate, phosphonodithioate, phosphonoselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • Nucleic acid monomers may comprise phosphorothioate linkages, phosphorodithioate linkages, or phosphoroselenoate linkages, or any combination thereof.
  • Nucleic acid molecules can be either single-stranded or double-stranded.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • a nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).
  • the terms "recombinant”, “engineered”, and “modified” refer to a cell, microorganism, nucleic acid molecule, polypeptide, protein, plasmid, or vector that has been modified by introduction of an exogenous nucleic acid molecule, or refers to a cell or microorganism that has been genetically engineered by human intervention — that is, modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering.
  • Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material.
  • modifications include those in coding regions or functional fragments thereof of heterologous or homologous polypeptides from a reference or parent molecule.
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a mutation is a substitution of one or three codons or amino acids, a deletion of one to about 5 codons or amino acids, or a combination thereof.
  • a “conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433 at page 10; Lehninger, Biochemistry, 2 nd Edition; Worth Publishers, Inc. NY, NY, pp.71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).
  • a variant of a reference amino acid sequence comprises one or more conservative amino acid substitution.
  • proteins e.g., binding protein, immunogenic peptide
  • proteins comprise a variant sequence as compared to a reference sequence (e.g., a variant TCR CDR3P as compared to a reference TCR CDR3P disclosed herein).
  • a "variant" amino acid sequence, peptide, or polypeptide refers to an amino acid sequence (or peptide or polypeptide) having one or two amino acid substitutions, deletions, or insertions as compared to a reference amino acid sequence.
  • a variant amino acid sequence, peptide, or polypeptide retains substantially a same functionality (e.g., binding specificity and affinity for a peptide:HLA complex) as the reference molecule; for example, a variant TCR fragment as disclosed herein retains about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, a least about 99%, or 100% of the antigen-binding specificity and affinity as compared to a reference TCR binding fragment.
  • altered domain refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild type TCRa chain, TCRP chain, TCRa constant domain, TCRP constant domain) of at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%).
  • Altered domains or altered proteins or derivatives can include those based on all possible codon choices for the same amino acid and codon choices based on conservative amino acid substitutions.
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (ala; A), serine (ser; S), threonine (thr; T); 2) aspartic acid (asp; D), glutamic acid (glu; E); 3) asparagine (asn; N), glutamine (gin; Q); 4) arginine (arg; R), lysine (lys; K); 5) Isoleucine (ile; I), leucine (L), methionine (met; M), valine (val; V); and 6) phenylalanine (phe; F), tyrosine (tyr; Y), tryptophan (trp; W).
  • construct refers to any polynucleotide that contains a recombinant nucleic acid molecule.
  • a "transgene” or “transgene construct” refers to a construct that contains two or more genes operably linked in an arrangement that is not found in nature.
  • operably- linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably-linked with a coding sequence when it can affect the expression of that coding sequence (z.e., the coding sequence is under the transcriptional control of the promoter).
  • Unlinked means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
  • the genes present in a transgene are operably linked to an expression control sequence (e.g., a promoter).
  • a construct e.g., a transgene
  • a vector e.g., a bacterial vector, a viral vector
  • a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
  • Vectors can be, for example, plasmids, cosmids, viruses, an RNA vector or a linear or circular DNA or RNA molecule that can include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules.
  • Exemplary vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acid molecules to which they are linked (expression vectors).
  • Vectors useful in the compositions and methods of this disclosure are described further herein.
  • expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof.
  • the term "introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection", or “transformation”, or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule can be incorporated into the genome of a cell (e.g., a chromosome, a plasmid, a plastid, or a mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a cell e.g., a chromosome, a plasmid, a plastid, or a mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • heterologous or exogenous nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell but can be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.
  • the source of the heterologous or exogenous nucleic acid molecule, construct or sequence can be from a different genus or species.
  • a heterologous or exogenous nucleic acid molecule is added (z.e., not endogenous, or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can be present in multiple copies.
  • heterologous refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.
  • a cell comprising a "modification” or a “heterologous" polynucleotide or binding protein includes progeny of that cell, regardless of whether the progeny were themselves transduced, transfected, or otherwise manipulated or changed.
  • heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
  • a host cell can be modified to express one or more heterologous or exogenous nucleic acid molecule encoding a desired TCR specific for a P53R175H antigen peptide (e.g., TCRa and TCR0) and optionally, as disclosed herein, also encoding a CD8 co-receptor polypeptide comprising an a chain, a P chain, or a portion thereof, such as an extracellular portion capable of binding to MHC.
  • a P53R175H antigen peptide e.g., TCRa and TCR0
  • CD8 co-receptor polypeptide comprising an a chain, a P chain, or a portion thereof, such as an extracellular portion capable of binding to MHC.
  • the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not necessarily to the number of separate nucleic acid molecules introduced into a host cell.
  • endogenous refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated, or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell.
  • an endogenous control sequence from a first gene e.g., a promoter, translational attenuation sequences
  • a second native gene or nucleic acid molecule can be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.
  • homologous refers to a molecule or activity found in or derived from a host cell, species, or strain.
  • a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene, and can optionally have an altered expression level, a different sequence, an altered activity, or any combination thereof.
  • Sequence identity refers to the percentage of amino acid residues or nucleobases in one sequence that are identical with the amino acid residues or nucleobases (respectively) in a reference 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.
  • the percentage sequence identity values can be generated using the NCBI BLAST 2.0 software as defined by Altschul et al. (1997), NucL Acids Res. 25:3389-3402, with the parameters set to default values. Additionally or alternatively, the degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs designed for this purpose, such as global or local alignment algorithms.
  • Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm.
  • a global alignment algorithm such as a Needleman and Wunsch algorithm, can be used to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Default settings can be used.
  • scoring matrices can be used that assign positive scores for some non-identical amino acids (e.g., conservative amino acid substitutions, amino acids with similar physio-chemical properties, and/or amino acids that exhibit frequent substitutions in orthologs, homologs, or paralogs).
  • Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.
  • Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and are preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C.
  • Nucleic acid molecule variants retain the capacity to encode a binding protein or a binding domain thereof having a functionality described herein, such as binding a target molecule.
  • the term "variant" as used herein refers to at least one fragment of the full-length sequence referred to, more specifically one or more amino acid or nucleic acid sequence which is, relative to the full-length sequence, truncated at one or both termini by one or more amino acids.
  • Such a fragment includes or encodes for a peptide having at least 6, least 7, least 8, least 10, least 12, least 15, least 20, least 25, least 50, least 75, least 100, least 150, or least 200 successive amino acids of the original sequence or a variant thereof.
  • the total length of the variant may be at least 6, least 7, least 8, least 9, least 10, least 11, least 12, least 20, least 25, least 30, least 40, least 50, least 60, least 70, least 80, least 90, least 100, or more amino acids.
  • the term "variant" relates not only to at least one fragment, but also to a polypeptide or a fragment thereof including amino acid sequences that are at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the reference amino acid sequence referred to or the fragment thereof, wherein amino acids other than those essential for the biological activity or the fold or structure of the polypeptide are deleted or substituted, one or more such essential amino acids are replaced in a conservative manner, and/or amino acids are added such that the biological activity of the polypeptide is preserved.
  • the state of the art includes various methods that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity (see, e.g., Arthur Lesk (2008), Introduction to Bioinformatics, Oxford University Press, 2008, 3rd edition).
  • the Clustal W software can be used using default settings (Larkin, M. A., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948). Other methods and programs for these purposes are known in the art and described herein.
  • variants may, in addition, include chemical modifications, for example, isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation and the like.
  • chemical modifications for example, isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation and the like.
  • the term "variant" of a nucleic acid molecule includes nucleic acids the complementary strand of which hybridizes, for example, under stringent conditions, to the reference or wild type nucleic acid.
  • Stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and in general is an empirical calculation dependent on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes less so.
  • Hybridization generally depends on the ability of denatured DNA to reanneal to complementary strands present in an environment below their melting temperature: the higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which may be used.
  • stringent conditions are applied for any hybridization, z.e., hybridization occurs only if the probe is 70% or more identical to the target sequence.
  • Probes having a lower degree of identity with respect to the target sequence may hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions, for example, lowering the concentration of salt to 2* SSC or, optionally and subsequently, to 0.5* SSC, while the temperature is, for example, about 50 °C-68 °C, about 52 °C-68 °C, about 54 °C-68 °C, about 56 °C-68 °C, about 58 °C-68 °C, about 60 °C-68 °C, about 62 °C-68 °C, about 64 °C-68 °C, or about 66 °C-68 °C.
  • the temperature is about 64 °C-68 °C or about 66 °C-68 °C. It is possible to adjust the concentration of salt to 0.2* SSC or even O.lx SSC. Nucleic acid sequences having a degree of identity with respect to the reference or wild type sequence of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% may be isolated.
  • variant of a nucleic acid sequence refers to any nucleic acid sequence that encodes the same amino acid sequence and variants thereof as the reference nucleic acid sequence, in line with the degeneracy of the genetic code.
  • a “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs, in some contexts slightly, in composition (e.g., one base, atom or functional group is different, added, or removed; or one or more amino acids are mutated, inserted, or deleted), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% level of activity of the parent polypeptide.
  • a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant), avidity, or activation of a host cell.
  • binding affinity e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant
  • a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, motif, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 at least %, 99.9%, or at least 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).
  • a biological benefit e.g., effector function
  • a “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity), such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release).
  • Functional variants of specifically disclosed binding proteins and polynucleotides are contemplated.
  • altered domain refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild type TCRa chain, TCRP chain, TCRa constant domain, or TCRP constant domain) of at least 85% e.g, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%).
  • a wild type motif, region, domain, peptide, polypeptide, or protein e.g., a wild type TCR
  • the present disclosure provides a binding protein, comprising a T cell receptor (TCR) a chain variable (Va) domain and a TCR P chain variable (VP) domain, wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:3.
  • the HLA comprises an HLA-A*2, optionally HLA-A*02:01.
  • the binding protein can be heterologously expressed by a human immune system cell, such as, for example, a T cell.
  • the Va domain and/or the VP domain are each independently human, humanized, or chimeric, and are preferably each human.
  • Binding proteins, compositions, and methods disclosed herein can utilize a Va domain, VP domain, or CDRs therefrom derived from a human subject, for example, from sequencing of an isolated T cell or population thereof from a human subject.
  • TCR Va domains, VP domains, and CDRs therefrom isolated from a human subject can have advantageous properties over variable domains and CDRs from other sources, such as mice transgenic for a single human HLA allele.
  • Va domains, VP domains, and CDRs derived from a human subject can have undergone negative thymic selection against substantially the whole human peptidome presented by a full set of human HLA molecules in vivo, which can reduce the likelihood that the binding protein is cross-reactive to other human self-antigens.
  • a binding protein comprises one or more variable domains or one or more CDRs derived from (e.g., identified in) a T cell of a subject (e.g., a human subject) having a disease, such as a cancer.
  • a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a human subject having a cancer as disclosed herein.
  • a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a subject (e.g., a human subject) having a disease associated with a P53 R175H mutation.
  • a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a subject (e.g., a human subject) with a cell that comprises a P53 R175H mutation.
  • a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a healthy subject (e.g., a healthy human subject).
  • a healthy subject lacks a specific pathological diagnosis (e.g., disease diagnosis, such as a cancer diagnosis).
  • a healthy subject lacks a specific pathological diagnosis, but comprises a different pathological diagnosis, for example, lacks a cancer diagnosis but comprises a diagnosis of hypertension or type II diabetes.
  • Presently disclosed binding proteins are capable of being heterologously expressed by host cells, such as, for example, human immune cells, such as T cells. Furthermore, expression of a presently disclosed binding protein can confer advantageous properties upon a host cell; e.g., having binding specificity for a P53 antigen:HLA complex of the present disclosure, improved activation, proliferation, or killing activity in the presence of a P53 antigen (e.g., containing an R175H mutation, e.g., SEQ ID N0:3):HLA presenting tumor cell, or the like.
  • a P53 antigen e.g., containing an R175H mutation, e.g., SEQ ID N0:3:HLA presenting tumor cell, or the like.
  • the binding protein when expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and/or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell is capable of specifically killing a HLA-A*02:01 + tumor cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3. Killing of a target cell can be determined, for example, with the Incucyte® bioimaging platform (Essen Bioscience).
  • this platform uses activated caspase and labelled (e.g., RapidRed or NucRed) tumor cell signals, wherein overlap is measured and increased overlap area equals tumor cell death by apoptosis. Killing can also be determined using a 4-hour assay in which target cells are loaded with labeled chromium ( 51 Cr), and 51 Cr in the supernatant is measured following 4-hour co-incubation with an immune cell expressing a binding protein of the present disclosure. In certain embodiments, a killing assay can be performed using an effectortarget cell ratio of 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10:1, 20: 1, 25: 1, 50: 1, or 100: 1, or the like.
  • a killing assay can be performed using an effectortarget cell ratio of 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10:1, 20: 1, 25: 1, 50: 1, or 100: 1, or the like.
  • the binding protein when expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and/or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell has elevated expression of Nur77 when in the presence of a HLA- A*02:01 + tumor cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3, optionally in the further presence of exogenous IFN-y, wherein the Nur77 expression is elevated as compared to: (i) Nur77 expression by a reference immune cell (i.e., of the same cell type as, and otherwise phenotypically and/or genotypically at least substantially identical or functionally equivalent to, the immune cell expressing the binding protein) not expressing the binding protein, when the reference immune cell is in the presence of the tumor cell; and/or (ii) Nur77 expression by the immune cell expressing the binding protein when not in the presence of the tumor cell and/or when a reference immune cell (i) a
  • Nur77 can be determined, for example, using a transgenic expression construct comprising a Nur77 locus operably linked to a sequence encoding a reporter construct, e.g., GFP or dTomato (see Ahsouri and Weiss, J Immunol 79S(2):657-668 (2017)).
  • a transgenic expression construct comprising a Nur77 locus operably linked to a sequence encoding a reporter construct, e.g., GFP or dTomato (see Ahsouri and Weiss, J Immunol 79S(2):657-668 (2017)).
  • the binding protein when expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and/or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell has elevated expression of CD137 (also known as 4-1BB) when in the presence of a HLA-A*02 + tumor cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3, optionally in the further presence of exogenous IFN-y, wherein the CD137 expression is elevated as compared to: (i) CD137 expression by a reference immune cell not expressing the binding protein, when the reference immune cell is in the presence of the tumor cell; and/or (ii) CD137 expression by the immune cell expressing the binding protein when not in the presence of the tumor cell and/or when not in the presence of an antigen-presenting cell expressing a peptide:HLA complex, wherein the peptide comprises or consists of the amino acid sequence
  • CD137 expression can be determined using, for example, flow cytometry using a labeled anti-CD137 antibody.
  • CD137 is measured following a 16-hour assay in which the immune cell is co-incubated with or stimulated with peptide or a target cell expressing the peptide.
  • the binding protein is encoded by a polynucleotide that is heterologous to the immune cell;
  • the immune cell comprises a human CD8 + T cell, a human CD4+ T cell, or both;
  • the tumor cell expressing a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3 is HLA-A*02:01 + ; and/or
  • the tumor cell comprises a KSM26 cell or a KLE cell.
  • the binding protein is capable of binding to the peptide:HLA complex independent of, or in the absence of, CD8.
  • CD8-independent binding can be determined by expressing the binding protein in a CD8-negative cell (e.g., a CD4 + T cell, a Jurkat cell, or the like) and identifying binding of the cell to a target.
  • CD8- independent binding can be determined by exposing the binding protein to a peptide HLA complex wherein the HLA has been engineered to abrogate binding to CD8.
  • a binding protein that comprises:
  • TCR T cell receptor
  • Va chain variable domain
  • CDR3a complementarity determining region 3 amino acid sequence set forth in any one of SEQ ID NOs:35, 34, 8, 9, 21, 22, 47, 48, 60, 61, 73, 74, 129, 130, 142, 143, 155, 156, 168, and 169, or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions; and/or
  • a TCR P chain variable (VP) domain comprising the CDR3P amino acid sequence set forth in any one of SEQ ID NOs:41, 40, 14, 15, 27, 28, 53, 54, 66, 67, 79, 80, 94, 95, 135, 136, 148, 149, 161, 162, 174, and 175, or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions, wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises or consists of the amino acid sequence HMTEVVRHC (SEQ ID NO:3) and wherein the HLA comprises an HLA-A*2.
  • the HLA comprises HLA-A*02:01.
  • the Va domain and/or the VP domain is human, humanized, or chimeric, and is preferably human.
  • the binding protein comprises the CDR3a and CDR3P amino acid sequences set forth in SEQ ID NOs: (i) 35 and 41, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (ii) 22 and 28, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iii) 9 and 15, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iv) 48 and 54, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (v) 61 and 67, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vi) 74 and 80, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vii) 8 and 14, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (viii) 21 and 27, respectively,
  • the binding protein comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO:32, 19, 6, 45, 58, 71, 127, 140, 153, or 166, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO:33, 7, 20, 46, 59, 72, 128, 141, 154, or 167, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 38, 12, 25, 51, 64, 77, 133, 146, 159, or 172, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO:39, 13, 26, 52, 65, or 78, 134, 147,
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs:
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 32, 33, 35, 38, 39, and 41, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 32, 33, 34, 38, 39, and 40, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 71, 72, 74, 77, 78, and 80, respectiv, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 71, 72, 73, 77, 78, and 79, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 127, 128, 129, 133, 134, and 135, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 127, 128, 130, 133, 134, and 136, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 140, 141, 142, 146, 147, and 148, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 140, 141, 143, 146, 147, and 149, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 153, 154, 155, 159, 160, and 161, respectively.
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 153, 154, 156, 159, 160, and 162, respectively. In some embodiments, the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 166, 167, 168,
  • the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 166, 167, 169, 172,
  • Table 1 shows CDR amino acid sequences (IMGT definition) of certain binding proteins (TCRs).
  • Table 2 CDR3 amino acid sequences (IMGT junction definition) of the binding proteins in
  • a consensus CDR3P (TCR5 and TCR6, IMGT junction definition) is provided in SEQ ID NO:94.
  • a consensus CDR3P (TCR5 and TCR6, IMGT definition) is provided in SEQ ID NO:95.
  • a binding protein comprises the CDR3P amino acid sequence set forth in SEQ ID NO.:94 or 95.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, as shown in Table 1.
  • the binding protein comprises the Va domain CDR1 and CDR2 and the VP domain CDR1 and CDR2 of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, as shown in Table 1, and further comprises the corresponding CDR3a and CDR3P as shown in Table 2.
  • the binding protein comprises the Va domain CDRs (1-3) and/or the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and/or the VP CDRs are as identified by the IMGT scheme.
  • CDR3a and CDR3P are as identified by the IMGT scheme.
  • CDR3a and CDR3P are as identified by the IMGT junction scheme.
  • the binding protein comprises the Va domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the IMGT scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR3 and FR4 are as identified by the IMGT junction scheme.
  • the binding protein comprises the VP domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the IMGT scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR3 and FR4 are as identified by the IMGT junction scheme.
  • the binding protein comprises the Va domain CDRs (1-3) and/or the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and/or the VP CDRs are as identified by the Kabat scheme.
  • the binding protein comprises the Va domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the Kabat scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR Va domain framework region
  • the binding protein comprises the VP domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the IMGT scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR VP domain framework region
  • the binding protein comprises the Va domain CDRs (1-3) and/or the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and/or the VP CDRs are as identified by the AHo scheme.
  • the binding protein comprises the Va domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the AHo scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR Va domain framework region
  • the binding protein comprises the VP domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the AHo scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR VP domain framework region
  • the binding protein comprises the Va domain CDRs (1-3) and/or the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and/or the VP CDRs are as identified by the Chothia scheme.
  • the binding protein comprises the Va domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the Chothia scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR Va domain framework region
  • the binding protein comprises the VP domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the Chothia scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR VP domain framework region
  • the binding protein comprises the Va domain CDRs (1-3) and/or the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and/or the VP CDRs are as identified by the Enhanced Chothia scheme.
  • the binding protein comprises the Va domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the Enhanced Chothia scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR Va domain framework region
  • the binding protein comprises the VP domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the Enhanced Chothia scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR VP domain framework region
  • the binding protein comprises the Va domain CDRs (1-3) and/or the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and/or the VP CDRs are as identified by the EU scheme.
  • the binding protein comprises the Va domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the EU scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR Va domain framework region
  • the binding protein comprises the VP domain framework region (FR)1, FR2, FR3, and/or FR4 (as identified by the EU scheme) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, or comprises a variant of the FR1, FR2, FR3, or FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the FR1, FR2, FR3, or FR4.
  • FR VP domain framework region
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and the VP CDRs are as identified by the IMGT scheme.
  • CDR3a and CDR3P are as identified by the IMGT scheme.
  • CDR3a and CDR3P are as identified by the IMGT junction scheme.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and the VP CDRs are as identified by the Kabat scheme.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and the VP CDRs are as identified by the AHo scheme.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and the VP CDRs are as identified by the Chothia scheme.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F38, TCR F40, TCR G41, or TCR G46, wherein the Va domain CDRs and the VP CDRs are as identified by the EU scheme.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR4, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR4 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR4 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR4 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR4, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR4 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR4 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR4 (according to IMGT), or
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR4, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR4 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR4 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR4 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR4, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR4 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR4 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR4 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR4, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR4 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR4 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR4 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR4, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR4 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR4 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR4 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR4, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR4 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR4 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR4 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR2 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR2 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR2 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR2 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR2 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR2 (according to IMGT), or
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR2 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR2 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR2 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR2 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR2 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR2 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR2 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR2 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR2 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR2 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR2 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR2 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR2, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR2 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR2 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR2 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR3, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR3 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR3 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR3 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR3, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR3 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR3 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR3 (according to IMGT), or
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR3, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR3 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR3 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR3 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR3, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR3 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR3 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR3 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR3, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR3 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR3 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR3 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR3, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR3 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR3 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR3 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR3, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR3 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR3 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR3 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR5, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR5 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR5 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR5 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR5, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR5 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR5 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR5 (according to IMGT), or
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR5, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR5 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR5 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR5 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR5, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR5 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR5 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR5 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR5, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR5 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR5 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR5 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR5, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR5 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR5 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR5 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR5, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR5 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR5 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR5 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR6, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR6 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR6 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR6 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR6, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR6 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR6 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR6 (according to IMGT), or
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR6, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR6 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR6 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR6 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR6, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR6 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR6 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR6 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR6, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR6 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR6 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR6 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR6, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR6 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR6 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR6 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR6, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR6 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR6 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR6 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR8, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR8 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR8 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR8 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR8, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR8 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR8 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR8 (according to IMGT), or
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR8, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR8 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR8 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR8 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR8, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR8 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR8 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR8 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR8, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR8 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR8 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR8 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR8, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR8 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR8 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR8 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five,
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR8, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR8 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR8 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR8 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F38, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F38 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F38 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F38 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F38, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F38 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F38 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F38 (
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F38, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F38 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F38 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F38 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F38, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F38 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F38 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F38 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F38, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F38 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F38 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F38 (according to Enhanced Chothia), or a variant thereof comprising at most one
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F38, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F38 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F38 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F38 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F38, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F38 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F38 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F38 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F41, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F41, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F41 (
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F41, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F41 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F41 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F41 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F41, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F41 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F41 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F41 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F41, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F41 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F41 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F41 (according to Enhanced Chothia), or a variant thereof comprising at most one
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F41, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F41 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F41 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F41 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR F41, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR F41 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR F41 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR F41 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G41, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G41, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G41 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G41 (
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G41, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G41 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G41 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G41 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G41, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G41 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G41 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G41 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G41, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G41 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G41 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G41 (according to Enhanced Chothia), or a variant thereof comprising at most one
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G41, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G41 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G41 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G41 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G41, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G41 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G41 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G41 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G46, wherein the Va CDRs and the VP CDRs are as identified by the IMGT scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G46 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G46 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G46 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G46, wherein the Va CDR 1 and 2 and the VP CDR 1 and 2 are as identified by the IMGT scheme and the CDR3a and CDR3P are as identified by the IMGT junction scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G46 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G46 (according to IMGT), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G46 (
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G46, wherein the Va CDRs and the VP CDRs are as identified by the Kabat scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G46 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G46 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G46 (according to Kabat), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G46, wherein the Va CDRs and the VP CDRs are as identified by the Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G46 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G46 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G46 (according to Chothia), or a variant thereof comprising at most one, at most two, at most three
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G46, wherein the Va CDRs and the VP CDRs are as identified by the Enhanced Chothia scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G46 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G46 (according to Enhanced Chothia), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G46 (according to Enhanced Chothia), or a variant thereof comprising at most one
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G46, wherein the Va CDRs and the VP CDRs are as identified by the AHo scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G46 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G46 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G46 (according to AHo), or a variant thereof comprising at most one, at most two, at most three, at most four
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • the binding protein comprises the Va domain CDRs (1-3) and the VP domain CDRs (1-3) of TCR G46, wherein the Va CDRs and the VP CDRs are as identified by the EU scheme, and the binding protein further comprises: in the Va domain, the Va FR1 of TCR G46 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and/or deletions relative to the Va FR1; in the Va domain, the Va FR2 of TCR G46 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions, insertions, and/or deletions relative to the Va FR2; in the Va domain, the Va FR3 of TCR G46 (according to EU), or a variant thereof comprising at most one, at most two, at most three, at most four, at most five
  • a variant CDRla, CDR2a, CDRip, and/or CDR2P comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid amino acid susbstitutions, insertions, and/or deletions relative to the CDRla, CDR2a, CDRip, or CDR2P may be used, and/or a CDRla, CDR2a, CDRip, and/or CDR2P from a different binding protein exemplified herein may be used.
  • Amino acid insertion(s), substitution(s), and/or deletions in a CDR, a FR, or a constant domain can be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.
  • Amino acid substitution(s) can comprise or consist of conservative amino acid substitution(s).
  • the Va domain and/or the VP domain can be human, humanized, or chimeric, and is preferably human.
  • Amino acids associated with "dominant” (vs. endogenous TCR) expression of engineered TCRs include:
  • VP o In framework region 1, a R at IMGT position 9 and/or a Y at IMGT position 10; and o In framework region 2, a Q at IMGT position 43.
  • introducing one or more of the above amino acids into a TCR V-region at the indicated position(s) can in some cases generate a variant with improved expression and optionally function.
  • the Va domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs:31, 5, 18, 44, 57, 70, 126, 139, 152, and 165; and/or
  • the VP domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs:37, 11, 24, 50, 63, 76, 132, 145, 158, and 171.
  • the Va and the VP domain comprise, consist essentially of, or consist of amino acid sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences set forth in SEQ ID NOs: (i) 31 and 37, respectively; (ii) 5 and 11, respectively; (iii) 18 and 24, respectively; (iv) 44 and 50, respectively; (v) 57 and 63, respectively; (vi) 70 and 76, respectively; (vii) 126 and 132, respectively; (viii) 139 and 145, respectively; (ix) 152 and 158, respectively; or (x) 165 and 171, respectively.
  • the Va domain and the VP domain comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOs: (i) 31 and 37, respectively; (ii) 5 and 11, respectively; (iii) 18 and 24, respectively; (iv) 44 and 50, respectively; (v) 57 and 63, respectively; (vi) 70 and 76, respectively; (vii) 126 and 132, respectively; (viii) 139 and 145, respectively; (ix) 152 and 158, respectively; or (x) 165 and 171, respectively.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:31 and the VP domain comprises, consists essentially of, or consists of amino acid sequence set forth in SEQ ID NO:37.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO:31 and the VP domain comprises amino acid sequence set forth in SEQ ID NO:37.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO:31 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO:37.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO:31 and the VP domain consists of amino acid sequence set forth in SEQ ID NO:37.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:70 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO:76.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO:70 and the VP domain comprises amino acid sequence set forth in SEQ ID NO:76.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO:70 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 76.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO:70 and the VP domain consists of amino acid sequence set forth in SEQ ID NO:76.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 5 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO: 11.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO:5 and the VP domain comprises amino acid sequence set forth in SEQ ID NO: 11.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 5 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 11.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO: 5 and the VP domain consists of amino acid sequence set forth in SEQ ID NO: 11.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 18 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO:24.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO: 18 and the VP domain comprises amino acid sequence set forth in SEQ ID NO:24.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 18 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO:24.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO: 18 and the VP domain consists of amino acid sequence set forth in SEQ ID NO:24.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:44 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO: 50.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO:44 and the VP domain comprises amino acid sequence set forth in SEQ ID NO:50.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO:44 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 50.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO:44 and the VP domain consists of amino acid sequence set forth in SEQ ID NO: 50.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:57 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO:63.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO:57 and the VP domain comprises amino acid sequence set forth in SEQ ID NO:63.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 57 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 63.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO:57 and the VP domain consists of amino acid sequence set forth in SEQ ID NO: 63.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 126 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO: 132.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO: 126 and the VP domain comprises amino acid sequence set forth in SEQ ID NO: 132.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 126 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 132.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO: 126 and the VP domain consists of amino acid sequence set forth in SEQ ID NO: 132.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 139 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO: 145.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO: 139 and the VP domain comprises amino acid sequence set forth in SEQ ID NO: 145.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 139 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 145.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO: 139 and the VP domain consists of amino acid sequence set forth in SEQ ID NO: 145.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 152 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO: 158.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO: 152 and the VP domain comprises amino acid sequence set forth in SEQ ID NO: 158.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 152 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 158.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO: 152 and the VP domain consists of amino acid sequence set forth in SEQ ID NO: 158.
  • the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 165 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO: 171.
  • the Va domain comprises the amino acid sequence set forth in SEQ ID NO: 165 and the VP domain comprises amino acid sequence set forth in SEQ ID NO: 171.
  • the Va domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 165 and the VP domain consists essentially of amino acid sequence set forth in SEQ ID NO: 171.
  • the Va domain consists of the amino acid sequence set forth in SEQ ID NO: 165 and the VP domain consists of amino acid sequence set forth in SEQ ID NO: 171.
  • the binding protein comprises the Va domain amino acid sequence and the VP domain amino acid sequence of TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR F41, TCR F40, TCR G41, or TCR G46, as shown in Table 3.
  • the binding protein can further comprise a TCR a chain constant domain (Ca) and/or a TCR P chain constant domain (CP).
  • a TCR a chain constant domain Ca
  • CP TCR P chain constant domain
  • the Va and the Ca together comprise a TCRa chain and the VP and the CP together comprise a TCRP chain.
  • the Ca comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs:96-98.
  • the CP comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs:99-102 and 245.
  • the Ca and the CP comprise, consist essentially of, or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequences set forth in SEQ ID NOs: 97 and 100, 102, or 245 respectively.
  • the binding protein comprises, consists essentially of, or consists of a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise, consist essentially of, or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequences set forth in: (i) SEQ ID NOs:86 and 87, respectively; (ii) SEQ ID NOs: 82 and 83, respectively; (iii) SEQ ID NOS.:84 and 85, respectively; (iv) SEQ ID NOs:88 and 89, respectively; (v) SEQ ID NOs:90 and 91, respectively; (vi) SEQ ID NOs: 92 and 93, respectively; (vii) SEQ ID NOs: 177 and
  • the binding protein comprises (i) a TCRa chain having the amino acid sequence of SEQ ID NO: 86 and (ii) a TCRP chain having the amino acid sequence of SEQ ID NO:87. In some embodiments, the binding protein comprises (i) a TCRa chain consisting essentially of the amino acid sequence of SEQ ID NO:86 and (ii) a TCRP chain consisting essentially of the amino acid sequence of SEQ ID NO:87. In some embodiments, the binding protein comprises (i) a TCRa chain consisting of the amino acid sequence of SEQ ID NO:86 and (ii) a TCRP chain consisting of the amino acid sequence of SEQ ID NO:87.
  • the binding protein comprises (i) a TCRa chain having the amino acid sequence of SEQ ID NO: 92 and (ii) a TCRP chain having the amino acid sequence of SEQ ID NO:93.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs:86 and 87, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs:86 and 87, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs:86 and 87, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs:82 and 83, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs:82 and 83, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs:82 and 83, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs:84 and 85, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs:84 and 85, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs:84 and 85, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs:88 and 89, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs:88 and 89, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs:88 and 89, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs:90 and 91, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs:90 and 91, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs:90 and 91, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs:92 and 83, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs:92 and 93, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs:92 and 93, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs: 177 and 178, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs: 177 and 178, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs: 177 and 178, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs: 179 and 180, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs: 179 and 180, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs: 179 and 180, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs: 181 and 182, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs: 181 and 182, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs: 181 and 182, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise the amino acid sequences of SEQ ID NOs: 183 and 184, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist essentially of the amino acid sequences of SEQ ID NOs: 183 and 184, respectively.
  • the binding protein comprises a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain consist of the amino acid sequences of SEQ ID NOs: 183 and 184, respectively.
  • the binding protein comprises a TCR, a single-chain TCR (scTCR), a single-chain T cell receptor variable fragment (scTv), or a chimeric antigen receptor (CAR).
  • scTCR single-chain TCR
  • scTv single-chain T cell receptor variable fragment
  • CAR chimeric antigen receptor
  • the binding protein comprises a TCR.
  • the binding protein is a TCR selected from Table 4. Table 4. TCR chain amino acid sequences (SEQ ID NOs.) of certain binding proteins according to Table 1. It will be understood that alternative TCR constant domains to those contained within the
  • TCR chain amino acid sequences shown in Table 4 may be used, e.g. a constant domain may be provided that does not contain a serine-to-cysteine or threonine-to-cysteine mutation, and/or that comprises an LVL mutation as described herein, and/or a TRBC1 constant domain (or cysteine- engineered variant thereof) may be used in place of a TRBC2 constant domain (or cysteine- engineered variant thereof), or a a TRBC2 constant domain (or cysteine-engineered variant thereof) may be used in place of a TRBC1 constant domain (or cysteine-engineered variant thereof).
  • Table 5 SEQ ID NOs. of amino acid sequences (TCRp-GSG-P2A-TCRa) encoded by expression constructs.
  • an expression construct encoding a binding protein e.g., any of TCRs 2-6 8, F38, F40, G41, and G46, e.g. as in Table 5) can comprise a different organization, encode a different self-cleaving peptide, or both.
  • an expression construct can encode TCRa-2A-TCRP, and/or can comprise a T2A, P2A, E2A, or F2A self-cleaving peptide, optionally with a N-terminal linker such as a GSG linker.
  • a binding protein can comprise a TCR, a single-chain TCR (scTCR), a scTv, or a chimeric antigen receptor (CAR).
  • TCR TCR
  • scTCR single-chain TCR
  • CAR chimeric antigen receptor
  • a binding protein comprises a soluble TCR, optionally fused to a binding domain (e.g., a scFv) specific for a CD3 protein. See Elie Dolgin, Nature Biotechnology 40 AM- 449 (2022).
  • a polynucleotide encoding a binding protein can further comprise: (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor p chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).
  • coexpression or concurrent expression of a binding protein and a CD8 co-receptor protein or portion thereof functional to bind to an HLA molecule may improve one or more desired activity of a host cell (e.g., immune cell, such as a T cell, optionally a CD4 + T cell) as compared to expression of the binding protein alone.
  • a host cell e.g., immune cell, such as a T cell, optionally a CD4 + T cell
  • the binding protein-encoding polynucleotide and the CD8 co-receptor polypeptide-encoding polynucleotide may be present on a single nucleic acid molecule (e.g., in a same expression vector), or may be present on separate nucleic acid molecules in a host cell.
  • a CD8 co-receptor alpha chain can comprise, consist essentially of, or consist of SEQ ID NO.:318, or SEQ ID NO.:318 with the signal peptide removed.
  • SEQ ID NO.:3118 An example of a polynucleotide encoding SEQ ID NO.:318 is provided in SEQ ID NO.:319.
  • a CD8 co-receptor alpha chain comprises, consists essentially of, or consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO.:318, or SEQ ID NO.:318 with the signal peptide removed.
  • a CD8 co-receptor beta chain can comprise, consist essentially of, or consist of SEQ ID NO.:320, or SEQ ID NO.:320 with the signal peptide removed.
  • An example of a polynucleotide encoding SEQ ID NO.: 320 is provided in SEQ ID NO.:321.
  • a CD8 co-receptor beta chain comprises, consists essentially of, or consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO.:320, or SEQ ID NO.:320 with the signal peptide removed.
  • a polynucleotide comprises: (a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).
  • a polynucleotide comprises a polynucleotide that encodes a self-cleaving peptide and is disposed between: (1) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; and/or (2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain.
  • a self-cleaving peptide can comprise a linker N-terminal and/or C-terminal to the self-cleaving (e.g., viral 2A) sequence.
  • linkers include GSG, GPP, PGP, and AAA.
  • a polynucleotide can comprise, operably linked in-frame: (i) (pnCD8a)-(pnSCPl)-(pnCD8P)-(pnSCP2)-(pnBP); (ii) (pnCD8P)-(pnSCPl)-(pnCD8a)- (pnSCP2)-(pnBP); (iii) (pnBP)-(pnSCPl)-(pnCD8a)-(pnSCP2)-(pnCD8P);
  • the encoded binding protein comprises a TCRa chain and a TCRP chain
  • the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain.
  • the polynucleotide comprises, operably linked in-frame: (i) (pnCD8a)-(pnSCPl)-(pnCD8P)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa); (ii) (pnCD8P)-(pnSCPl)-(pnCD8a)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa); (iii) (pnCD8a)-(pnSCPl)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP); (iv) (pnCD8P)-(pnSCPl)-(pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP); (v) (pnTCRP)-(pn
  • an encoded polypeptide of the present disclosure comprises one or more junction amino acids.
  • Junction amino acids or “junction amino acid residues” refer to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide.
  • Junction amino acids can result from the design of a construct that encodes a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein), or from cleavage of, for example, a self-cleaving peptide adjacent one or more domains of an encoded binding protein of this disclosure (e.g., a P2A peptide disposed between a TCR a-chain and a TCR P-chain, the self-cleavage of which can leave one or more junction amino acids in the a-chain, the TCR P-chain, or both).
  • a fusion protein e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein
  • cleavage of, for example, a self-cleaving peptide adjacent one or more domains of an encoded binding protein of this disclosure e.g.
  • a binding protein is expressed as part of a transgene construct that encodes, and/or a host cell of the present disclosure can encode: one or more additional accessory protein, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor P-chain; a CD8 co-receptor a-chain or both; or any combination thereof.
  • additional accessory protein such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor P-chain; a CD8 co-receptor a-chain or both; or any combination thereof.
  • polynucleotides and transgene constructs useful for encoding and expressing binding proteins and accessory components are described in PCT application PCT/US2017/053112, the polynucleotides, transgene constructs, and accessory components, including the nucleotide and amino acid sequences, of which are hereby incorporated by reference.
  • any or all of a binding protein of the present disclosure, a safety switch protein, a tag, a selection marker, a CD8 co-receptor P-chain, or a CD8 co-receptor a-chain may be encoded by a single nucleic acid molecule or may be encoded by polynucleotide sequences that are, or are present on, separate nucleic acid molecules.
  • Exemplary safety switch proteins include, for example, a truncated EGF receptor polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity, but that retains its native amino acid sequence, has type I transmembrane cell surface localization, and has a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 118: 1255-1263, 2011); a caspase polypeptide (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; Di Stasi et al., N.
  • huEGFRt truncated EGF receptor polypeptide
  • accessory components useful for modified host cells of the present disclosure comprise a tag or selection marker that allows the cells to be identified, sorted, isolated, enriched, or tracked.
  • marked host cells having desired characteristics e.g., an antigen-specific TCR and a safety switch protein
  • selection marker comprises a nucleic acid construct (and the encoded gene product) that confers an identifiable change to a cell permitting detection and positive selection of immune cells transduced with a polynucleotide comprising a selection marker.
  • RQR is a selection marker that comprises a major extracellular loop of CD20 and two minimal CD34 binding sites.
  • an RQR-encoding polynucleotide comprises a polynucleotide that encodes the 16-amino-acid CD34 minimal epitope.
  • the CD34 minimal epitope is incorporated at the amino terminal position of a CD8 co-receptor stalk domain (Q8).
  • the CD34 minimal binding site sequence can be combined with a target epitope for CD20 to form a compact marker/suicide gene for T cells (RQR8) (Philip et al., 2014, incorporated by reference herein).
  • This construct allows for the selection of host cells expressing the construct, with for example, CD34 specific antibody bound to magnetic beads (Miltenyi) and that utilizes clinically accepted pharmaceutical antibody, rituximab, that allows for the selective deletion of a transgene expressing engineered T cell (Philip et al., 2014).
  • selection markers also include several truncated type I transmembrane proteins normally not expressed on T cells: the truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (see for example, Di Stasi et al., N. Engl. J. Med. 365: 1673-1683, 2011; Mavilio et al., Blood 83 : 1988-1997 , 1994; Fehse et al., Mol. Ther. 7:448- 456, 2000; each incorporated herein in their entirety).
  • a useful feature of CD 19 and CD34 is the availability of the off-the-shelf Miltenyi CliniMACsTM selection system that can target these markers for clinical-grade sorting.
  • CD 19 and CD34 are relatively large surface proteins that may tax the vector packaging capacity and transcriptional efficiency of an integrating vector.
  • Surface markers containing the extracellular, non-signaling domains or various proteins e.g., CD19, CD34, LNGFR
  • Any selection marker may be employed and should be acceptable for Good Manufacturing Practices.
  • selection markers are expressed with a polynucleotide that encodes a gene product of interest (e.g., a binding protein of the present disclosure, such as a TCR or CAR).
  • selection markers include, for example, reporters such as GFP, EGFP, P-gal or chloramphenicol acetyltransferase (CAT).
  • a selection marker such as, for example, CD34 is expressed by a cell and the CD34 can be used to select enrich for, or isolate (e.g., by immunomagnetic selection) the transduced cells of interest for use in the methods described herein.
  • a CD34 marker is distinguished from an anti-CD34 antibody, or, for example, a scFv, TCR, or another antigen recognition moiety that binds to CD34.
  • a selection marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD 19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.
  • tNGFR truncated low-affinity nerve growth factor
  • tCD19 truncated CD 19
  • tCD34 truncated CD34
  • the encoded RQR polypeptide is contained in a P-chain, an a-chain, or both, or a fragment or variant of either or both, of the encoded CD8 co-receptor.
  • a modified host cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor protein that comprises a P-chain containing a RQR polypeptide and further comprises a CD8 a-chain.
  • An encoded CD8 co-receptor includes, in some embodiments, an a-chain or a fragment or variant thereof.
  • An amino acid sequence of the human CD8 co-receptor a-chain precursor is known and is provided at, for example, UniProtKB -P30433 (see also UniProtKB - P31783; - P10732; and -P10731).
  • An encoded CD8 co-receptor includes, in some embodiments, a P-chain or a fragment or variant thereof.
  • An amino acid sequence of the human CD8 co-receptor P-chain precursor is known and is provided at, for example, UniProtKB -Pl 0966 (see also UniProtKB - Q9UQ56; -E9PD41; Q8TD28; and -P30434; and -P05541).
  • An isolated polynucleotide of this disclosure may further comprise a polynucleotide encoding a safety switch protein, a selection marker, a CD8 co-receptor beta chain, or a CD8 co- receptor alpha chain as disclosed herein, or may comprise a polynucleotide encoding any combination thereof.
  • a polynucleotide can be codon optimized for expression in a host cell.
  • the host cell comprises a human immune system cell, such as a T cell, a NK cell, or a NK-T cell (Scholten et al.. Clin. Immunol. 119: 135, 2006).
  • Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGeneTM tool, or GeneArt® (Life Technologies). Codon-optimized sequences include sequences that are partially codon-optimized (z.e., one or more of the codons is optimized for expression in the host cell) and those that are fully codon-optimized.
  • each polypeptide can independently fully codon optimized, partially codon optimized, or not codon optimized.
  • the present disclosure provides an expression vector, comprising any polynucleotide as provided herein operably linked to an expression control sequence.
  • vectors that comprise a polynucleotide or transgene construct of the instant disclosure.
  • Some examples of vectors include plasmids, viral vectors, cosmids, and others.
  • Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g., lentiviral vector, retroviral vector).
  • vectors are capable of directing the expression of genes to which they are operably linked (these vectors may be referred to as "expression vectors").
  • expression vectors e.g., polynucleotides encoding polypeptides as described herein
  • agents e.g., polynucleotides encoding polypeptides as described herein
  • each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.
  • polynucleotides of the present disclosure may be operably linked to certain elements of a vector.
  • polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operably linked.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (z.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion.
  • Expression control sequences may be operably linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from lentiviral vector or a y-retroviral vector).
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • herpesvirus
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • “Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome.
  • “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
  • "Lentiviral vector,” as used herein, means lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types.
  • Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a doublestranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells. In some embodiments, a lentiviral vector is a self-inactivating lentiviral vector.
  • a selfinactivating lentiviral vector can comprise a modification to prevent the transfer of enhancer and promoter elements in the 5' long terminal repeat (LTR) of the vector to transduced cells, for example, comprising a deletion in the 3'LTR of the viral genome that is transferred into the 5'LTR after one round of reverse transcription, resulting in a provirus that contains no LTR derived enhancer or promoter elements.
  • a lentiviral vector is a third generation lentiviral vector.
  • a third generation lentiviral vector can utilize a packaging system split into two or more plasmids, e.g., one encoding Rev and one encoding Gag and Pol.
  • a third generation lentiviral vector can utilize a packaging system that lacks Tat or does not require Tat expression, and instead comprises, e.g., a chimeric 5' LTR fused to a heterologous promoter on the transfer plasmid.
  • the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors.
  • the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. 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 (ovine lentivirus).
  • Retroviral and lentiviral vector constructs and expression systems are also commercially available.
  • Other viral 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., Gene Ther. 5: 1517, 1998).
  • HSVs herpes simplex viruses
  • vectors developed for gene therapy uses can also be used with the compositions and methods of this disclosure.
  • Such vectors include those derived from baculoviruses and a- viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).
  • the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi ci stronic expression.
  • additional sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.
  • a vector is capable of delivering the polynucleotide or transgene construct to a host cell (e.g, a hematopoietic progenitor cell or a human immune system cell).
  • a vector is capable of delivering a polynucleotide or transgene construct to human immune system cell, such as, for example, a CD4 + T cell, a CD8 + T cell, a CD4' CD8' double negative T cell, a stem cell memory T cell, a y5 T cell, a natural killer cell, a dendritic cell, or any combination thereof.
  • a vector is capable of delivering a transgene construct to a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.
  • a vector that encodes a polynucleotide or transgene construct of the present disclosure may further comprise a polynucleotide that encodes a nuclease that can be used to perform a chromosomal knockout in a host cell (e.g, a CRISPR-Cas endonuclease or another endonuclease as disclosed herein) or that can be used to deliver a therapeutic polynucleotide or transgene or portion thereof to a host cell in a gene therapy replacement or gene repair therapy.
  • a host cell e.g, a CRISPR-Cas endonuclease or another endonuclease as disclosed herein
  • nuclease used for a chromosomal knockout or a gene replacement or gene repair therapy can be delivered to a host cell independent of a vector that encodes a polynucleotide or transgene construct of this disclosure.
  • the vector is capable of delivering the polynucleotide to a host cell.
  • the host cell is a hematopoietic progenitor cell or a human immune system cell.
  • the human immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a y5 T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof.
  • the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector or a y-retroviral vector.
  • transposon-based systems examples include, but are not limited to, sleeping beauty (e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and/or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Tol2 (e.g., derived from medaka fish); and spinON.
  • sleeping beauty e.g., derived from the genome of salmonid fish
  • piggyback e.g., derived from lepidopteran cells and/or the Myotis lucifugus
  • mariner e.g., derived from Drosophila
  • frog prince e.g., derived from Rana pipiens
  • Tol2 e.g., derived from medaka fish
  • spinON Host Cells
  • host cells that encode and/or express a binding protein (and, optionally, one or more accessory protein, such as a transduction marker, a CD8 co-receptor polypeptide, or the like, as provided herein).
  • a host cell is provided that is modified to comprise a polynucleotide and/or an expression vector of the present disclosure, and/or to express a binding protein of the present disclosure.
  • a host cell expresses a binding protein (e.g., TCR, scTCR, CAR), and, optionally, one or more accessory protein, at a cell surface.
  • a host cell secretes a soluble binding protein (e.g., scTv, soluble TCR).
  • Any suitable host cell may be modified to include a heterologous polynucleotide encoding a binding protein of this disclosure, including, for example, an immune cell, such as T cell, a NK cell, or a NK-T cell.
  • a modified immune cell comprises a CD4 + T cell, a CD8 + T cell, or both.
  • Any appropriate method can be used to transfect or transduce the cells, for example, T cells, or to administer the polynucleotides or compositions of the present methods.
  • Known methods for delivering polynucleotides to host cells include, for example, use of cationic polymers, lipid-like molecules, and certain commercial products such as, for example, IN-VIVO- JET PEI.
  • Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, and the like.
  • Still further methods of transfecting or transducing host cells employ vectors, described in further detail herein.
  • the host cell or modified cell comprises a hematopoietic progenitor cell, stem cell (e.g., iPSC), and/or or human immune cell
  • the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof.
  • the immune cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y5 T cell, or any combination thereof.
  • the host cell comprises peripheral blood mononuclear cells (PBMCs).
  • the host cell comprises a CD8+ T cell.
  • the host cell comprises a CD4+ T cell.
  • the host cell comprises bulk T cells, bulk CD4+ T cells, bulk CD 8+ T cells, CD4+ central memory T cells, CD8+ central memory T cells, stem cell memory T cells, or a combination of CD4+ central memory (TCM) and CD4+ naive (TN) T cells.
  • TCM CD4+ central memory
  • TN CD4+ naive
  • the immune cell comprises a CD4+ T cell and a CD8+ T cell.
  • the CD4+ T cell, the CD8+ T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).
  • a host cell can be a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • a host cell can be a lymphoid cell.
  • a host cell can be a lymphocyte.
  • a host cell can be a T cell.
  • a host cell can be an alpha beta T cell (whether expressing or not expressing an endogenous alpha-beta TCR).
  • a host cell can be a gamma delta T cell (whether expressing or not expressing an endogenous gammadelta TCR).
  • a host cell can be a B cell.
  • a host cell can be a natural killer (NK) cell.
  • a host cell can be a Natural Killer T (NKT) cell.
  • a host cell can be a mammalian cell.
  • a host cell can be a human cell.
  • a host cell can be a primary cell.
  • a host cell can be an immortalized cell.
  • a host cell can be of a cell line.
  • a host cell can be differentiated from a stem cell, for example, an induced pluripotent stem cell (iPSC), embryonic stem cell, hematopoietic stem cell (HSC), or the like.
  • iPSC induced pluripotent stem cell
  • HSC hematopoietic stem cell
  • a host cell e.g., an immune cell
  • a host cell may modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide involved in immune signaling or other related activities.
  • exemplary gene knockouts include those that encode PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA molecule, a TCR molecule, or the like.
  • certain endogenously expressed immune cell proteins may be recognized as foreign by an allogeneic host receiving the modified immune cells, which may result in elimination of the modified immune cells (e.g., an HLA allele), or may downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, FasL, TIGIT, TIM3), or may interfere with the binding activity of a heterologously expressed binding protein of the present disclosure (e.g., an endogenous TCR of a modified T cell that binds a non-P53 antigen (or non-P53 R175H antigen) and thereby interferes with the modified immune cell binding a cell that expresses a P53 antigen).
  • a heterologously expressed binding protein of the present disclosure e.g., an endogenous TCR of a modified T cell that binds a non-P53 antigen (or non-P53 R175H antigen) and thereby interferes with the modified immune cell binding a cell that expresses
  • a modified cell is a donor cell (e.g., allogeneic) or an autologous cell.
  • a modified cell of this disclosure comprises a chromosomal gene knockout of one or more of a gene that encodes PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA component (e.g., a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a pi microglobulin, or a P2 microglobulin), or a TCR component (e.g., a gene that encodes a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep.
  • HLA component e.g., a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a pi microglobulin, or a P2 microglobulin
  • TCR component e.g., a gene that encodes a TCR variable region or a TCR constant region
  • chromosomal gene knockout refers to a genetic alteration or introduced inhibitory agent in a host cell that prevents e.g., reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.
  • a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell.
  • Chromosomal editing can be performed using, for example, endonucleases.
  • endonucleases refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain.
  • an endonuclease is capable of cleaving a targeted gene thereby inactivating or "knocking out" the targeted gene.
  • An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease.
  • the nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non- homologous end joining (NHEJ).
  • NHEJ non- homologous end joining
  • a donor nucleic acid molecule may be used for a donor gene "knock-in”, for target gene "knock-out”, and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event.
  • NHEJ is an error-prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide.
  • NHEJ may be used to "knock-out" a target gene.
  • Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.
  • a "zinc finger nuclease” refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease.
  • ZFN zinc finger nuclease
  • Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter triplet sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285: 1917-1934, 1999).
  • ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair.
  • DSB DNA double strand break
  • a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site.
  • NHEJ non-homologous end joining
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.
  • TALEN transcription activator-like effector nuclease
  • a "TALE DNA binding domain” or “TALE” is composed of one or more TALE repeat domains/units, each generally having a highly conserved 33-35 amino acid sequence with divergent 12th and 13th amino acids.
  • the TALE repeat domains are involved in binding of the TALE to a target DNA sequence.
  • the divergent amino acid residues referred to as the Repeat Variable Diresidue (RVD), correlate with specific nucleotide recognition.
  • RVD Repeat Variable Diresidue
  • the natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagineisoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagineglycine) binds to a T nucleotide.
  • Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No.
  • TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells.
  • Non- homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression.
  • homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene.
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.
  • CRISPR/Cas nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence.
  • CRISPR/Cas systems are classified into three types (z.e., type I, type II, and type III) based on the sequence and structure of the Cas nucleases.
  • the crRNA-guided surveillance complexes in types I and III need multiple Cas subunits.
  • Type II system the most studied, comprises at least three components: an RNA- guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA).
  • the tracrRNA comprises a duplex forming region.
  • a crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the Cas9/crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM.
  • Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and/or deletions which disrupt expression of the targeted locus.
  • a transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair.
  • the crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012).
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR/Cas nuclease system.
  • Exemplary gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.
  • Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, Pl-Sce, 1-SceIV, I-CsmI, I- Panl, I-Scell, I-Ppol, I-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res . 25:3379-3388, 1997; Dujon et al., Gene 82:115-118, 1989; Perl er et al., Nucleic Acids Res.
  • naturally occurring meganucleases may be used to promote sitespecific genome modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, an HLA-encoding gene, or a TCR component-encoding gene.
  • a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, an HLA-encoding gene, or a TCR component-encoding gene.
  • an engineered meganuclease having a novel binding specificity for a target gene is used for sitespecific genome modification (see, e.g., Porteus et al., Nat. BiotechnoL 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res.
  • a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL. MegaTALs can be utilized to not only knockout one or more target genes, but to also introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.
  • a chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of PD-1, TIM3, LAG3, CTLA4, TIGIT, FasL, an HL A component, or a TCR component, or any combination thereof) in the host cell.
  • a host cell e.g., an immune cell
  • a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen
  • the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR/Cas nuclease system or base editing system (Komor, A. C.; Kim, Y. B.; Packer, M. S.; Zuris, J. A.; Liu, D. R. Nature 533, 420-424 (2016).
  • base editing is a genome-editing approach that uses components from CRISPR systems together with other enzymes to directly introduce point mutations into cellular DNA or RNA without making double-stranded DNA breaks.
  • DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor.
  • RNA base editors function similarly, using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing byproducts. See e.g., Rees H el al. Nature Reviews Genetics (2018).
  • Chromosomal gene knockout can be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent. Chromosomal gene knockouts can also be inferred from the absence of gene expression (e.g., the absence of an mRNA or polypeptide product encoded by the gene) following the knockout.
  • a chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.
  • a chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.
  • a polynucleotide encoding a binding protein of the present disclosure, or encoding a component thereof is knocked-in to a host cell genome, optionally at an endogenous HLA component gene locus or TCR component gene locus.
  • a population of host cells comprising a binding protein disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or at least 5000 fold increased functional avidity for a target anti
  • the host cells can comprise a binding protein (e.g., a TCR comprising Va and VP regions and/or CDRs disclosed herein) that binds a target antigen (for example, a P53 R175H mutant peptide e.g., present in a peptide:HLA complex).
  • a binding protein e.g., a TCR comprising Va and VP regions and/or CDRs disclosed herein
  • a target antigen for example, a P53 R175H mutant peptide e.g., present in a peptide:HLA complex.
  • the increase in avidity can be, for example, as determined by an assay for determining expression an activation marker (e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, a cytokine, an interleukin, an interferon) upon exposure to target cells that express or present the target antigen, or and/or an assay to determine EC50 (e.g., peptide dose at which a half-maximal activation of a T cell population is reached).
  • an activation marker e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, a cytokine, an interleukin, an interferon
  • EC50 e.g., peptide dose at which a half-maximal activation of a T cell population is reached.
  • the host cells and the control cells are both T cells, and the host cell and control cell populations can comprise the same, about the same, or substantially the same composition or amount(s) of T cell type(s) (e.g., CD4+, CD8+, or both).
  • T cell type(s) e.g., CD4+, CD8+, or both.
  • a population of host cells comprising a binding protein disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold, at least 250 fold, or at least 1000 fold increased killing of target cells as compared to a population of control cells (for example, cells expressing a control binding protein specific for the same target antigen).
  • the killing of target cells can be, for example, as determined by an in vitro cytotoxicity assay, for example, at an effector to target ratio of about 0.5: 1, 1 :1, 2: 1, 3:1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 20: 1, 25: 1, 50: 1, or 100:1.
  • the host cells and the control cells are both T cells, and the host cell and control cell populations can comprise the same, about the same, or substantially the same composition or amount(s) of T cell type(s) (e.g., CD4+, CD8+, or both).
  • a population of host cells comprising a binding protein disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or at least 5000 fold increased activation as compared to a
  • the activation can be, for example, as determined by an assay for determining expression an activation marker (e.g., CD137, CD69, Granzyme B, CD107a, IFN- gamma, TNF-a, IL-12, a cytokine, an interleukin, an interferon) upon exposure to target cells that express or present the target antigen.
  • an activation marker e.g., CD137, CD69, Granzyme B, CD107a, IFN- gamma, TNF-a, IL-12, a cytokine, an interleukin, an interferon
  • the host cells and the control cells are both T cells, and the host cell and control cell populations can comprise the same, about the same, or substantially the same composition or amount(s) of T cell type(s) (e.g., CD4+, CD8+, or both).
  • compositions and unit doses are provided herein that comprise a host cell of the present disclosure (e.g. a modified host cell, such as an immune cell engineered to encode a binding protein as provided herein) and a pharmaceutically acceptable carrier, diluent, or excipient.
  • a host cell of the present disclosure e.g. a modified host cell, such as an immune cell engineered to encode a binding protein as provided herein
  • a pharmaceutically acceptable carrier diluent, or excipient.
  • a host cell composition or unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4 + T cells, combined with (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells (i.e., has less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less then about 1% the population of naive T cells present in a unit dose as compared to a patient sample having a comparable number of PBMCs).
  • a host cell composition or unit dose comprises (i) a composition comprising at least about 50% modified CD4 + T cells, combined with (ii) a composition comprising at least about 50% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the host cell composition or unit dose contains a reduced amount or substantially no naive T cells.
  • a host cell composition or unit dose comprises (i) a composition comprising at least about 60% modified CD4 + T cells, combined with (ii) a composition comprising at least about 60% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a host cell composition or unit dose comprises (i) a composition comprising at least about 70% engineered CD4 + T cells, combined with (ii) a composition comprising at least about 70% engineered CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a host cell composition or unit dose comprises (i) a composition comprising at least about 80% modified CD4 + T cells, combined with (ii) a composition comprising at least about 80% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the host cell composition or unit dose contains a reduced amount or substantially no naive T cells.
  • a host cell composition or unit dose comprises (i) a composition comprising at least about 85% modified CD4 + T cells, combined with (ii) a composition comprising at least about 85% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the host cell composition or unit dose contains a reduced amount or substantially no naive T cells.
  • a host cell composition or unit dose comprises (i) a composition comprising at least about 90% modified CD4 + T cells, combined with (ii) a composition comprising at least about 90% modified CD8 + T cells, in about a 1 : 1 ratio, wherein the host cell composition or unit dose contains a reduced amount or substantially no naive T cells.
  • a host cell composition or unit dose comprises about a 1 : 1 ratio, about a 1 :2 ratio, about a 1 :3 ratio, about a 1 :4 ratio, about a 1 :5 ratio, about a 1 :6 ratio, about a 1 :7 ratio, about a 1 :8 ratio, about a 1 :9 ratio, about a 1 : 10 ratio, about a 2: 1 ratio, about a 3 : 1 ratio, about a 4: 1 ratio, about a 5: 1 ratio, about a 6: 1 ratio, about a 7: 1 ratio, about an 8: 1 ratio, about a 9: 1 ratio, about a 10: 1 ratio, about a 3 :2 ratio, or about a 2:3 ratio of CD4+ to CD8+ T cells (for example, of CD4+ T cells modified to comprise or express a binding protein disclosed herein to CD8+ T cells modified to comprise or express a binding protein disclosed herein).
  • a host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells that is at least 1 : 1, at least 1 :2, at least 1 :3, at least 1 :4, at least 1 :5, at least 1 :6, at least 1 :7, at least 1 :8, at least 1 :9, at least 1 : 10, at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 3:2, or at least 2:3.
  • a host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells that is at most 1 : 1, at most 1 :2, at most 1 :3, at most 1 :4, at most 1 :5, at most 1 :6, at most 1 :7, at most 1 :8, at most 1 :9, at most 1 : 10, at most 2: l, at most 3: l, at most 4: 1, at most 5: 1, at most 6: 1, at most 7: 1, at most 8: 1, at most 9: 1, at most 10: 1, at most 3:2, or at most 2:3.
  • a host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells that is between about 1:10 and 10:1, 1:10 and 8:1, 1:10 and 7:1, 1:10 and 6:1, 1:10 and5:l, l:10and4:l, l:10and3:l, l:10and2:l, ElOand 1:1, ElOand 1:2, ElOand 1:3, 1:10 and 1:4, 1:10 and 1:5, 1:10 and 1:7, 1:5 and 10:1, 1:5 and 8:1, 1:5 and 7:1, 1:5 and 6:1, 1:5 and 5:1, 1:5 and4:l, 1:5 and3:l, 1:5 and 2:1, 1:5 and 1:1, 1:5 and 1:2, 1:5 and 1:3, 1:5 and 1:4, 1:3 and 10:1, 1:3 and 8:1, 1:3 and 7:1, 1:3 and 6:1, 1:3 and 5:1, 1:3 and 4:1, 1:3 and 3:1, 1:3 and 3:1
  • CD4+ T cells in a composition, host cell composition, or unit dose can be CD4+ T cells that are modified or engineered to express a CD8 co-receptor disclosed herein, for example, using a vector or polynucleotide disclosed herein.
  • a host cell composition or unit dose of the present disclosure may comprise any host cell as described herein, or any combination of host cells.
  • a host cell composition or unit dose comprises modified CD8+ T cells, modified CD4+ T cells, or both, wherein these T cells are modified to encode a binding protein specific for a P53 peptide antigen (e.g., SEQ ID NO:3):HLA-A*02:01 complex.
  • a host cell composition or unit dose of the present disclosure can comprise any host cell or combination of host cells as described herein, and can further comprise a modified cell (e.g., immune cell, such as a T cell) expressing a binding protein specific for a different antigen (e.g., a different P53 antigen, or an antigen from a different protein or target, such as, for example, BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gpl30, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L
  • a unit dose can comprise modified CD8 + T cells expressing a binding protein that specifically binds to a P53 R175H (e.g., SEQ ID N0:3)-HLA complex and modified CD4 + T cells (and/or modified CD8 + T cells) expressing a binding protein (e.g., a CAR) that specifically binds to a different antigen.
  • a binding protein e.g., a CAR
  • any of the host cells disclosed herein may be administered in a combination therapy.
  • a host cell composition or unit dose comprises equal, or approximately equal numbers of engineered CD45RA' CD3 + CD8 + and modified CD45RA' CD3 + CD4 + T M cells.
  • a host cell composition or unit dose comprises one or more populations of cells (e.g., CD4+ or CD8+ cells) that have undergone CD62L positive selection, for example, to improve in vivo persistence.
  • populations of cells e.g., CD4+ or CD8+ cells
  • Host cells can be genetically engineered to comprise or express a binding protein ex vivo, in vitro, or in vivo. In some embodiments, a host cell is genetically engineered ex vivo to express a binding protein. In some embedments, a host cell is genetically engineered in vitro to express a binding protein. In some embodiments, a host cell is genetically engineered in vivo to express a binding protein.
  • the present disclosure provides methods for treating or for preventing a relapse of a disease or disorder associated with a P53 mutation (e.g, a P53 R175H mutation, such as in a peptide comprising or consisting of SEQ ID NO:3) in a subject.
  • a disease or disorder associated with a P53 mutation e.g, a P53 R175H mutation, such as in a peptide comprising or consisting of SEQ ID NO:3
  • diseases or disorders include, for example, cancers, such as solid cancers and hematological malignancies.
  • the disease or disorder comprises a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal mel
  • Treatment refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
  • a subject e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat.
  • an appropriate dose or treatment regimen comprising a composition (e.g., comprising a binding protein, polynucleotide, vector, host cell, host cell composition, unit dose, and/or immunogenic polypeptide) of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof.
  • a “therapeutically effective amount” or “effective amount”, as used herein, refers to an amount of a composition sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner.
  • a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone.
  • a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
  • a combination may also be a cell expressing more than one active ingredient.
  • pharmaceutically acceptable excipient or carrier or “physiologically acceptable excipient or carrier” refer to biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject and generally recognized as safe or not causing a serious adverse event.
  • statically significant refers to a p value of 0.050 or less when calculated using the Students t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.
  • Subjects that can be treated by the present invention are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes.
  • the subject may be a human subject.
  • the subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • Compositions according to the present disclosure may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art.
  • a modified host cell, host cell composition, or unit dose as described herein is administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid so as to encounter target cells (e.g., leukemia cells).
  • target cells e.g., leukemia cells.
  • An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.
  • adoptive immune therapy refers to administration of naturally occurring or genetically engineered, disease- or antigen-specific immune cells (e.g., T cells).
  • adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
  • the subject comprises a P53 R175H antigen. In some embodiments, the subject expresses a P53 antigen comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3.
  • the subject is HLA-A*02:01 +
  • a method comprises determining the HLA type or types of a subject and/or identifying the presence of a P53 (e.g., R175H) antigen (e.g., a peptide comprising or consisting of SEQ ID NO:3, or a polynucleotide encoding the same in the subject), prior to administering therapy according to the present disclosure.
  • a P53 e.g., R175H
  • antigen e.g., a peptide comprising or consisting of SEQ ID NO:3, or a polynucleotide encoding the same in the subject
  • HLA typing This genetic determination of the HLA expression is referred to herein as “HLA typing” and can determined though molecular approaches in a clinical laboratory licensed for HLA typing.
  • HLA typing is performed using PCR amplification followed by high throughput NGS and subsequent HLA determination.
  • the HLA haplotype can be determined at the major HLA loci (e.g., HLA- A, HLA-B, HLA-C, etc ).
  • HLA typing can be performed using any known method, including, for example, protein or nucleic acid testing.
  • nucleic acid testing include sequence-based typing (SBT) and use of sequence-specific oligonucleotide probes (SSOP) or sequence-specific primers (SSP).
  • SBT sequence-based typing
  • SSP sequence-specific primers
  • HLA typing is performed using PCR amplification followed by high throughput Next Generation Sequencing (NGS) and subsequent HLA determination.
  • NGS Next Generation Sequencing
  • sequence typing is performed using a system available through Scisco Genetics (sciscogenetics.com/pages/technology.html, the contents of which is incorporated herein by reference in its entirety).
  • Other methods for HLA typing include, e.g., those disclosed in Mayor et al. PLoS One 10(5y.eG ⁇ 21 ⁇ 53 (2015), which methods and reagents are incorporated herein by reference.
  • a method comprises administering a composition comprising immune cells (e.g. T cells, such as modified CD8+ and/or modified CD4+ T cells) that comprise a heterologous polynucleotide encoding a second binding protein as provided herein, when the subject expresses HL A- A* 02:01.
  • immune cells e.g. T cells, such as modified CD8+ and/or modified CD4+ T cells
  • the amount of cells therein is at least one cell (for example, one modified CD8 + T cell subpopulation (e.g., optionally comprising memory and/or naive CD8 + T cells); one modified CD4 + T cell subpopulation (e.g., optionally comprising memory and/or naive CD4 + T cells)) or is more typically greater than 10 2 cells, for example, up to 10 4 , up to 10 5 , up to 10 6 , up to 10 7 , up to 10 8 , up to 10 9 , or more than IO 10 cells.
  • one modified CD8 + T cell subpopulation e.g., optionally comprising memory and/or naive CD8 + T cells
  • one modified CD4 + T cell subpopulation e.g., optionally comprising memory and/or naive CD4 + T cells
  • the cells are administered in a range from about 10 4 to about IO 10 cells/m 2 , preferably in a range of about 10 5 to about 10 9 cells/m 2 .
  • an administered dose comprises up to about 3.3 x 10 5 cells/kg.
  • an administered dose comprises up to about 1 x 10 6 cells/kg.
  • an administered dose comprises up to about 3.3 x 10 6 cells/kg.
  • an administered dose comprises up to about 1 x 10 7 cells/kg.
  • a modified immune cell is administered to a subject at a dose comprising up to about 5 x 10 4 cells/kg, 5 x 10 5 cells/kg, 5 x 10 6 cells/kg, or up to about 5 x 10 7 cells/kg. In certain embodiments, a modified immune cell is administered to a subject at a dose comprising at least about 5 x 10 4 cells/kg, 5 x 10 5 cells/kg, 5 x 10 6 cells/kg, or up to about 5 x 10 7 cells/kg. The number of cells will depend upon the ultimate use for which the composition is intended as well as the type of cells included therein.
  • cells modified to contain a binding protein will comprise a cell population containing at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of such cells.
  • cells are generally in a volume of a liter or less, 500 mis or less, 250 mis or less, or 100 mis or less.
  • the density of the desired cells is typically greater than 10 4 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the cells may be administered as a single infusion or in multiple infusions over a range of time.
  • a clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 cells.
  • a unit dose of the modified immune cells can be co-administered with (e.g., simultaneously or contemporaneously with) hematopoietic stem cells from an allogeneic donor.
  • one or more of the modified immune cells comprised in the unit dose is autologous to the subject.
  • a unit dose comprises, consists essentially of, or consists of, or a plurality of unit doses comprises, consists essentially of, or consists of, at least 5 xlO A 7, at least 1 xl0 A 8, at least 5 xl0 A 8, at least lxlO A 9, at least 2.5 xlO A 9, at least 5 xlO A 9, at least 1 xl0 A 10, at least 1.5 xl0 A 10, at least 2 xl0 A 10, at least 3 xl0 A 10, at least 5 xl0 A 10, or at least 1 xl0 A l 1 viable host cells, e.g., that encode, comprise, or express a binding protein as disclosed herein.
  • a unit dose comprises, consists essentially of, or consists of, or a plurality of unit doses comprises, consists essentially of, or consists of, at most 1 xl0 A 8, at most 5 xlO A 9, at most 1 xl0 A 10, at most 1.5 xl0 A 10, at most 2 xl0 A 10, at most 2.5 xl0 A 10, at most 3 xl0 A 10, at most 4 xl0 A 10, at most 5 xl0 A 10, at most 1 xl0 A l 1, at most 5 xl0 A l 1, or at most 2 xlO A 12 viable host cells, e.g., that encode, comprise, or express a binding protein as disclosed herein.
  • a unit dose comprises, consists essentially of, or consists of, or a plurality of unit doses comprises, consists essentially of, or consists of, about lxl0 A 8, about 5xl0 A 8, about lx!0 A 9, about 2xlO A 9, about 3xlO A 9, about 4xlO A 9, about 5xlO A 9, about 6xlO A 9, about 7xlO A 9, about 8xlO A 9, about 9xlO A 9, about lxl0 A 10, about l.lxl0 A 10, about 1.2xl0 A 10, about 1.3xl0 A 10, about 1.4xl0 A 10, about 1.5xl0 A 10, about 1.6xl0 A 10, about 1.7xl0 A 10, about 1.8xl0 A 10, about 1.9xl0 A 10, about 2xl0 A 10, about 3xl0 A 10, about 4xl0 A 10, about 5xl0 A 10, about 7.5xl0 A 10, about 10xl0 A 10, or about lxlO A
  • a unit dose comprises, consists essentially of, or consists of, or a plurality of unit doses comprises, consists essentially of, or consists of, about 1 xlO A 8 to about 1 xlO A l 1, about 1 xlO A 8 to about 5 xl0 A 10, about 1 xlO A 8 to about 2 xl0 A 10, about 1 xlO A 8 to about 1.5 xl0 A 10, about 1 xlO A 8 to about 1 xl0 A 10, about 1 xlO A 8 to about 5 xlO A 9, about 1 xlO A 9 to about 1 xlO A l 1, about 1 xlO A 9 to about 5 xl0 A 10, about 1 xlO A 9 to about 2 xl0 A 10, about 1 xlO A 9 to about 1.5 xl0 A 10, about 1 xlO A 9 to about 1 xl0 A 10, about 1 xlO A 9 to about 5 xlO A 10, about 1 xlO A
  • the subject receiving the modified immune cell has previously received lymphodepleting chemotherapy.
  • the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof.
  • the method further comprises administering an inhibitor of an immune checkpoint molecule, as disclosed herein, to the subject.
  • compositions that comprise a composition (binding protein, polynucleotide, vector, host cell, host cell composition, unit dose, and/or immunogenic polypeptide) as disclosed herein and a pharmaceutically acceptable carrier, diluents, or excipient.
  • Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof.
  • compositions comprising fusion proteins or host cells as disclosed herein further comprise a suitable infusion media.
  • Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma- Lyte A (Baxter), 5% dextrose in water, Ringer's lactate can be utilized.
  • An infusion medium can be supplemented with human serum albumin or other human serum components.
  • Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (z.e., weight, mass, or body area), the type and severity of the patient's condition, the particular form of the active ingredient, and the method of administration.
  • an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • An effective amount of a pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein.
  • An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
  • a disease or disease-state e.g., recurrence
  • compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until infusion into the patient.
  • Doses will vary, but a preferred dose for administration of a modified immune cell as described herein is about 10 4 cells/m 2 , about 5 x 10 4 cells/m 2 , about 10 5 cells/m 2 , about 5 x 10 5 cells/m 2 , about 10 6 cells/m 2 , about 5 x 10 6 cells/m 2 , about 10 7 cells/m 2 , about 5 x 10 7 cells/m 2 , about 10 8 cells/m 2 , about 5 x 10 8 cells/m 2 , about 10 9 cells/m 2 , about 5 x 10 9 cells/m 2 , about IO 10 cells/m 2 , about 5 x IO 10 cells/m 2 , or about 10 11 cells/m 2 .
  • a unit dose comprises a modified immune cell as described herein at a dose of about 10 4 cells/m 2 to about 10 11 cells/m 2 .
  • the composition may also include sterile aqueous or oleaginous solution or suspension.
  • suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer’s solution, isotonic salt solution, 1,3 -butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water.
  • Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate.
  • any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained- release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of engineered immune cells or active compound calculated to produce the desired effect in association with an appropriate pharmaceutical carrier.
  • an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide a benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects.
  • Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine.
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
  • Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • administration of a composition refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state. Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., modified immune cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof). In certain embodiments, a plurality of doses of a composition described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks.
  • Treatment or prevention methods of this disclosure may be administered to a subject as part of a treatment course or regimen, which may comprise additional treatments prior to, or after, administration of the instantly disclosed unit doses, cells, or compositions.
  • a subject receiving a unit dose of the modified immune cell is receiving or had previously received a hematopoietic cell transplant (HCT; including myeloablative and non- myeloablative HCT).
  • HCT hematopoietic cell transplant
  • Techniques and regimens for performing HCT are known in the art and can comprise transplantation of any suitable donor cell, such as a cell derived from umbilical cord blood, bone marrow, or peripheral blood, a hematopoietic stem cell, a mobilized stem cell, or a cell from amniotic fluid.
  • a modified immune cell of the present disclosure can be administered with or shortly after hematopoietic stem cells in a modified HCT therapy.
  • the HCT comprises a donor hematopoieitic cell comprising a chromosomal knockout of a gene that encodes an HL A component, a chromosomal knockout of a gene that encodes a TCR component, or both.
  • a lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof.
  • Methods according to this disclosure may further include administering one or more additional agents to treat the disease or disorder in a combination therapy.
  • a combination therapy comprises administering a composition of the present disclosure with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor.
  • a combination therapy comprises administering a composition of the present disclosure with an agonist of a stimulatory immune checkpoint agent.
  • a combination therapy comprises administering a composition of the present disclosure with a secondary therapy, such as chemotherapeutic agent, a radiation therapy, a surgery, an antibody, or any combination thereof.
  • immune suppression agent refers to one or more cells, proteins, molecules, compounds or complexes providing inhibitory signals to assist in controlling or suppressing an immune response.
  • immune suppression agents include those molecules that partially or totally block immune stimulation; decrease, prevent or delay immune activation; or increase, activate, or up regulate immune suppression.
  • immunosuppression agents to target include PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-IRA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.
  • immunosuppression agents to target include PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2
  • An immune suppression agent inhibitor may be a compound, an antibody, an antibody fragment or fusion polypeptide (e.g., Fc fusion, such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme or RNAi molecule, or a low molecular weight organic molecule.
  • a method may comprise a composition of the present disclosure with one or more inhibitor of any one of the following immune suppression components, singly or in any combination.
  • a composition of the present disclosure is used in combination with a PD-1 inhibitor, for example a PD-1 -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS- 936558 or any combination thereof.
  • a composition of the present disclosure is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.
  • cemiplimab IBI-308; nivolumab + relatlimab; BCD-100; camrelizumab; JS-OO1; spartalizumab; tislelizumab; AGEN-2034; BGBA-333 + tislelizumab; CBT-501; dostarlimab; durvalumab + MED 1-0680; JNJ-3283; pazopanib hydrochloride + pembrolizumab; pidilizumab; REGN-1979 + cemiplimab; ABBV-181; ADUS-100 + spartalizumab; AK-104; AK-105; AMP-224; BAT-1306; BI-754091; CC-90006; cemiplimab + REGN-3767; CS-1003; GLS-010; LZM-009; MEDI-5752; MGD-013; PF-06801591
  • composition of the present disclosure of the present disclosure is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS- 986016, or any combination thereof.
  • a LAG3 inhibitor such as LAG525, IMP321, IMP701, 9H12, BMS- 986016, or any combination thereof.
  • a composition of the present disclosure is used in combination with an inhibitor of CTLA4.
  • a composition of the present disclosure is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.
  • a composition of the present disclosure is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both.
  • a B7-H4 antibody binding fragment may be a scFv or fusion protein thereof, as described in, for example, Dangaj et al., Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO /201740724A1 and WO 2013/025779A1.
  • a composition of the present disclosure is used in combination with an inhibitor of CD244.
  • composition of the present disclosure is used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof.
  • Anti CD-160 antibodies are described in, for example, PCT Publication No. WO 2010/084158.
  • composition of the present disclosure cell is used in combination with an inhibitor of TIM3.
  • composition of the present disclosure is used in combination with an inhibitor of Gal9.
  • composition of the present disclosure is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor.
  • composition of the present disclosure is used in combination with an inhibitor of A2aR.
  • composition of the present disclosure is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015).
  • composition of the present disclosure is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFP) or Treg development or activity.
  • an inhibitor of an inhibitory cytokine typically, a cytokine other than TGFP
  • Treg development or activity typically, a cytokine other than TGFP
  • a composition of the present disclosure is used in combination with an IDO inhibitor, such as levo-l-methyl tryptophan, epacadostat (INCB024360; Liu etal., Blood 775:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (l-MT)-tira-pazamine, or any combination thereof.
  • an IDO inhibitor such as levo-l-methyl tryptophan, epacadostat (INCB024360; Liu etal., Blood 775:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American
  • a composition of the present disclosure is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N- omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
  • an arginase inhibitor such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N- omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
  • composition of the present disclosure is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).
  • a composition of the present disclosure is used in combination with an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both.
  • a composition of the present disclosure is used in combination with an inhibitor of PVRIG, PVRL2, or both.
  • Anti-PVRIG antibodies are described in, for example, PCT Publication No. WO 2016/134333.
  • Anti-PVRL2 antibodies are described in, for example, PCT Publication No. WO 2017/021526.
  • composition of the present disclosure is used in combination with a LAIR1 inhibitor.
  • composition of the present disclosure is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.
  • a composition of the present disclosure is used in combination with an agent that increases the activity (z.e., is an agonist) of a stimulatory immune checkpoint molecule.
  • a composition of the present disclosure can be used in combination with a CD137 (4-1BB) agonist (such as, for example, urelumab), a CD134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-1127), a CD28 agonist (such as, for example, TGN1412, CD80, or CD86), a CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodies described in PCT Patent Publication No.
  • a method may comprise administering a composition of the present disclosure with one or more agonist of a stimulatory immune checkpoint molecule, including any of the foregoing, singly or in any combination.
  • a combination therapy comprises a composition of the present disclosure and a secondary therapy comprising one or more of: an antibody or antigen bindingfragment thereof that is specific for a cancer antigen expressed by the non-inflamed solid tumor, a radiation treatment, a surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.
  • a combination therapy method comprises administering a composition of the present disclosure and further administering a radiation treatment or a surgery.
  • Radiation therapy is well-known in the art and includes X-ray therapies, such as gamma-irradiation, and radiopharmaceutical therapies. Surgeries and surgical techniques appropriate to treating a given cancer in a subject are well-known to those of ordinary skill in the art.
  • a combination therapy method comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent.
  • a chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar- modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor.
  • Illustrative chemotherapeutic agents include, without limitation, the following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busul
  • Cytokines may be used to manipulate host immune response towards anticancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting immune anticancer or antitumor response include, for example, IFN-a, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, singly or in any combination with a composition of the present disclosure.
  • Also provided herein are methods for modulating an adoptive immunotherapy wherein the methods comprise administering, to a subject who has previously received a modified host cell of the present disclosure that comprises a heterologous polynucleotide encoding a safety switch protein, a cognate compound of the safety switch protein in an amount effective to ablate in the subject the previously administered modified host cell.
  • the safety switch protein comprises tEGFR and the cognate compound is cetuximab, or the safety switch protein comprises iCasp9 and the cognate compound is AP1903 (e.g., dimerized AP1903), or the safety switch protein comprises a RQR polypeptide and the cognate compound is rituximab, or the safety switch protein comprises a myc binding domain and the cognate compound is an antibody specific for the myc binding domain.
  • methods are provided for manufacturing a composition, or a unit dose of the present disclosure.
  • the methods comprise combining (i) an aliquot of a host cell transduced with a vector of the present disclosure with (ii) a pharmaceutically acceptable carrier.
  • vectors of the present disclosure are used to transfect/transduce a host cell (e.g., a T cell) for use in adoptive transfer therapy (e.g., targeting a cancer antigen).
  • the methods further comprise, prior to the aliquotting, culturing the transduced host cell and selecting the transduced cell as having incorporated (z.e., expressing) the vector.
  • the methods comprise, following the culturing and selection and prior to the aliquotting, expanding the transduced host cell.
  • the manufactured composition or unit dose may be frozen for later use. Any appropriate host cell can be used for manufacturing a composition or unit dose according to the instant methods, including, for example, a hematopoietic stem cell, PBMCs, a T cell, a primary T cell, a T cell line, a NK cell, or a NK-T cell.
  • the methods comprise a host cell which is a CD8 + T cell, a CD4 + T cell, or both.
  • binding proteins e.g., a P53 R175H mutation, such as in a peptide comprising or consisting of SEQ ID NO:3
  • a P53 mutation e.g., a P53 R175H mutation, such as in a peptide comprising or consisting of SEQ ID NO:3
  • binding proteins e.g., a P53 R175H mutation, such as in a peptide comprising or consisting of SEQ ID NO:3
  • a P53 mutation e.g., a P53 R175H mutation, such as in a peptide comprising or consisting of SEQ ID NO:3
  • the disease or disorder comprises a cancer.
  • the cancer is a solid cancer or a hematological malignancy.
  • the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma;
  • PDAC pancreatic ductal
  • the method comprises parenteral or intravenous administration of the subject composition. In some embodiments, the method comprises administering a plurality of doses of the binding protein, polynucleotide, expression vector, host cell, host cell composition, unit dose, and/or immunogenic polypeptide the subject.
  • the plurality of doses is administered at intervals between administrations of about two to about four weeks.
  • the composition comprises the modified host cell.
  • the method comprises administering the modified host cell to the subject at a dose of about 10 4 cells/kg to about 10 11 cells/kg.
  • the method further comprises administering a cytokine to the subject.
  • the cytokine comprises IL-2, IL-15, or IL-21.
  • the subject has received or is receiving an immune checkpoint inhibitor and/or an agonist of a stimulatory immune checkpoint agent.
  • the present disclosure also provides the following, non-limiting, enumerated Embodiments.
  • a binding protein comprising:
  • a T cell receptor (TCR) a chain variable (Va) domain comprising the complementarity determining region 3 (CDR3a) amino acid sequence set forth in any one of SEQ ID NOs:35, 34, 8, 9, 21, 22, 47, 48, 60, 61, 73, 74, 129, 130, 142, 143, 155, 156, 168, and 169, or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions; and/or (b) a TCR P chain variable (VP) domain comprising the CDR3P amino acid sequence set forth in any one of SEQ ID NOs:41, 40, 14, 15, 27, 28, 53, 54, 66, 67, 79, 80, 94, 95, 135, 136, 148, 149, 161, 162, 174, and 175, or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions, wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the binding protein is
  • Embodiment 2 The binding protein of Embodiment 1, wherein the HLA comprises HLA-A*02:01.
  • Embodiment 3 The binding protein of Embodiment 1 or 2, wherein the Va domain and/or the VP domain is human, humanized, or chimeric, and is preferably human.
  • Embodiment 4 The binding protein of any one of Embodiments 1-3, comprising the CDR3a and CDR3P amino acid sequences set forth in SEQ ID NOs: (i) 35 and 41, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (ii) 22 and 28, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iii) 9 and 15, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iv) 48 and 54, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (v) 61 and 67, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vi) 74 and 80, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vii) 8 and 14, respectively, or variants thereof having one, two, or three, optionally conservative, amino
  • Embodiment 5 The binding protein of any one of Embodiments 1-4, comprising: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO:32, 19, 6, 45, 58, 71, 127, 140, 153, or 166, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO:33, 7, 20, 46, 59, 72, 128, 141, 154, or 167 or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 38, 12, 25, 51, 64, 77, 133, 146, 159, or 172 or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO:39, 13, 26, 52, 65
  • Embodiment 6 The binding protein of any one of Embodiments 1-5, comprising the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs:
  • Embodiment 7 The binding protein of any one of Embodiments 1-6, comprising the
  • Embodiment 8 The binding protein of any one of Embodiments 1-6, comprising the
  • Embodiment 9 The binding protein of any one of Embodiments 1-6, comprising the
  • Embodiment 10 The binding protein of any one of Embodiments 1-6, comprising the
  • Embodiment 11 The binding protein of any one of Embodiments 1-10, wherein:
  • the Va domain comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs:31, 5, 18, 44, 57, 70, 126, 139, 152, and 165; and/or
  • the VP domain comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs:37, 11, 24, 50, 63, 76, 132, 145, 158, and 171.
  • Embodiment 12 The binding protein of any one of Embodiments 1-11, wherein the Va and the VP domain comprise or consist of amino acid sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences set forth in SEQ ID NOs:
  • Embodiment 13 The binding protein of any one of Embodiments 1-12, wherein the Va domain and the VP domain comprise or consist of the amino acid sequences set forth in SEQ ID NOs:
  • Embodiment 14 The binding protein of any one of Embodiments 1-13, wherein the Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:31 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO:37.
  • Embodiment 15 The binding protein of any one of Embodiments 1-13, wherein the Va domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:70 and the VP domain comprises or consists of amino acid sequence set forth in SEQ ID NO:76.
  • Embodiment 16 The binding protein of any one of Embodiments 1-15, further comprising a TCR a chain constant domain (Ca) and/or a TCR P chain constant domain (CP), preferably comprising a Ca and a CP wherein the Va and the Ca comprise a TCRa chain and the VP and the CP comprise a TCRP chain.
  • a TCR a chain constant domain Ca
  • CP TCR P chain constant domain
  • Embodiment 17 The binding protein of Embodiment 16, wherein the Ca comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs:96-98.
  • Embodiment 18 The binding protein of Embodiment 16 or 17, wherein the CP comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs:99-102.
  • Embodiment 19 The binding protein of any one of Embodiments 16-18, wherein the Ca and the CP comprise or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequences set forth in SEQ ID NOs: 97 and 100, 102, or 245, respectively.
  • Embodiment 20 The binding protein of any one of Embodiments 1-19, comprising a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequences set forth in:
  • Embodiment 21 The binding protein of any one of Embodiments 1-20, comprising
  • Embodiment 22 The binding protein of any one of Embodiments 1-20, comprising (i) a TCRa chain having the amino acid sequence of SEQ ID NO:92 and (ii) a TCRP chain having the amino acid sequence of SEQ ID NO:93.
  • Embodiment 23 The binding protein of any one of Embodiments 1-22, wherein the binding protein comprises a TCR, a single-chain TCR (scTCR), a single-chain T cell receptor variable fragment (scTv), or a chimeric antigen receptor (CAR).
  • scTCR single-chain TCR
  • scTv single-chain T cell receptor variable fragment
  • CAR chimeric antigen receptor
  • Embodiment 24 The binding protein of any one of Embodiments 1-23, wherein the binding protein comprises a TCR.
  • Embodiment 25 An isolated polynucleotide encoding the binding protein of any one of Embodiments 1-24.
  • Embodiment 26 The polynucleotide of Embodiment 25, comprising a polynucleotide having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the polynucleotide sequence set forth in any one of SEQ ID NOs:107-124 or 185-196, or any combination thereof.
  • Embodiment 27 The polynucleotide of Embodiment 25 or 26, further comprising:
  • Embodiment 28 The polynucleotide of Embodiment 27, comprising:
  • Embodiment 29 a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).
  • Embodiment 29 The polynucleotide of Embodiment 27 or 28, further comprising a polynucleotide that encodes a self-cleaving peptide and is disposed between:
  • polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain;
  • Embodiment 30 The polynucleotide of any one of Embodiments 27-29, comprising, operably linked in-frame:
  • Embodiment 31 The polynucleotide of any one of Embodiments 27-30, wherein the encoded binding protein comprises a TCRa chain and a TCRP chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain.
  • Embodiment 32 The polynucleotide of Embodiment 31, comprising, operably linked in-frame:
  • Embodiment 33 The polynucleotide of any one of Embodiments 25-32, encoding an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs: 42, 16, 29, 55, 68, 81, 137, 150, 163, and 176.
  • Embodiment 34 The polynucleotide of any one of Embodiments 25-33, which is or comprises a polynucleotide sequence that is codon optimized for expression in a host cell, wherein, optionally, the host cell is a human immune system cell, and wherein, further optionally, is a T cell.
  • Embodiment 35 An isolated recombinant polynucleotide comprising an expression control element operably coupled to a sequence encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:34, 35, 8, 9, 21, 22, 47, 48, 60, 61, 73, and 74, wherein, optionally, the polypeptide is or comprises a T cell receptor (TCR) beta chain variable domain (VP).
  • TCR T cell receptor
  • VP beta chain variable domain
  • Embodiment 36 An expression vector, comprising the polynucleotide of any one of Embodiments 25-35 operably linked to an expression control sequence, wherein, optionally, the polynucleotide is codon-optimized for expression in a host cell.
  • Embodiment 37 The expression vector of Embodiment 36, wherein the vector is capable of delivering the polynucleotide to a host cell.
  • Embodiment 38 The expression vector of Embodiment 37, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.
  • Embodiment 39 The expression vector of Embodiment 38, wherein the human immune system cell is a CD4 + T cell, a CD8 + T cell, a CD4 CD8' double negative T cell, a y5 T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, bulk T cells, PBMCs, or any combination thereof.
  • Embodiment 40 The expression vector of Embodiment 39, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.
  • Embodiment 41 The expression vector of any one of Embodiments -36-40, wherein the vector is a viral vector.
  • Embodiment 42 The expression vector of Embodiment 41, wherein the viral vector is a lentiviral vector or a y-retroviral vector.
  • Embodiment 43 A host cell modified to comprise the polynucleotide of any one of Embodiments 25-35 and/or modified to comprise the expression vector of any one of Embodiments 36-42 and/or modified to express the binding protein of any one of Embodiments 1-24.
  • Embodiment 44 A host cell expressing the binding protein of any one of Embodiments 1-24.
  • Embodiment 45 The host cell of Embodiment 43 or 44, wherein the modified cell comprises a hematopoietic progenitor cell and/or or human immune cell.
  • Embodiment 46 The host cell of Embodiment 45, wherein the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, PBMCs, bulk T cells, or any combination thereof.
  • Embodiment 47 The host cell of Embodiment 46, wherein the immune cell comprises a CD4 + T cell, a CD8 + T cell, a CD4' CD8' double negative T cell, a y5 T cell, a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof, wherein, optionally, the immune cell comprises a CD4 + T cell and a CD8 + T cell, wherein, further optionally, the CD4 + T cell, the CD8 + T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optional
  • Embodiment 48 The host cell of any one of Embodiments 43-47, wherein the modified cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TIGIT gene; a TCR component gene, a FasL gene, or any combination thereof.
  • Embodiment 49 The host cell of Embodiment 48, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.
  • an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.
  • Embodiment 50 The host cell of Embodiment 48 or 49, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.
  • Embodiment 51 A composition comprising the host cell of any one of Embodiments 43-50 and a pharmaceutically acceptable carrier, diluent, or excipient.
  • Embodiment 52 The composition of Embodiment 51, comprising at least about 30% modified CD4 + T cells, combined with (ii) a composition comprising at least about 30% modified CD8 + T cells, in about a 1 : 1 ratio.
  • Embodiment 53 The composition of Embodiment 51 or 52, wherein the composition contains substantially no naive T cells.
  • Embodiment 54 A composition comprising:
  • Embodiment 55 A method for treating a disease or disorder associated with a P53 mutation (e.g., a P53 R175H mutation, optionally comprised in the peptide of SEQ ID NO:3) in a subject, the method comprising administering to the subject an effective amount of:
  • a disease or disorder associated with a P53 mutation e.g., a P53 R175H mutation, optionally comprised in the peptide of SEQ ID NO:3
  • the expression vector of any one of Embodiments 36-42 (iii) the expression vector of any one of Embodiments 36-42; (iv) the host cell of any one of Embodiments 43-50, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and wherein, optionally, the host cell is autologous, allogeneic, or syngeneic to the subject; and/or
  • Embodiment 56 The method of Embodiment 55, wherein the disease or disorder comprises a cancer, wherein the cancer is optionally a solid cancer or a hematological malignancy.
  • Embodiment 57 The method of Embodiment 55 or 56, wherein the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a nonsmall-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal
  • Embodiment 58 The method of any one of Embodiments 55-57, wherein the binding protein, polynucleotide, vector, host cell, or composition is administered to the subject parenterally or intravenously.
  • Embodiment 59 The method of any one of Embodiments 55-58, wherein the method comprises administering a plurality of doses of any one or more of (i)-(v) to the subject.
  • Embodiment 60 The method of Embodiment 59, wherein the plurality of doses are administered at intervals between administrations of about two to about four weeks.
  • Embodiment 61 The method of any one of Embodiments 55-60, wherein the composition comprises the host cell or the composition comprising the host cell, and wherein the method comprises administering the host cell or composition to the subject at a dose of about 10 4 cells/kg to about 10 11 cells/kg.
  • Embodiment 62 The method of any one of Embodiments 55-61, further comprising determining that the subject expresses HLA-A*2, optionally HLA-A*02:01, prior to administering the binding protein, polynucleotide, vector, host cell, or composition.
  • Embodiment 63 The method of any one of Embodiments 55-62, wherein the method further comprises administering a cytokine to the subject.
  • Embodiment 64 The method of Embodiment 63, wherein the cytokine comprises IL-2, IL-15, or IL-21.
  • Embodiment 65 The method of any one of Embodiments 55-64, wherein the subject has received or is receiving an immune checkpoint inhibitor and/or an agonist of a stimulatory immune checkpoint agent.
  • Embodiment 66 The binding protein of any one of Embodiments 1-24, the polynucleotide of any one of Embodiments 25-35, the expression vector of any one of Embodiments 36-42, the host cell of any one of Embodiments 43-50, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and/or the composition of any one of Embodiments 51-54, for use in a method for treating a disease or disorder associated with a P53 mutation (e.g.
  • a P53 R175H mutation e.g., a disease or disorder encoding and/or expressing the peptide of SEQ ID NO:3) in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Mely
  • Embodiment 67 The binding protein of any one of Embodiments 1-24, the polynucleotide of any one of Embodiments 25-35, the expression vector of any one of Embodiments 36-42, the host cell of any one of Embodiments 43-50, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and/or the composition of any one of Embodiments 51-54, for use the manufacture of a medicament for treating a disease or disorder associated with a P53 mutation (e.g., a P53 R175H mutation; e.g., a disease or disorder encoding and/or expressing the peptide of SEQ ID NO:3) in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy.
  • a P53 mutation e.g., a P53 R175H mutation; e.
  • the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucos
  • PDAC pancreatic
  • a binding protein comprising:
  • TCR T cell receptor
  • Va chain variable domain
  • CDR3a complementarity determining region 3 amino acid sequence set forth in any one of SEQ ID NOs:35, 34, 8, 9, 21, 22, 47, 48, 60, 61, 73, 74, 129, 130, 142, 143, 155, 156, 168, and 169, or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions; and/or
  • a TCR P chain variable (VP) domain comprising the CDR3P amino acid sequence set forth in any one of SEQ ID NOs:41, 40, 14, 15, 27, 28, 53, 54, 66, 67, 79, 80, 94, 95, 135, 136, 148, 149, 161, 162, 174, and 175, or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions, wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises or consists of the amino acid sequence HMTEVVRHC (SEQ ID NO:3) and wherein the HLA comprises an HLA-A*2.
  • Embodiment 2a The binding protein of Embodiment la, wherein the HLA comprises HL A- A* 02:01.
  • Embodiment 3a The binding protein of Embodiment la or 2a, wherein the Va domain and/or the VP domain is human, humanized, or chimeric, and is preferably human.
  • Embodiment 4a The binding protein of any one of Embodiments la-3 a, comprising the CDR3a and CDR3P amino acid sequences set forth in SEQ ID NOs: (i) 35 and 41, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (ii) 22 and 28, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iii) 9 and 15, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iv) 48 and 54, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (v) 61 and 67, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vi) 74 and 80, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vii) 8 and 14, respectively, or variants thereof having one, two, or three, optionally
  • Embodiment 5a The binding protein of any one of Embodiments la-4a, comprising: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO:32, 19, 6, 45, 58, 71, 127, 140, 153, or 166, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO:33, 7, 20, 46, 59, 72, 128, 141, 154, or 167, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 38, 12, 25, 51, 64, 77, 133, 146, 159, or 172 or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO:39, 13, 26,
  • Embodiment 6a The binding protein of any one of Embodiments la-5a, comprising the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs:
  • Embodiment 7a The binding protein of any one of Embodiments la-6a, comprising the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 32, 33, 35, 38, 39, and 41, respectively.
  • Embodiment 8a The binding protein of any one of Embodiments la-6a, comprising the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 32, 33, 34, 38, 39, and 40, respectively.
  • Embodiment 9a The binding protein of any one of Embodiments la-6a, comprising the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 71, 72, 74, 77, 78, and 80, respectiv, respectively.
  • Embodiment 10a The binding protein of any one of Embodiments la-6a, comprising the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs: 71, 72, 73, 77, 78, and 79, respectively.
  • Embodiment I la The binding protein of any one of Embodiments la-lOa, wherein:
  • the Va domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs:31, 5, 18, 44, 57, 70, 126, 139, 152, and 165; and/or
  • the VP domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs:37, 11, 24, 50, 63, 76, 132, 145, 158, and 171.
  • Embodiment 12a The binding protein of any one of Embodiments la-1 la, wherein the Va and the VP domain comprise, consist essentially of, or consist of amino acid sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences set forth in SEQ ID NOs: (i) 31 and 37, respectively; (ii) 5 and 11, respectively; (iii) 18 and 24, respectively; (iv) 44 and 50, respectively; (v) 57 and 63, respectively; (vi) 70 and 76, respectively; (vii) 126 and 132, respectively;
  • Embodiment 13 a The binding protein of any one of Embodiments la- 12a, wherein the Va domain and the VP domain comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOs: (i) 31 and 37, respectively; (ii) 5 and 11, respectively;
  • Embodiment 14a The binding protein of any one of Embodiments la- 13 a, wherein the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID N0:31 and the VP domain comprises, consists essentially of, or consists of amino acid sequence set forth in SEQ ID NO:37.
  • Embodiment 15a The binding protein of any one of Embodiments la-13a, wherein the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:70 and the VP domain comprises, consist essentially of, or consists of amino acid sequence set forth in SEQ ID NO:76.
  • Embodiment 16a The binding protein of any one of Embodiments la-15a, further comprising a TCR a chain constant domain (Ca) and/or a TCR P chain constant domain (CP), preferably comprising a Ca and a CP wherein the Va and the Ca comprise a TCRa chain and the VP and the CP comprise a TCRP chain.
  • a TCR a chain constant domain Ca
  • CP TCR P chain constant domain
  • Embodiment 17a The binding protein of Embodiment 16a, wherein the Ca comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs:96-98.
  • Embodiment 18a The binding protein of Embodiment 16a or 17a, wherein the CP comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs:99-102 and 245.
  • Embodiment 19a The binding protein of any one of Embodiments 16a- 18a, wherein the Ca and the CP comprise, consist essentially of, or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequences set forth in SEQ ID NOs: 97 and 100, 102, or 245, respectively.
  • Embodiment 20a The binding protein of any one of Embodiments la- 19a, comprising a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise, consist essentially of, or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequences set forth in: (i) SEQ ID NOs:86 and 87, respectively; (ii) SEQ ID NOs:82 and 83, respectively; (iii) SEQ ID NOS.:84 and 85, respectively; (iv) SEQ ID NOs:88 and 89, respectively; (v)
  • SEQ ID NOs:90 and 91 respectively;
  • SEQ ID NOs:92 and 93 respectively;
  • SEQ ID NOs: 177 and 178 respectively;
  • SEQ ID NOs: 179 and 180 respectively;
  • SEQ ID NOs: 183 and 184 respectively.
  • Embodiment 21a The binding protein of any one of Embodiments la-20a, comprising, consisting essentially of, or consisting of: (i) a TCRa chain having the amino acid sequence of SEQ ID NO: 86 and (ii) a TCRP chain having the amino acid sequence of SEQ ID NO:87.
  • Embodiment 22a The binding protein of any one of Embodiments la-20a, comprising, consisting essentially of, or consisting of: (i) a TCRa chain having the amino acid sequence of SEQ ID NO: 92 and (ii) a TCRP chain having the amino acid sequence of SEQ ID NO:93.
  • Embodiment 23 a The binding protein of any one of Embodiments la-22a, wherein the binding protein comprises a TCR, a single-chain TCR (scTCR), a single-chain T cell receptor variable fragment (scTv), or a chimeric antigen receptor (CAR).
  • scTCR single-chain TCR
  • scTv single-chain T cell receptor variable fragment
  • CAR chimeric antigen receptor
  • Embodiment 24a The binding protein of any one of Embodiments la-23 a, wherein the binding protein comprises a TCR.
  • Embodiment 25a An isolated polynucleotide encoding the binding protein of any one of Embodiments la-24a.
  • Embodiment 26a The polynucleotide of Embodiment 25a, comprising a polynucleotide having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the polynucleotide sequence set forth in any one of SEQ ID NOs: 107-124 and 185-196, or any combination thereof.
  • Embodiment 27a The polynucleotide of Embodiment 25a or 26a, further comprising:
  • Embodiment 28a The polynucleotide of Embodiment 27a, comprising: (a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor p chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).
  • Embodiment 29a The polynucleotide of Embodiment 27a or 28a, further comprising a polynucleotide that encodes a self-cleaving peptide and is disposed between:
  • polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain;
  • Embodiment 30a The polynucleotide of any one of Embodiments 27a-29a, comprising, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnBP); (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnBP); (iii) (pnBP)-(pnSCPi)-(pnCD8a)- (pnSCP2)-(pnCD8P); (iv) (pnBP)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnCD8a); (v)
  • pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain
  • pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain
  • pnBP is the polynucleotide encoding a binding protein
  • pnSCPi and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptide
  • Embodiment 31a The polynucleotide of any one of Embodiments 27a-30a, wherein the encoded binding protein comprises a TCRa chain and a TCRP chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain.
  • Embodiment 32a The polynucleotide of Embodiment 31a, comprising, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRP)-(pnSCP3)- (pnTCRa); (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa); (iii) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP); (iv) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTC
  • Embodiment 33a The polynucleotide of any one of Embodiments 25a-32a, encoding an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs: 42, 16, 29, 55, 68, 81, 137, 150, 163, and 176.
  • Embodiment 34a The polynucleotide of any one of Embodiments 25a-33a, which is or comprises a polynucleotide sequence that is codon optimized for expression in a host cell, wherein, optionally, the host cell is a human immune system cell, and wherein, further optionally, is a T cell.
  • Embodiment 35a An isolated recombinant polynucleotide comprising an expression control element operably coupled to a sequence encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:34, 35, 8, 9, 21, 22, 47, 48, 60, 61, 73, 74, 15, 28, 41, 54, 67, 80, 136, 149, 162, 175, 14, 27, 40, 53, 66, 79, 135, 148, 161, and 174, wherein, optionally, the polypeptide is or comprises a T cell receptor (TCR) beta chain variable domain (VP).
  • TCR T cell receptor
  • VP beta chain variable domain
  • Embodiment 36a An expression vector, comprising the polynucleotide of any one of Embodiments 25a-35a operably linked to an expression control sequence, wherein, optionally, the polynucleotide is codon-optimized for expression in a host cell.
  • Embodiment 37a The expression vector of Embodiment 36a, wherein the vector is capable of delivering the polynucleotide to a host cell.
  • Embodiment 38a The expression vector of Embodiment 37a, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.
  • Embodiment 39a The expression vector of Embodiment 38a, wherein the human immune system cell is a CD4 + T cell, a CD8 + T cell, a CD4 CD8' double negative T cell, a y5 T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, bulk T cells, PBMCs, or any combination thereof.
  • Embodiment 40a The expression vector of Embodiment 39a, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.
  • Embodiment 41a The expression vector of any one of Embodiments 36a-40a, wherein the vector is a viral vector.
  • Embodiment 42a The expression vector of Embodiment 41a, wherein the viral vector is a lentiviral vector or a y-retroviral vector.
  • Embodiment 43a A host cell modified to comprise the polynucleotide of any one of Embodiments 25a-35a and/or modified to comprise the expression vector of any one of Embodiments 36a-42a and/or modified to express the binding protein of any one of Embodiments la-24a.
  • Embodiment 44a A host cell expressing the binding protein of any one of Embodiments la-24a.
  • Embodiment 45a The host cell of Embodiment 43a or 44a, wherein the modified cell comprises a hematopoietic progenitor cell and/or or human immune cell.
  • Embodiment 46a The host cell of Embodiment 45a, wherein the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, PBMCs, bulk T cells, or any combination thereof.
  • Embodiment 47a The host cell of Embodiment 46a, wherein the immune cell comprises a CD4 + T cell, a CD8 + T cell, a CD4' CD8' double negative T cell, a y5 T cell, a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof, wherein, optionally, the immune cell comprises a CD4 + T cell and a CD8 + T cell, wherein, further optionally, the CD4 + T cell, the CD8 + T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein
  • Embodiment 48a The host cell of any one of Embodiments 43a-47a, wherein the modified cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TIGIT gene; a TCR component gene, a FasL gene, or any combination thereof.
  • Embodiment 49a The host cell of Embodiment 48a, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.
  • an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.
  • Embodiment 50a The host cell of Embodiment 48a or 49a, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.
  • Embodiment 51a A composition comprising the host cell of any one of Embodiments 43a-50a and a pharmaceutically acceptable carrier, diluent, or excipient.
  • Embodiment 52a The composition of Embodiment 51a, comprising at least about 30% modified CD4 + T cells, combined with (ii) a composition comprising at least about 30% modified CD8 + T cells, in about a 1 : 1 ratio.
  • Embodiment 53a The composition of Embodiment 51a or 52a, wherein the composition contains substantially no naive T cells.
  • Embodiment 54a A composition comprising:
  • Embodiment 55a A method for treating a disease or disorder associated with a P53 mutation (e.g., a P53 R175H mutation, optionally comprised in the peptide of SEQ ID NO:3) in a subject, the method comprising administering to the subject an effective amount of:
  • a disease or disorder associated with a P53 mutation e.g., a P53 R175H mutation, optionally comprised in the peptide of SEQ ID NO:3
  • the expression vector of any one of Embodiments 36a-42a (iii) the expression vector of any one of Embodiments 36a-42a; (iv) the host cell of any one of Embodiments 43a-50a, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and wherein, optionally, the host cell is autologous, allogeneic, or syngeneic to the subject; and/or
  • Embodiment 56a The method of Embodiment 55a, wherein the disease or disorder comprises a cancer, wherein the cancer is optionally a solid cancer or a hematological malignancy.
  • Embodiment 57a The method of Embodiment 55a or 56a, wherein the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a nonsmall-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an
  • Embodiment 58a The method of any one of Embodiments 55a-57a, wherein the binding protein, polynucleotide, vector, host cell, or composition is administered to the subject parenterally or intravenously.
  • Embodiment 59a The method of any one of Embodiments 55a-58a, wherein the method comprises administering a plurality of doses of any one or more of (i)-(v) to the subject.
  • Embodiment 60a The method of Embodiment 59a, wherein the plurality of doses are administered at intervals between administrations of about two to about four weeks.
  • Embodiment 61a The method of any one of Embodiments 55a-60a, wherein the composition comprises the host cell or the composition comprising the host cell, and wherein the method comprises administering the host cell or composition to the subject at a dose of about 10 4 cells/kg to about 10 11 cells/kg.
  • Embodiment 62a The method of any one of Embodiments 55a-61a, further comprising determining that the subject expresses HLA-A*2, optionally HLA-A*02:01, prior to administering the binding protein, polynucleotide, vector, host cell, or composition.
  • Embodiment 63a The method of any one of Embodiments 55a-62a, wherein the method further comprises administering a cytokine to the subject.
  • Embodiment 64a The method of Embodiment 63a, wherein the cytokine comprises IL-2, IL-15, or IL-21.
  • Embodiment 65a The method of any one of Embodiments 55a-64a, wherein the subject has received or is receiving an immune checkpoint inhibitor and/or an agonist of a stimulatory immune checkpoint agent.
  • Embodiment 66a The binding protein of any one of Embodiments la-24a, the polynucleotide of any one of Embodiments 25a-35a, the expression vector of any one of Embodiments 36a-42a, the host cell of any one of Embodiments 43a-50a, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and/or the composition of any one of Embodiments 51a-54a, for use in a method for treating a disease or disorder associated with a P53 mutation (e.g.
  • a P53 R175H mutation e.g., a disease or disorder encoding and/or expressing the peptide of SEQ ID NO:3) in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Mely
  • Embodiment 67a The binding protein of any one of Embodiments la-24a, the polynucleotide of any one of Embodiments 25a-35a, the expression vector of any one of Embodiments 36a-42a, the host cell of any one of Embodiments 43a-50a, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and/or the composition of any one of Embodiments 5 la-54a, for use the manufacture of a medicament for treating a disease or disorder associated with a P53 mutation (e.g., a P53 R175H mutation; e.g., a disease or disorder encoding and/or expressing the peptide of SEQ ID NO:3) in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and, wherein, optionally, the disease or disorder is selected from

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  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation concerne des compositions et des procédés permettant de cibler un antigène P53 pour, par exemple, traiter ou prévenir le cancer. Des modes de réalisation de la divulgation comprennent des protéines de liaison, telles que des récepteurs de lymphocytes T se liant à un complexe antigène P53 : HLA. Dans certains modes de réalisation, un antigène P53 est un peptide comprenant une mutation R175H, telle qu'un peptide comprenant ou constitué de la séquence d'acides aminés de SEQ ID NO : 3. Les protéines de liaison divulguées sont dans certains modes de réalisation hautement sensibles à l'antigène et sont capables d'induire l'activation de lymphocytes T hôtes à de faibles concentrations d'antigène peptidique. Des polynucléotides codant pour une telle protéine de liaison peuvent être introduits dans une cellule hôte, telle qu'un lymphocyte T, et la cellule peut être utilisée en immunothérapie pour traiter divers cancers.
PCT/US2024/013407 2023-01-30 2024-01-29 Protéines de liaison spécifiques pour p53 mutant et leurs utilisations Ceased WO2024163371A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020227091A1 (fr) * 2019-05-03 2020-11-12 Gigamune, Inc. Cellules modifiées exprimant des récepteurs des lymphocytes t antitumoraux et leurs méthodes d'utilisation
WO2020264269A1 (fr) * 2019-06-27 2020-12-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs des lymphocytes t reconnaissant la mutation r175h ou y220c dans p53
WO2022183167A1 (fr) * 2021-02-25 2022-09-01 Alaunos Therapeutics, Inc. Vecteurs recombinants comprenant des cassettes d'expression polycistronique et leurs procédés d'utilisation
WO2022236050A1 (fr) * 2021-05-07 2022-11-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs de lymphocytes t reconnaissant les mutations c135y, r175h, ou m237i dans p53

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020227091A1 (fr) * 2019-05-03 2020-11-12 Gigamune, Inc. Cellules modifiées exprimant des récepteurs des lymphocytes t antitumoraux et leurs méthodes d'utilisation
WO2020264269A1 (fr) * 2019-06-27 2020-12-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs des lymphocytes t reconnaissant la mutation r175h ou y220c dans p53
WO2022183167A1 (fr) * 2021-02-25 2022-09-01 Alaunos Therapeutics, Inc. Vecteurs recombinants comprenant des cassettes d'expression polycistronique et leurs procédés d'utilisation
WO2022236050A1 (fr) * 2021-05-07 2022-11-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs de lymphocytes t reconnaissant les mutations c135y, r175h, ou m237i dans p53

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