WO2019133443A1 - Récepteurs de lymphocytes t modifiés - Google Patents

Récepteurs de lymphocytes t modifiés Download PDF

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Publication number
WO2019133443A1
WO2019133443A1 PCT/US2018/066866 US2018066866W WO2019133443A1 WO 2019133443 A1 WO2019133443 A1 WO 2019133443A1 US 2018066866 W US2018066866 W US 2018066866W WO 2019133443 A1 WO2019133443 A1 WO 2019133443A1
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Prior art keywords
tcr
polypeptide
domain
modified
extracellular domain
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PCT/US2018/066866
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English (en)
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David Campbell
Ramesh Bhatt
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Janux Therapeutics, Inc.
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Publication of WO2019133443A1 publication Critical patent/WO2019133443A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464452—Transcription factors, e.g. SOX or c-MYC
    • A61K39/464453—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • A61K39/464456—Tyrosinase or tyrosinase related proteinases [TRP-1 or TRP-2]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464486—MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TCRs modified T cell receptors
  • TCRs modified T cell receptors
  • the heterodimer comprises a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide each comprise three hyper variable complementarity determining (CDR) regions, wherein at least one CDR region of either the first polypeptide or the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment.
  • CDR hyper variable complementarity determining
  • the first polypeptide comprises a TCR alpha extracellular domain, or fragment thereof, and the second polypeptide comprises a TCR beta extracellular domain, or a fragment thereof.
  • the first polypeptide comprises a TCR gamma extracellular domain, or fragment thereof, and the second polypeptide comprises a TCR delta extracellular domain, or a fragment thereof.
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to a glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the tumor microenvironment comprises a pH that is about 5.8 to about 6.8.
  • the non-tumor environment comprises a pH that is about 7.2 to about 7.8.
  • the tumor microenvironment comprises an elevated lactic acid concentration relative to the non-tumor environment.
  • the tumor microenvironment comprises an elevated pyruvate concentration relative to the non-tumor environment.
  • the tumor microenvironment is hypoxic.
  • the target antigen is selected from the group consisting of: MAGE-A3, NY-ESO-l, gplOO, WT1, and tyrosinase.
  • the first polypeptide and the second polypeptide each comprise a transmembrane domain.
  • the first polypeptide and the second polypeptide each comprise a cytoplasmic domain.
  • the modified TCR is expressed on a surface of a cell.
  • the modified TCR is a soluble TCR.
  • the first polypeptide or the second polypeptide further comprise an effector domain.
  • the effector domain comprises an anti-CD3 single chain variable fragment effector.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, H- bonding interactions, or a combination thereof.
  • the heterodimer comprises a non natural amino acid, a modified non-natural amino acid, or a combination thereof.
  • isolated or non-naturally occurring cells comprising a modified TCR described above.
  • the isolated or non-naturally occurring cell is a T cell.
  • compositions comprising: (a) the isolated or non-naturally occurring cells described above; and (b) a pharmaceutically acceptable excipient.
  • compositions comprising: (a) the modified TCR described above; and (b) a pharmaceutically acceptable excipient.
  • vectors comprising a nucleic acid molecule encoding a modified TCR described above.
  • FIG. 1 is an exemplary schematic of a T cell receptor (TCR) .
  • FIG 2A-2C shows exemplary schematics of modified TCRs in the soluble format in which the modified TCR is further conjugated to an effector domain.
  • the effector domain is an anti-CD3 moiety.
  • FIGs. 2A-2C are exemplary schematics of modified TCRs with an effector domain.
  • FIG. 2A depicts the modified TCR heterodimer conjugated to an anti-CD3 single-chain variable fragment (scFv) effector.
  • FIG. 2B illustrates a format in which the modified TCR heterodimer is bound to an Fc that is also bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • 2C illustrates a single polypeptide TCR format comprising a variable region of a TCR alpha extracellular domain and a variable region of the TCR beta extracellular domain wherein the single polypeptide is bound to an Fc that is also bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • scFv anti-CD3 single-chain variable fragment
  • Transmembrane domain refers to the region of a receptor which crosses the plasma membrane. Examples include the transmembrane region of a transmembrane protein (for example a Type 1 transmembrane protein), an artificial hydrophobic sequence, and a combination thereof.
  • “Fragment” as used herein refers to a peptide or a polypeptide that comprises less than the full length amino acid sequence.
  • “Target antigen” as used herein refers to a molecule that binds to a variable region of the first polypeptide of the TCR, or the second polypeptide of the TCR, or both.
  • TCR T Cell Receptor
  • Native TCRs are transmembrane receptors expressed on the surface of T cells that recognize antigens bound to major histocompatibility complex molecules (MHC).
  • Native TCRs are heterodimeric, and comprise either an alpha polypeptide chain and a beta polypeptide chain linked through a disulfide bond (FIG. 1) or a gamma polypeptide chain and a delta polypeptide chain linked through a disulfide bond.
  • the heterodimeric polypeptide chain is expressed as part of a complex with accessory proteins which include, for example, two CD3 epsilon polypeptides, one CD3 gamma polypeptide, one CD3 delta polypeptide, and two CD3 zeta polypeptides.
  • the polypeptide chain (alpha, beta, gamma, and delta) comprise an extracellular domain, a transmembrane domain, and a cytoplasmic domain.
  • Each extracellular domain comprises a variable region (V), a joining region (J), and a constant region (C).
  • the constant region is N-terminal to the transmembrane domain, and the transmembrane domain is N-terminal to the cytoplasmic domain.
  • the variable regions of the peptide chain (alpha, beta, gamma, and delta) comprise three hypervariable or complementarity determining regions (CDRs). The three CDRs are embedded into a framework
  • variable region also identifies the hypervariable region.
  • TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature.
  • the Va in IMGT nomenclature is referred to by a unique“TRAV” number.
  • nb is referred to by a unique“TRBV” number.
  • the corresponding joining and constant regions are referred to as TRAJ and TRAC, respectively for the a joining and constant regions, and TRBJ and TRBC, respectively for the b joining and constant regions.
  • Ug is referred to be a unique“TRGV” number and the corresponding joining and constant regions are referred to as TRGJ and TRGC.
  • V5 is referred to be a unique“TRDV” number and the corresponding joining and constant regions are referred to as TRDJ and TRDC.
  • the sequences defined by the IMGT nomenclature are known in the art, and are contained within the online IMGT public database.
  • TCRs T Cell Receptors
  • TCRs modified T cell receptors
  • the heterodimer comprises a first polypeptide and a second polypeptide wherein either the first polypeptide or the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment.
  • TCRs modified T cell receptors
  • the heterodimer comprises a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide are mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor
  • the first polypeptide and the second polypeptide comprise a hyper-variable complementarity determining (CDR) region, a variable domain interface region, and a surface-exposed framework region.
  • CDR region is mutated.
  • variable domain interface region is mutated.
  • surface-exposed framework region is mutated.
  • the CDR region, the variable domain interface region, and the surface-exposed framework region are mutated.
  • the CDR region and the variable domain interface region are mutated.
  • the CDR region and the surface-exposed framework region are mutated.
  • the variable domain interface region and the surface-exposed framework region are mutated.
  • TCRs modified T cell receptors
  • the heterodimer comprises a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide each comprise three hyper variable complementarity determining (CDR) regions, wherein at least one CDR region of either the first polypeptide or the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment.
  • CDR hyper variable complementarity determining
  • the greater affinity of the modified TCRs within tumor microenvironments creates specificity for the modified TCR to engage with the target antigen in the tumor microenvironment, while minimizing engagement with the target antigen outside the tumor microenvironment thus creating an improved safety profile of the modified TCR.
  • the modified TCR is a heterodimer of an alpha polypeptide chain and a beta polypeptide chain (a/b heterodimer).
  • the first polypeptide comprises a TCR alpha extracellular domain, or fragment thereof
  • the second polypeptide comprises a TCR beta extracellular domain, or a fragment thereof.
  • the first polypeptide and the second polypeptide each comprise a transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide each comprise a cytoplasmic domain. In some embodiments, the modified TCR is expressed on a surface of a cell.
  • the TCR alpha extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR alpha extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR alpha extracellular domain is a full length TCR alpha extracellular domain. In some embodiments, the TCR alpha extracellular domain, or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs) within the variable region. In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR alpha extracellular domain, or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR alpha extracellular domain, or fragment thereof comprises a non-natural amino acid or a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the TCR beta extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR beta extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR beta extracellular domain is a full length TCR beta extracellular domain. In some embodiments, the TCR beta extracellular domain or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR beta extracellular domain or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR beta extracellular domain, or fragment thereof comprises a non-natural amino acid, a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the modified TCR contains a hinge region linking the TCR extracellular domain with the transmembrane domain.
  • the transmembrane domain provides for insertion of the modified TCR to be expressed on the surface of a cell.
  • transmembrane sequences include, but are not limited to: a) CD8 beta derived: GLLVAGVLVLLV SLGVAIHLCC (SEQ ID NO: 1); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFF CVRC (SEQ ID NO: 2); c) CD3 zeta derived:
  • ALPAALAVISFLLGLGLGVACVLA SEQ ID NO: 6
  • g native TCR alpha polypeptide chain transmembrane sequences
  • h native TCR beta polypeptide chain transmembrane sequences, or a combination thereof.
  • the polypeptide comprising the TCR alpha extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the cytoplasmic domain comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is 4-1BB or CD28. In some embodiments, the cytoplasmic domain comprises two costimulatory domains. In some embodiments, the cytoplasmic domain comprises more than two costimulatory domains. In some embodiments, the costimulatory domain, includes, but is not limited to C27, CD28, ICOS, 4-1BB, 0X40 or CD3z. In some embodiments, the cytoplasmic domain includes ZAP70. In some embodiments, the cytoplasmic domain includes LAT. In some embodiments, the cytoplasmic domain comprises CD3z, ZAP70, and LAT.
  • isolated or non-naturally occurring cell comprising a modified TCR as disclosed herein.
  • the isolated or non-naturally occurring cell is a T cell.
  • the modified TCR is a soluble TCR.
  • the first polypeptide comprising the TCR alpha extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the first polypeptide comprising the TCR alpha extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the TCR alpha extracellular domain, or fragment thereof and TCR beta extracellular domain, or fragment thereof are mutated to delete the native cysteines which form the native disulfide linkage of the heterodimer.
  • the TCR alpha extracellular domain, or fragment thereof, and the TCR beta extracellular domain, or fragment thereof, are connected by a disulfide bond.
  • the TCR alpha extracellular domain comprises an alpha chain TRAC constant domain sequence and the TCR beta extracellular domain comprises a beta chain TRBC1 or TRBC2 constant domain sequence.
  • Cys4 of the alpha chain TRAC constant domain sequence is modified by truncation or substitution and Cys2 of exon 2 of the beta chain TRBC1 or TRBC2 constant domain sequence is modified by truncation or substitution, thereby deleting a native disulfide bond.
  • Thr48 of the alpha chain TRAC constant domain sequence is mutated to Cys and Ser57 of the beta chain TRBC1 or TRBC2 constant domain sequence is mutated to Cys.
  • the TCR alpha extracellular domain, or fragment thereof further comprises an effector domain.
  • the TCR beta extracellular domain, or fragment thereof further comprises an effector domain.
  • the modified TCR heterodimer comprises an effector domain.
  • the effector domain is an anti-CD3 moiety.
  • the TCR alpha extracellular domain or the TCR beta extracellular domain is bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • the TCR alpha extracellular domain or the TCR beta extracellular domain is bound to an Fc that is also bound to an anti-CD3 scFv.
  • the modified TCR comprises a single polypeptide comprising a variable region of a TCR alpha extracellular domain (Va), or a fragment thereof, and a variable region of a TCR beta extracellular domain (nb), or a fragment thereof, instead of an a/b heterodimer.
  • the single polypeptide further comprises a sequence that connects Va and nb.
  • the single polypeptide comprises a constant region of the TCR alpha extracellular domain (Ca) or a constant region of the TCR beta extracellular domain ( ⁇ b) or a combination thereof.
  • the modified TCRs described herein further comprise modifications in the TCR alpha extracellular domain or the TCR beta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs. In some embodiments, the modified TCRs described herein further comprise modifications in the TCR alpha extracellular domain and the TCR beta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs. In some embodiments, the modifications are in the TCR alpha constant domain or in the TCR beta constant domain. In some embodiments, the modifications are in the TCR alpha constant domain and in the TCR beta constant domain.
  • the modifications comprise interchanging the TCR alpha constant domain and the TCR beta constant domain. In some embodiments, the modifications comprise replacing the TCR alpha constant domain and the TCR beta constant domain with the corresponding domains from TCR gamma and delta.
  • the modified TCR is a heterodimer of a gamma polypeptide chain and a delta polypeptide chain (g/d heterodimer).
  • the first polypeptide comprises a TCR gamma extracellular domain, or fragment thereof
  • the second polypeptide comprises a TCR delta extracellular domain, or a fragment thereof.
  • the first polypeptide and the second polypeptide each comprise a transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide each comprise a cytoplasmic domain. In some embodiments, the modified TCR is expressed on a surface of a cell.
  • the TCR gamma extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR gamma extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR gamma extracellular domain is a full length TCR gamma extracellular domain. In some embodiments, the TCR gamma extracellular domain, or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs) within the variable region. In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR gamma extracellular domain, or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR gamma extracellular domain, or fragment thereof comprises a non-natural amino acid or a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the TCR delta extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR delta extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR delta extracellular domain is a full length TCR delta extracellular domain. In some embodiments, the TCR delta extracellular domain or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR delta extracellular domain or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR delta extracellular domain, or fragment thereof comprises a non-natural amino acid, a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the modified TCR contains a hinge region linking the TCR extracellular domain with the transmembrane domain.
  • the transmembrane domain provides for insertion of the modified TCR to be expressed on the surface of a cell.
  • transmembrane sequences include, but are not limited to: a) CD8 beta derived: GLLVAGVLVLLV SLGVAIHLCC (SEQ ID NO: 1); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFF CVRC (SEQ ID NO: 2); c) CD3 zeta derived:
  • ALPAALAVISFLLGLGLGVACVLA SEQ ID NO: 6
  • g native TCR gamma polypeptide chain transmembrane sequences
  • h native TCR delta polypeptide chain transmembrane sequences, or a combination thereof.
  • the polypeptide comprising the TCR gamma extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the cytoplasmic domain comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is 4-1BB or CD28. In some embodiments, the cytoplasmic domain comprises two costimulatory domains. In some embodiments, the cytoplasmic domain comprises more than two costimulatory domains. In some embodiments, the costimulatory domain, includes, but is not limited to C27, CD28, ICOS, 4-1BB, 0X40 or CD3z. In some embodiments, the cytoplasmic domain includes ZAP70. In some embodiments, the cytoplasmic domain includes LAT. In some embodiments, the cytoplasmic domain comprises CD3z, ZAP70, and LAT.
  • the isolated or non-naturally occurring cell comprising a modified TCR as disclosed herein.
  • the isolated or non-naturally occurring cell is a T cell.
  • the modified TCR is a soluble TCR.
  • the first polypeptide comprising the TCR gamma extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the first polypeptide comprising the TCR gamma extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof comprises a truncated transmembrane domain. In some embodiments, the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof, lacks a transmembrane domain. In some embodiments, the TCR gamma extracellular domain, or fragment thereof and TCR delta extracellular domain, or fragment thereof, are mutated to delete the native cysteines which form the native disulfide linkage of the heterodimer.
  • the TCR gamma extracellular domain, or fragment thereof, and the TCR delta extracellular domain, or fragment thereof, are connected by a disulfide bond.
  • the TCR gamma extracellular domain comprises a gamma chain TRGC constant domain sequence and the TCR delta extracellular domain comprises a delta chain TRDC constant domain sequence.
  • the TCR gamma extracellular domain, or fragment thereof, and the TCR delta extracellular domain, or fragment thereof have a sequence modification (e.g. truncation or substitution), thereby deleting a native disulfide bond.
  • the TCR gamma extracellular domain, or fragment thereof further comprises an effector domain.
  • the TCR delta extracellular domain, or fragment thereof further comprises an effector domain.
  • the modified TCR heterodimer comprises an effector domain.
  • the effector domain is an anti-CD3 moiety.
  • the TCR gamma extracellular domain or the TCR delta extracellular domain is bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • the TCR gamma extracellular domain or the TCR delta extracellular domain is bound to an Fc that is also bound to an anti-CD3 scFv.
  • the modified TCR comprises a single polypeptide comprising a variable region of a TCR gamma extracellular domain (Ug). or a fragment thereof, and a variable region of a TCR delta extracellular domain (V5), or a fragment thereof, instead of an g/d heterodimer.
  • the single polypeptide further comprises a sequence that connects V g and V5.
  • the single polypeptide comprises a constant region of the TCR gamma extracellular domain (C g) or a constant region of the TCR delta extracellular domain (C5) or a combination thereof.
  • the modified TCRs described herein further comprise modifications in the TCR gamma extracellular domain or the TCR delta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs.
  • the modified TCRs described herein further comprise modifications in the TCR gamma extracellular domain and the TCR delta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs.
  • the modifications are in the TCR gamma constant domain or in the TCR delta constant domain.
  • the modifications are in the TCR gamma constant domain and in the TCR delta constant domain.
  • the modifications comprise interchanging the TCR gamma constant domain and the TCR delta constant domain. In some embodiments, the modifications comprise replacing the TCR gamma constant domain and the TCR delta constant domain with the corresponding domains from TCR alpha and beta.
  • Cancer cells in a solid tumor are able to form a tumor microenvironment in their surroundings to support the growth and metastasis of the cancer cells.
  • a tumor microenvironment is often hypoxic.
  • the tumor mass increases, the interior of the tumor grows farther away from existing blood supply, which leads to difficulties in fully supplying oxygen to the tumor microenvironment.
  • the tumor cells tend to rely on energy generated from lactic acid fermentation, which does not require oxygen.
  • lactic acid fermentation is that the tumor microenvironment is acidic in contrast to other parts of the body which are typically either neutral or slightly basic.
  • human blood plasma has a pH of about 7.4.
  • physiological pH is about pH 7.4.
  • acidic pH is about pH 5.8 to about pH 6.8.
  • conditions that exist in a tumor microenvironment include conditions that exist therein compared to a non-tumor microenvironment (e.g. a healthy or non -diseased cell or tissue). Conditions that exist in a tumor microenvironment include increased vascularization, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure and altered metabolites or metabolism indicative of a tumor.
  • the pH of the tumor microenvironment is more acidic from the normal microenvironment (i.e. non-tumor environment), such as any pH from about 5.6 to 6.8.
  • the tumor microenvironment comprises a pH that is about 5.8 to about 6.8.
  • the pH of the tumor microenvironment is about 5.6.
  • the pH of the tumor microenvironment is about 5.7.
  • the pH of the tumor microenvironment is about 5.8.
  • the pH of the tumor microenvironment is about 5.9.
  • the pH of the tumor microenvironment is about 6.0.
  • the pH of the tumor microenvironment is about 6.1.
  • the pH of the tumor microenvironment is about 6.2. In some embodiments, the pH of the tumor microenvironment is about 6.3. In some embodiments, the pH of the tumor microenvironment is about 6.4. In some embodiments, the pH of the tumor microenvironment is about 6.5. In some embodiments, the pH of the tumor microenvironment is about 6.6. In some embodiments, the pH of the tumor microenvironment is about 6.7. In some embodiments, the pH of the tumor microenvironment is about 6.8. In some embodiments, the pH of the non-tumor microenvironment is more basic than the tumor microenvironment. The pH of the non-tumor microenvironment is any pH that exists under physiologic conditions, such as any pH from about 7.0 to about 7.8.
  • the non-tumor environment comprises a pH that is about 7.2 to about 7.8. In some embodiments, the pH of the non-tumor environment is about 7.2. In some embodiments, the pH of the non-tumor environment is about 7.3. In some embodiments, the pH of the non-tumor environment is about 7.4. In some embodiments, the pH of the non-tumor environment is about 7.5. In some embodiments, the pH of the non-tumor environment is about 7.6. In some embodiments, the pH of the non-tumor environment is about 7.7. In some embodiments, the pH of the non-tumor environment is about 7.8.
  • the condition that differs between a non-tumor environment and a diseased environment, such as a tumor microenvironment comprises the concentration of lactate.
  • the tumor microenvironment comprises an elevated lactic acid concentration relative to the non-tumor environment.
  • the tumor microenvironment comprises an elevated lactic acid concentration of about 5 mM to about 20 mM lactic acid.
  • the tumor microenvironment comprises an elevated lactic acid concentration of about 10 mM to about 20 mM lactic acid.
  • the tumor microenvironment comprises an elevated lactic acid concentration of about 15 mM to about 18 mM lactic acid.
  • the tumor microenvironment comprises an elevated lactic acid concentration of at least about 16 mM lactic acid. In some embodiments, the tumor microenvironment comprises an elevated lactic acid concentration of at least about 16.5 mM lactic acid. In some embodiments, the tumor microenvironment comprises an elevated lactic acid concentration of at least about 17 mM lactic acid. In some embodiments, the non-tumor environment comprises a lactate concentration of about 0.5 mM to about 5 mM lactic acid. In some embodiments, the non-tumor environment comprises a lactate concentration of about 0.2 mM to about 4 mM lactic acid. In some embodiments, the non-tumor environment comprises a lactate concentration of about 0.5 mM lactic acid.
  • the non-tumor environment comprises a lactate concentration of about 1.0 mM lactic acid. In some embodiments, the non-tumor environment comprises a lactate concentration of about 2.0 mM lactic acid. In some embodiments, the non-tumor environment comprises a lactate concentration of about 3.0 mM lactic acid. In some embodiments, the non-tumor environment comprises a lactate concentration of about 4.0 mM lactic acid. In some embodiments, the non-tumor environment comprises a lactate concentration of about 5.0 mM lactic acid.
  • the tumor microenvironment comprises an elevated pyruvate concentration relative to the non-tumor environment.
  • the tumor microenvironment is hypoxic.
  • hypoxic conditions include any conditions in which oxygen concentration is less than the equilibrium
  • the non-tumor environment comprises an oxygen concentration of about 40 mM to about400 pM. In some embodiments, the non-tumor environment comprises an oxygen concentration of about 40 pM to 200 pM. In some embodiments, the non-tumor environment comprises an oxygen concentration of about 40 pM to 140 pM.
  • the target antigen includes, but is not limited to MAGE-A3, NY-ESO-l, gplOO, WT1, and tyrosinase.
  • the target antigen is MAGE -A3.
  • the target antigen is NY-ESO-l.
  • the target antigen is gplOO.
  • the target antigen is WT1.
  • the target antigen is tyrosinase.
  • TCRs modified T cell receptors
  • TCRs modified T cell receptors
  • the heterodimer comprises a first polypeptide and a second polypeptide wherein either the first polypeptide or the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment.
  • TCRs modified T cell receptors
  • the heterodimer comprises a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide are mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment.
  • the first polypeptide and the second polypeptide comprise a hyper-variable complementarity determining (CDR) region, a variable domain interface region, and a surface-exposed framework region.
  • the CDR region is mutated.
  • the variable domain interface region is mutated.
  • the surface-exposed framework region is mutated.
  • the CDR region, the variable domain interface region, and the surface-exposed framework region are mutated. In some embodiments, the CDR region and the variable domain interface region are mutated. In some embodiments, the CDR region and the surface-exposed framework region are mutated. In some embodiments, the variable domain interface region and the surface-exposed framework region are mutated.
  • TCRs modified T cell receptors
  • the heterodimer comprises a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide each comprise three hyper-variable complementarity determining (CDR) regions, wherein at least one CDR region of either the first polypeptide or the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment.
  • CDR hyper-variable complementarity determining
  • the modified TCR is a heterodimer of an alpha polypeptide chain and a beta polypeptide chain (a/b heterodimer).
  • the first polypeptide comprises a TCR alpha extracellular domain, or fragment thereof
  • the second polypeptide comprises a TCR beta extracellular domain, or a fragment thereof.
  • the first polypeptide and the second polypeptide each comprise a transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide each comprise a cytoplasmic domain. In some embodiments, the modified TCR is expressed on a surface of a cell. [0072] In some embodiments, the TCR alpha extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR alpha extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR alpha extracellular domain is a full length TCR alpha extracellular domain.
  • the TCR alpha extracellular domain, or fragment thereof comprises three hyper-variable complementarity determining regions (CDRs) within the variable region.
  • CDRs hyper-variable complementarity determining regions
  • at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen.
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR alpha extracellular domain, or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR alpha extracellular domain, or fragment thereof comprises a non-natural amino acid or a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the TCR beta extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR beta extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR beta extracellular domain is a full length TCR beta extracellular domain. In some embodiments, the TCR beta extracellular domain or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR beta extracellular domain or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR beta extracellular domain, or fragment thereof comprises a non-natural amino acid, a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the modified TCR contains a hinge region linking the TCR extracellular domain with the transmembrane domain.
  • the transmembrane domain provides for insertion of the modified TCR to be expressed on the surface of a cell.
  • transmembrane sequences include, but are not limited to: a) CD8 beta derived: GLLVAGVLVLLV SLGVAIHLCC (SEQ ID NO: 1); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFF CVRC (SEQ ID NO: 2); c) CD3 zeta derived:
  • ALPAALAVISFLLGLGLGVACVLA SEQ ID NO: 6
  • g native TCR alpha polypeptide chain transmembrane sequences
  • h native TCR beta polypeptide chain transmembrane sequences, or a combination thereof.
  • the polypeptide comprising the TCR alpha extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the cytoplasmic domain comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is 4-1BB or CD28. In some embodiments, the cytoplasmic domain comprises two costimulatory domains. In some embodiments, the cytoplasmic domain comprises more than two costimulatory domains. In some embodiments, the costimulatory domain, includes, but is not limited to C27, CD28, ICOS, 4-1BB, 0X40 or CD3z. In some embodiments, the cytoplasmic domain includes ZAP70. In some embodiments, the cytoplasmic domain includes LAT. In some embodiments, the cytoplasmic domain comprises CD3z, ZAP70, and LAT.
  • the modified TCR is a soluble TCR.
  • the first polypeptide comprising the TCR alpha extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the first polypeptide comprising the TCR alpha extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the TCR alpha extracellular domain, or fragment thereof and TCR beta extracellular domain, or fragment thereof are mutated to delete the native cysteines which form the native disulfide linkage of the heterodimer.
  • the TCR alpha extracellular domain, or fragment thereof, and the TCR beta extracellular domain, or fragment thereof, are connected by a disulfide bond.
  • the TCR alpha extracellular domain comprises an alpha chain TRAC constant domain sequence and the TCR beta extracellular domain comprises a beta chain TRBC1 or TRBC2 constant domain sequence.
  • Cys4 of the alpha chain TRAC constant domain sequence is modified by truncation or substitution and Cys2 of exon 2 of the beta chain TRBC1 or TRBC2 constant domain sequence is modified by truncation or substitution, thereby deleting a native disulfide bond.
  • Thr48 of the alpha chain TRAC constant domain sequence is mutated to Cys and Ser57 of the beta chain TRBC1 or TRBC2 constant domain sequence is mutated to Cys.
  • the TCR alpha extracellular domain, or fragment thereof further comprises an effector domain.
  • the TCR beta extracellular domain, or fragment thereof further comprises an effector domain.
  • the modified TCR heterodimer comprises an effector domain.
  • the effector domain is an anti-CD3 moiety.
  • the TCR alpha extracellular domain or the TCR beta extracellular domain is bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • the TCR alpha extracellular domain or the TCR beta extracellular domain is bound to an Fc that is also bound to an anti-CD3 scFv.
  • the modified TCR comprises a single polypeptide comprising a variable region of a TCR alpha extracellular domain (Va), or a fragment thereof, and a variable region of a TCR beta extracellular domain (nb), or a fragment thereof, instead of an a/b heterodimer.
  • the single polypeptide further comprises a sequence that connects Va and nb.
  • the single polypeptide comprises a constant region of the TCR alpha extracellular domain (Ca) or a constant region of the TCR beta extracellular domain ( ⁇ b) or a combination thereof.
  • the modified TCRs described herein further comprise modifications in the TCR alpha extracellular domain or the TCR beta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs. In some embodiments, the modified TCRs described herein further comprise modifications in the TCR alpha extracellular domain and the TCR beta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs. In some embodiments, the modifications are in the TCR alpha constant domain or in the TCR beta constant domain. In some embodiments, the modifications are in the TCR alpha constant domain and in the TCR beta constant domain.
  • the modifications comprise interchanging the TCR alpha constant domain and the TCR beta constant domain. In some embodiments, the modifications comprise replacing the TCR alpha constant domain and the TCR beta constant domain with the corresponding domains from TCR gamma and delta.
  • the modified TCR is a heterodimer of a gamma polypeptide chain and a delta polypeptide chain (g/d heterodimer).
  • the first polypeptide comprises a TCR gamma extracellular domain, or fragment thereof
  • the second polypeptide comprises a TCR delta extracellular domain, or a fragment thereof.
  • the first polypeptide and the second polypeptide each comprise a transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide each comprise a cytoplasmic domain. In some embodiments, the modified TCR is expressed on a surface of a cell. [0090] In some embodiments, the TCR gamma extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR gamma extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR gamma extracellular domain is a full length TCR gamma extracellular domain.
  • the TCR gamma extracellular domain, or fragment thereof comprises three hyper-variable complementarity determining regions (CDRs) within the variable region.
  • CDRs hyper-variable complementarity determining regions
  • at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen.
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR gamma extracellular domain, or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR gamma extracellular domain, or fragment thereof comprises a non-natural amino acid or a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the TCR delta extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR delta extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR delta extracellular domain is a full length TCR delta extracellular domain. In some embodiments, the TCR delta extracellular domain or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR delta extracellular domain or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR delta extracellular domain, or fragment thereof comprises a non-natural amino acid, a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the modified TCR contains a hinge region linking the TCR extracellular domain with the transmembrane domain.
  • the transmembrane domain provides for insertion of the modified TCR to be expressed on the surface of a cell.
  • transmembrane sequences include, but are not limited to: a) CD8 beta derived: GLLVAGVLVLLV SLGVAIHLCC (SEQ ID NO: 1); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFF CVRC (SEQ ID NO: 2); c) CD3 zeta derived:
  • ALPAALAVISFLLGLGLGVACVLA SEQ ID NO: 6
  • g native TCR gamma polypeptide chain transmembrane sequences
  • h native TCR delta polypeptide chain transmembrane sequences, or a combination thereof.
  • the polypeptide comprising the TCR gamma extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the cytoplasmic domain comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is 4-1BB or CD28. In some embodiments, the cytoplasmic domain comprises two costimulatory domains. In some embodiments, the cytoplasmic domain comprises more than two costimulatory domains. In some embodiments, the costimulatory domain, includes, but is not limited to C27, CD28, ICOS, 4-1BB, 0X40 or CD3z. In some embodiments, the cytoplasmic domain includes ZAP70. In some embodiments, the cytoplasmic domain includes LAT. In some embodiments, the cytoplasmic domain comprises CD3z, ZAP70, and LAT.
  • the modified TCR is a soluble TCR.
  • the first polypeptide comprising the TCR gamma extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the first polypeptide comprising the TCR gamma extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the TCR gamma extracellular domain, or fragment thereof and TCR delta extracellular domain, or fragment thereof are mutated to delete the native cysteines which form the native disulfide linkage of the heterodimer.
  • the TCR gamma extracellular domain, or fragment thereof, and the TCR delta extracellular domain, or fragment thereof, are connected by a disulfide bond.
  • the TCR gamma extracellular domain comprises a gamma chain TRGC constant domain sequence and the TCR delta extracellular domain comprises a delta chain TRDC constant domain sequence.
  • the TCR gamma extracellular domain, or fragment thereof, and the TCR delta extracellular domain, or fragment thereof have a sequence modification (e.g. truncation or substitution), thereby deleting a native disulfide bond.
  • the TCR gamma extracellular domain, or fragment thereof further comprises an effector domain.
  • the TCR delta extracellular domain, or fragment thereof further comprises an effector domain.
  • the modified TCR heterodimer comprises an effector domain.
  • the effector domain is an anti-CD3 moiety.
  • the TCR gamma extracellular domain or the TCR delta extracellular domain is bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • the TCR gamma extracellular domain or the TCR delta extracellular domain is bound to an Fc that is also bound to an anti-CD3 scFv.
  • the modified TCR comprises a single polypeptide comprising a variable region of a TCR gamma extracellular domain (Ug). or a fragment thereof, and a variable region of a TCR delta extracellular domain (V5), or a fragment thereof, instead of an g/d heterodimer.
  • the single polypeptide further comprises a sequence that connects V g and V5.
  • the single polypeptide comprises a constant region of the TCR gamma extracellular domain (C g) or a constant region of the TCR delta extracellular domain (C5) or a combination thereof.
  • the modified TCRs described herein further comprise modifications in the TCR gamma extracellular domain or the TCR delta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs.
  • the modified TCRs described herein further comprise modifications in the TCR gamma extracellular domain and the TCR delta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs.
  • the modifications are in the TCR gamma constant domain or in the TCR delta constant domain.
  • the modifications are in the TCR gamma constant domain and in the TCR delta constant domain.
  • the modifications comprise interchanging the TCR gamma constant domain and the TCR delta constant domain. In some embodiments, the modifications comprise replacing the TCR gamma constant domain and the TCR delta constant domain with the corresponding domains from TCR alpha and beta.
  • the isolated recombinant nucleic acid molecules encoding modified T cell receptors are provided as a DNA construct. In other embodiments, the isolated recombinant nucleic acid molecules encoding modified T cell receptors (TCRs) are provided as a messenger RNA transcript.
  • the polynucleotide molecules are constructed by known methods such as by combining the genes encoding the domains either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells.
  • a suitable promoter operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • the promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.
  • the nucleic acid molecule encoding a modified TCR disclosed herein is inserted into a vector, preferably an expression vector, which represents a further embodiment.
  • This recombinant vector can be constructed according to known methods.
  • Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.
  • a variety of expression vector/host systems may be utilized to contain and express the polynucleotide encoding the modified TCRs.
  • Examples of expression vectors for expression in E.coli are pSKK (Le Gall et ah, J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.
  • the modified TCRs as described herein are produced by introducing a vector encoding the polypeptides described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified.
  • compositions comprising: (a) modified T cell receptors (TCRs) as disclosed herein; and (b) a pharmaceutically acceptable carrier or excipient.
  • TCRs modified T cell receptors
  • a pharmaceutical composition disclosed herein comprises (a) a modified T cell receptors (TCR) comprising a heterodimer that binds to a target antigen, wherein the heterodimer comprises a first polypeptide and a second polypeptide wherein either the first polypeptide or the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment; and (b) a pharmaceutically acceptable carrier or excipient.
  • TCR modified T cell receptors
  • a pharmaceutical composition disclosed herein comprises (a) a modified T cell receptors (TCR) comprising a heterodimer that binds to a target antigen, wherein the heterodimer comprises a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment; and (b) a pharmaceutically acceptable carrier or excipient.
  • the first polypeptide and the second polypeptide comprise a hyper-variable complementarity determining (CDR) region, a variable domain interface region, and a surface-exposed framework region.
  • CDR hyper-variable complementarity determining
  • the CDR region is mutated. In some embodiments, the variable domain interface region is mutated. In some embodiments, the surface- exposed framework region is mutated. In some embodiments, the CDR region, the variable domain interface region, and the surface-exposed framework region are mutated. In some embodiments, the CDR region and the variable domain interface region are mutated. In some embodiments, the CDR region and the surface-exposed framework region are mutated. In some embodiments, the variable domain interface region and the surface-exposed framework region are mutated.
  • a pharmaceutical composition disclosed herein comprises (a) a modified T cell receptors (TCR) comprising a heterodimer that binds to a target antigen, wherein the heterodimer comprises a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide each comprise three hyper-variable complementarity determining (CDR) regions, wherein at least one CDR region of either the first polypeptide or the second polypeptide is mutated such that the heterodimer binds with greater affinity to the target antigen in a tumor microenvironment compared to a non-tumor environment; and (b) a pharmaceutically acceptable carrier or excipient.
  • TCR modified T cell receptors
  • the modified TCR is a heterodimer of an alpha polypeptide chain and a beta polypeptide chain (a/b heterodimer).
  • the first polypeptide comprises a TCR alpha extracellular domain, or fragment thereof
  • the second polypeptide comprises a TCR beta extracellular domain, or a fragment thereof.
  • the first polypeptide and the second polypeptide each comprise a transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide each comprise a cytoplasmic domain. In some embodiments, the modified TCR is expressed on a surface of a cell.
  • the TCR alpha extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR alpha extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR alpha extracellular domain is a full length TCR alpha extracellular domain. In some embodiments, the TCR alpha extracellular domain, or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs) within the variable region. In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR alpha extracellular domain, or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR alpha extracellular domain, or fragment thereof comprises a non-natural amino acid or a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the TCR beta extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR beta extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR beta extracellular domain is a full length TCR beta extracellular domain. In some embodiments, the TCR beta extracellular domain or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR beta extracellular domain or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR beta extracellular domain, or fragment thereof comprises a non-natural amino acid, a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the modified TCR contains a hinge region linking the TCR extracellular domain with the transmembrane domain.
  • the transmembrane domain provides for insertion of the modified TCR to be expressed on the surface of a cell.
  • transmembrane sequences include, but are not limited to: a) CD8 beta derived: GLLVAGVLVLLV SLGVAIHLCC (SEQ ID NO: 1); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFF CVRC (SEQ ID NO: 2); c) CD3 zeta derived:
  • ALPAALAVISFLLGLGLGVACVLA SEQ ID NO: 6
  • g native TCR alpha polypeptide chain transmembrane sequences
  • h native TCR beta polypeptide chain transmembrane sequences, or a combination thereof.
  • the polypeptide comprising the TCR alpha extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the cytoplasmic domain comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is 4-1BB or CD28. In some embodiments, the cytoplasmic domain comprises two costimulatory domains. In some embodiments, the cytoplasmic domain comprises more than two costimulatory domains. In some embodiments, the costimulatory domain, includes, but is not limited to C27, CD28, ICOS, 4-1BB, 0X40 or CD3z. In some embodiments, the cytoplasmic domain includes ZAP70. In some embodiments, the cytoplasmic domain includes LAT. In some embodiments, the cytoplasmic domain comprises CD3z, ZAP70, and LAT.
  • the modified TCR is a soluble TCR.
  • the first polypeptide comprising the TCR alpha extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the first polypeptide comprising the TCR alpha extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the second polypeptide comprising the TCR beta extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the TCR alpha extracellular domain, or fragment thereof and TCR beta extracellular domain, or fragment thereof are mutated to delete the native cysteines which form the native disulfide linkage of the heterodimer.
  • the TCR alpha extracellular domain, or fragment thereof, and the TCR beta extracellular domain, or fragment thereof, are connected by a disulfide bond.
  • the TCR alpha extracellular domain comprises an alpha chain TRAC constant domain sequence and the TCR beta extracellular domain comprises a beta chain TRBC1 or TRBC2 constant domain sequence.
  • Cys4 of the alpha chain TRAC constant domain sequence is modified by truncation or substitution and Cys2 of exon 2 of the beta chain TRBC1 or TRBC2 constant domain sequence is modified by truncation or substitution, thereby deleting a native disulfide bond.
  • Thr48 of the alpha chain TRAC constant domain sequence is mutated to Cys and Ser57 of the beta chain TRBC1 or TRBC2 constant domain sequence is mutated to Cys.
  • the TCR alpha extracellular domain, or fragment thereof further comprises an effector domain.
  • the TCR beta extracellular domain, or fragment thereof further comprises an effector domain.
  • the modified TCR heterodimer comprises an effector domain.
  • the effector domain is an anti-CD3 moiety.
  • the TCR alpha extracellular domain or the TCR beta extracellular domain is bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • the TCR alpha extracellular domain or the TCR beta extracellular domain is bound to an Fc that is also bound to an anti-CD3 scFv.
  • the modified TCR comprises a single polypeptide comprising a variable region of a TCR alpha extracellular domain (Va), or a fragment thereof, and a variable region of a TCR beta extracellular domain (nb), or a fragment thereof, instead of an a/b heterodimer.
  • the single polypeptide further comprises a sequence that connects Va and nb.
  • the single polypeptide comprises a constant region of the TCR alpha extracellular domain (Ca) or a constant region of the TCR beta extracellular domain ( ⁇ b) or a combination thereof.
  • the modified TCRs described herein further comprise modifications in the TCR alpha extracellular domain or the TCR beta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs. In some embodiments, the modified TCRs described herein further comprise modifications in the TCR alpha extracellular domain and the TCR beta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs. In some embodiments, the modifications are in the TCR alpha constant domain or in the TCR beta constant domain. In some embodiments, the modifications are in the TCR alpha constant domain and in the TCR beta constant domain.
  • the modifications comprise interchanging the TCR alpha constant domain and the TCR beta constant domain. In some embodiments, the modifications comprise replacing the TCR alpha constant domain and the TCR beta constant domain with the corresponding domains from TCR gamma and delta.
  • the modified TCR is a heterodimer of a gamma polypeptide chain and a delta polypeptide chain (g/d heterodimer).
  • the first polypeptide comprises a TCR gamma extracellular domain, or fragment thereof
  • the second polypeptide comprises a TCR delta extracellular domain, or a fragment thereof.
  • the first polypeptide and the second polypeptide each comprise a transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide each comprise a cytoplasmic domain. In some embodiments, the modified TCR is expressed on a surface of a cell.
  • the TCR gamma extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR gamma extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR gamma extracellular domain is a full length TCR gamma extracellular domain. In some embodiments, the TCR gamma extracellular domain, or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs) within the variable region. In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR gamma extracellular domain, or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR gamma extracellular domain, or fragment thereof comprises a non-natural amino acid or a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the TCR delta extracellular domain, or fragment thereof comprises a variable region. In some embodiments, the TCR delta extracellular domain, or fragment thereof comprises a variable region, a joining region, and a constant region. In some embodiments, the TCR delta extracellular domain is a full length TCR delta extracellular domain. In some embodiments, the TCR delta extracellular domain or fragment thereof, comprises three hyper-variable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the target antigen or to increase binding affinity and binding specificity to the target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutations in one or two CDRs.
  • CDRs hyper-variable complementarity determining regions
  • the at least one CDR region is mutated to a histidine.
  • the histidine binds to glutamic acid, aspartic acid, or tryptophan, or a combination thereof, of the target antigen.
  • the heterodimer binds to the target antigen through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, cation-Pi interactions, H-bonding interactions, or a combination thereof.
  • the TCR delta extracellular domain or fragment thereof comprises a modified amino acid.
  • the modified amino acid comprises a post-translational modification.
  • the TCR delta extracellular domain, or fragment thereof comprises a non-natural amino acid, a modified non-natural amino acid, or combination thereof.
  • the modified non-natural amino acid comprises a post-translational modification.
  • the modified TCR contains a hinge region linking the TCR extracellular domain with the transmembrane domain.
  • the transmembrane domain provides for insertion of the modified TCR to be expressed on the surface of a cell.
  • transmembrane sequences include, but are not limited to: a) CD8 beta derived: GLLVAGVLVLLV SLGVAIHLCC (SEQ ID NO: 1); b) CD4 derived: ALIVLGGV AGLLLFIGLGIFF C VRC (SEQ ID NO: 2); c) CD3 zeta derived:
  • ALPAALAVISFLLGLGLGVACVLA SEQ ID NO: 6
  • g native TCR gamma polypeptide chain transmembrane sequences
  • h native TCR delta polypeptide chain transmembrane sequences, or a combination thereof.
  • the polypeptide comprising the TCR gamma extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof further comprises a cytoplasmic domain C-terminal to the transmembrane domain.
  • the cytoplasmic domain comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is 4-1BB or CD28. In some embodiments, the cytoplasmic domain comprises two costimulatory domains. In some embodiments, the cytoplasmic domain comprises more than two costimulatory domains. In some embodiments, the costimulatory domain, includes, but is not limited to C27, CD28, ICOS, 4-1BB, 0X40 or CD3z. In some embodiments, the cytoplasmic domain includes ZAP70. In some embodiments, the cytoplasmic domain includes LAT. In some embodiments, the cytoplasmic domain comprises CD3z, ZAP70, and LAT.
  • the modified TCR is a soluble TCR.
  • the first polypeptide comprising the TCR gamma extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the first polypeptide comprising the TCR gamma extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof comprises a truncated transmembrane domain.
  • the second polypeptide comprising the TCR delta extracellular domain, or fragment thereof lacks a transmembrane domain.
  • the TCR gamma extracellular domain, or fragment thereof and TCR delta extracellular domain, or fragment thereof are mutated to delete the native cysteines which form the native disulfide linkage of the heterodimer.
  • the TCR gamma extracellular domain, or fragment thereof, and the TCR delta extracellular domain, or fragment thereof, are connected by a disulfide bond.
  • the TCR gamma extracellular domain comprises a gamma chain TRGC constant domain sequence and the TCR delta extracellular domain comprises a delta chain TRDC constant domain sequence.
  • the TCR gamma extracellular domain, or fragment thereof, and the TCR delta extracellular domain, or fragment thereof have a sequence modification (e.g. truncation or substitution), thereby deleting a native disulfide bond.
  • the TCR gamma extracellular domain, or fragment thereof further comprises an effector domain.
  • the TCR delta extracellular domain, or fragment thereof further comprises an effector domain.
  • the modified TCR heterodimer comprises an effector domain.
  • the effector domain is an anti-CD3 moiety.
  • the TCR gamma extracellular domain or the TCR delta extracellular domain is bound to an anti-CD3 single-chain variable fragment (scFv) effector.
  • the TCR gamma extracellular domain or the TCR delta extracellular domain is bound to an Fc that is also bound to an anti-CD3 scFv.
  • the modified TCR comprises a single polypeptide comprising a variable region of a TCR gamma extracellular domain (Ug). or a fragment thereof, and a variable region of a TCR delta extracellular domain (V5), or a fragment thereof, instead of an g/d heterodimer.
  • the single polypeptide further comprises a sequence that connects V g and V5.
  • the single polypeptide comprises a constant region of the TCR gamma extracellular domain (C g) or a constant region of the TCR delta extracellular domain (C5) or a combination thereof.
  • the modified TCRs described herein further comprise modifications in the TCR gamma extracellular domain or the TCR delta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs.
  • the modified TCRs described herein further comprise modifications in the TCR gamma extracellular domain and the TCR delta extracellular domain, wherein the modifications inhibit mispairing of the modified TCRs with the endogenous TCRs.
  • the modifications are in the TCR gamma constant domain or in the TCR delta constant domain.
  • the modifications are in the TCR gamma constant domain and in the TCR delta constant domain.
  • the modifications comprise interchanging the TCR gamma constant domain and the TCR delta constant domain. In some embodiments, the modifications comprise replacing the TCR gamma constant domain and the TCR delta constant domain with the corresponding domains from TCR alpha and beta.
  • compositions comprising: (a)
  • TCRs modified T cell receptors
  • the isolated or non- naturally occurring cells are T cells.
  • compositions comprising: (a)
  • TCRs modified T cell receptors
  • the TCRs as described herein may be provided in a pharmaceutical composition together with one or more
  • pharmaceutically acceptable carriers or excipients.
  • pharmaceutically acceptable carrier any pharmaceutically acceptable carrier or excipients.
  • compositions include, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose.
  • the compositions are sterile.
  • These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.
  • Soluble TCRs, or cells, in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier.
  • composition may be in any suitable form, (depending upon the desired method of administration). It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include instructions for use. It may include a plurality of said unit dosage forms.
  • the pharmaceutical composition may be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route.
  • a parenteral route e.g., subcutaneous, intramuscular, or intravenous
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.
  • the modified TCRs described herein are introduced into a cytotoxic cell.
  • the cytotoxic cell is a T cell.
  • the T cell is a naive T cell, a
  • central memory cell or an effector memory T cell.
  • a source of T-cells is obtained from a subject.
  • the term“subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals).
  • T-cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T-cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T-cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis are washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg- free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • buffers such as, for example, Ca-free, Mg- free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T-cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T-cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T-cells, can be further isolated by positive or negative selection techniques.
  • Enrichment of a T-cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • a monoclonal antibody cocktail may include antibodies to CD 14, CD20, CD1 lb, CD16, HLA-DR, and CD8.
  • a T-cell population can be selected that expresses one or more of IFN-g, TNFa, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines.
  • T-cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • cryopreserved cells are thawed and washed and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
  • a blood sample or an apheresis product is taken from a generally healthy subject.
  • a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T-cells may be expanded, frozen, and used at a later time.
  • the T-cells of the disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a
  • T-cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen binding fragment thereof, or an anti- CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T-cells.
  • a population of T-cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T-cells.
  • an anti-CD3 antibody and an anti- CD28 antibody is used as described herein, such as by contact with an anti-CD3 antibody, or antigen binding fragment thereof, or an anti- CD2 antibody immobilized on a surface, or by contact with a protein
  • the primary stimulatory signal and the costimulatory signal for the T-cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in“cis” formation) or to separate surfaces (i.e., in“trans” formation).
  • one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.
  • the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • a surface such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • the two agents are immobilized on beads, either on the same bead, i.e.,“cis,” or to separate beads, i.e.,“trans.”
  • the cells such as T-cells
  • the beads and the cells are subsequently separated, and then the cells are cultured.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.
  • a library of candidate TCRs is generated and screened for desirable activity in a tumor micro environment (for e.g. binding an antigen at pH of about 5.8 to 6.8 but not at pH of about 7.2 to about 7.8, or binding an antigen in a microenvironment with elevated lactic acid concentration, elevated pyruvate concentration or in a hypoxic microenvironment) with each candidate having a mutation or a combination of mutations in one of the CDRs.
  • Those candidate TCRs that bind to the antigen in a normal physiological environment for e.g. non-tumor environment
  • Candidate TCRs that bind to the antigen in a tumor micro-environment but not in a non-tumor environment are sequenced and motifs are analyzed.
  • the libraries are introduced via expression vectors resulting in display of the modified TCR on the surface of phage, bacterial cells, yeast cells, or mammalian cells.
  • cells displaying the modified TCR are tested for their abilities to bind to target antigens at pH range of about 5.8 to about 6.8 and about 7.2 to about 7.8.
  • Cells are contacted with fluorescently labeled target antigen and the cells are sorted by FACs to isolate those cells which can bind to the fluorescently labeled target antigen of about 5.8 to about 6.8, but not at about 7.2 to about 7.8.
  • the cells can be subjected to additional cycles by expansion by growth in culture and again by subjecting the culture to all or part of the screening steps.
  • Additional conditionals also tested for the screening of modified TCR libraries include, but are not limited to: lactic acid concentration (higher in a tumor microenvironment), pyruvate concentration (higher in a tumor microenvironment), and a hypoxic microenvironment, such as in a tumor.
  • TDA immunoabsorbant target displacement assay
  • the target antigen is adsorbed to the wells of an ELISA plate overnight at 4° C.
  • the plate is blocked by addition of 2% non-fat dry milk in PBS, about 0.5% (v/v) Tween20 (PBST), and incubation at room temperature for about 1 hour.
  • PBST 0.5% Tween20
  • the plate is then washed about three times with PBST.
  • About 50 m ⁇ of superblock is added.
  • About 50 m ⁇ of the modified TCR is dissolved in superblock and incubated at about 37 ° C for different periods of time.
  • the plate is washed about three times with PBST.
  • About 100 ml of anti-huTCR-HRP is added in about 2% NEM/PBST and incubated at room temperature for about 1 hour.
  • the plate is washed about four times with PBST and about twice with PBS.
  • the assay is developed using TMB (Thermo Scientific) as per manufacturer’s instructions.
  • Modified TCRs are selected which demonstrate binding to its target antigen at pH of about 5.8 to about 6.8, but with minimal or no binding to its target antigen at pH of about 7.2 to about 7.8 conditions.
  • a TCR, and a modified candidate TCR as validated in Example 3, is tested in a cell based cytotoxicity assay.
  • A549 human epithelial cell line derived from a lung carcinoma tissue
  • an alternative cancer cell line (DU145, LNCaP, or PC-3 cells) is obtained from, for example, the ATCC.
  • Human umbilical vein endothelial cells (HUVEC) are isolated from human umbilical veins as described. (Grant et al.,“Matrigel induces thymosin h 4 gene in differentiating endothelial cells”, J Cell Sci 1995; 108:3685-94).
  • HUVEC cells are used as a positive control as a cell line that express ATP synthase on the cell surface.
  • DMEM fetal calf serum
  • serum replacement medium 3 serum replacement medium 3
  • Low-pH (6.7) medium is prepared by reducing bicarbonate to 10 mmol/L at 5% C0 2 and supplementing with 34 mmol/L NaCl to maintain osmolality or incubation of 22 mmol/L bicarbonate medium under 17% C0 2 conditions. The method of lowering pH used is varied by experimental constraints and assay.
  • Propidium iodide (BD Biosciences, San Jose, Calif.) is included with all samples to discriminate cells with compromised membranes.
  • the mean fluorescent intensity of LITC in 10,000 cells is quantified by LACSCalibur flow cytometer (Becton Dickinson, Lranklin Lakes, N.J.) and cells with propidium iodide uptake are excluded to eliminate detection of mitochondrial ATP synthase on CELLQuest software (BD Biosciences).
  • Cell surface ATP generation assay A549 or l-LN cells (60,000 per well) in 96-well plates are refreshed with medium and treated with a TCR, a modified candidate TCR, anti-beta-subunit antibody, rabbit IgG raised to bovine serum albumin (Organon Teknika, West Chester, Pa.), piceatannol (a known inhibitor of ATP synthase Ll used as a positive control, Sigma), or medium alone for 30 minutes at 37 ° C., 5% C0 2 . Cells are then incubated with 0.05 mmol/L ADP for 20 seconds.
  • Cell proliferation assay The effect of the candidate modified TCR on cancer cell lines is assessed with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) proliferation assay in serum-free medium. Relative cell numbers in each well of a 96-well microplate after incubation for 20 hours, 37 °C., and 5% C0 2 in the presence or absence of the candidate TCR is determined using the AQueous One Cell Proliferation Assay (Promega) per protocol of the
  • Cancer cells e.g. A549 cells(5,000 per well) treated with a candidate TCR, a modified candidate TCR, anti-beta-subunit antibody, rabbit IgG, cariporide, and Triton X (a detergent used to permeabilize cells as a positive control) are incubated at 37 ° C. and 5% C0 2 or
  • TCR, anti-beta-subunit antibody, rabbit IgG, and cariporide on A549 cells is determined using an ELISA apoptosis and necrosis assay (Roche) that is dependent on detection of extranuclear histone-DNA fragments.
  • Apoptosis or necrosis is determined from, respectively, the cell lysates or
  • apoptotic or necrotic indices are calculated by dividing the average absorbance from treated samples in quadruplicate by the average absorbance from untreated samples in quadruplicate
  • Medium pH can be regulated by incubation at 5% C0 2 or 17%
  • Lentivirus encoding the appropriate constructs are prepared as follows. 5xl0 6 HEK293FT-cells are seeded into a 100 mm dish and allowed to reach 70-90% confluency overnight. 2.5 pg of the indicated DNA plasmids and 20 pL Lentivirus Packaging Mix are diluted in 0.5 mL DMEM or Opti-MEM I Medium without serum and mixed gently. In a separate tube, 30 pL of transfection reagent is diluted in 0.5 mL DMEM or Opti- MEM I Medium without serum and mixed gently.
  • NanoFect/DMEM and DNA/DMEM solutions are mixed together and Codexed for 10-15 seconds prior to incubation of the DMEM-plasmid-reagent mixture at room temperature for 15 minutes.
  • the complete transfection complex from the previous step is added dropwise to the plate of cells and rocked to disperse the transfection complex evenly in the plate.
  • the plate is then incubated overnight at 37°C in a humidified 5% C0 2 incubator. The following day, the supernatant is replaced with 10 mL fresh media and supplemented with 20 pL ⁇ of ViralBoost (500x, ALSTEM). The plates are then incubated at
  • the lentivirus containing supernatant is then collected into a 50 mL sterile, capped conical centrifuge tube and put on ice. After centrifugation at 3000 rpm for 15 minutes at 4°C, the cleared supernatant is filtered with a low -protein binding 0.45 pm sterile filter and virus is subsequently isolated by ultracentrifugation at 25,000 rpm for 1.5 hours, at 4°C. The pellet is removed and re-suspended in DMEM media and Lentivirus concentrations/titers are established by quantitative RT-PCR. Any residual plasmid DNA is removed by treatment with DNase 1. The virus stock preparation is either used for infection immediately or aliquoted and stored at -80°C for future use.
  • PBMCs Peripheral Blood Mononuclear Cells
  • Whole blood is collected in 10 mL Heparin vacutainers and either processed immediately or stored overnight at 4°C.
  • Approximately 10 mL of whole anti -coagulated blood is mixed with sterile phosphate buffered saline (PBS) buffer for a total volume of 20 mL in a 50 mL conical centrifuge tube.
  • PBS sterile phosphate buffered saline
  • 20 mL of this blood/PBS mixture is then gently overlayed onto the surface of 15 mL of Licoll reagent prior to centrifugation at 400 x g for 30-40 min at room temperature with no brake application.
  • the layer of cells containing PBMCs is removed carefully to minimize contamination by Licoll.
  • Residual Licoll, platelets, and plasma proteins are then removed by washing the PBMCs three times with 40 mL of PBS by centrifugation at 200 x g for 10 minutes at room temperature. The cells are then counted with a hemocytometer. The washed PBMCs are transferred to insulated vials and frozen at -80°C for 24 hours before storing in liquid nitrogen for later use.
  • Lentivirus is thawed on ice and 5xl0 6 lentivirus, along with 2 pL of viral transduction enhancer per mL of media is added to each well of lxlO 6 cells. Cells are incubated for an additional 24 hours before repeating addition of virus.
  • lentivirus is thawed on ice and the virus is added at 5 or 50 MOI in presence of 5 pg/mL Polybrene. Cells are spinoculated at 100 x g for 100 minutes at room temperature.
  • Cells are then grown in the continued presence of 300 IU/mL of human IL-2 for a period of 6-14 days. Cell concentrations are analyzed every 2-3 days, with media being added at that time to maintain the cell suspension at lxlO 6 cells/mL.
  • Expression plasmids encoding the TCR alpha and beta chains or the TCR gamma and delta chains are produced using standard molecular biology techniques. Plasmids are transformed into chemically- competent cells and grown overnight at 37°C. Protein expression is induced by the addition of Isopropyl b-D-l -thiogalactopyranoside (IPTG) to 1 mM and bacteria are grown for a further 3 hours at 37°C.
  • IPTG Isopropyl b-D-l -thiogalactopyranoside
  • Bacteria are harvested by centrifugation at 4000 x g for 15 minutes and lysed in a protein extraction
  • Lysis proceeds for 1 hour at room temperature with agitation before inclusion bodies are harvested by centrifugation at 10000 x g for 5 minutes. Pellets are washed twice with a
  • Soluble TCRs are prepared by dissolving alpha and beta inclusion bodies or the TCR gamma and delta inclusion bodies in 6M guanidine-HCI containing 10 mM dithiothreitol and incubating at 37°Cfor 30 minutes.
  • Samples are diluted into 50 ml urea folding buffer (5 M urea; 0.4 M L-arginine; 0.1 M Tris-CI, pH 8.1; 2 mM EDTA; 6.5 mM b-mercapthoethylamine; 1.9 mM cystamine) and dialysed against eight volumes of water overnight at 4°C, followed by dialysis for a further 24 hours in eight volumes of 10 mM Tris (8.1 ), with one buffer change. Dialysate (30 ml) is concentrated to 1 ml. Concentrated protein is diluted to 5 ml in phosphate-buffered saline and concentrated to 0.5 ml.
  • 50 ml urea folding buffer 5 M urea; 0.4 M L-arginine; 0.1 M Tris-CI, pH 8.1; 2 mM EDTA; 6.5 mM b-mercapthoethylamine; 1.9 mM cystamine
  • TCR fusion constructs are produced in mammalian cells according to known methods.
  • Example 7 In vitro Screening of a Modified TCR produced in Examples 5 or 6 for Antigen Recognition
  • a modified TCR produced in Example 5 is tested for its ability to recognize antigens when separately expressed in CD8 + T cells and CD4 + T cells.
  • PBMC from a subject is transfected as described in Zhao et al. (2006), et al., Mol. Ther. 13: 151-159 (2006) with (i) RNA encoding the WT alpha chain of the TCR and (ii) RNA encoding the WT beta chain of the TCR, or DNA encoding Green Fluorescence Protein (GFP).
  • GFP Green Fluorescence Protein
  • Transfected cells are washed and stimulated with or without (T alone) one of the following cells: T2+ pulsed with antigen.
  • Responder cells (1 c 10 5 electroporated PBLs) and 1 c 10 5 stimulator cells are incubated in a 0.2-ml culture volume in individual wells of 96-well plates. Stimulator cells and responder cells are co-cultured for 16 to 24 h.
  • Cytokine secretion of culture supernatants diluted to the linear range of the assay is measured using commercially available ELISA kits (IFN-g Endogen, Cambridge, Mass.). The amount of IFN-g (pg/ml) produced by transfected CD8 + T cells is determined, while the amount of IFN-g (pg/ml) produced by transfected CD4 + T cells is determined.

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Abstract

L'invention concerne des récepteurs de lymphocytes T (TCR) modifiés, des compositions pharmaceutiques associées, ainsi que des acides nucléiques, et leurs procédés de préparation et de découverte.
PCT/US2018/066866 2017-12-28 2018-12-20 Récepteurs de lymphocytes t modifiés WO2019133443A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021050948A1 (fr) * 2019-09-12 2021-03-18 TCR2 Therapeutics Inc. Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090105133A1 (en) * 2004-11-23 2009-04-23 Medigene Limited Gamma-Delta T Cell Receptors
US20160130319A1 (en) * 2013-06-26 2016-05-12 Yi Li High-stability t-cell receptor and preparation method and application thereof
WO2017109496A1 (fr) * 2015-12-22 2017-06-29 Immunocore Limited Récepteurs de lymphocytes t spécifiques du complexe de l'antigène tumoral ny-eso-1 avec hla-a*02
WO2017174822A1 (fr) * 2016-04-08 2017-10-12 Adaptimmune Limited Récepteurs de lymphocytes t
WO2017175006A1 (fr) * 2016-04-08 2017-10-12 Immunocore Limited Récepteurs des lymphocytes t

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090105133A1 (en) * 2004-11-23 2009-04-23 Medigene Limited Gamma-Delta T Cell Receptors
US20160130319A1 (en) * 2013-06-26 2016-05-12 Yi Li High-stability t-cell receptor and preparation method and application thereof
WO2017109496A1 (fr) * 2015-12-22 2017-06-29 Immunocore Limited Récepteurs de lymphocytes t spécifiques du complexe de l'antigène tumoral ny-eso-1 avec hla-a*02
WO2017174822A1 (fr) * 2016-04-08 2017-10-12 Adaptimmune Limited Récepteurs de lymphocytes t
WO2017175006A1 (fr) * 2016-04-08 2017-10-12 Immunocore Limited Récepteurs des lymphocytes t

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021050948A1 (fr) * 2019-09-12 2021-03-18 TCR2 Therapeutics Inc. Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion
CN114828862A (zh) * 2019-09-12 2022-07-29 T细胞受体治疗公司 使用融合蛋白进行tcr重编程的组合物和方法
EP4028033A4 (fr) * 2019-09-12 2023-10-04 TCR2 Therapeutics Inc. Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion

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