US20150191524A1 - Engineering t cell receptors - Google Patents

Engineering t cell receptors Download PDF

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US20150191524A1
US20150191524A1 US14/417,434 US201314417434A US2015191524A1 US 20150191524 A1 US20150191524 A1 US 20150191524A1 US 201314417434 A US201314417434 A US 201314417434A US 2015191524 A1 US2015191524 A1 US 2015191524A1
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cell receptor
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hla
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Sheena N. Smith
David M. Kranz
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University of Illinois
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Definitions

  • TCR T cell receptor
  • T cell receptors and antibodies are molecules that have evolved to recognize different classes of antigens (ligands)((Murphy (2012), xix, 868 p.)).
  • TCRs are antigen-specific molecules that are responsible for recognizing antigenic peptides presented in the context of a product of the major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) or any nucleated cell (e.g., all human cells in the body, except red blood cells).
  • MHC major histocompatibility complex
  • APCs antigen presenting cells
  • nucleated cell e.g., all human cells in the body, except red blood cells.
  • antibodies typically recognize soluble or cell-surface antigens, and do not require presentation of the antigen by an MHC.
  • This system endows T cells, via their TCRs, with the potential ability to recognize the entire array of intracellular antigens expressed by a cell (including virus proteins) that are processed intracellularly into short peptides, bound to an intracellular MHC molecule, and delivered to the surface as a peptide-MHC complex (pepMHC).
  • This system allows virtually any foreign protein (e.g., mutated cancer antigen or virus protein) or aberrantly expressed protein to serve a target for T cells (reviewed in (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012), xix, 868 p.)).
  • the interaction of a TCR and a pepMHC can drive the T cell into various states of activation, depending on the affinity (or dissociation rate) of binding.
  • the TCR recognition process allows a T cell to discriminate between a normal, healthy cell and, e.g., one that has become transformed via a virus or malignancy, by providing a diverse repertoire of TCRs, wherein there is a high probability that one or more TCRs will be present with a binding affinity for the foreign peptide bound to an MHC molecule that is above the threshold for stimulating T cell activity (Manning and Kranz (1999) Immunology Today, 20, 417-422).
  • T cells have evolved a co-receptor system in which the cell surface molecules CD4 and CD8 bind to the MHC molecules (class II and class I, respectively) and synergize with the TCR in mediating signaling activity.
  • Directed evolution has been used to generate TCRs with higher affinity for a specific pepMHC.
  • the three different display methods that have been used are yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), and T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84).
  • the process involves engineering, or modifying, a TCR that exhibits the normal, low affinity of the wild-type TCR, so that affinity of mutants of the TCR have increased affinity for the cognate pepMHC (the original antigen that the T cells were specific for).
  • the wild-type TCR was used as a template for producing mutagenized libraries in one or more of the CDRs, and mutants with higher affinity were selected by binding to the cognate peptide-MHC antigen.
  • TCR-engineering approaches require a different TCR isolated from a T cell clone with reactivity towards a specific peptide antigen in order to develop a higher affinity TCR mutant specific for the peptide antigen (cognate antigen), or structurally similar variants thereof.
  • TCR TCR isolated from a T cell clone with reactivity towards a specific peptide antigen in order to develop a higher affinity TCR mutant specific for the peptide antigen (cognate antigen), or structurally similar variants thereof.
  • non-cognate antigens structurally very different antigens
  • the present invention relates to T cell receptor (TCR) scaffolds useful, for example and by way of example only, for the generation of products having novel binding specificities. More specifically, the present invention relates to a library of T cell receptor proteins displayed on the surface of yeast, phage, or mammalian cells; to TCR proteins that are selected from the library for binding to a non-cognate antigen not recognized by the original TCR; and to the use of the TCR proteins selected in vitro for therapeutic, diagnostic, or imaging applications.
  • TCR T cell receptor
  • One aspect of the invention relates to a modified T cell receptor, or antigen binding fragment thereof, comprising a V ⁇ and a V ⁇ derived from a wild type T cell receptor, wherein the V ⁇ , the V ⁇ , or both, comprise a mutation in one or more complementarity determining regions (CDRs) relative to the wild type T cell receptor, wherein the modified T cell receptor binds to a non-cognate peptide-MHC not bound by the wild type T cell receptor.
  • CDRs complementarity determining regions
  • the wild type T cell receptor comprises the V ⁇ amino acid sequence set forth in SEQ ID NO:1 and the V ⁇ amino acid sequence set forth in SEQ ID NO:2.
  • the modified T cell receptor comprises a modified V ⁇ comprising an amino acid sequence having at least 80% identity to the V ⁇ amino acid sequence set forth in SEQ ID NO:1 and a modified V ⁇ comprising an amino acid sequence having at least 80% identity to the V ⁇ amino acid sequence set forth in SEQ ID NO:2, wherein the modified T cell receptor does not bind to the cognate peptide-MHC bound by the wild type T cell receptor.
  • the modified T cell receptor comprises an amino acid substitution at one or more of CDR1 ⁇ 31, CDR3 ⁇ 98, CDR3 ⁇ 99, CDR3 ⁇ 97, CDR3 ⁇ 102, CDR3 ⁇ 99, CDR3 ⁇ 100, CDR3 ⁇ 101, CDR1 ⁇ 32, CDR1 ⁇ 30, CDR3 ⁇ 98.
  • the modified T cell receptor comprises the wild type amino acid at position CDR2 ⁇ 51.
  • the modified T cell receptor further comprises the wild type amino acid at position CDR1 ⁇ 31.
  • the modified T cell receptor further comprises the wild type amino acid at position CD1 ⁇ 28 and CD1 ⁇ 52.
  • the wild type T cell receptor is a single-chain T cell receptor A6-X15 comprising the amino acid sequence set forth in SEQ ID NO:3.
  • the non-cognate peptide-MHC comprises Mart1:HLA.A2, SL9 HIV:HLA.A2, WT-1:HLA.A2, or SURV:HLA.A2.
  • the modified T cell receptor comprises 1) a modified V ⁇ region comprising an amino acid sequence having at least 90% identity to the V ⁇ region of the amino acid sequence set forth in one of SEQ ID NOs:33, 41, or 42 and 2) a modified V ⁇ region comprising an amino acid sequence having at least 90% identity to the V ⁇ region of the amino acid sequence set forth in one of SEQ ID NOs:33, 41, or 42.
  • the amino acid sequence set forth in one of SEQ ID NOs:33, 41, or 42 is selected from one of SEQ ID NOs:33, 41, or 42.
  • the modified T cell receptor is generated by in vitro selection of a yeast display library of mutant T cell receptors.
  • the wild type T cell receptor is human.
  • the modified T cell receptor is a single chain T cell receptor.
  • the wild type T cell receptor binds HLA-A2.
  • a polypeptide encoding the modified T cell receptor is provided.
  • a polynucleotide encoding the polypeptide is provided.
  • Another aspect of the invention provides a modified T cell receptor, or antigen binding fragment thereof, comprising a V ⁇ and a V ⁇ derived from a wild type T cell receptor, wherein the V ⁇ comprises amino acid residues 140 to 256 of SEQ ID NO:34, and wherein the V ⁇ comprises amino acid residues 1 to 122 of SEQ ID NO:34.
  • Another aspect of the invention provides a modified T cell receptor, or antigen binding fragment thereof, comprising a V ⁇ and a V ⁇ derived from a wild type T cell receptor, wherein the V ⁇ comprises amino acid residues 140 to 255 of SEQ ID NO:43, and wherein the V ⁇ comprises amino acid residues 1 to 122 of SEQ ID NO:43.
  • One aspect of the invention provides a method for engineering a T cell receptor, or an antigen binding fragment thereof, with a desired specificity comprising: a) isolating a polynucleotide that encodes a wild type T cell receptor, or an antigen binding fragment thereof; b) generating a library of mutant T cell receptors, or antigen binding fragments thereof, wherein the mutant T cell receptors, or antigen-binding fragment thereof, comprise a mutation in one or more complementarity determining regions relative to the wild type T cell receptor; c) expressing the mutant T cell receptors in a surface display system; and d) selecting mutant T cell receptors that bind to a non-cognate peptide-MHC.
  • the wild type T cell receptor comprises the V ⁇ amino acid sequence set forth in SEQ ID NO:1 and the V ⁇ amino acid sequence set forth in SEQ ID NO:2.
  • the wild type T cell receptor is a single-chain T cell receptor A6-X15 comprising the amino acid sequence set forth in SEQ ID NO:3.
  • the surface display system is a yeast display system.
  • the non-cognate peptide-MHC is Mart1:HLA.A2, SL9 HIV:HLA.A2, WT-1:HLA.A2, or SURV:HLA.A2.
  • the method further comprises a step of affinity maturation.
  • FIG. 1 is a diagram that shows a method for the rational design of a single scaffold for engineering higher affinity TCRs specific for non-cognate antigens.
  • FIG. 2A is a 3-dimensional diagram that shows a structural view of the A6 TCR:pepMHC complex (A6; PDB:1AO7).
  • the variable (V) and constant (C) regions of the ⁇ -chain and ⁇ -chain are indicated.
  • the structure shown does not include the C ⁇ region of the A6 TCR.
  • HLA-A2 ( ⁇ 1, ⁇ 2, ⁇ 3, and ⁇ 2m) is shown in gray, and the Tax peptide (LLFGYPVYV; SEQ ID NO:5) is shown in black.
  • FIG. 2B is a 3-dimensional diagram that shows the CDR footprint over the peptide-MHC (Tax-HLA.A2).
  • FIG. 3 is an overlay of the footprint of the 5 most encountered residues within 3.0 ⁇ of corresponding peptide in the A6 wt and 5 predicted structures.
  • FIG. 4A depicts the crystal structures of V ⁇ 2-containing TCRs and shows predicted key MHC contact positions in the TCR CDR1 ⁇ and CDR2 ⁇ loops (modified from (Borbulevych et al. (2011) J Immunol, 187, 2453-63)).
  • FIG. 4B shows the binding of high-affinity A6 TCR X15 and 4 variants, each having an alanine substitution at one of four residues, to various concentrations of Tax (LLFGYPVYV, SEQ ID NO:5):HLA-A2 dimer (DimerX; obtained from BD Pharmingen).
  • FIG. 5 shows the amino acid sequence of the A6 V ⁇ (SEQ ID NO:2) and V ⁇ (SEQ ID NO:1) regions, and the positions shaded in gray indicate where degenerate libraries were constructed in the stabilized variant A6-X15 (SEQ ID NO:3).
  • the CDRs of each V domain are labeled, and the sequence of the linker that joins the two V regions in the yeast display vector is also shown.
  • FIG. 6 is a schematic of a single-chain T cell receptor (scTCR) using yeast display.
  • FIG. 7 shows the amino acid sequence alignment of ten clones chosen from a degenerate library of the human A6 X15 scTCR, the RD1 library.
  • FIG. 8 shows a flow cytometry histogram of the RD1 library after sorting with the cognate antigen (Tax:HLA.A2). Gray indicates yeast cells stained with secondary antibody only.
  • FIG. 9 shows a flow cytometry histogram of the RD1 library after sorting with the non-cognate antigen (Mart1:HLA.A2). Gray indicates yeast cells stained with secondary antibody only.
  • FIG. 10A shows the sequence alignments of six clones isolated following the 4th sort with the cognate ligand, Tax (LLFGYPVYV; SEQ ID NO:5):HLA-A2 dimer.
  • FIG. 10B shows the DNA sequence alignments of the six RD1 scaffold variants at degenerate positions. Below each codon is the amino acid encoded by that codon with the number of possible codon combinations within an NNS library.
  • FIG. 11A is a histogram depicting positive staining with the anti-HA antibody for the N-terminal tag, thus indicating surface expression of the AGA2 fusion.
  • Cells were stained with the anti-HA antibody and goat anti-mouse IgG alexa 647 secondary antibody (black line histogram) or secondary only as a control (gray filled histogram).
  • the negative peak for the HA stained cells (black line) is observed in all yeast display experiments and is due to yeast that have lost plasmid, and serves as an internal control for each induced yeast sample.
  • FIG. 11B is a histogram that shows negative staining with c-myc as this clone lacked the C-terminal c-myc tag.
  • Cells were stained with chicken anti-c-myc antibody and goat anti-chicken IgY alexa 647 secondary antibody or secondary only as a control (gray).
  • FIG. 11C is a histogram showing staining of the A6-X15 clone with various concentrations of the selecting cognate antigen, Tax:HLA.A2, dimer at the indicated concentrations.
  • FIG. 11D is a histogram showing staining of the A6-X15 clone with various concentrations of the non-selecting non-cognate antigen, Mart1:HLA.A2.
  • FIG. 11E is a plot of the mean fluorescence intensity (MFI) from staining with various concentrations of the peptide:HLA.A2 dimers Tax or Mart1 peptide:HLA.A2 dimer at 4-500 nM.
  • MFI mean fluorescence intensity
  • FIG. 12A shows the sequence alignments of five clones isolated following the 5th sort with a non-cognate ligand, Mart1 (ELAGIGILTV; SEQ ID NO:7):HLA-A2 dimer.
  • FIG. 12B shows the DNA sequence alignments of the five RD1 scaffold variants at degenerate positions. Below each codon is the amino acid encoded by that codon with the number of possible codon combinations within an NNS library.
  • FIG. 13A is a histogram that shows positive staining of the clone with the anti-HA antibody for the N-terminal tag, and thus indicating surface expression of the AGA2 fusion.
  • Cells were stained with anti-HA antibody and goat anti-mouse IgG alexa 647 secondary antibody (black line histogram) or secondary only as a control (gray filled histogram).
  • FIG. 13B is a histogram that shows positive staining with c-myc as this clone contained the C-terminal c-myc tag.
  • Cells were stained with chicken anti-c-myc antibody and goat anti-chicken IgY Alexa 647 secondary antibody or secondary only as a control (gray).
  • FIG. 13C is a histogram that shows staining of the A6-S5-4 clone with various concentrations of the selecting non-cognate antigen, Mart1:HLA.A2 dimer, at the indicated concentrations.
  • FIG. 13D is a histogram that shows the staining of the A6-S5-4 clone with various concentrations of the non-selecting cognate antigen, Tax:HLA.A2.
  • FIG. 13E is a plot of the mean fluorescence intensity (MFI) from staining with various concentrations of the peptide:HLA.A2 dimers Mart1 or Tax peptide:HLA.A2 dimer at 4-500 nM.
  • MFI mean fluorescence intensity
  • FIG. 14 shows the amino acid sequences of the scaffold A6 single-chain TCR wild type A6 V ⁇ and V ⁇ regions and two high-affinity variants isolated from the RD1 library, including the five positions of degeneracy in the library.
  • the two clones were isolated from the selection with Tax (clone S4-3; identical to the single-chain stabilized TCR A6-X15; SEQ ID NO:3), and Mart1 (clone S5-4; SEQ ID NO:33).
  • X represents any amino acid.
  • the asterisk is used to indicate where no linker is present in the wild-type A6 structure.
  • FIG. 15A shows flow cytometry histograms of the RD1-MART1-S5-4 CDR3 libraries after sorting with the non-cognate, selecting antigen, MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2.
  • FIG. 15B is a histogram that shows staining of the RD1-MART1-S5-4 clone, which was used as a template for CDR3 affinity maturation libraries, with 200 nM, 1 ⁇ M, and 5 ⁇ M MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2 UV-exchanged monomers followed by PE-conjugated streptavidin.
  • FIG. 15C is a histogram that shows staining of the RD1-MART1 HIGH clone isolated after the second sort of the RD1-MART1-S5-4 CDR3 libraries with 200 nM, 1 ⁇ M, and 5 ⁇ M MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2 UV-exchanged monomers followed by PE-conjugated streptavidin.
  • FIG. 16A is a histogram that shows the RD1-MART1 HIGH clone stained with 10 nM MART1/HLA-A2 dimer (DimerX; obtained from BD Pharmingen) and APC-conjugated goat anti-mouse secondary antibody as a positive control. Gray indicates yeast cells stained with secondary antibody only.
  • FIG. 16B is a histogram that shows the RD1-MART1 HIGH clone stained with 500 nM null Tax/HLA-A2 dimer (DimerX; obtained from BD Pharmingen) and APC-conjugated goat anti-mouse secondary antibody.
  • FIG. 16C is a histogram that shows the RD1-MART1 HIGH clone stained with 500 nM null WT1/HLA-A2 dimer (DimerX; obtained from BD Pharmingen) and APC-conjugated goat anti-mouse secondary antibody.
  • FIG. 16D is a histogram that shows the RD1-MART1 HIGH clone stained with 500 nM null Survivin/HLA-A2 dimer (DimerX; obtained from BD Pharmingen) and APC-conjugated goat anti-mouse secondary antibody.
  • FIGS. 17A-D are a series of histograms that show flow cytometry analysis of human T2 (HLA-A2+) cells incubated first with no peptide ( FIG. 17A ), Tax ( FIG. 17B ), WTI ( FIG. 17C ), or MART1 ( FIG. 17D ), followed by incubation with biotin-labeled RD1-MART1 HIGH TCR.
  • FIG. 17E is a line graph that depicts the titration showing that the RD1-MART1 HIGH TCR had an affinity (K D value) of at least 28 nM.
  • FIG. 18 shows the amino acid sequences of the scaffold A6 single-chain TCR (wild-type) and high-affinity variants isolated and affinity matured from the RD1 library, including the five positions of degeneracy in the library.
  • Two of the clones were isolated from the selection with Tax (clone RD1-Tax-S4-3; identical to the single-chain stabilized TCR A6-X15) and MART1 (clone RD1-MART1-S5-4).
  • Tax clone RD1-Tax-S4-3; identical to the single-chain stabilized TCR A6-X15
  • MART1 clone RD1-MART1-S5-4
  • the high affinity clone selected from the RD1-MART1-S5-4 CDR3 affinity maturation libraries is shown (RD1-MART1 HIGH ).
  • X represents any amino acid. The asterisk indicates where no linker is present in the wild-type A6 structure.
  • FIG. 19A is 3-dimensional diagram of the A6:Tax (LLFGYPVYV; SEQ ID NO:5)/HLA.A2 crystal structure (PDB: 1AO7) in close proximity to an overlay of MART1 (ELAGIGILTV; SEQ ID NO:7)/HLA.A2 (PDB: 1JF1) (Sliz et al. (2001) J Immunol, 167, 3276-84) and WT1 (RMFPNAPYL; SEQ ID NO:9)/HLA.A2 (PDB: 3HPJ) (Borbulevych et al. (2010) Mol Immunol, 47, 2519-24) crystal structures.
  • TCR ⁇ D27, G29, and S99; TCR ⁇ L99 and W100 were made degenerate based on NNK nucleic acid composition.
  • TCR ⁇ Q31 is a binary position where either the wild type residue glutamine or threonine may be selected.
  • Positions 100-103 in CDR3 ⁇ are binary where the four adjacent residues may be selected as A6 wild type (AGGR, SEQ ID NO:44) or A6-X15 (MSAQ, SEQ ID NO:45).
  • FIG. 19B is a flow cytometry histogram of the RD2 library after sorting with the selecting cognate antigen Tax/HLA.A2. Gray indicates yeast cells stained with secondary antibody only.
  • FIG. 19C is a flow cytometry histogram of the RD2 library after sorting with the non-cognate antigen MART1/HLA.A2.
  • FIG. 20 shows the sequence alignment of five clones isolated from the second generation degenerate library (RD2) of the human A6 scTCR that shows diversity prior to selection.
  • RD2 second generation degenerate library
  • FIG. 21A is a histogram that shows the RD2-MART1-S3-3 clone, selected following the 3 rd sort of the RD2 library, stained with 2 ⁇ M MART1/HLA.A2 UV-exchanged monomers, PE-conjugated streptavidin.
  • FIG. 21B is a histogram that shows the RD2-MART1-S3-4 clone, selected following the 3 rd sort of the RD2 library, stained with 2 ⁇ M MART1/HLA.A2 UV-exchanged monomers, PE-conjugated streptavidin.
  • FIG. 21C is a histogram that shows the RD2-MART1-S3-3 clone, selected following the 3 rd sort of the RD2 library, stained with 2 ⁇ M null Tax/HLA.A2 UV-exchanged monomers, PE-conjugated streptavidin.
  • FIG. 21D is a histogram that shows the RD2-MART1-S3-4 clone, selected following the 3 rd sort of the RD2 library, stained with 2 ⁇ M null Tax/HLA.A2 UV-exchanged monomers, PE-conjugated streptavidin.
  • FIG. 21E shows the sequences of the scaffold A6 single-chain TCR and high-affinity variants isolated from the RD2 library selection with MART1. Sequences of the wild type A6 V ⁇ and V ⁇ regions of the A6 TCR (Garboczi et al. (1996) Nature, 384, 134-141), the high affinity single-chain variant A6-X15 (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72), and two of the clones isolated from the selection with MART1 (clone RD2-MART1-S3-3 and clone RD2-MART1-S3-4) are shown.
  • FIG. 22 shows the amino acid sequence of an alternative scaffold, human TCR T1-S18.45.
  • FIG. 23A is a histogram that shows the titration of biotinylated T1-S18.45 scTv on antigen-presenting cell line T2 (HLA-A2+) pre-loaded with MART-1 peptide (1 ⁇ M) or null peptide, SL9 (1 ⁇ M).
  • Cells were stained with 3.9 nM, 7.8 nM, 15.2 nM, 31.1 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 ⁇ M, and 5 ⁇ M biotinylated T1-S18.45 scTv as indicated and followed by SA:PE. Data shown is representative of 4 experiments.
  • FIG. 23B is a line graph showing the mean fluorescence unit (MFU) values of histograms in FIG. 23A plotted versus scTv-biotin concentration.
  • MFU mean fluorescence unit
  • FIG. 24 is a diagram that illustrates exemplary therapeutic applications of the high-affinity, single-chain TCRs isolated from the scaffold libraries.
  • FIG. 24A shows five examples of TCR formats for use as soluble therapeutic products: 1) single-chain TCR in either a V ⁇ -V ⁇ orientation or V ⁇ -V ⁇ orientation (mutated high-affinity V domains are shown with an asterisk); 2) single-chain TCR fused in frame with the constant region domains of an antibody; 3) in-frame immunoglobulin fusion to either the constant region of the light chain or the heavy chain; 4) single-chain TCR (or the immunoglobulin fusions shown in 2 and 3) directly coupled to a drug; and 5) single-chain TCR linked in-frame with a single-chain Fv (VL-linker-VH).
  • FIG. 24B shows the variable domains (V) isolated by yeast display for high-affinity binding using the TCR scaffold inserted into mammalian cell vectors for expression by T cells in adoptive T cell therapy as 1) single-chain receptors in chimeric antigen receptors (CARs) and 2) full length ⁇ and ⁇ TCRs.
  • V variable domains isolated by yeast display for high-affinity binding using the TCR scaffold inserted into mammalian cell vectors for expression by T cells in adoptive T cell therapy as 1) single-chain receptors in chimeric antigen receptors (CARs) and 2) full length ⁇ and ⁇ TCRs.
  • SEQ ID NO:1 is the amino acid sequence of the V ⁇ region of the A6 TCR.
  • SEQ ID NO:2 is the amino acid sequence of the V ⁇ region of the A6 TCR.
  • SEQ ID NO:3 is the amino acid sequence of the single chain TCR A6-X15 and the identical clones RD1-Tax-S4-3 and RD1-Tax-S4-5.
  • SEQ ID NO:4 is the amino acid sequence of the RD1 library.
  • SEQ ID NO:5 is the amino acid sequence of the Tax antigen.
  • SEQ ID NO:6 is the amino acid sequence of the Mart1-9 mer antigen.
  • SEQ ID NO:7 is the amino acid sequence of the Mart1-10 mer antigen.
  • SEQ ID NO:8 is the amino acid sequence of the SL9 HIV antigen.
  • SEQ ID NO:9 is the amino acid sequence of the WT-1 antigen.
  • SEQ ID NO:10 is the amino acid sequence of the Survivin antigen.
  • SEQ ID NO:11 is the amino acid sequence of the NY-ESO-1 antigen.
  • SEQ ID NO:12 is the amino acid sequence of the PPI antigen.
  • SEQ ID NO:13 is the amino acid sequence of the MDM2 antigen.
  • SEQ ID NO:14 is the amino acid sequence of the HBE183 antigen.
  • SEQ ID NO:15 is the amino acid sequence of the gp100 antigen.
  • SEQ ID NO:16 is the amino acid sequence of the MUC1 antigen.
  • SEQ ID NO:17 is the amino acid sequence of the MAGE A3 antigen.
  • SEQ ID NO:18 is the amino acid sequence of the HER-2/neu antigen.
  • SEQ ID NO:19 is the amino acid sequence of the EGFRvIII antigen.
  • SEQ ID NO:20 is the amino acid sequence of the CEA antigen.
  • SEQ ID NO:21 is the amino acid sequence of the linker of the RD1 library.
  • SEQ ID NOs:22-31 are the amino acid sequences of clones #1-10 of the RD1 library.
  • SEQ ID NO:32 is the amino acid sequence of the clone RD1-Tax-S4-1, and the identical clones RD1-Tax-S4-2, RD1-Tax-S4-4, and RD1-Tax-S4-6.
  • SEQ ID NO:33 is the amino acid sequence of the clone RD1-Mart1-S5-1, and the identical clones RD1-Mart1-S5-2, RD1-Mart1-S5-3, RD1-Mart1-S5-4, RD1-Mart1-S5-5, and RD1-Mart1-S5-6.
  • SEQ ID NO:34 is the amino acid sequence of the clone RD1-Mart1 HIGH .
  • SEQ ID NO:35 is the amino acid sequence of the RD2 library.
  • SEQ ID NOs:36-40 are the amino acid sequences of clones #1-5 of the RD2 library.
  • SEQ ID NO:41 is the amino acid sequence of the clone RD2-Mart1-S3-3.
  • SEQ ID NO:42 is the amino acid sequence of the clone RD2-Mart1-S3-4.
  • SEQ ID NO:43 is the amino acid sequence of the clone T1-S18.45.
  • SEQ ID NO:44 is the amino acid sequence of positions 100-103 in CDR3 ⁇ of the A6 wild type TCR.
  • SEQ ID NO:45 is the amino acid sequence of positions 100-103 in CDR3 ⁇ of A6-X15.
  • SEQ ID NO:46 is the amino acid sequence of the cognate antigen of the TCR modified by Kessels et al. ((2000) Proc Natl Acad Sci USA, 97, 14578-14583).
  • SEQ ID NO:47 is the amino acid sequence of the structurally similar peptide of the TCR modified by Kessels et al. ((2000) Proc Natl Acad Sci USA, 97, 14578-14583).
  • SEQ ID NO:48 is the polynucleotide sequence of the 5′ region of the RD1 gene optimized for both yeast and E. coli.
  • SEQ ID NO:49 is the polynucleotide sequence of the 3′ region of the RD1 gene optimized for both yeast and E. coli.
  • SEQ ID NO:50 is the polynucleotide sequence of the forward primer used to add pCT302 overhangs.
  • SEQ ID NO:51 is the polynucleotide sequence of the reverse primer used to add pCT302 overhangs.
  • SEQ ID NO:52 is the polynucleotide sequence of the forward primer used to generate the CDR3 ⁇ 1 library (Splice 4L).
  • SEQ ID NO:53 is the polynucleotide sequence of the reverse primer used to generate a CDR3 ⁇ 1 library (Splice 4L).
  • SEQ ID NO:54 is the polynucleotide sequence of the forward primer used to generate a CDR3 ⁇ 1 library (T7).
  • SEQ ID NO:55 is the polynucleotide sequence of the reverse primer used to generate a CDR3 ⁇ 1 library (T7).
  • SEQ ID NO:56 is the polynucleotide sequence of the forward primer used to generate a CDR3 ⁇ 2 library.
  • SEQ ID NO:57 is the polynucleotide sequence of the reverse primer used to generate a CDR3 ⁇ 2 library.
  • SEQ ID NO:58 is the polynucleotide sequence of the forward primer used to generate a CDR3 ⁇ library.
  • SEQ ID NO:59 is the polynucleotide sequence of the reverse primer used to generate a CDR3 ⁇ library.
  • SEQ ID NO:60 is the polynucleotide sequence of the N-terminal DNA flanking sequence added to the RD2 library sequence.
  • SEQ ID NO:61 is the polynucleotide sequence of the C-terminal DNA flanking sequence added to the RD2 library sequence.
  • linked refers to an association between two groups, which can be a covalent or non-covalent association.
  • Groups may be linked using a variable length peptide chain, a non-amino acid chemical group or other means as known in the art.
  • a linker region can be an amino acid sequence that operably links two functional or structural domains of a protein or peptide.
  • chemotherapeutic agent refers to any substance capable of reducing or preventing the growth, proliferation, or spread of a cancer cell, a population of cancer cells, tumor, or other malignant tissue.
  • the term is intended also to encompass any antitumor or anticancer agent.
  • the term “effective amount” is intended to encompass contexts such as a pharmaceutically effective amount or therapeutically effective amount.
  • the effective amount is capable of achieving a beneficial state, beneficial outcome, functional activity in a screening assay, or improvement of a clinical condition.
  • cancer cell is intended to encompass definitions as broadly understood in the art.
  • the term refers to an abnormally regulated cell that can contribute to a clinical condition of cancer in a human or animal.
  • the term can refer to a cultured cell line or a cell within or derived from a human or animal body.
  • a cancer cell can be of a wide variety of differentiated cell, tissue, or organ types as is understood in the art. Particular examples of cancer cells include breast cancer, colon cancer, skin cancer, ovarian cancer, leukemia, lung cancer, liver cancer, testicular cancer, esophageal cancer, and other types of cancer.
  • cognate antigen refers to the antigen for which the original TCR was shown to bind to and have specificity for.
  • non-cognate antigen refers to an antigen for which the TCR did not bind to nor have specificity for.
  • the “cognate” peptide refers to the antigenic peptide that the original TCR bound to, when it was part of a complex with a protein encoded by the major histocompatibilty complex (MHC).
  • MHC major histocompatibilty complex
  • non-cognate” peptide refers to a peptide that the original TCR did not bind to, when it was part of a complex with a protein encoded by the MHC.
  • wild type and wt are used interchangeably herein and are used in reference to a TCR having an amino acid sequence or a polynucleotide encoding the variable regions isolated from a naturally occurring or non-modified TCR, e.g., the original or parent T cell clone, with specificity for the cognate antigen.
  • modified T cell receptor refers to TCR sequences of the variable regions as isolated from the original T cell clone having one or more mutations.
  • modified TCRs include higher affinity TCRs and TCRs having binding specificity for a non-cognate antigen.
  • a coding sequence is the part of a gene or cDNA which codes for the amino acid sequence of a protein, or for a functional RNA such as a tRNA or rRNA.
  • Complement or complementary sequence means a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-pairing rules.
  • Downstream refers to a relative position in DNA or RNA and is the region toward the 3′ end of a strand.
  • Expression refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and subsequent translation of an mRNA into a protein.
  • Two nucleic acid sequences are heterologous to one another if the sequences are derived from separate organisms, whether or not such organisms are of different species, as long as the sequences do not naturally occur together in the same arrangement in the same organism.
  • Homology refers to the extent of identity between two nucleotide or amino acid sequences.
  • an amino acid sequence that is functionally equivalent to a specifically exemplified TCR sequence is an amino acid sequence that has been modified by single or multiple amino acid substitutions, by addition and/or deletion of amino acids, or where one or more amino acids have been chemically modified, but which nevertheless retains the binding specificity and high affinity binding activity of a cell-bound or a soluble TCR protein of the present disclosure.
  • Functionally equivalent nucleotide sequences are those that encode polypeptides having substantially the same biological activity as a specifically exemplified cell-bound or soluble TCR protein.
  • a soluble TCR protein lacks the portions of a native cell-bound TCR and is stable in solution (i.e., it does not generally aggregate in solution when handled as described herein and under standard conditions for protein solutions).
  • isolated refers to a composition, compound, substance, or molecule altered by the hand of man from the natural state.
  • a composition or substance that occurs in nature is isolated if it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is isolated, as the term is employed herein.
  • a nucleic acid construct is a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
  • Nucleic acid molecule means a single- or double-stranded linear polynucleotide containing either deoxyribonucleotides or ribonucleotides that are linked by 3′-5′-phosphodiester bonds.
  • Two DNA sequences are operably linked if the nature of the linkage does not interfere with the ability of the sequences to effect their normal functions relative to each other.
  • a promoter region would be operably linked to a coding sequence if the promoter were capable of effecting transcription of that coding sequence.
  • a polypeptide is a linear polymer of amino acids that are linked by peptide bonds.
  • promoter refers to a cis-acting DNA sequence, generally 80-120 base pairs long and located upstream of the initiation site of a gene, to which RNA polymerase may bind and initiate correct transcription. There can be associated additional transcription regulatory sequences which provide on/off regulation of transcription and/or which enhance (increase) expression of the downstream coding sequence.
  • a recombinant nucleic acid molecule for instance a recombinant DNA molecule, is a novel nucleic acid sequence formed in vitro through the ligation of two or more nonhomologous DNA molecules (for example a recombinant plasmid containing one or more inserts of foreign DNA cloned into at least one cloning site).
  • transformation and “transfection” refer to the directed modification of the genome of a cell by the external application of purified recombinant DNA from another cell of different genotype, leading to its uptake and integration into the subject cell's genome.
  • the recombinant DNA is not typically integrated into the bacterial chromosome, but instead replicates autonomously as a plasmid.
  • transformed and “transfected” are used interchangeably herein.
  • Upstream means on the 5′ side of any site in DNA or RNA.
  • a vector is a nucleic acid molecule that is able to replicate autonomously in a host cell and can accept foreign DNA.
  • a vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases which can be used for the insertion of foreign DNA, and usually selectable markers such as genes coding for antibiotic resistance, and often recognition sequences (e.g., promoter) for the expression of the inserted DNA.
  • Common vectors include plasmid vectors and phage vectors.
  • High affinity T cell receptor is an engineered TCR with stronger binding to a target ligand than the wild type TCR.
  • Some examples of high affinity include an equilibrium binding constant for a target ligand of between about 10 ⁇ 6 M and 10 ⁇ 12 M and all individual values and ranges therein. This range encompasses affinities between those reported to be wild type affinities 10 ⁇ 4 to 10 ⁇ 6 M, and those which have been isolated by directed evolution (about 10 ⁇ 12 M).
  • a cytokine is a protein, peptide or glycoprotein made by cells that affect other cells.
  • Mammal includes both human and non-human mammals.
  • the T cell receptor is composed of two chains ( ⁇ or ⁇ ) that pair on the surface of the T cell to form a heterodimeric receptor.
  • the ⁇ TCR is expressed on most T cells in the body and is known to be involved in the recognition of MHC-restricted antigens.
  • the molecular genetics, structure, and biochemistry of ⁇ TCRs have now been studied thoroughly.
  • Each ⁇ and ⁇ chain is composed of two domains: Constant domains (C) that anchor the protein in the cell membrane and that associate with invariant subunits of the CD3 signaling apparatus, and Variable domains (V) that confer antigen recognition through six loops, called complementarity determining regions (CDR).
  • CDR Constant domains
  • V Variable domains
  • Each of the V domains has three CDRs.
  • CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pepMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012), xix, 868 p.).
  • pepMHC major histocompatibility complex
  • the molecular genetics of the TCR have revealed a process of genetic recombination between multiple genes that combine to form the coding region of the V domains.
  • the process is analogous to antibody development in which the heavy and light chain genes rearrange to generate the tremendous diversity exhibited by B cell-derived antibodies (Tonegawa (1988) In Vitro Cell Dev Biol, 24, 253-65).
  • the ⁇ chain V domain is formed by the rearrangement of one V region (among about 75 in humans) to one Joining (J) gene segment (among about 61 in humans) (FIG. 5.8, Janeway, 8 th edition).
  • the 13 chain V domain is formed by the rearrangement of one V region (among about 52 in humans) to one Diversity (D) gene (among 2 in humans) to one Joining (J) gene segment (among 13 in humans) (FIG. 5.8, (Murphy (2012), xix, 868 p.)).
  • the junctions of the V ⁇ J ⁇ and J ⁇ D ⁇ J ⁇ gene rearrangements encode the CDR3 loops of each chain, and they contribute to the tremendous diversity of the ⁇ TCR, with a theoretical limit of over 10 15 different TCRs (Davis and Bjorkman (1988) Nature, 334, 395-402), well above the achievable diversity in a human because there are only about 10 11 T cells total (Mason (1998) Immunol Today, 19, 395-404).
  • the possible CDR1 and CDR2 diversity of each chain is represented by the number of V genes, as these loops are encoded within the V gene, and TCRs do not undergo somatic mutation in vivo.
  • the diversity of CDR1 and CDR2 loops are relatively limited compared to CDR3 loops, there have been a number of examples shown where there has been selection for particular V regions based on the peptide antigen and/or MHC product.
  • Class I MHC products bind to peptides of 8 to 10 amino acids in length and they are expressed on all nucleated cells in the body (reviewed by (Rock and Goldberg (1999) Annu Rev Immunol, 17, 739-79)). Whereas all the binding energy of an antibody-antigen interaction is focused on the foreign antigen, a substantial fraction of the binding energy of the TCR-peptide:MHC is directed at the self-MHC molecule (Manning and Kranz (1999) Immunology Today, 20, 417-422).
  • TCRs that have affinities for a peptide-MHC antigen (class I) above the normal range (so called higher affinity TCRs) in order to: 1) drive the activity of CD4 helper T cells (which lack the CD8 co-receptor) or 2) develop soluble TCRs that could be used for direct targeting of a cell, by attaching an “effector” molecule (e.g., antibody Fc regions, a toxic drug, or an antibody scFv such as an anti-CD3 antibody, to form a bispecific protein)((Ashfield and Jakobsen (2006) Drugs, 9, 554-9; Foote and Eisen (2000) Proc Natl Acad Sci USA, 97, 10679-81; Holler et al.
  • an effector molecule
  • tumor antigens include mutated peptides, differentiation antigens, and overexpressed antigens, all of which could serve as targets for therapies. Because the majority of the cancer antigens described to date were derived from intracellular proteins that can only be targeted at the cell surface in the context of an MHC molecule, TCRs make the ideal candidate for therapeutics as they have evolved to recognize this class of antigen.
  • TCRs can detect peptides derived from viral proteins that have been naturally processed in infected cells and displayed by an MHC molecule on the cell surface.
  • Many viral antigen targets have been identified over the past 25 years, including peptides derived from viral genomes in HIV and HTLV (e.g., Addo et al. (2007) PLoS ONE, 2, e321; Tsomides et al. (1994) J Exp Med, 180, 1283-93; Utz et al. (1996) J Virol, 70, 843-51).
  • patients with these diseases may lack the optimal TCRs for binding and destruction of the infected cells.
  • TCRs could be used as receptor antagonists of autoimmune targets, or as delivery agents to immunosuppress the local immune cell response, in a process that would be highly specific, thereby avoiding general immune suppression ((Molloy et al. (2005) Curr Opin Pharmacol, 5, 438-43; Stone et al. (2012) Protein Engineering)).
  • Directed evolution has been used to generate TCRs with higher affinity for a specific pepMHC.
  • the three different display methods that have been used are yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), and T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84).
  • the process involves the engineering of a TCR that exhibits the normal, low affinity of the wild-type TCR, so that affinity of mutants of the TCR had increased affinity for the cognate pepMHC (i.e., the original antigen that the T cells were specific for).
  • the wild-type TCR was used as a template for producing mutagenized libraries in one or more of the CDRs, followed by selection of mutants with higher affinity, by binding to the cognate peptide-MHC antigen.
  • Yeast display allows for the protein of interest to be expressed on the surface as an Aga2-fusion (Boder and Wittrup (1997) Nat. Biotech., 15, 553-557; Boder and Wittrup (2000) Methods Enzymol, 328, 430-44).
  • This system has been used successfully in the engineering of higher affinity TCRs, single-chain antibodies, fibronectin, and other proteins.
  • the TCR has been displayed as a stabilized single-chain protein, in V ⁇ -linker-V ⁇ or V ⁇ -linker-V ⁇ forms (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72; Holler et al.
  • T cell receptors in vitro engineered, high-affinity T cell receptors in a single-chain format were used to isolate human stabilized scTv fragments (V ⁇ -linker-V ⁇ ), which could be expressed as stable proteins, both on the surface of yeast and in soluble form from E. coli .
  • the TCRs included two stabilized, human scTv fragments, the A6 scTv that is specific for a peptide derived from the human T cell lymphotrophic virus Tax protein (peptide: Tax 11-19 , SEQ ID NO:5), and the 868 scTv that is specific for a peptide derived from the human immunodeficiency virus Gag protein (peptide: SL9 77-85 , SEQ ID NO:8).
  • TCRs Both of these TCRs used the V ⁇ 2 gene (IMGT: TRAV12 family), but they had CDR3 ⁇ , CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ residues derived from the original T cell clone from which the TCRs were isolated. Thus, the higher affinity mutants of these scTCRs were each derived from their original (parental) TCR against their cognate peptide-MHC antigens.
  • phage display the protein of interest is fused to the N-terminus of a viral coat protein (Scott and Smith (1990) Science, 249, 386-90).
  • TCRs including those called A6, 868, and 1G4 (MHC class-I restricted), have been engineered for higher affinity using this method (Li et al. (2005) Nat Biotechnol, 23, 349-54; Sami et al. (2007) Protein Eng Des Sel, 20, 397-403; Varela-Rohena et al. (2008) Nat Med, 14, 1390-5).
  • Phage display of these TCRs was enabled by introduction of a non-native disulfide bond between the two C domains in order to promote pairing of the ⁇ and ⁇ chains.
  • This system thus uses full-length (V ⁇ C ⁇ /V ⁇ C ⁇ ) heterodimeric proteins derived from the original T cell clones for engineering against their cognate peptide-MHC.
  • a third system that has been reported for the engineering of TCRs is mammalian cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84; Kessels et al. (2000) Proc Natl Acad Sci USA, 97, 14578-83).
  • This system uses a retroviral vector to introduce the TCR ⁇ and ⁇ -chains into a TCR-negative T cell hybridoma.
  • vessels et al. shows that has been reported for the engineering of TCRs.
  • the selected mutant TCR was shown to bind to a peptide that was structurally very similar to the cognate peptide (ASNENMDAM, SEQ ID NO:46, versus ASNENMETM, SEQ ID NO:47).
  • affinity of the mutant TCR was shown to be increased for the cognate pepMHC (Chervin et al. (2008) J Immunol Methods, 339, 175-84). It has been shown in many studies that such higher affinity TCRs also exhibit higher affinities against structurally similar variants of the cognate peptide (e.g., (Holler et al. (2003) Nat Immunol, 4, 55-62)).
  • TCRs were expressed on the surface in its native conformation, complexed with CD3 subunits, allowing for a fully functional T cell (signaling competent). Full-length, heterodimeric TCRs in their native host were thus engineered using this method.
  • the present invention provides for the use of a single, e.g., human TCR as a “platform” for engineering higher affinity TCRs against desired antigens (e.g., cognate or non-cognate antigens).
  • desired antigens e.g., cognate or non-cognate antigens
  • the TCR scaffold-based TCR engineering methods described herein can include, for example, generating site-directed, mutated libraries of the single TCR, followed by selections for binding to a non-cognate antigen. Engineering is guided by structural analysis of the original, single, or parent TCR.
  • the engineered TCRs can be used in soluble form for targeted delivery in vivo, or as recombinantly expressed by T cells in an adoptive transfer method or treatment.
  • a TCR scaffold that can be used to engineer TCR mutants against specific antigens.
  • the TCRs are useful for many purposes including, e.g., but not limited to, the treatment of cancer, viral diseases and autoimmune diseases.
  • a single-chain V ⁇ V ⁇ TCR (scTCR) scaffold can be prepared and used with a payload such as a cytokine, toxin, radioisotope, chemotherapeutic agent, or drug (similar to antibody-drug conjugates) to deliver the effector molecule to the location where the TCR binds (e.g., tumor).
  • the TCR can also be used in cell therapies, such as adoptive transfer of CD4+ T cells, CD8+ T cells, and/or natural killer (NK) cells, to mediate a response against, e.g., a cancer cell or virus-infected cell.
  • NK natural killer
  • the scTCR scaffolds provided herein can also be used for diagnosis of, e.g., malignant or viral-infected cells through identification of, e.g., neoplastic or viral-associated cell-surface antigens by covalent linkage, for example through amine-reactive or sulfhydryl-reactive amino acid side chains of the TCR, to a detectable group, such as a radioisotope or fluorescent moiety.
  • the scTCR scaffold described herein is displayable on the surface of yeast, phage, or mammalian cells and can be used to engineer TCRs with higher affinity to a non-cognate antigen.
  • the scTCR scaffold described herein can be expressed in a prokaryotic cell, such as Escherichia coli, Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Mucor miehei, Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis or Bacillus licheniformis , insect cells (e.g., Drosophila ), mammalian cells including cell lines such as Chinese hamster ovary cell lines (CHO), or plant species (e.g., canola, soybean, corn, potato, barley, rye, wheat)
  • CHO Chinese
  • the TCR scaffold can be generated against a particular antigen, and used, for example and by way of example only, to detect a specific peptide/MHC on the surface of a cell.
  • the scTCR genes disclosed can be linked by use of suitable peptide sequences, encoded within the DNA construct, to the genes for signaling domains and introduced into T cells that can eliminate the targeted cells. These constructs have been termed chimeric antigen receptors (CARs), which are now widely used in the field, including the use of CARs that contain a scTCR.
  • CARs chimeric antigen receptors
  • the current disclosure provides the amino acid sequences and the form of a single-chain V ⁇ V ⁇ T cell receptor (sc V ⁇ V ⁇ TCR) scaffold.
  • sc V ⁇ V ⁇ TCR single-chain V ⁇ V ⁇ T cell receptor
  • the variable alpha and variable beta chains are connected using any suitable peptide linker, including those known in the art such as with antibody single-chain Fv linkages (Bird et al. (1988) Science, 242, 423-426; Holliger et al. (1993) Proc Natl Acad Sci USA, 90, 6444-8; Hoogenboom (2005) Nat Biotechnol, 23, 1105-16; Turner et al. (1997) J Immunol Methods, 205, 43-54).
  • a soluble human single-chain TCR having the structure: V ⁇ -L-V ⁇ or V ⁇ -L-V ⁇ , wherein L is a linker peptide that links V ⁇ with V ⁇ , V ⁇ is a TCR variable ⁇ region, and V ⁇ is a TCR variable ⁇ region is provided.
  • the model V ⁇ V ⁇ TCR is called A6 where V ⁇ is a TCR variable ⁇ region of group 13, and V ⁇ 2 is a TCR variable ⁇ region of group 2 (Utz, U., et al., 1996).
  • the model V ⁇ V ⁇ TCR is a stabilized single-chain variant of A6 known as A6 X15 (Aggen, D. A., et al., 2011).
  • the linker peptide contains more than 5 lysine residues. In one embodiment, the linker peptide contains between 5 and 30 amino acids. In one embodiment, the linker peptide has an amino acid sequence of GSADDAKKDAAKKDGKS (SEQ ID NO:21). In one embodiment, the sc V ⁇ V ⁇ TCR scaffold provided does not contain a constant region. When the terminology sc V ⁇ V ⁇ TCR scaffold is used herein, it is understood that sc V ⁇ V ⁇ TCR scaffold is also included as the terminology is understood and used in the art. Thus, the V ⁇ and V ⁇ chains can be connected to each other in any configuration through the linker.
  • the scV ⁇ V ⁇ TCR scaffold of the disclosure binds specifically to a ligand with an equilibrium binding constant K D of between about 10 ⁇ 6 M and 10 ⁇ 12 M.
  • the ligand is a peptide/MHC ligand.
  • the sc V ⁇ V ⁇ TCR of the disclosure has enhanced affinity toward a ligand compared to the affinities of normal, wild type TCRs.
  • TCRs that bind to a collection of HLA-A, B, and C alleles could be used to treat diseases that encompass a large fraction of the human population. For example, the frequency of many HLA alleles in the population has been determined, and there are many cancer peptide antigens that have been described in association with these alleles (Marsh, Parham, and Barber, The HLA Facts Book, copyright 2000 by Academic Press, ISBN 0-12-545025-7).
  • the average frequency (and range) among Caucasian populations are: HLA-A1, 14%; HLA-A2, 25%; HLA-A3, 12%; HLA-A11, 7%; HLA-A24, 10%; HLA-B7, 9%; HLA-B44, 11%; HLA-Cw4, 12%; HLA-Cw7, 23%.
  • the range found within these populations are: HLA-A1 (5-28%); HLA-A2 (7-40%); HLA-A3 (3-20%); HLA-A11 (2-25%); HLA-A24 (5-18%); HLA-B7 (1-16%); HLA-B44 (5-22%); HLA-Cw4 (6-19%); HLA-Cw7 (13-39%).
  • the TCR scaffold approach can be extended to other human HLA alleles in various ways. For example, using structure-based design of the TCR scaffolds described here, it is possible to focus mutated libraries in the CDR loops that contact the MHC helices in order to generate leads against other alleles. For example, from the structure of the A6 TCR in complex with HLA-A2, it is known that CDR2alpha libraries would generate variants that bind in the region of the alpha2 helix of HLA-A2.
  • the TCR A 6 residue Y51 resides near HLA-A2 alpha2 helix position(s) E154, Q155, and A158.
  • the HLA-A1 allele differs only at position 158, with a valine rather than an alanine.
  • the A6 TCR may have a basal affinity for the HLA-A1 allele, which could be improved by generating libraries of CDR2 mutants that encompass position 51, followed by selections for higher affinity binding to the HLA-A1 all
  • Another example makes use of a scaffold that is derived from a TCR specific for a different allele (i.e., peptide bound to the product of that MHC allele).
  • a scaffold that is derived from a TCR specific for a different allele (i.e., peptide bound to the product of that MHC allele).
  • CDR libraries as shown for the A6 TCR scaffold, which will react with alternative non-cognate peptide-MHC complexes of that allele.
  • a cancer antigen peptide from MAGE-A3 binds to HLA-A1 and this could be used for selection of the TR libraries.
  • biologically active group is a group that causes a measurable or detectable effect in a biological system.
  • the biologically active group is selected from: an anti-tumor agent such as, but not limited to, angiogenesis inhibitors, enzyme inhibitors, microtubule inhibitors, DNA intercalators or cross-linkers, DNA synthesis inhibitors; a cytokine such as, but not limited to IL-2, IL-15, GM-CSF, IL-12, TNF- ⁇ , IFN- ⁇ or LT- ⁇ (Schrama et al.
  • an anti-inflammatory group such as, but not limited to, TGF- ⁇ , IL-37, IL-10 (Nold et al. (2010) Nat Immunol, 11, 1014-22; Stone et al. (2012) Protein Engineering), a radioisotope such as, but not limited to, 90 Y or 131 I (Reichert and Valge-Archer (2007) Nat Rev Drug Discov, 6, 349-56); a toxin such as, but not limited to, Pseudomonas exotoxin A, diphtheria toxin, or the A chain of ricin (Pastan et al. (2006) Nat Rev Cancer, 6, 559-65; Schrama et al. (2006) Nat Rev Drug Discov, 5, 147-59); a drug, or an antibody such as a single-chain Fv.
  • an anti-inflammatory group such as, but not limited to, TGF- ⁇ , IL-37, IL-10 (Nold et al. (2010) Nat Immunol, 11, 1014-22; Stone et
  • the biologically active group is a cytotoxic molecule, sometimes referred to as a drug (e.g., in the term “antibody drug conjugate”).
  • cytotoxic means toxic to cells.
  • examples of cytotoxic molecules include, but are not limited to, doxorubicin, methotrexate, mitomycin, 5-fluorouracil, duocarmycin, auristatins, maytansines, calicheamicins and analogs of the above molecules (Jarvis (2012) Chemical and Engineering News, 90, 12-18; Litvak-Greenfeld and Benhar (2012) Adv Drug Deliv Rev; Ricart and Tolcher (2007) Nat Clin Pract Oncol, 4, 245-55). Cytotoxic molecules do not need to cause complete cell death, but rather, a measurable or detectable inhibition of growth or decrease in cell activity.
  • a TCR described herein is linked to an enzyme capable of converting a prodrug into a drug. This is useful, for example, by allowing the active form of the drug to be created at the location targeted by the TCR (e.g., at the site of a tumor).
  • the biologically active group is bound to the single-chain TCR through a linker, which may be accomplished through standard chemical reactions such as with free amine groups or sulfhydryl groups of the TCR.
  • the TCR is attached to a single-chain antibody fragment (scFv) to generate a bispecific agent.
  • scFv single-chain antibody fragment
  • Bispecific antibodies that contain one scFv against a tumor antigen, and one against the CD3 molecule of the T cell have now been used successfully in the clinic (Bargou et al. (2008) Science, 321, 974-7).
  • a bispecific agent containing a TCR and a scFv against CD3 has also been reported (Liddy et al. (2012) Nat Med, 18, 980-7).
  • the detectable group is one that can be detected by spectroscopic or enzyme-based methods.
  • the detectable group is a fluorescent group, such as, but not limited to fluorescein, R-phycoerythrin (PE), PE-Cy5, PE-Cy7, Texas red, or allophycocyanin (APC); a radiolabeled group such as, but not limited to, 125 I, 32 P, 99m Tc; an absorbing group, or an enzyme with properties that generate detectable products such as, but not limited to, horseradish peroxidase, or alkaline phosphatase.
  • a biologically active group, detectable group or other group attached to the TCR can be attached using a flexible peptide linker or by chemical conjugation, and can be covalently or noncovalently attached to the TCR.
  • sc V ⁇ V ⁇ TCR scaffolds that recognize (or target) a specific antigen.
  • the TCR is specific for recognition of a virus or fragment thereof.
  • the TCR is specific for recognition of a cancer-specific epitope.
  • the TCR is specific for recognition of autoimmune associated epitope.
  • targets include those listed in The HLA Factsbook (Marsh et al. (2000)) and others known in the art.
  • Specific target antigens include tumor-associated antigens (van der Bruggen P et al. Peptide database: T cell-defined tumor antigens. Cancer Immun 2013. cancerimmunity.org/peptide/; (Cheever et al.
  • the target antigen is one of MART-1 (Kawakami Y et al. J Exp Med 1994; 180:347-352; Romero et al. 2002. Immunol. Rev.
  • the ranking score in Table 1 is from Cheever et al. (2009) Clin Cancer Res, 15, 5323-37. Also provided is a scV ⁇ V ⁇ TCR mutant derived from the A6 scaffold that recognizes the specific non-cognate antigen called MART-1/HLA-A2 (peptide ELAGIGILTV, SEQ ID NO:7, bound to the HLA molecule A2).
  • a human TCR for use in a method of treating or preventing a disease or disorder in a mammal, comprising administering an effective amount of a modified TCR linked to a therapeutically effective molecule to a mammal.
  • the mammal is human.
  • the mammal is a companion animal (e.g., a dog, cat, rabbit, rodent, horse) or a livestock animal (e.g., a cow, horse, pig).
  • a “disease state” is an abnormal function or condition of an organism.
  • the disease state is selected from the group consisting of: cancer, viral, bacterial or autoimmune disease.
  • a pharmaceutical composition comprising a scTCR as described herein and a pharmaceutically acceptable carrier.
  • the sc V ⁇ V ⁇ TCR described herein which has been linked to signaling domains that yields an active TCR on the surface of a T cell.
  • this scTCR can be used in a method of treating a disease state in a mammal, comprising: cloning the TCR into a vector, introducing the vector into T cells of a patient, and adoptive transferring of the T cells back into a patient.
  • the disclosure contemplates a DNA vector that includes at least one DNA segment encoding a single-chain T cell receptor (scTCR).
  • scTCR single-chain T cell receptor
  • mutagenesis techniques include, without limitation, de novo gene synthesis, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (see e.g., Sambrook et al. (1989) and Ausubel et al. (1999)).
  • TCR-derived proteins may be modified by certain amino acid substitutions, additions, deletions, and post-translational modifications, without loss or reduction of biological activity.
  • conservative amino acid substitutions that is, substitution of one amino acid for another amino acid of similar size, charge, polarity and conformation, are unlikely to significantly alter protein function.
  • the 20 standard amino acids that are the constituents of proteins can be broadly categorized into four groups of conservative amino acids as follows: the nonpolar (hydrophobic) group includes alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine; the polar (uncharged, neutral) group includes asparagine, cysteine, glutamine, glycine, serine, threonine and tyrosine; the positively charged (basic) group contains arginine, histidine and lysine; and the negatively charged (acidic) group contains aspartic acid and glutamic acid. Substitution in a protein of one amino acid for another within the same group is unlikely to have an adverse effect on the biological activity of the protein.
  • a scTCR of the disclosure may contain additional mutations in any region or regions of the variable domain that results in a stabilized protein.
  • one or more additional mutations is in one or more of CDR1, CDR2, HV4, CDR3, FR2, and FR3.
  • the regions used for mutagenesis can be determined by directed evolution, where crystal structures or molecular models are used to generate regions of the TCR which interact with the ligand of interest (antigen, for example).
  • the variable region can be reshaped, by adding or deleting amino acids to engineer a desired interaction between the scTCR and the ligand.
  • Polypeptides of the invention include modified TCRs, and antigen-binding fragments thereof (e.g., scTCR), and chimeric antigen receptors (CARs).
  • scTCR antigen-binding fragments thereof
  • CARs chimeric antigen receptors
  • polypeptide protein and peptide and “glycoprotein” are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences.
  • polypeptide or “protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
  • polypeptide and protein specifically encompass the modified TCRs, or antigen-binding fragments thereof, of the present disclosure, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of a modified TCR, or antigen binding fragment thereof.
  • a “polypeptide” or a “protein” can comprise one (termed “a monomer”) or a plurality (termed “a multimer”) of amino acid chains.
  • isolated protein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a protein with which the “isolated protein” is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof.
  • the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • a subject modified TCR may have: a) a TCR alpha chain variable region having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90%, at least 95% or at least 98% or 99% identical, to the alpha chain variable region of a modified TCR described herein; and b) a beta chain variable region having an amino acid sequence that is at least 80% identical, at least 85%, at least 90%, at least 95% or at least 98% or 99% identical, to the light chain variable region of a modified TCR described herein.
  • the modified TCR may comprise: a) a TCR alpha chain variable region comprising: i. a CDR1 region that is identical in amino acid sequence to the alpha chain CDR1 region of a selected TCR described herein; ii. a CDR2 region that is identical in amino acid sequence to the alpha chain CDR2 region of the selected TCR; and iii. a CDR3 region that is identical in amino acid sequence to the alpha chain CDR3 region of the selected TCR; and b) a beta chain variable region comprising: i. a CDR1 region that is identical in amino acid sequence to the beta chain CDR1 region of the selected TCR; ii.
  • the modified TCR, or antigen-binding fragment thereof is a variant modified TCR wherein the variant comprises an alpha chain and a beta chain identical to the selected modified TCR except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions in the CDR regions of the V alpha and V beta regions.
  • substitutions may be in CDRs either in the V alpha and/or the V beta regions. (See e.g., Muller, 1998, Structure 6:1153-1167).
  • a polynucleotide encoding a modified TCR, or an antigen-binding fragment thereof is provided.
  • the polynucleotide may be a variant of a polynucleotide encoding the modified TCR.
  • Polynucleotide variants may have substantial identity to a polynucleotide sequence encoding a modified TCR described herein.
  • a polynucleotide may be a polynucleotide comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a reference polynucleotide sequence such as a sequence encoding an antibody described herein, using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below).
  • BLAST analysis using standard parameters, as described below.
  • polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the binding affinity of the antibody encoded by the variant polynucleotide is not substantially diminished relative to an antibody encoded by a polynucleotide sequence specifically set forth herein.
  • two sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters.
  • This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods in Enzymology vol.
  • optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.
  • BLAST and BLAST 2.0 are described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively.
  • BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity among two or more the polynucleotides.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • nucleotide sequences that encode an antibody as described herein there are many nucleotide sequences that encode an antibody as described herein. Some of these polynucleotides bear minimal sequence identity to the nucleotide sequence of the native or original polynucleotide sequence that encode modified TCRs that bind to, e.g., the same non-cognate antigen. Nonetheless, polynucleotides that vary due to differences in codon usage are expressly contemplated by the present disclosure. In certain embodiments, sequences that have been codon-optimized for mammalian expression are specifically contemplated.
  • Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art.
  • a number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning , Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning , Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al.
  • Homology between nucleotide sequences can be determined by DNA hybridization analysis, wherein the stability of the double-stranded DNA hybrid is dependent on the extent of base pairing that occurs. Conditions of high temperature and/or low salt content reduce the stability of the hybrid, and can be varied to prevent annealing of sequences having less than a selected degree of homology.
  • hybridization and wash conditions 40-50° C., 6 ⁇ SSC (sodium chloride/sodium citrate buffer) and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology
  • hybridization and wash conditions of 50-65° C., 1 ⁇ SSC and 0.1% SDS indicate about 82-97% homology
  • hybridization and wash conditions of 52° C., 0.1 ⁇ SSC and 0.1% SDS indicate about 99-100% homology.
  • a wide range of computer programs for comparing nucleotide and amino acid sequences (and measuring the degree of homology) are also available, and a list providing sources of both commercially available and free software is found in Ausubel et al.
  • BLAST Basic Local Alignment Search Tool
  • Industrial strains of microorganisms e.g., Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Mucor miehei, Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis or Bacillus licheniformis ), insect ( Drosophila ), mammalian (e.g., Chinese hamster ovary cell lines, CHO), or plant species (e.g., canola, soybean, corn, potato, barley, rye, wheat) may be used as host cells for the recombinant production of the TCR proteins.
  • microorganisms e.g., Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Trichoderma
  • the first step in the heterologous expression of a high affinity TCR protein or soluble protein an expression construct is assembled to include the TCR or soluble TCR coding sequence and control sequences such as promoters, enhancers and terminators. Other sequences such as signal sequences and selectable markers may also be included.
  • the expression construct may include a secretory signal sequence.
  • the signal sequence is not included on the expression construct if cytoplasmic expression is desired.
  • the promoter and signal sequence are functional in the host cell and provide for expression and secretion of the TCR or soluble TCR protein. Transcriptional terminators may be included to ensure efficient transcription. Ancillary sequences enhancing expression or protein purification may also be included in the expression construct.
  • promoters transcriptional initiation regulatory region
  • the selection of the appropriate promoter may be dependent upon the proposed expression host. Promoters from heterologous sources may be used as long as they are functional in the chosen host.
  • Promoter selection is also dependent upon the desired efficiency and level of peptide or protein production.
  • Inducible promoters such as tac are often employed in order to dramatically increase the level of protein expression in E. coli . Overexpression of proteins may be harmful to the host cells. Consequently, host cell growth may be limited.
  • the use of inducible promoter systems allows the host cells to be cultivated to acceptable densities prior to induction of gene expression, thereby facilitating higher product yields.
  • signal sequences may be used according to the disclosure.
  • a signal sequence which is homologous to the TCR coding sequence may be used.
  • a signal sequence which has been selected or designed for efficient secretion and processing in the expression host may also be used.
  • suitable signal sequence/host cell pairs include the B. subtilis sacB signal sequence for secretion in B. subtilis , and the Saccharomyces cerevisiae ⁇ -mating factor or P. pastoris acid phosphatase phoI signal sequences for P. pastoris secretion.
  • the signal sequence may be joined directly through the sequence encoding the signal peptidase cleavage site to the protein coding sequence, or through a short nucleotide bridge consisting of usually fewer than ten codons, where the bridge ensures correct reading frame of the downstream TCR sequence.
  • Elements for enhancing transcription and translation have been identified for eukaryotic protein expression systems. For example, positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp on either side of a heterologous promoter may elevate transcriptional levels by 10- to 400-fold in plant cells.
  • the expression construct should also include the appropriate translational initiation sequences. Modification of the expression construct to include a Kozak consensus sequence for proper translational initiation may increase the level of translation by 10 fold.
  • a selective marker is often employed, which may be part of the expression construct or separate from it (e.g., carried by the expression vector), so that the marker may integrate at a site different from the gene of interest.
  • markers that confer resistance to antibiotics (e.g., bla confers resistance to ampicillin for E. coli host cells, nptII confers kanamycin resistance to a wide variety of prokaryotic and eukaryotic cells) or that permit the host to grow on minimal medium (e.g., HIS4 enables P. pastoris or His ⁇ S. cerevisiae to grow in the absence of histidine).
  • the selectable marker has its own transcriptional and translational initiation and termination regulatory regions to allow for independent expression of the marker. If antibiotic resistance is employed as a marker, the concentration of the antibiotic for selection will vary depending upon the antibiotic, generally ranging from 10 to 600 ⁇ g of the antibiotic/mL of medium.
  • the expression construct is assembled by employing known recombinant DNA techniques (Sambrook et al., 1989; Ausubel et al., 1999). Restriction enzyme digestion and ligation are the basic steps employed to join two fragments of DNA. The ends of the DNA fragment may require modification prior to ligation, and this may be accomplished by filling in overhangs, deleting terminal portions of the fragment(s) with nucleases (e.g., ExoIII), site directed mutagenesis, or by adding new base pairs by PCR. Polylinkers and adaptors may be employed to facilitate joining of selected fragments.
  • the expression construct is typically assembled in stages employing rounds of restriction, ligation, and transformation of E. coli .
  • cloning vectors suitable for construction of the expression construct are known in the art ( ⁇ ZAP and pBLUESCRIPT SK-1, Stratagene, LaJolla, Calif.; pET, Novagen Inc., Madison, Wis.—cited in Ausubel et al., 1999) and the particular choice is not critical to the disclosure.
  • the selection of cloning vector will be influenced by the gene transfer system selected for introduction of the expression construct into the host cell. At the end of each stage, the resulting construct may be analyzed by restriction, DNA sequence, hybridization and PCR analyses.
  • the expression construct may be transformed into the host as the cloning vector construct, either linear or circular, or may be removed from the cloning vector and used as is or introduced onto a delivery vector.
  • the delivery vector facilitates the introduction and maintenance of the expression construct in the selected host cell type.
  • the expression construct is introduced into the host cells by any of a number of known gene transfer systems (e.g., natural competence, chemically mediated transformation, protoplast transformation, electroporation, biolistic transformation, transfection, or conjugation) (Ausubel et al., 1999; Sambrook et al., 1989). The gene transfer system selected depends upon the host cells and vector systems used.
  • the expression construct can be introduced into S. cerevisiae cells by protoplast transformation or electroporation. Electroporation of S. cerevisiae is readily accomplished, and yields transformation efficiencies comparable to spheroplast transformation.
  • Monoclonal or polyclonal antibodies preferably monoclonal, specifically reacting with a TCR protein at a site other than the ligand binding site may be made by methods known in the art, and many are commercially available. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratories; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, New York; and Ausubel et al. (1999) Current Protocols in Molecular Biology , John Wiley & Sons, Inc., New York.
  • TCRs in cell-bound or soluble form which are specific for a particular target ligand are useful, for example, as diagnostic probes for screening biological samples (such as cells, tissue samples, biopsy material, bodily fluids and the like) or for detecting the presence of the target ligand in a test sample.
  • the TCRs are labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal. Suitable labels include but are not limited to radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like.
  • the TCR can be coupled to a ligand for a second binding molecules: for example, the TCR can be biotinylated.
  • Detection of the TCR bound to a target cell or molecule can then be effected by binding of a detectable streptavidin (a streptavidin to which a fluorescent, radioactive, chemiluminescent, or other detectable molecule is attached or to which an enzyme for which there is a chromophoric substrate available).
  • a detectable streptavidin a streptavidin to which a fluorescent, radioactive, chemiluminescent, or other detectable molecule is attached or to which an enzyme for which there is a chromophoric substrate available.
  • Labeled TCRs can be detected using a monitoring device or method appropriate to the label used. Fluorescence microscopy or fluorescence activated cell sorting can be used where the label is a fluorescent moiety, and where the label is a radionuclide, gamma counting, autoradiography or liquid scintillation counting, for example, can be used with the proviso that the method is appropriate to the sample being analyzed and the radionuclide used. In addition, there can be secondary detection molecules or particle employed where there is a detectable molecule or particle which recognized the portion of the TCR which is not part of the binding site for the target ligand in the absence of a MHC component as noted herein.
  • Radionuclides useful for therapy and/or imaging in vivo include 111 Indium, 97 Rubidium, 125 Iodine, 131 Iodine, 123 Iodine, 67 Gallium, 99 Technetium.
  • Toxins include diphtheria toxin, ricin and castor bean toxin, among others, with the proviso that once the TCR-toxin complex is bound to the cell, the toxic moiety is internalized so that it can exert its cytotoxic effect.
  • Immunotoxin technology is well known to the art, and suitable toxic molecules include, without limitation, chemotherapeutic drugs such as vindesine, antifolates, e.g., methotrexate, cisplatin, mitomycin, .anthrocyclines such as daunomycin, daunorubicin or adriamycin, and cytotoxic proteins such as ribosome inactivating proteins (e.g., diphtheria toxin, pokeweed antiviral protein, abrin, ricin, pseudomonas exotoxin A or their recombinant derivatives. See, generally, e.g., Olsnes and Pihl (1982) Pharmac. Ther. 25:355-381 and Monoclonal Antibodies for Cancer Detection and Therapy , Eds. Baldwin and Byers, pp. 159-179, Academic Press, 1985.
  • chemotherapeutic drugs such as vindesine
  • antifolates e.g., methotre
  • TCR molecules The general structure of TCR molecules and methods of making and using, including binding to a peptide:Major Histocompatibility Complex have been disclosed. See, for example PCT/US98/04274; PCT/US98/20263; WO99/60120.
  • scTCRs specific for a particular target ligand are useful in treating animals and mammals, including humans believed to be suffering from a disease associated with the particular antigen.
  • Therapeutic products can be made using the materials shown herein. Effective amounts of therapeutic products are the minimum dose that produces a measurable effect in a subject. Therapeutic products are easily prepared by one of ordinary skill in the art.
  • a scTCR of the disclosure is administered directly to a patient.
  • a scTCR of the disclosure is linked to PEG or to immunoglobulin constant regions, as known in the art. This embodiment lengthens the serum clearance.
  • the scTCR is linked to a chemotherapeutic agent or drug in order to deliver the drug to a target cell such as a cancer cell.
  • the scTCR is linked to a biologic effector molecule such as a cytokine (Tayal and Kalra (2008) Eur J Pharmacol, 579, 1-12).
  • the scTCR is linked to a cytokine with anti-tumor activity, such as IL-2, IL-12, or TNF ⁇ (Wong et al. (2011) Protein Eng Des Sel, 24, 373-83).
  • the scTCR is linked to an immune-inhibitory cytokine, such as IL-10 or IL-13 (Stone et al. (2012) Protein Engineering).
  • the scTCR is linked to another antigen binding molecule to form a bispecific agent (Miller et al.
  • the bispecific molecule is comprised of a scTCR linked to a single chain Fv, such as an anti-CD3 ((Bargou et al. (2008) Science, 321, 974-7; Liddy et al. (2012) Nat Med, 18, 980-7), to crosslink T cells and diseased cells.
  • the scTCR is linked to TCR signaling domains, such as CD3, to form a chimeric antigen receptor ((Porter et al. (2011) N Engl J Med, 365, 725-33; Sadelain et al.
  • the scTCR compositions can be formulated by any of the means known in the art. They can be typically prepared as injectables, especially for intravenous, intraperitoneal or synovial administration (with the route determined by the particular disease) or as formulations for intranasal or oral administration, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection or other administration may also be prepared. The preparation may also, for example, be emulsified, or the protein(s)/peptide(s) encapsulated in liposomes.
  • the active ingredients are often mixed with optional pharmaceutical additives such as excipients or carriers which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • concentration of the scTCR in injectable, aerosol or nasal formulations is usually in the range of 0.05 to 5 mg/ml. The selection of the particular effective dosages is known and performed without undue experimentation by one of ordinary skill in the art. Similar dosages can be administered to other mucosal surfaces.
  • vaccines that could include a scTCR may contain minor amounts of pharmaceutical additives such as auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE); and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween® 80 emulsion.
  • the scTCRs of the present disclosure and/or binding fragments having primary structure similar (more than 90% identity) to the TCR variable regions and which maintain the high affinity for the target ligand may be formulated into vaccines as neutral or salt forms.
  • Pharmaceutically acceptable salts include but are not limited to the acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic acids, e.g., acetic, oxalic, tartaric, or maleic acid.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
  • inorganic bases e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides
  • organic bases e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
  • scTCRs for therapeutic use are administered in a manner compatible with the dosage formulation, and in such amount and manner as are prophylactically and/or therapeutically effective, according to what is known to the art.
  • the quantity to be administered which is generally in the range of about 100 to 20,000 ⁇ g of protein per dose, more generally in the range of about 1000 to 10,000 ⁇ g of protein per dose.
  • Similar compositions can be administered in similar ways using labeled scTCRs for use in imaging, for example, to detect cells to which a target ligand is bound. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician or veterinarian and may be peculiar to each individual, but such a determination is within the skill of such a practitioner.
  • the vaccine or other immunogenic composition may be given in a single dose; two dose schedule, for example two to eight weeks apart; or a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as required to maintain and/or reinforce the immune response, e.g., at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • Humans (or other animals) immunized with the retrovirus-like particles of the present disclosure are protected from infection by the cognate retrovirus.
  • Autoimmune diseases are those diseases in which the immune system produces an immune response against an antigen that is normally present in the host.
  • Autoimmune diseases include rheumatoid arthritis, adjuvant arthritis, myasthenia gravis, encephalomyelitis, multiple sclerosis, thyroiditis, inflammatory bowel disease or systemic lupus erythematosus, type I diabetes, non-obese diabetes, Grave's disease, Hashimoto's disease, osteoarthritis, dermatitis, hepatitis, pemphigus vulgaris, celiac disease, Sjogren's syndrome, Addison's disease, primary myxedema, Goodpasture's syndrome, tuberculoid leprosy, ankylosing spondylitis, Reiter's disease, uveitis, amyloidosis, psoriasis vulgaris, idiopathic hemochromatosis and psorasis.
  • Every formulation or combination of components described or exemplified can be used to practice the disclosure, unless otherwise stated.
  • Specific names of substances are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same substances differently.
  • a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination.
  • methods, target ligands, biologically active groups, starting materials, and synthetic methods other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation.
  • the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above also may be used in veterinary medicine.
  • Suitable agents may be formulated and administered systemically or locally.
  • Techniques for formulation and administration may be found in Alfonso and Gennaro (1995). Suitable routes may include, for example, oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, or intramedullary injections, as well as intrathecal, intravenous, or intraperitoneal injections.
  • the agents of the disclosure may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present disclosure in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • Appropriate compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, and then administered as described above.
  • Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
  • compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions, including those formulated for delayed release or only to be released when the pharmaceutical reaches the small or large intestine.
  • compositions of the present disclosure may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • TCRs all adopt a similar Ig-fold and docking angle, and TCR recognition of pepMHC is mediated entirely by specific residues on CDR loops (Garcia et al. (2009) Nat Immunol, 10, 143-7; Marrack et al. (2008) Annu Rev Immunol, 26, 171-203; Rudolph et al. (2006) Annu Rev Immunol, 24, 419-66)).
  • a single TCR with known structure provides a scaffold for in vitro engineering with specificity and high affinity against non-cognate peptides displayed on MHC.
  • mutants with degenerate residues within CDR loop residues that are most likely to directly contact peptide libraries of mutants within a single TCR were generated in order to provide TCRs that can be developed having high affinity against non-cognate peptide-MHC antigens.
  • FIG. 1 The general strategy used to discover, or generate, novel TCRs against non-cognate antigens from a single scaffold is shown in FIG. 1 .
  • the process involves: selecting a single TCR with a known structure.
  • the human TCR called A6 was used.
  • the A6 TCR was the first human TCR whose structure was solved (Garboczi et al. (1996) Nature, 384, 134-141), and it has been the topic of a long series of structural and biochemical studies (e.g., (Armstrong et al. (2008) Biochem J, 415, 183-96; Ding et al. (1999) Immunity, 11, 45-56; Hawse et al. (2012) J Immunol, 188, 5819-23).
  • Another step in the process is identifying residues within the TCR (e.g., A6) binding site that are most likely to contribute to peptide specificity.
  • This step involved modeling a collection of peptides into the HLA.A2 molecule, docking of the A6 TCR, analysis of the frequency by which A6 TCR residues reside within 3 angstroms of each peptide.
  • mutants that bind to specific, non-cognate pep-MHC ligands are selected.
  • Various methods have been used for selecting variants, including affinity-based binding to immobilized ligands (phage display) or magnetic particle selections with antigens (yeast display), or fluorescent activated cell sorting with labeled-peptide-MHC antigens (yeast display). Examples of each of these steps are described further below.
  • the A6 TCR was selected as the single TCR having a known structure.
  • the structure of the A6:Tax peptide:HLA-A2 complex (PDB: 1AO7) (Garboczi et al. (1996) Nature, 384, 134-141), was published in 1996.
  • the side view of the complex showed that the ends of the variable domains that contained the six CDRs docked onto the Tax:HLA.A2 molecule, with the central region of the binding site positioned over the peptide Tax ( FIG. 2A )
  • This view shows that the TCR adopts a diagonal position over the peptide-MHC, a finding which has now been observed for all TCR:peptide-MHC structures.
  • the two CDR3 loops are positioned over the peptide, while there are various residues from CDR1 and CDR2 loops that interact predominantly with the helices of the MHC molecule.
  • This diagonal docking orientation with the V ⁇ region positioned over the ⁇ 2 MHC helix and the N-terminal end of the peptide, and the V ⁇ region positioned over the ⁇ 1 MHC helix and C-terminal end of the peptide has been observed in virtually all complexes to date.
  • the conserved features of these interactions suggested that it may be possible to use a single TCR as a scaffold, in which mutagenesis of various key “peptide-interacting” residues allowed the generation and discovery of novel TCR specificities ( FIG. 2B ).
  • the Rosetta Backrub modeling program was used to model the HLA.A2 restricted peptides (i.e., Tax, Mart1-9 mer, Mart1-10 mer, SL9 HIV, WT1, and Survivin) into the HLA.A2 groove using Rosetta Backrub flexible backbone modeling algorithms ( FIG. 3 ) ((Lauck et al. (2010) Nucleic Acids Res, 38, W569-75); kortemmelab.ucsf.edu/backrub/).
  • Candidate residues for degeneracy were then determined by measuring which CDR loop positions would be most likely to allow for contacts with these peptides in the lowest energy conformation model for each peptide.
  • Table 3 lists the A6 TCR residues found within 3.0 ⁇ of peptide in the 6 structures and the percentage of structures in which it was found. Positions in bold type were used to construct a degenerate library, but other positions can be used either separately or in combination to construct additional libraries for developing TCRs against many different peptide/HLA complexes.
  • A6-X15 SEQ ID NO:3
  • This sequence also includes five framework mutations (S33A, E59D, N63D, N66K, K121I, all in the V ⁇ domain) and two CDR mutations (A52V and Q106L, both in the V ⁇ domain) that were isolated previously in a stability screen of the scTCR.
  • This mutant also contained four CDR3 ⁇ mutations (A100M, G101S, G102A, and R103Q) that yielded higher affinity binding to Tax:HLA.A2 (Li et al. (2005) Nat Biotechnol, 23, 349-54).
  • A6-X15 also uses the highly stable V ⁇ 2 segment (IMGT: TRAV12-2), but contains a Phe49Ser V ⁇ mutation for improved stability.
  • V ⁇ 2-containing TCRs have described in detail putative conserved resides in the V ⁇ 2 segment that have evolved to recognize the HLA.A2 helices.
  • FIG. 4 shows the analysis of selected A6 TCR residues for their contribution to binding of Tax:HLA.A2.
  • CDR1 or CDR2 are important in maintaining MHC restriction
  • four residues of the (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72) were substituted with alanine and tested for binding.
  • Table3 shows approximate affinities of A6 X15 alanine mutants and fold changes in binding affinity relative to wild type. The results showed that the tyrosine at position 51 of CDR2 ⁇ , which contacts the ⁇ helix of HLA.A2, was most important in binding. Thus, this residue was maintained in the library described here.
  • CDR2 ⁇ residues Y51 and S52 are conserved across different ⁇ -chains and frequently bind HLA.A2 at the same position (Marrack et al. (2008) Annu Rev Immunol, 26, 171-203). Crystal structure analysis of the V ⁇ 2-containing TCRs A6, DMF4, and DMF5 have shown that CDR1 ⁇ residues R28 and Q31 are important in making MHC contacts, although the exact HLA residues contacted vary. Q31 has also been shown to make contacts with peptide in addition to HLA-A2 (Borbulevych et al. (2011) J Immunol, 187, 2453-63).
  • V ⁇ 2 residues R28, Q31, Y51, and S52 are important in contacting the HLA.A2 molecule, the binding energy contribution of each contact had not been previously described.
  • site-directed alanine mutants were made in the A6 X15 construct at each position and stained with Tax (LLFGYPVYV, SEQ ID NO:5):HLA-A2 dimer ( FIG. 4B ).
  • the residue that contributed the most significant binding energy was CDR2 ⁇ position Y51. Since this residue exclusively contacted MHC helices in the A6 wt structure, this residue was retained as wild type in the library.
  • This example describes the preparation of a library of mutant TCRs.
  • a display system in which a library of TCR mutants can be screened for the rare mutant(s) that bind to the non-cognate antigen.
  • Three display systems have been used for engineering TCRs for higher affinity, and could be used for this process: yeast display, phage display, and T cell (mammalian cell) display.
  • Alternative display methods such as ribosome, RNA, DNA, and CIS display, may also be suitable for this process.
  • the wild type TCR with low affinity for the cognate antigen was cloned into the system, and used as a template for engineering higher affinity TCRs against the cognate antigen. Any of these systems could be applied to the approach described here, in which a single scaffold TCR (A6 in the present case) is used as a template for rational design of libraries and the selection of TCRs against non-cognate antigens.
  • yeast display was used as the platform.
  • the single-chain A6-X15 TCR was used as the template as it is stably expressed in properly folded form on the surface of yeast ( FIG. 6 ).
  • the gene containing degenerate codons at each of the top five positions shown in FIG. 3C and FIG. 4 was synthesized by Genscript (Piscataway, N.J., USA), and was designated the RD1 library. Genes with complete or partial codon degeneracy can now be readily synthesized by commercial sources, or can be generated by PCR using multiple synthetic oligonucleotide primers.
  • the synthetic genes can be used as templates after cloning into a common plasmid (e.g., pET vectors) or directly as PCR product templates.
  • Two flanking oligonucleotide primers with homology to the linearized yeast display vector were synthesized and PCR was performed in order to generate double stranded PCR products that could be inserted by homologous recombination into the yeast display vector, pCT302.
  • the human A6-X15 scTCR library was thus introduced into the yeast display vector by combining the linearized pCT302 vector, A6-X15 RD1 library PCR product, and competent EBY100 yeast cells.
  • the resultant RD1 library contained about 6 ⁇ 10 6 independent clones, was judged by plating limiting dilution aliquots of yeast after electroporation. As expected due to the diversity at each of the residues that are near the Tax peptide in the complex, the resultant library did not show positive staining with either the cognate antigen Tax:HLA-A2 or the Mart1:HLA-A2 complex as determined using peptide-HLA.A2 DimerX preparations.
  • a V ⁇ -Linker-V ⁇ scTCR is shown with an N-terminal hemagglutinin (HA) and C-terminal c-myc tags, used to detect expression of the scTCR on the surface of yeast (Stone et al. (2012) Methods Enzymol, 503, 189-222).
  • the scTCR construct was expressed as a fusion with the AGA-2 yeast mating protein which allows the scTCR to be expressed on the surface of yeast and rapidly analyzed via flow cytometry and screened by fluorescent activated cell sorting (FACS).
  • This example describes selecting mutant TCRs that bind to target ligands.
  • the library was selected with the non-cognate antigen Mart1-10 mer:HLA.A2, in addition to the cognate antigen, Tax:HLA.A2.
  • Mart1-10 mer in addition to the cognate antigen
  • Tax:HLA.A2 in addition to the cognate antigen
  • these two peptides differ not only in length, but when aligned only the leucine at position 2 of Mart1-10 mer (position 1 of Tax) and the valine at position 10 of Mart1 (position 9 of Tax) are identical.
  • the valine is considered an HLA.A2 anchor residue, such that the side chains exposed to the TCR are distinctly different between the two cases.
  • FACS fluorescent-activated cell sorting
  • the RD1 library did not show a detectable positive peak, but after the second sorting, a positive population began to emerge, and cells were plated after the fourth sorting for additional analyses.
  • the amino acid and nucleotide sequences of the six clones analyzed are in FIG. 10 .
  • FACS fluorescent-activated cell sorting
  • yeast cells were also stained with chicken anti-c-myc antibody, goat anti-chicken IgY alexa 647 secondary antibody and double positives were isolated in order to exclude truncated clones.
  • (B) Aliquots of yeast cells after each sort were then incubated with 100 nM Mart1 (ELAGIGILTV, SEQ ID NO:7):HLA-A2 dimer (DimerX; obtained from BD Pharmingen), followed by APC-conjugated goat anti-mouse secondary antibody.
  • the RD1 library and the first three sorts did not show detectable positive peaks, but after the fourth sorting, a positive population began to emerge, and cells were plated after the fifth sorting for additional analyses.
  • Five clones revealed that they all had identical amino acid sequences, indicating that these residues were important in conferring high-affinity binding.
  • the nucleotide sequences do not vary between clones of the same amino acid sequence, only a small amount of possible codon combinations are possible with this amino acid sequence.
  • the amino acid and nucleotide sequences are shown in FIG. 12 .
  • all five TCR residues derived by sorting of the A6 library, differed between the original high-affinity TCR and the Mart1 selection.
  • the wild type sequence was encountered in 2 of 6 sequences (e.g., Tax-S4-3) and a variant containing threonine at position 31 occurred in 4 of 6 sequences (e.g., Tax-S4-1).
  • the five residues were: Leu99 ⁇ , Ala102 ⁇ , Gln31 ⁇ or Thr31 ⁇ , Thr97 ⁇ , and Asp98 ⁇ .
  • the residues were: Trp99 ⁇ , Gly102 ⁇ , Thr31 ⁇ , Lys97 ⁇ , and Tyr98 ⁇ .
  • the Thr31 ⁇ residue was found in both the Mart1-specific TCR and one variant of the Tax-specific TCR (e.g., Tax-S4-1)
  • the A6 crystal structure shows this position as being important in contacting both the peptide and MHC (see FIG. 4 ). Due to its structural similarity to glutamine, threonine may allow for MHC contacts to be maintained without leading to cross-reactivity with other peptides.
  • TCRs In order to use TCRs in the present disclosure for specific targeting of cells that express the antigens, it is critical that they exhibit peptide specificity. To determine if the TCRs selected for binding to the Tax (SEQ ID NO:5) and Mart1 (SEQ ID NO:7) peptides exhibited specificity in their reaction with the selecting peptides, representative clones for each were titrated with various concentrations of both the Tax and Mart1:HLA.A2 dimers.
  • RD1-MART1-S5-4 isolated scTv SEQ ID NO:33
  • degenerate libraries were made in the CDR3 loops of RD1-MART1-S5-4 in order to select for mutants for increased affinity to peptide MART1/HLA-A2/Ig dimers.
  • the RD1-MART1-S5-4 CDR3 libraries were sorted sequentially with 1-200 nM MART1/HLA-A2 dimer (DimerX; obtained from BD Pharmingen), APC-conjugated goat anti-mouse secondary antibody, for a total of two sorts.
  • FIG. 15A Aliquots of yeast cells after each sort were then incubated with 50 nM MART1/HLA-A2 dimer (DimerX; obtained from BD Pharmingen), followed by APC-conjugated goat anti-mouse secondary antibody.
  • MART1/HLA-A2 dimer DimerX; obtained from BD Pharmingen
  • APC-conjugated goat anti-mouse secondary antibody One clone, RD1-MART1 HIGH (SEQ ID NO:34), showed a significant binding increase from the template clone, RD1-MART1-S5-4, when stained with MART1/HLA.A2 monomers.
  • RD1-MART1 HIGH contained TCR ⁇ mutations Q31T, T97K, and D98Y, and TCR ⁇ mutations L99W, A100M, G101A, R103G, P104V (SEQ ID NO:34).
  • RD1-MART1 HIGH scTv specifically bound MART1/HLA-A2 with high-affinity
  • a soluble form of RD1-MART1 HIGH scTv was expressed and refolded from E. coli inclusion bodies and biotinylated via a C-terminal BirA tag (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72; Zhang et al. (2007) J Exp Med, 204, 49-55).
  • the human cell line T2 (HLA-A2+) was incubated with 1 ⁇ M MART1, Tax, or WT1 peptides and washed.
  • Biotinylated RD1-MART1 HIGH scTv was titrated on T2 cells pre-loaded with MART-1 peptide (1 ⁇ M), null peptide, tax (1 ⁇ M), or without peptide. The cells were washed and incubated with SA-PE and analyzed by flow cytometry. Only cells loaded with MART1 peptide were bound by the RD1-MART1 HIGH TCR. The results showed that the soluble TCR was specific for MART-1 and that it exhibited low nanomolar binding affinity ( FIG. 17 ).
  • the Rosetta modeling information used to generate the RD1 library ( FIG. 3 ), and the A6:Tax/HLA.A2 crystal structure (PDB: 1AO7) overlaid with the crystal structures of MART1/HLA.A2 (PDB: 1JF1) (Sliz et al. (2001) J Immunol, 167, 3276-84) and WT1/HLA.A2 (PDB: 3HPJ) (Borbulevych et al. (2010) Mol Immunol, 47, 2519-24) ( FIG. 19A ), were used to guide the generation of a second generation library, called RD2.
  • RD2 a second generation library
  • This library included 5 degenerate positions (TCR ⁇ D27, G29, and S99; TCR ⁇ L99 and W100) based on NNK nucleic acid composition, one binary position at TCR ⁇ Q31 where either the wild type residue glutamine or threonine could be selected, and a binary sequence in position s100-103 in CDR3 ⁇ where the four adjacent residues could be selected as A6 wild type (AGGR, SEQ ID NO:44) or A6-X15 (MSAQ, SEQ ID NO:45) ( FIG. 20 ).
  • A6 wild type AGGR, SEQ ID NO:44
  • A6-X15 MSAQ, SEQ ID NO:45
  • Two sequential magnetic bead selections of the RD2 library were performed following incubation with 5 ⁇ M Tax/HLA.A2 UV-exchanged monomers and streptavidin MACS beads (obtained from Miltenyi Biotec) ( FIG. 19B ).
  • yeast cells were incubated with 1 nM Tax/HLA-A2 dimer (DimerX; obtained from BD Pharmingen) and APC-conjugated goat anti-mouse secondary antibody. Aliquots of yeast cells after each selection were then incubated with 50 nM Tax/HLA-A2 dimer (DimerX; obtained from BD Pharmingen), APC-conjugated goat anti-mouse secondary antibody.
  • Two sequential magnetic bead selections of the RD2 library were also performed following incubation with 5 ⁇ M MART1/HLA-A2 UV-exchanged monomers and streptavidin MACS beads (obtained from Miltenyi Biotec) ( FIG. 19C ).
  • yeast cells were incubated with 100 nM MART1/HLA-A2 dimer (DimerX; obtained from BD Pharmingen) and APC-conjugated goat anti-mouse secondary antibody. Aliquots of yeast cells after each selection were then incubated with 50 nM MART1/HLA-A2 dimer (DimerX; obtained from BD Pharmingen), APC-conjugated goat anti-mouse secondary antibody.
  • the RD2 library was selected with two peptide/MHC ligands, Tax/HLA.A2 (cognate) and MART1/HLA.A2 (non-cognate) via two MACS magnetic bead selections followed by one round of FACS. Selections with the cognate antigen, Tax, showed the emergence of a positively staining population after the first magnetic selection with Tax/HLA.A2 monomers ( FIG. 19B ). Selections with MART1/HLA.A2 revealed the emergence of a positively staining population following the third FACS selections ( FIG. 19C ).
  • Sequencing analysis revealed the selection of the A6 wild-type CDR3 ⁇ loop sequence AGGR (SEQ ID NO:44) for both RD2-MART1-S3-3 and RD2-MART1-S3-4. Additionally, both RD2-MART1-S3-3 and RD2-MART1-S3-4 selected TCR- ⁇ M99, TCR- ⁇ S27 and H29, and two PCR-based mutations in TCR- ⁇ (S34 and P40). Whereas RD2-MART1-S3-3 selected threonine at position 31 in CDR1 ⁇ binary position, the RD2-MART1-S3-4 clone retained the wild-type Q31. Additionally, RD2-MART1-S3-3 selected TCR ⁇ R99 and S100, whereas RD2-MART1-S3-4 selected L99 and W100 ( FIG. 21E ).
  • RD2-MART1-S3-3 contained TCR ⁇ mutations D27S, G29H, Q31T, F34S, 540P, S99R, W100S, and TCR ⁇ mutation L99M (SEQ ID NO:41) and RD2-MART1-S3-4 contained TCR ⁇ mutations D27S, G29H, F34S, S40P, S99L, and TCR ⁇ mutation L99M (SEQ ID NO:42).
  • T1-S18.45 another engineered high affinity scTCR known as T1-S18.45 was used as a template TCR for a single chain scaffold in the yeast display system.
  • T1-518.45 uses V ⁇ 2 and V ⁇ 16 and was isolated against the MART1/HLA-A2 antigen (Fleischer et al. (2004) J Immunol, 172, 162-9).
  • the wild-type single-chain TCR was used as a template for CDR3 libraries, and affinity maturation was performed as described above.
  • the high-affinity mutant T1-518.45 was sequenced ( FIG. 22 ), showing that it contained the TCR ⁇ mutations N92S, D99S, N100S, A101D, and R102F (SEQ ID NO:43).
  • T1-518.45 scTv bound MART1/HLA-A2 with high-affinity
  • a soluble form of T1-518.45 scTv was produced in E. coli and biotinylated (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72; Zhang et al. (2007) J Exp Med, 204, 49-55).
  • Biotinylated T1-S18.45 scTv was titrated on antigen-presenting cell line T2 (HLA-A2+) pre-loaded with MART-1 peptide (1 ⁇ M) or null peptide, 5L9 (1 ⁇ M). The results showed that the soluble TCR was specific for MART-1 and that it exhibited low nanomolar binding affinity ( FIG. 23 ).
  • TCRs generated from the scaffold strategy could be used either in soluble form or in TCR gene therapy for adoptive T cell therapies.
  • the TCRs can be readily formatted for use as soluble therapeutic products, which carry a “payload” to the target cell expressing the specific peptide-MHC antigen.
  • the formats include those already practiced in the art, including immunoglobulin fusions, chemotherapeutic or drug conjugates, and bispecific antibodies: 1) single-chain TCRs in either a V ⁇ -V ⁇ orientation or V ⁇ -V ⁇ orientation, expressed in soluble form for binding applications or as a platform for the other applications shown; mutated, high-affinity V domains are shown with an asterisk (*); 2) the single-chain TCR can be fused in frame with the constant regions domains of an antibody to produce an immunoglobulin fusion with effector functions and other properties of the Fc regions, as has been done with the extracellular domain of the TNF- ⁇ receptor in the product Enbrel (Brower (1997) Nat Biotechnol, 15, 1240); 3)
  • the individual V ⁇ and V ⁇ domains can be configured like a conventional antibody, as in
  • FIG. 24B shows the variable domains (V) isolated by yeast display for high-affinity binding using the TCR scaffold can be inserted into mammalian cell vectors for expression in T cells in an adoptive T cell therapies.
  • the TCRs can be used either as 1) single-chain receptors in chimeric antigen receptors (CAR) as is now well known, or 2) they can be cloned as full length ⁇ and ⁇ TCRs for conventional TCR gene therapy in adoptive T cell formats.
  • CAR chimeric antigen receptors
  • Antibodies used to detect yeast surface expression included: anti-HA epitope tag (Clone HA.11; Covance), anti-Cmyc epitope tag (Clone 9E10; Molecular Probes), Goat-anti-mouse IgG F(ab′) 2 AlexaFluor 647 secondary antibody (Invitrogen), and Goat-anti-chicken IgG F(ab′) 2 AlexaFluor 647 secondary antibody (Invitrogen).
  • HLA-A2 [Tax 11-19 : SEQ ID NO:5, Mart1 26-35 A27L : SEQ ID NO:7] were synthesized by standard F-moc (N-(9-fluorenyl)methoxycarbonyl) chemistry at the Macromolecular Core Facility at Penn State University College of Medicine (Hershey, Pa., USA).
  • F-moc N-(9-fluorenyl)methoxycarbonyl
  • BD DimerX recombinant soluble dimeric HLA-A2:Ig fusion protein
  • Candidate residues for degeneracy were determined by measuring which CDR loop positions would be most likely to allow for contacts with a variety of peptides using Rosetta Backrub flexible backbone modeling algorithms ((Lauck et al. (2010) Nucleic Acids Res, 38, W569-75; Smith and Kortemme (2008) J Mol Biol, 380, 742-56), kortemmelab.ucsf.edu/backrub/).
  • Rosetta Backrub flexible backbone modeling algorithms (Lauck et al. (2010) Nucleic Acids Res, 38, W569-75; Smith and Kortemme (2008) J Mol Biol, 380, 742-56), kortemmelab.ucsf.edu/backrub/).
  • PDB 1AO7
  • Rosetta was used to model in a variety of HLA-A2 restricted peptides of interest (SL9, Mart1, WT1, Survivin) into the peptide binding groove of HLA-A2 by using the Multiple Mutation Mutagenesis module.
  • CDR loop residues that were within 3 ⁇ of peptide residues were determined for the lowest energy conformation of each model, and the 5 most frequently encountered positions were made degenerate using A6 X15 scTCR, which contains stabilizing mutations as previously described, as a template (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72).
  • the high affinity DMF5 variant contained 2 mutations that increased the affinity 250-fold (Hawse et al. (2012) J Immunol, 188, 5819-23).
  • the use of computational approaches to engineer T cell receptors with de novo affinities for non-cognate ligands has not been described.
  • the present invention provides for the first time the use of computational modeling to guide scaffold generation to be used for directed evolution.
  • Candidate residues for degeneracy were determined by measuring which CDR loop positions would be most likely to allow for contacts with a variety of peptides using Rosetta Backrub flexible backbone modeling algorithms (Lauck, 2010 #7691 ⁇ ; kortemmelab.ucsf.edu/backrub/). Using the A6:Tax peptide:HLA-A2 crystal structure (PDB: 1AO7), Rosetta was used to model in a variety of HLA-A2 restricted peptides of interest (SL9, Mart1, WT1, Survivin).
  • the A6 RD1 Library was expressed in yeast display plasmid pCT302 (V ⁇ -L-V ⁇ ) (Boder and Wittrup (1997) Nat. Biotech., 15, 553-557; Boder and Wittrup (2000) Methods Enzymol, 328, 430-44), which contains a galactose-inducable AGA2 fusion allowing for growth in Trp media.
  • Induction of the scTv gene involves growth of the transformed EBY100 yeast cells to stationary phase in selection media followed by transfer to galactose-containing media.
  • the A6 RD1 Library was synthesized by Genscript (Piscataway, N.J., USA) using A6 X15 as a template (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72; Li et al. (2005) Nat Biotechnol, 23, 349-54).
  • Genscript Procataway, N.J., USA
  • A6 X15 As a template
  • the construct consisted of the variable fragments attached by the linker region GSADDAKKDAAKKDGKS (SEQ ID NO: 21) (Aggen et al. (2011) Protein Engineering, Design, & Selection, 24, 361-72; Kieke et al. (1999) Proc Natl Acad Sci USA, 96, 5651-6; Soo Hoo et al. (1992) Proc. Natl. Acad.
  • pCT302 overhangs were added to the synthesized library using forward primer 5′-CAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTC TGCTAGCAATGCTGGTGTAACACAAACGCCAA-3′ (SEQ ID NO: 50) and reverse primer 5′-GGAACAAAGTCGATTTTGTTACATCTACACTGTTGTTAACAGATCTCG AGTCATTATAAATCTTCTTCAGAGATC-3′ (SEQ ID NO: 51).
  • Yeast libraries were generated by homologous recombination in EBY100 yeast by electroporating PCR products along with NheI and XhoI digested pCT302 (Benatuil et al. (2010) Protein Eng Des Sel, 23, 155-9; Colby et al. (2004) Methods Enzymol, 388, 348-58; Starwalt et al. (2003) Protein Eng, 16, 147-56; Swers et al. (2004) Nucleic Acids Res, 32, e36).
  • the resultant library size was 6 ⁇ 10 6 .
  • the library was induced in galactose-containing media (SG-CAA) for 48 h, washed with 1 mL 1% PBS/BSA, and stained with the following: anti-HA epitope tag (1:50), anti-V ⁇ 2 antibody (1:50), and tax or Mart1 peptide:HLA-A2 DimerX (100 nM) along with goat-anti-mouse IgG F(ab′) 2 AlexaFluor 647 secondary antibody (1:100), and anti-cmyc (1:50) along with goat-anti-chicken IgG F(ab′) 2 AlexaFluor 647 secondary antibody (1:100).
  • CDR3 libraries were generated by splicing by overlap extension (SOE) PCR spanning 5 adjacent codons at a time (2 libraries in the CDR3 ⁇ loop; 1 in the CDR3 ⁇ loop) (Horton et al. (1990) Biotechniques, 8, 528-35) using the RD1-MART1 scTV clone selected from the RD1 library as a template.
  • SOE overlap extension
  • Yeast libraries were generated by homologous recombination in EBY100 yeast by electroporation of PCR products along with NheI and XhoI digested yeast display vector pCT302 (Benatuil et al. (2010) Protein Eng Des Sel, 23, 155-9; Colby et al. (2004) Methods Enzymol, 388, 348-58; Starwalt et al. (2003) Protein Eng, 16, 147-56; Swers et al. (2004) Nucleic Acids Res, 32, e36).
  • the resultant library sizes were ⁇ 1: 2.1 ⁇ 10 7 , ⁇ 2: 1.7 ⁇ 10 7 , and ⁇ : 1.1 ⁇ 10 7 . Libraries were pooled in equal cell numbers in ratios reflecting relative diversity, and expanded in SD-CAA media.
  • the combined library was induced in galactose-containing media (SG-CAA) for 48 hours, washed with 1 mL 1% PBS/BSA, and stained with MART1/HLA.A2 DimerX, goat-anti-mouse IgG F(ab′)2 AlexaFluor 647 secondary antibody (1:100). Cells were washed (1 ml, 1% PBS/BSA), and the most fluorescent cells were selected using a FACS Aria (BD Bioscience) high-speed sorter. Selection was performed with MART1/HLA-A2 dimer (1-200 nM).
  • PyMOL software (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC) was used to overlay crystal structures of the A6:Tax/HLA.A2 complex (PDB: 1AO7) with crystal structures of the MART1/HLA.A2 (PDB: 1JF1) (Sliz et al. (2001) J Immunol, 167, 3276-84) and WT1/HLA.A2 (PDB: 3HPJ) (Borbulevych et al. (2010) Mol Immunol, 47, 2519-24).
  • the TCR ⁇ Position Q31 was a binary position where either the wild type residue glutamine or threonine could be selected, and the positions 100-103 in CDR3 ⁇ , were binary where the four adjacent residues could be selected as A6 wild type (AGGR, SEQ ID NO:44) or A6-X15 (MSAQ, SEQ ID NO:45).
  • the A6 RD2 library was expressed in yeast display plasmid pCT302 (V ⁇ -L-V ⁇ ) (Boder and Wittrup (1997) Nat. Biotech., 15, 553-557; Boder and Wittrup (2000) Methods Enzymol, 328, 430-44), which contains a galactose-inducable AGA2 fusion allowing for growth in Trp media. Induction of the scTv gene involves growth of the transformed EBY100 yeast cells to stationary phase in selection media followed by transfer to galactose-containing media.
  • the A6 RD2 Library was synthesized by DNA2.0 (Menlo Park, Calif., USA) using the A6-X15 as a template.
  • the construct consisted of the variable fragments attached by the linker region GSADDAKKDAAKKDGKS (SEQ ID NO:21) and N-terminal HA and C-terminal Cmyc epitope tags.
  • the following gene was synthesized where positions indicated by “X” were made degenerate by NNK codons, the positions labeled “1234” were binary allowing for A6 wild type CDR3 ⁇ loop AGGR (SEQ ID NO:44) or A6-X15 CDR3 ⁇ loop MSAQ (SEQ ID NO:45), the position indicated by “#” was binary allowing for either wild type residue Q or mutated T, and positions indicated by “*” were stop codons: NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMAWYRQDPGMGLRLIHYSVGVGI TDQGDVPDGYKVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGX1234PELYFGPGT RLTVTEDLINGSADDAKKDAAKKDGKSK
  • the delivered PCR product was amplified via PCR using the Splice4L and T7 primers, and yeast libraries were generated by homologous recombination in EBY100 yeast by electroporation of amplified PCR products along with NheI and XhoI digested yeast display vector pCT302 (Benatuil et al. (2010) Protein Eng Des Sel, 23, 155-9; Colby et al. (2004) Methods Enzymol, 388, 348-58; Starwalt et al. (2003) Protein Eng, 16, 147-56; Swers et al. (2004) Nucleic Acids Res, 32, e36).
  • the resultant library size was 2.4 ⁇ 10 8 .
  • the RD2 library was induced in galactose-containing media (SG-CAA) for 48 hours, washed with 1 mL 1% PBS/BSA, and stained with 5 ⁇ m Tax (SEQ ID NO:5) or MART1 (SEQ ID NO:7) peptide/HLA.A2 UV-exchanged HLA.A2 monomers (Rodenko et al. (2006) Nat Protoc, 1, 1120-32; Toebes et al. (2006) Nat Med, 12, 246-51).
  • Magnetic bead selections were performed utilizing streptavidin MACS microbeads (Miltenyl Biotec), for a total of two selections using MACS LS columns on a QuadroMACSTM Separator (Miltenyl Biotec). Following two selections, the selected libraries was stained with the following: selecting peptide/HLA.A2 DimerX, goat-anti-mouse IgG F(ab′)2 AlexaFluor 647 secondary antibody (1:100). Cells were washed (1 ml, 1% PBS/BSA), and the most fluorescent cells were selected using a FAGS Aria (BD Bioscience) high-speed sorter.
  • Selections were performed with 1 nM and 100 nM peptide/HLA.A2 for selecting cognate antigen Tax, and selecting, non-cognate antigen MART1, respectively. Expression was monitored with anti-HA epitope tag (1:50), goat-anti-mouse IgG F(ab′)2 AlexaFluor 647 secondary antibody (1:100), and anti-cmyc (1:50), goat-anti-chicken IgG F(ab′)2 AlexaFluor 647 secondary antibody (1:100).
  • Colonies were expanded and induced in galactose-containing media (SG-CAA) for 48 h, washed with 1 mL 1% PBS/BSA, and stained with the following: anti-HA epitope tag (1:50), anti-V ⁇ 2 antibody (1:50), and tax or Mart1 peptide:HLA-A2 DimerX (100 nM) along with goat-anti-mouse IgG F(ab′) 2 AlexaFluor 647 secondary antibody (1:100), and anti-cmyc (1:50) along with goat-anti-chicken IgG F(ab′) 2 AlexaFluor 647 secondary antibody (1:100).
  • Cells were washed (1 ml, 1% PBS/BSA) and analyzed on an Accuri C6 flow cytometer.
  • Plasmids were recovered using ZymoprepTM Yeast Plasmid Miniprep II (Zymo Research) and introduced back into E. coli via heat shock transformation into Subcloning EfficiencyTM DH5 ⁇ TM Competent Cells (Invitrogen). E. coli cells were expanded and plasmids were isolated using QIAprep Spin Miniprep Kit (Qiagen). Sequences of individual clones were determined by Sanger sequencing.

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