EP4028033A1 - Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion - Google Patents

Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion

Info

Publication number
EP4028033A1
EP4028033A1 EP20862242.3A EP20862242A EP4028033A1 EP 4028033 A1 EP4028033 A1 EP 4028033A1 EP 20862242 A EP20862242 A EP 20862242A EP 4028033 A1 EP4028033 A1 EP 4028033A1
Authority
EP
European Patent Office
Prior art keywords
tcr
nucleic acid
domain
recombinant nucleic
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20862242.3A
Other languages
German (de)
English (en)
Other versions
EP4028033A4 (fr
Inventor
Patrick Baeuerle
Robert Hofmeister
Daniel Getts
Dario Gutierrez
Philippe KIEFFER-KWON
Julie DONAGHEY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TCR2 Therapeutics Inc
Original Assignee
TCR2 Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by TCR2 Therapeutics Inc filed Critical TCR2 Therapeutics Inc
Publication of EP4028033A1 publication Critical patent/EP4028033A1/fr
Publication of EP4028033A4 publication Critical patent/EP4028033A4/fr
Pending legal-status Critical Current

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector

Definitions

  • cancer immunotherapy Most patients with hematological malignancies or with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.
  • CAR chimeric antigen receptor
  • CTL019 The clinical results with CD19-specific CAR T cells (called CTL019) have shown complete remissions in patients suffering from chronic lymphocytic leukemia (CLL) as well as in childhood acute lymphoblastic leukemia (ALL) (see, e.g., Kalos et ah, Sci Transl Med 3:95ra73 (2011), Porter et ah, NEJM 365:725-733 (2011), Grupp et ah, NEJM 368:1509-1518 (2013)).
  • An alternative approach is the use of T cell receptor (TCR) alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells.
  • TCR T cell receptor
  • TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity.
  • Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma.
  • successful patient therapy with engineered T cells may require the T cells to be capable of strong activation, expansion, persistence over time, and, in case of relapsing disease, to enable a ‘memory’ response.
  • High and manageable clinical efficacy of CAR T cells is currently limited to CD19-positive B cell malignancies and to NY-ESO-1 -peptide expressing synovial sarcoma patients expressing HLA- A2.
  • modified T cells comprising fusion proteins of TCR subunits, including CD3 epsilon, CD3gamma, CD3 delta, TCR gamma, TCR delta, TCR alpha and TCR beta chains with binding domains specific for cell surface antigens that have the potential to overcome limitations of existing approaches. Additionally, these modified T cells may have functional disruption of an endogenous TCR (e.g., TCR alpha, beta or both). These modified T cells may have the ability to kill target cells more efficiently than CARs, but release comparable or lower levels of pro-inflammatory cytokines. These modified T cells and methods of their use may represent an advantage for these cells relative to CARs because elevated levels of these cytokines have been associated with dose-limiting toxicities for adoptive CAR T therapies.
  • endogenous TCR e.g., TCR alpha, beta or both.
  • modified T cells comprising T-cell receptor (TCR) fusion protein (TFP) and a TCR constant domain
  • TCR constant domain described herein can be a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR constant domain.
  • the TFP described herein can comprise a TCR subunit and an antibody or fragment thereof.
  • the TFP described herein can comprise a TCR subunit and a binding ligand or fragment thereof.
  • the TCR subunit can be derived from any of the TCR chains such as TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 epsilon, CD3 delta, or CD3 gamma.
  • the antibody or fragment thereof can be murine, human, or humanized.
  • the present disclosure provides a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain, and a transmembrane domain, and an antibody comprising an antigen binding domain; and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a modified T cell comprising a functional disruption of an endogenous TCR.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the present disclosure provides a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain, a transmembrane domain, and a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the binding ligand or fragment thereof are operatively linked, and wherein the TFP functionally incorporates into TCR complex when expressed in a modified T cell comprising a functional disruption of an endogenous TCR.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the TCR subunit further comprises an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta.
  • the TCR constant domain is a TCR delta constant domain.
  • the TCR delta constant domain comprises SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the intracellular domain is an intracellular domain of TCR gamma.
  • the sequence encoding the TCR delta constant domain further encodes a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR delta constant domain.
  • the second antigen binding domain or ligand binding domain is the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the second antigen binding domain or ligand binding domain is operatively linked to the sequence encoding the TCR delta constant domain via a linker.
  • the TCR constant domain is a TCR gamma constant domain.
  • the TCR gamma constant domain comprises SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the intracellular domain is an intracellular domain of TCR delta.
  • the sequence encoding the TCR gamma constant domain further encodes a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR gamma constant domain.
  • the second antigen binding domain or ligand binding domain is the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the second antigen binding domain or ligand binding domain is operatively linked to the sequence encoding the TCR gamma constant domain via a linker.
  • the recombinant nucleic acid comprises a sequence encoding a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR gamma constant domain comprises SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR gamma constant domain further encodes a TCR gamma variable domain, thereby encoding a full TCR gamma domain.
  • the full TCR gamma domain is gamma 9 or gamma 4.
  • the full TCR gamma domain comprises SEQ ID NO:255, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR delta constant domain comprises SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR delta constant domain further encodes a TCR delta variable domain, thereby encoding a full TCR delta domain.
  • the full TCR delta domain is delta 2 or delta 1.
  • the full TCR delta constant domain comprises SEQ ID NO:256, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the intracellular signaling domain is CD3 epsilon, CD3 gamma, or CD3 delta. In some embodiments, the intracellular signaling domain is CD3 epsilon.
  • the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, the recombinant nucleic acid further comprises a portion of a TCR alpha constant domain, a portion of a TCR beta domain, or both.
  • the sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRGC gene sequence. In some embodiments, the sequence comprises, from 5 ’-3’, a first leader sequence, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker sequence, and a TRGC gene sequence.
  • the sequence comprises, from 5 ’-3’, a first leader sequence, an antigen binding domain sequence, a first linker sequence, a TRDC gene sequence, a cleavable linker, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, and a TRGC gene sequence.
  • the sequence comprises, from 5 ’-3’, a first leader sequence, a TRDC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRGC gene sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence.
  • the sequence comprises, from 5 ’-3’, a first leader sequence, a first antigen binding domain sequence, a first linker sequence, a TRDC gene sequence or fragment thereof, a TRAC gene sequence or fragment thereof, a cleavable linker sequence, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, a TRGC gene sequence or fragment thereof, and a TRBC gene sequence or fragment thereof.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO: 1. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:2. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:3. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:4. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:5. In some embodiments, the sequence encodes a sequence of SEQ ID NO:242. In some embodiments, the sequence encodes a sequence of SEQ ID NO:244. In some embodiments, the sequence encodes a sequence of SEQ ID NO:245.
  • the sequence encodes a sequence of SEQ ID NO:246. In some embodiments, the sequence encodes a sequence of SEQ ID NO:248. In some embodiments, the sequence encodes a sequence of SEQ ID NO:250. In some embodiments, the sequence encodes a sequence of SEQ ID NO:252. In some embodiments, the sequence encodes a sequence of SEQ ID NO:257. In some embodiments, the sequence encodes a sequence of SEQ ID NO:263. In some embodiments, the sequence encodes a sequence of SEQ ID NO:264. [0013] In some embodiments, the binding ligand is capable of binding an Fc domain of the antibody. In some embodiments, the binding ligand is capable of selectively binding an IgGl antibody.
  • the binding ligand is capable of specifically binding an IgG4 antibody.
  • the antibody or fragment thereof binds to a cell surface antigen.
  • the antibody or fragment thereof is murine, human or humanized.
  • the antibody or fragment thereof binds to a cell surface antigen on the surface of a tumor cell.
  • the binding ligand comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the binding ligand does not comprise an antibody or fragment thereof.
  • the binding ligand comprises a CD 16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD 16-binding polypeptide. In some embodiments, the binding ligand is human or humanized. In some embodiments, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof capable of being bound by the binding ligand. In some embodiments, the antibody or fragment thereof is capable of being secreted from a cell.
  • the present disclosure provides a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain, and a transmembrane domain, and an antigen binding domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain; or a sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a modified T cell comprising a functional disruption of an endogenous TCR.
  • TCR T cell receptor
  • TFP T cell receptor
  • the TCR subunit further comprises an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta.
  • the recombinant nucleic acid further comprises at least a partial sequence encoding a TCR alpha constant domain, a TCR beta constant domain, or a partial sequence of both a TCR alpha constant domain and a TCR beta constant domain.
  • the antigen binding domain comprises a ligand.
  • the ligand binds to the receptor of a cell.
  • the ligand binds to the polypeptide expressed on a surface of a cell.
  • the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide.
  • the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4 and combinations thereof.
  • the antigen binding domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the antigen binding domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the ligand or fragment thereof is a monomer or a dimer. In some embodiments, the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not comprise a variable region. In some embodiments, the antigen binding domain does not comprise a CDR. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • NVG2D Natural Killer Group 2D
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some embodiments, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within a same nucleic acid molecule. In some embodiments, the encoded TFP and the encoded TCR constant domains are operatively linked by a first linker sequence. In some embodiments, the first linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A, e.g., a T2A or a P2A cleavage site.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within different nucleic acid molecules.
  • the TCR subunit and the antibody domain, the antigen binding domain or the binding ligand or fragment thereof of the TFP are operatively linked by a second linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR delta, or TCR gamma.
  • the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain of a TCR gamma chain or a TCR delta chain.
  • the TCR extracellular domain comprises the extracellular portion of a constant domain of a TCR gamma chain or a TCR delta chain, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a delta constant domain, or a fragment thereof.
  • the delta constant domain has the sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a gamma constant domain.
  • the gamma constant domain has the sequence of SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the extracellular domain of the TFP does not comprise the variable domain of a gamma chain or a delta chain.
  • the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit of the TFP comprises the extracellular, transmembrane and intracellular domain of CD3 epsilon.
  • the TCR subunit of CD3 epsilon comprises the sequence of SEQ ID NO:258, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TFP, the TCR gamma constant domain, the TCR delta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR delta,
  • the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; or the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the antibody is an antibody fragment In some embodiments, the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain.
  • an antigen binding domain is selected from a group consisting of an anti-CD 19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain, an anti- HER2 binding domain, an anti-PMSA binding domain, an anti-MUC16 binding domain, an anti- CD22 binding domain, an anti-PD-Ll binding domain, an anti BAFF or BAFF receptor binding domain, an anti-Nectin-4 binding domain, an anti-TROP-2 binding domain, an anti-GPC3 binding domain, and an anti-ROR-1 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:60, a CDR2 of SEQ ID NO:61, and a CDR3 of SEQ ID NO:62. In some embodiments, the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:63, a CDR2 of SEQ ID NO:64, and a CDR3 of SEQ ID NO:65. In some embodiments, the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:66, a CDR2 of SEQ ID NO:67, and a CDR3 of SEQ ID NO:68.
  • the anti-MSLN binding domain comprises a sequence with at least about 80% sequence identity to a sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.
  • the anti-CD19 binding domain comprises a CDR1 of SEQ ID NO:73, a CDR2 of SEQ ID NO:75, and a CDR3 of SEQ ID NO:77.
  • the anti-CD19 binding domain comprises a CDR1 of SEQ ID NO: 79, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO: 83.
  • the anti-CD19 binding domain comprises a sequence with at least about 80% sequence identity to a sequence of SEQ ID NO:85 and/or SEQ ID NO:87.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR beta transmembrane domain.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain.
  • the present disclosure provides a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, and a murine TCR alpha or murine TCR beta transmembrane domain, and an antibody or fragment thereof comprising an antigen binding domain; and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain; or a sequence encoding a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a modified T cell comprising a functional disruption of an endogenous TCR.
  • TCR T cell receptor
  • TFP T cell receptor
  • the present disclosure provides a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, and a murine TCR alpha or murine TCR beta transmembrane domain, and a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain; or a sequence encoding a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the binding ligand or fragment thereof are operatively linked, and wherein the TFP functionally incorporates into TCR complex when expressed in a modified T cell comprising a functional disruption of an endogenous
  • TCR T cell receptor
  • the TCR subunit comprises an intracellular domain of murine TCR alpha or murine TCR beta.
  • the TCR constant domain is a TCR alpha constant domain.
  • the TCR alpha constant domain comprises SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO:207, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR alpha constant domain comprises a murine TCR alpha constant domain.
  • the murine TCR alpha constant domain comprises amino acids 2-137 of the murine TCR alpha constant domain.
  • the murine TCR alpha constant domain comprises amino acids 2-137 of SEQ ID NO: 146. In some embodiments, the murine TCR alpha constant domain comprises a sequence of SEQ ID NO:207. In some embodiments, the murine TCR alpha constant domain comprises amino acids 82-137 of SEQ ID NO: 146. In some embodiments, the murine TCR alpha constant domain comprises a sequence of SEQ ID NO: 17. In some embodiments, the intracellular domain is an intracellular domain of TCR beta. In some embodiments, the sequence encoding the TCR alpha constant domain further encodes a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR alpha constant domain.
  • the second antigen binding domain or ligand binding domain is the same or different as the antigen binding domain or ligand binding domain of the TFP. In some embodiment, the second antigen binding domain or ligand binding domain is operatively linked to the sequence encoding the TCR alpha constant domain via a linker.
  • the TCR constant domain is a TCR beta constant domain. In some embodiments, the TCR beta constant domain comprises SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, TCR beta constant domain comprises a murine TCR beta constant domain.
  • murine TCR beta constant domain comprises amino acids 2-173 of the murine TCR beta constant domain. In some embodiments, murine TCR beta constant domain comprises amino acids 2-173 of SEQ ID NO: 152. In some embodiments, murine TCR beta constant domain comprises SEQ ID NO:209.
  • the TCR beta constant domain comprises amino acids 123-173 of SEQ ID NO: 152. In some embodiments, the TCR beta constant domain comprises SEQ ID NO: 18. In some embodiments, the intracellular domain is an intracellular domain of TCR alpha. In some embodiments, the sequence encoding the TCR beta constant domain further encodes a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR beta constant domain. In some embodiments, the second antigen binding domain or ligand binding domain is the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the second antigen binding domain or ligand binding domain is operatively linked to the sequence encoding the TCR beta constant domain via a linker.
  • the recombinant nucleic acid comprise sequence encoding a TCR alpha constant domain and a TCR beta constant domain.
  • the TCR alpha constant domain comprises SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO:207, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR beta constant domain comprises SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the intracellular signaling domain is CD3 epsilon, CD3 gamma, or CD3 delta. In some embodiments, the intracellular signaling domain is CD3 epsilon.
  • the sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence comprises, from 5’ to 3’, a first leader sequence, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence. In some embodiments, the sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence.
  • the sequence comprises, from 5’-3’, a first leader sequence, a TRAC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRBC gene sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO: 10.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:204.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:206.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:210. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:211. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:217. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:218. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:219. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:220. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:259.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:261. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:262. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, the binding ligand is capable of binding an Fc domain of the antibody. In some embodiments, the binding ligand is capable of selectively binding an IgGl antibody. In some embodiments, the binding ligand is capable of specifically binding an IgG4 antibody. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen. In some embodiments, the antibody or fragment thereof is murine, human or humanized.
  • the antibody or fragment thereof binds to a cell surface antigen on the surface of a tumor cell.
  • the binding ligand comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the binding ligand does not comprise an antibody or fragment thereof.
  • the binding ligand comprises a CD 16 polypeptide or fragment thereof.
  • the binding ligand comprises a CD 16- binding polypeptide.
  • the binding ligand is human or humanized.
  • the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof capable of being bound by the binding ligand.
  • the antibody or fragment thereof is capable of being secreted from a cell.
  • the present disclosure provides a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, and a murine TCR alpha or murine TCR beta transmembrane domain, and an antigen binding domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain; or a sequence encoding a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in
  • the TCR subunit comprises an intracellular domain of murine TCR alpha or murine TCR beta.
  • the extracellular domain comprises the extracellular portion of a TCR alpha constant domain or TCR beta constant domain, or a fragment thereof.
  • the recombinant nucleic acid further comprises at least a partial sequence encoding a TCR gamma constant domain, a TCR delta constant domain, or a partial sequence of both a TCR gamma constant domain and a TCR delta constant domain.
  • the antigen binding domain comprises a ligand.
  • the ligand binds to the receptor of a cell.
  • the ligand binds to the polypeptide expressed on a surface of a cell.
  • the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide.
  • the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4 and combinations thereof.
  • the antigen binding domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the antigen binding domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the ligand or fragment thereof is a monomer or a dimer. In some embodiments, the antigen binding domain does not comprise an antibody or fragment thereof In some embodiments, the antigen binding domain does not comprise a variable region. In some embodiments, the antigen binding domain does not comprise a CDR. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some embodiments, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within a same nucleic acid molecule.
  • the TFP and the TCR constant domains are operatively linked by a first linker sequence.
  • the first linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A, e.g., a T2A or a P2A cleavage site.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within different nucleic acid molecules.
  • the TCR subunit and the antibody domain, the antigen binding domain or the binding ligand or fragment thereof of the TFP are operatively linked by a second linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from TCR alpha or TCR beta, e.g., murine TCR alpha or TCR beta.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, of TCR alpha or TCR beta.
  • the TCR extracellular domain comprises the extracellular portion of a constant domain TCR alpha chain or a TCR beta chain, e.g., a murine TCR alpha chain or a TCR beta chain, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a TCR alpha chain or a TCR beta chain, e.g., a murine TCR alpha chain or a TCR beta chain, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a TCR intracellular domain of a TCR alpha chain or a TCR beta chain, e.g., a murine TCR alpha chain or a TCR beta chain, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises an alpha constant domain.
  • the alpha constant domain has the sequence of SEQ ID NO : 17, SEQ ID NO : 142, SEQ ID NO : 143 , SEQ ID NO: 146, or SEQ ID NO:207 functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the encoded TCR comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a beta constant domain.
  • the beta constant domain has the sequence of SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the extracellular domain of the TCR subunit does not comprise the variable domain of an alpha chain or a beta chain.
  • the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit of the TFP comprises the extracellular, transmembrane and intracellular domain of CD3 epsilon.
  • the TCR subunit of CD3 epsilon comprises the sequence of SEQ ID NO:258, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TFP, the TCR alpha constant domain, the TCR beta domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR alpha constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof; or the TCR constant domain is a TCR alpha constant domain and a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the antibody is an antibody fragment.
  • the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain.
  • an antigen binding domain is selected from a group consisting of an anti-CD 19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti- 79b binding domain, an anti-HER2 binding domain, an anti-PMSA binding domain, an anti- MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti BAFF or BAFF receptor binding domain, an anti-Nectin-4 binding domain, an anti-TROP-2 binding domain, an anti-GPC3 binding domain, and an anti-ROR-1 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:60, a CDR2 of SEQ ID NO:61, and a CDR3 of SEQ ID NO:62. In some embodiments, the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO:63, a CDR2 of SEQ ID NO:64, and a CDR3 of SEQ ID NO:65. In some embodiments, the anti-MSLN binding domain comprises a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ED NO: 67, and a CDR3 of SEQ ID NO: 68.
  • the anti-MSLN binding domain comprises a sequence with at least about 80% sequence identity to a sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.
  • the anti- CD19 binding domain comprises a CDR1 of SEQ ID NO:73, a CDR2 of SEQ ID NO:75, and a CDR3 of SEQ ID NO:77.
  • the anti-CD19 binding domain comprises a CDR1 of SEQ ID NO:79, a CDR2 of SEQ ID NO:81, and a CDR3 of SEQ ED NO:83 In some embodiments, the anti-CD 19 binding domain comprises a sequence with at least about 80% sequence identity to a sequence of SEQ ID NO:85 and/or SEQ ID NO:87.
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a circRNA. In some embodiments, the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2 ’-O-methyl, 2 ’-O-m ethoxy ethyl (2’-0-MOE), 2’-0-aminopropyl, 2’-deoxy, T- deoxy-2’-fluoro, 2’-0-aminopropyl (2’-0-AP), 2'-0-dimethylaminoethyl (2’-0-DMAOE), 2’-0- dimethylaminopropyl (2’-0-DMAP), T-O-dimethylaminoethyloxyethyl (2’-0-DMAEOE), 2’-0- N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphon
  • the recombinant nucleic acid further comprises a leader sequence. In some embodiments, the recombinant nucleic acid further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3’UTR sequence. In some embodiments, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid. In another aspect, the present disclosure provides a vector comprising the recombinant nucleic acid.
  • the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno- associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector is an AAV6 vector.
  • the method further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • the present disclosure provides a modified T cell comprising the recombinant nucleic acid, or the vector, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • the present disclosure provides a modified T cell comprising the sequence encoding the TFP of the nucleic acid or a TFP encoded by the sequence of the nucleic acid encoding the TFP, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • the present disclosure provides a modified allogenic T cell comprising the sequence encoding the TFP or a TFP encoded by the sequence of the nucleic acid encoding the TFP.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain.
  • the TCR constant domain e g., the TCR alpha constant domain, the TCR beta constant domain or the TCR alpha constant domain and the TCR beta constant domain
  • is a murine TCR constant domain e.g., a murine TCR alpha constant domain, a murine TCR beta constant domain or a murine TCR alpha constant domain and a murine TCR beta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain.
  • the endogenous TCR that is functionally disrupted has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell.
  • the functional disruption is a disruption of a gene encoding the endogenous TCR.
  • the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
  • the T cell is a human T cell selected from CD4 cells, CD8 cells, naive T-cells, memory stem T-cells, central memory T- cells, double negative T-cells, effector memory T-cells, effector T-cells, ThO cells, TcO cells, Thl cells, Tel cells, Th2 cells, Tc2 cells, Thl7 cells, Th22 cells, alpha/beta T cells, gamma/delta T cells, natural killer (NK) cells, natural killer T ( KT) cells, hematopoietic stem cells and pluripotent stem cells.
  • the T cell is a CD8+ or CD4+ T cell
  • the T cell is an allogenic T cell.
  • the method further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: the modified T cells; and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of producing the modified T cell, the method comprising disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid or the vector.
  • the method further comprises disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • the present disclosure provides a method of producing the modified T cell, the method comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid, or the vector.
  • the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • the T cell is a human T cell.
  • the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell.
  • the nuclease is a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
  • ZFN zinc-finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR/Cas nuclease CRISPR/Cas nuclease
  • megaTAL nuclease a megaTAL nuclease
  • the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit.
  • the nuclease is a meganuclease.
  • the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence.
  • the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified T cell produced according to the method described herein; and (b) a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified T cell produced according to the method described herein; and (b) a pharmaceutically acceptable carrier.
  • the modified T cell is an allogeneic T cell. In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell. In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of a modified T cell comprising the recombinant nucleic acid, or the vector. In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition. In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition. In some embodiments, the cancer is a solid cancer, a lymphoma or a leukemia.
  • the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer.
  • less cytokines are released in the subject compared to a subject administered an effective amount of an autologous T cell expressing the TFP described herein.
  • the method does not induce graft versus host disease.
  • the subject has a reduced risk of developing graft versus host disease compared to a subject administered an effective amount of an autologous T cell expressing the TFP described herein.
  • the present disclosure provides the recombinant nucleic acid, the vector, the modified T cell, or the pharmaceutical composition, for use as a medicament or in the preparation of a medicament.
  • Figure l is a series of graphs showing surface expression of CD3 (SK7) vs TCRa3 (IP26) in TRA-edited (left) and TRB-edited (right) cells. Wild type Jurkat cells were edited at either the TRAC or TRBC genes to disrupt TRA or TRB surface expression. Cells negative for CD3 and TCRap were purified using Magnetic- Activated Cell Sorting (MACS). The gates on the plots were drawn to delineate CD3 and TCRap negative-negative population of cells and the percentages of cells remaining in each quadrant are shown in the corners.
  • CD3 SK7
  • TCRa3 IP26
  • Figure 2 is a series of graphs showing surface expression of CD3 (SK7) vs TCRap (IP26) in TRA-/- (left) and TRB-/- (right) Jurkat cells.
  • the first panel from the left is a non- transduced negative control.
  • the second panel shows TRB-/- cells transduced with a TCRp Full Length TFP.
  • the third and fourth panels show TRA-/- and TRB-/- transduced with a TCRyd TFP construct.
  • Figure 3 is a drawing showing various strategies to generate allogeneic TFP T cells.
  • Figure 4 is a schematic showing elements of the constructs used to generate allogeneic TFP T cells.
  • Figure 5 is a series of graphs showing surface expression of CD3 vs TCRaP in T cells transduced with TCRyd TFP constructs after editing the TRAC locus versus wild type cells and TRAC edited, non-transduced T cells.
  • the graphs depict cell populations prior to MACS purification.
  • Figure 6 is a series of graphs showing surface expression of CD3 vs TCRaP in T cells transduced with TCRyd TFP constructs after editing the TRAC locus versus wild type cells and TRAC edited, non-transduced T cells. Graphs depict cell populations after MACS purification.
  • Figure 7 is a series of graphs showing surface expression of allogeneic TFP transduced T cells (TFP+). The left-hand panels show TFP+ percentage of T cells transduced with TCRyd TFP constructs. The right-hand panels show TFP+ percentage of T cells transduced with TCRJ3 full length positive control constructs compared to non-transduced control cells.
  • Figure 8 is a series of graphs showing surface expression of CD4 versus CD8 populations within T cells transduced with TCRyd TFP constructs compared to T cells transduced with TCR Full length positive control constructs.
  • Figure 9 is a series of graphs showing surface expression of CD45RA versus CCR7 populations in CD4 or CD8 T cells transduced with TCRyd TFP constructs compared to T cells transduced with TCR Full length positive control constructs.
  • Figure 10 is two graphs showing Luc-Cyto analysis of allogeneic T effector cells cultured with tumor target cells at 3-to-l, 1-to-l, or l-to-3 and l-to-9 ratios.
  • Target Nalm-6 (CD19 positive) cells are shown in the left panel while CD19 negative cells (K562 cells) are shown in the right panel.
  • the x-axes represent percentage of tumor cell lysis.
  • Figure 12 is a schematic diagram showing TFP constructs in TRAC or TRBC edited cells, and whether or not the TCR is reconstituted with expression of the construct.
  • Figure 12 shows that murine TCRa or TCRP TFP constructs are able to reconstitute the TCR, and human TCRy or TCR5 TFP constructs are able to reconstitute the TCR.
  • Figures 13A and 13B show cell surface expression of human TCR TFP constructs.
  • Figures 13A is a schematic diagram showing the constructs expressed in Figure 13B. Human anti-CD19 TCRp TFP and human anti-CD19 TCRP (constant domain) TFP are shown.
  • Figure 13B shows surface expression of CD3 vs TCRc/.b in TRBC knockout Jurkat cells transduced with the constructs of Figure 13 A.
  • Figure 14 is a series of graphs showing surface expression of (i) SSC-A versus CD19;
  • TRAC was not knocked out in cells transduced with anti-CD 19-CD3s.
  • Figure 15 is a series of graphs showing showing Luc-Cyto analysis of TRAC knockout T cells transduced with the constructs shown cultured with tumor target cells at 3-to-l, 1-to-l, or 1- to-3 and l-to-9 ratios from left to right. TRAC was not knocked out in cells transduced with anti-CD19-CD3s.
  • Target Nalm-6 (CD19 positive) cells are shown in the top panel while CD19 negative cells (K562 cells) are shown in the bottom panel.
  • the x-axes represent percentage of tumor cell lysis.
  • Figure 17 is a series of graphs showing secretion (in pg/mL) of GM-CSF and IFNy of the TRAC knockout T cells transduced with the constructs shown without or with (from left to right) non-HLA-matched dendritic cells. TRAC was not knocked out in cells transduced with anti- CD 19-CD3S.
  • Figures 18A-18C are a series of graphs showing tumor burden as measured by luminescence in Nalm6-luc tumor mouse model mice injected with the TRAC knockout T cells transduced with the constructs shown. TRAC was not knocked out in cells transduced with anti- CD 19-CD3S.
  • Figure 19 shows infiltration of CD7+ cells into the liver of non-tumor bearing mice from the in vivo assay shown in Figure 18
  • Surface expression of CD45RA versus CD7 in the mouse liver is shown as well as immunohistochemistry of CD7 expression in the murine liver.
  • Figure 20 is a series of graphs showing showing Luc-Cyto analysis of TRAC knockout T cells transduced with the constructs shown cultured with tumor target cells at 3-to-l, 1-to-l, or 1- to-3 ratios from left to right.
  • Target Nalm-6 (CD 19 positive) cells are shown in the left panel while CD19 negative cells (K562 cells) are shown in the right panel.
  • the x-axes represent percentage of tumor cell lysis.
  • Figure 21 is a series of four graphs showing cytokine secretion (in pg/mL) of supernatants taken from the Nalm-6 co-culture assay in Figure 20 in which allogeneic T effector cells were cultured with tumor target cells at 3-to-l, 1-to-l, or l-to-3 ratios from left to right. Graphs show cytokine secretion for GM-CSF, IFNy, IL2 and TNFa.
  • Figure 22 is a series of graphs showing showing Luc-Cyto analysis of TRAC knockout T cells transduced with the constructs shown cultured with tumor target cells at 3-to-l, 1-to-l, or 0.1:1 ratios from left to right.
  • Target Nalm-6 (CD 19 positive) cells are shown in the left panel while CD19 negative cells (K562 cells) are shown in the right panel.
  • the x-axes represent percentage of tumor cell lysis.
  • Figure 23 is a series of four graphs showing cytokine secretion (in pg/mL) of supernatants taken from the Nalm-6 co-culture assay in Figure 20 in which allogeneic T effector cells were cultured with tumor target cells at 3-to-l, 1-to-l, or 0.1 : 1 ratios from left to right.
  • Graphs show cytokine secretion for GM-CSF, IFNy, IL2 and TNFa
  • a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, and (2) a transmembrane domain, and (ii) an antibody comprising an antigen binding domain, and (b) a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a modified T cell comprising a functional disruption of an endogenous TCR.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain, or a sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the binding ligand or fragment thereof are operatively linked, and wherein the TFP functionally incorporates into TCR complex when expressed in a modified T cell comprising a functional disruption of an endogenous TCR.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, and (2) a transmembrane domain, and (ii) an antigen binding domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and (b) a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a TCR gamma constant domain or a TCR delta constant domain; or a sequence encoding a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a modified T cell comprising
  • a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, and (2) a murine TCR alpha or murine TCR beta transmembrane domain, and (ii) an antibody or fragment thereof comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain; or a sequence encoding a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a modified T cell comprising a functional disruption of
  • a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, and (2) a murine TCR alpha or murine TCR beta transmembrane domain, and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain; or a sequence encoding a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the binding ligand or fragment thereof are operatively linked, and wherein the TFP functionally incorporates into TCR complex when expressed in a modified
  • recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising TCR alpha, TCR beta, TCR gamma, or TCR delta or a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, and (ii) a human or humanized antibody comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR delta constant domain and a TCR gamma constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TCR subunit and the antibody are operatively linked, and where
  • recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising TCR alpha, TCR beta, TCR gamma, or TCR delta or a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the binding
  • recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising TCR alpha, TCR beta, TCR gamma, or TCR delta or a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, and (ii) an antigen domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta
  • recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising TCR alpha, TCR beta, TCR gamma, or TCR delta or a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, , and (ii) a human or humanized antibody comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TFP
  • recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising TCR alpha, TCR beta, TCR gamma, or TCR delta or a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain; wherein the TCR subunit and the antibody are operatively linked
  • recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising TCR alpha, TCR beta, TCR gamma, or TCR delta or a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, and (ii) an antigen domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain;
  • vectors comprising the recombinant nucleic acid disclosed herein.
  • modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein; wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.
  • compositions comprising: (a) the modified T cells of the disclosure; and (b) a pharmaceutically acceptable carrier.
  • a method of producing the modified T cell of the disclosure comprising (a) disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of the disclosure, or the vectors disclosed herein.
  • a method of producing the modified T cell of the disclosure comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid disclosed herein, or the vectors disclosed herein.
  • compositions disclosed herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions disclosed herein.
  • a pharmaceutical composition comprising (a) a modified T cell produced according to the methods disclosed herein; and (b) a pharmaceutically acceptable carrier.
  • an element means one element or more than one element.
  • “about” can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art.
  • subject or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g ., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals.
  • “Patients” are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
  • treating refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.
  • treat or prevent is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely.
  • preventing refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
  • the disease or condition e.g., tumor formation
  • a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered.
  • therapeutically effective dose herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols.
  • TCR T cell receptor
  • TCP T cell receptor
  • TCP T cell receptor
  • TCP T cell receptor
  • TCR tumor cell receptor
  • TCP T cell receptor fusion protein
  • TCP includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.
  • stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
  • Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
  • the term “stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “IT AM”.
  • IT AM immunoreceptor tyrosine-based activation motif
  • the term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g ., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface.
  • T cells may recognize these complexes using their T cell receptors (TCRs).
  • TCRs T cell receptors
  • MHC molecules are typically bound by TCRs as part of peptide:MHC complex.
  • the MHC molecule may be an MHC class I or II molecule.
  • the complex may be on the surface of an antigen presenting cell, such as a dendritic cell or a B cell, or any other cell, including cancer cells, or it may be immobilized by, for example, coating on to a bead or plate.
  • HLA human leukocyte antigen system
  • MHC major histocompatibility complex
  • A, B & C HLA class I antigens
  • DP, DQ, & DR HLA class II antigens
  • T cells undergo a positive selection step to ensure recognition of self MHCs followed by a negative step to remove T cells that bind too strongly to MHC which present self-antigens.
  • a positive selection step to ensure recognition of self MHCs
  • a negative step to remove T cells that bind too strongly to MHC which present self-antigens.
  • certain T cells and the TCRs they express will only recognize peptides presented by certain types of MHC molecules - i.e. those encoded by particular HLA alleles. This is known as HLA restriction.
  • HLA-A*0201 One HLA allele of interest is HLA-A*0201, which is expressed in the vast majority (>50%) of the Caucasian population. Accordingly, TCRs which bind WT1 peptides presented by MHC encoded by HLA-A*0201 (i.e. are HLA-A*0201 restricted) are advantageous since an immunotherapy making use of such TCRs will be suitable for treating a large proportion of the Caucasian population.
  • HLA-A alleles of interest are HLA-A*0101, HLA-A*2402, and HLA-A*0301.
  • Widely expressed HLA-B alleles of interest are HLA-B*3501, HLA-B*0702 and HLA- B*3502.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a modified T-T cell.
  • immune effector function e.g., in a modified T-T cell
  • the intracellular signaling domain can comprise a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise an IT AM (“immunoreceptor tyrosine-based activation motif’).
  • IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Costimulatory molecules include, but are not limited to an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18) and 4-1BB (CD137).
  • a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD 7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • 4- IBB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No.
  • AAA62478.2 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a “4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • antibody refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen.
  • Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
  • antibody fragment refers to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope.
  • antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • “Heavy chain variable region” or “VH” with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • a camelid “VHH” domain is a heavy chain comprising a single variable antibody domain.
  • a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • the portion of the TFP composition of the disclosure comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • scFv single chain antibody
  • the antigen binding domain of a TFP composition of the disclosure comprises an antibody fragment.
  • the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
  • recombinant antibody refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • antigen or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene.
  • an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
  • CD19 refers to the Cluster of Differentiation 19 protein, which is an antigenic determinant detectable on B cell leukemia precursor cells, other malignant B cells and most cells of the normal B cell lineage.
  • BCMA refers to the B-cell maturation antigen also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17) and Cluster of Differentiation 269 protein (CD269) is a protein that in humans is encoded by the TNFRSF17 gene.
  • TNFRSF17 is a cell surface receptor of the TNF receptor superfamily which recognizes B- cell activating factor (BAFF) (see, e.g ., Laabi et al., EMBO 11 (11): 3897-904 (1992). This receptor is expressed in mature B lymphocytes, and may be important for B-cell development and autoimmune response.
  • BAFF B- cell activating factor
  • CD 16 also known as Fc /RIII refers to a cluster of differentiation molecule found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages. CD16 has been identified as Fc receptors FcyRIIIa (CD16a) and FcyRIIIb (CD16b), which participate in signal transduction.
  • CD 16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC).
  • IgSF immunoglobulin superfamily
  • NKG2D refers to a transmembrane protein belonging to the CD94/NKG2 family of C-type lectin-like receptors. In humans, NKG2D is expressed by NK cells, gd T cells and CD8+ ab T cells. KG2D recognizes induced-self proteins from MIC and RAET1/ULBP families which appear on the surface of stressed, malignant transformed, and infected cells.
  • MSLN Mesothelin
  • NTRKR1 neurotrophic tyrosine kinase receptor-related 1
  • MUC16 also known as “mucin 16, cell-surface associated” or “ovarian cancer-related tumor marker CA125” is a membrane-tethered mucin that contains an extracellular domain at its amino terminus, a large tandem repeat domain, and a transmembrane domain with a short cytoplasmic domain. Products of this gene have been used as a marker for different cancers, with higher expression levels associated with poorer outcomes.
  • CD22 also known as sialic acid binding Ig-like lectin 2, SIGLEC-2, T cell surface antigen leu-14, and B cell receptor CD22, is a protein that mediates B cell/B cell interactions, and is thought to be involved in the localization of B cells in lymphoid tissues, and is associated with diseases including refractory hematologic cancer and hairy cell leukemia.
  • M971 human anti-CD22 monoclonal antibody
  • Programmed cell death protein 1 also known as “PD-1” and CD279 (cluster of differentiation 279), is a protein on the surface of cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.
  • PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).
  • PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2.
  • P-L1 Programmed death-ligand 1
  • PD-L1 is a 40kDa type 1 transmembrane protein that has been speculated to play a major role in suppressing the adaptive arm of immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis.
  • the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals.
  • clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated.
  • the binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM) motif.
  • SHP-1 or SHP-2 phosphatases
  • IRS Immunoreceptor Tyrosine-Based Switch Motif
  • the “CD79a” and “CD79P” genes encode proteins that make up the B lymphocyte antigen receptor, a multimeric complex that includes the antigen-specific component, surface immunoglobulin (Ig).
  • Ig surface immunoglobulin
  • Surface Ig non-covalently associates with two other proteins, Ig-alpha and Ig-beta (encoded by CD79a and its paralog CD79p, respectively) which are necessary for expression and function of the B-cell antigen receptor.
  • Functional disruption of this complex can lead to, e g., human B-cell chronic lymphocytic leukemias.
  • B cell activating factor is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for receptors TNFRSF13B/TACI,
  • TNFRSF 17 BCMA TNFRSF 13C BAFF-R.
  • This cytokine is expressed in B cell lineage cells, and acts as a potent B cell activator. It has been also shown to play an important role in the proliferation and differentiation of B cells.
  • anti-tumor effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure in prevention of the occurrence of tumor in the first place.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic or, alternatively, “allogenic,” refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • cancer refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g ., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
  • effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • a functional disruption refers to a physical or biochemical change to a specific (e.g., target) nucleic acid (e.g., gene, RNA transcript, of protein encoded thereby) that prevents its normal expression and/or behavior in the cell.
  • a functional disruption refers to a modification of the gene via a gene editing method.
  • a functional disruption prevents expression of a target gene (e.g., an endogenous gene).
  • transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTORTM gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g, 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • “Humanized” forms of non-human (e.g, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’) 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Human or “fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the present disclosure by standard techniques known in the art, such as site- directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g ., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g, threonine, valine, isoleucine
  • aromatic side chains e.g, tyrosine, phenylalanine, tryptophan, histidine
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • promoter refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • linker and “flexible polypeptide linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
  • the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3.
  • the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser). Also included within the scope of the present disclosure are linkers described in WO2012/138475 (incorporated herein by reference).
  • a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other.
  • RNA polymerase Shortly after the start of transcription, the 5’ end of the mRNA being synthesized is bound by a cap- synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • in vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
  • the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400.
  • Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • the poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
  • adenosine residues are added to the free 3’ end at the cleavage site.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
  • subject is intended to include living organisms in which an immune response can be elicited (e.g ., mammals, human).
  • a “substantially purified” cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • terapéutica as used herein means a treatment.
  • a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • prophylaxis means the prevention of or protective treatment for a disease or disease state.
  • tumor antigen or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
  • the hyperproliferative disorder antigens of the present disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “specifically binds,” refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., CD 19) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
  • a cognate binding partner e.g., CD 19
  • the term “meganuclease” refers to an endonuclease that binds double- stranded DNA at a recognition sequence that is greater than 12 base pairs.
  • the recognition sequence for a meganuclease of the present disclosure is 22 base pairs.
  • a meganuclease can be an endonuclease that is derived from I-Crel and can refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties.
  • Methods for producing such modified variants of I-Crel are known in the art (e.g.,
  • a meganuclease as used herein binds to double-stranded DNA as a heterodimer or as a "single-chain meganuclease" in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker.
  • the term "homing endonuclease” is synonymous with the term “meganuclease.”
  • Meganucleases of the present disclosure are substantially non-toxic when expressed in cells, particularly in human T cells, such that cells can be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.
  • single-chain meganuclease refers to a polypeptide comprising a pair of nuclease subunits joined by a linker.
  • a single-chain meganuclease has the organization: N-terminal subunit - Linker - C-terminal subunit.
  • the two meganuclease subunits will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences.
  • single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences.
  • a single-chain meganuclease may be referred to as a "single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric.
  • the term “meganuclease” can refer to a dimeric or single-chain meganuclease.
  • TALEN refers to an endonuclease comprising a DNA-binding domain comprising 16-22 TAL domain repeats fused to any portion of the Fokl nuclease domain.
  • Compact TALEN refers to an endonuclease comprising a DNA-binding domain with 16-22 TAL domain repeats fused in any orientation to any catalytically active portion of nuclease domain of the I-Tevl homing endonuclease.
  • CRISPR refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA.
  • megaTAL refers to a single-chain nuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequence- specific homing endonuclease.
  • TALE transcription activator-like effector
  • T cell receptor and “T cell receptor complex” are used interchangeably to refer to a molecule found on the surface of T cells that is, in general, responsible for recognizing antigens.
  • the TCR comprises a heterodimer consisting of a TCR alpha and TCR beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of TCR gamma and TCR delta chains.
  • the TCR further comprises one or more of CD3s, CD3y, and CD35.
  • the TCR comprises CD3e.
  • the TCR comprises CD3y.
  • the TCR comprises CD36.
  • the TCR comprises CD3z.
  • the constant domain of human TCR alpha has a sequence of SEQ ID NO: 142.
  • the constant domain of human TCR alpha has an IgC domain having a sequence of SEQ ID NO: 143, a transmembrane domain having a sequence of SEQ ID NO: 144, and an intracellular domain having a sequence of SS.
  • the constant domain of murine TCR alpha has a sequence of SEQ ID NO: 147.
  • the constant domain of murine TCR alpha has a transmembrane domain having a sequence of SEQ ID NO: 144, and an intracellular domain having a sequence of SS.
  • the constant domain of human TCR beta has a sequence of SEQ ID NO: 148.
  • the constant domain of human TCR beta has an IgC domain having a sequence of SEQ ID NO: 149, a transmembrane domain having a sequence of SEQ ID NO: 150, and an intracellular domain having a sequence of SEQ ID NO:
  • the constant domain of murine TCR beta has a sequence of SEQ ID NO: 152. In some embodiments, the constant domain of murine TCR beta has a transmembrane domain having a sequence of SEQ ID NO: 152, and an intracellular domain having a sequence of SEQ ID NO: 153. In some embodiments, the constant domain of human TCR delta has a sequence of SEQ ID NO: 243. In some embodiments, the constant domain of human TCR delta has an IgC domain having a sequence of SEQ ID NO: 265, a transmembrane domain having a sequence of SEQ ID NO: 159, and an intracellular domain having a sequence of L.
  • the constant domain of human TCR gamma has a sequence of SEQ ID NO: 21. In some embodiments, the constant domain of human TCR gamma has an IgC domain having a sequence of SEQ ID NO: 155, a transmembrane domain having a sequence of SEQ ID NO: 156, and an intracellular domain having a sequence of SEQ ID NO: 157. [0185] In some embodiments, human CD3 epsilon has a sequence of SEQ ID NO: 258.
  • human CD3 epsilon has an extracellular domain having a sequence of SEQ ID NO: 126, a transmembrane domain having a sequence of SEQ ID NO: 127, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 128.
  • human CD3 delta has a sequence of SEQ ID NO: 136.
  • human CD3 delta has an extracellular domain having a sequence of SEQ ID NO: 138, a transmembrane domain having a sequence of SEQ ID NO: 139, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 140.
  • human CD3 gamma has a sequence of SEQ ID NO: 130. In some embodiments, human CD3 gamma has an extracellular domain having a sequence of SEQ ID NO: 132, a transmembrane domain having a sequence of SEQ ID NO: 133, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 134.
  • Ranges throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
  • compositions of matter and methods of use for the treatment of a disease such as cancer using modified T cells comprising a T cell receptors (TCR) fusion protein (TFP and a TCR constant domain, wherein the modified T cell also has a functionally disrupted endogenous TCR subunit.
  • TCR T cell receptor
  • TCP T cell receptor
  • TCR constant domain TCR constant domain
  • TFPs provide substantial benefits as compared to Chimeric Antigen Receptors.
  • the term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a recombinant polypeptide comprising an extracellular antigen binding domain in the form of, e.g., a single domain antibody or scFv, a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as “intracellular signaling domains”) comprising a functional signaling domain derived from a stimulatory molecule as defined below.
  • the central intracellular signaling domain of a CAR is derived from the CD3 zeta chain that is normally found associated with the TCR complex.
  • the CD3 zeta signaling domain can be fused with one or more functional signaling domains derived from at least one co-stimulatory molecule such as 4-1BB (i.e., CD137), CD27 and/or CD28.
  • T cell receptor (TCR) fusion proteins (TFPs) T cell receptor (TCR) fusion proteins
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises a binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, wherein the sequence of the binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • a binding domain e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD 19, e.g., human CD 19, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to mesothelin, e.g., human mesothelin, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to MUC16, e.g., human MUC16, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD20, e.g., human CD20, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD70, e.g., human CD70, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD79B, e.g., human CD79B, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to HER2, e.g., human HER2, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to PSMA, e.g., human PSMA, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to BCMA, e.g., human BCMA, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to ROR1, e.g., human ROR1, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD22, e.g., human CD22, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to GPC3, e.g., human GPC3, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to Nectin-4, e.g. , human Nectin-4, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to Trop-2, e.g., human Trop-2, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
  • the TFP of the present disclosure comprises a target-specific binding element otherwise referred to as an antigen binding domain.
  • the choice of moiety depends upon the type and number of target antigen that define the surface of a target cell.
  • the antigen binding domain may be chosen to recognize a target antigen that acts as a cell surface marker on target cells associated with a particular disease state.
  • examples of cell surface markers that may act as target antigens for the antigen binding domain in a TFP of the present disclosure include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases ( e.g ., malignant diseases).
  • the TFP-mediated T cell response can be directed to an antigen of interest by way of engineering an antigen-binding domain into the TFP that specifically binds a desired antigen.
  • the antigen binding domain can be any domain that binds to the antigen including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of a camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticalin, DARPIN and the like.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH variable domain of a camelid derived nanobody
  • a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP.
  • the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment.
  • the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-CD 19 binding domain described herein, e.g., a humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.
  • the humanized or human anti-CD19 binding domain comprises one or more (e.g. , all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., the humanized or human anti-TAA binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
  • the humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein.
  • the humanized or human anti-TAA binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein.
  • the anti-TAA binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
  • a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g, substitutions) but not more than 30, 20 or 10 modifications (e.g, substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence
  • the humanized or human anti-TAA binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a linker, e.g, a linker described herein.
  • the humanized anti-TAA binding domain includes a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.
  • the light chain variable region and heavy chain variable region of a scFv can be, e.g, in any of the following orientations: light chain variable region -linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • the antigen-binding domain comprises an anti-CD19 humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment having a light chain CDR1 of SEQ ID NO:73, a CDR2 of SEQ ID NO:75, and a CDR3 of SEQ ID NO:77 and a heavy chain CDR1 of SEQ ID NO: 79, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO: 83.
  • the anti-CD 19 antibody is a murine scFv.
  • the anti-CD-19 antibody comprises a VL of SEQ ID NO:85 and a VH of SEQ ID NO:87 [0194]
  • the antigen-binding domain comprises an anti-mesothelin humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO 60, a CDR2 of SEQ ID NO:61, and a CDR3 of SEQ ID NO:62 or a CDR1 of SEQ ID NO 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65 or a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQ ID NO: 68.
  • the anti-mesothelin antibody has a variable domain of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.
  • the antigen-binding domain comprises an anti-CD70 humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO:88, a CDR2 of SEQ ID NO:89, and a CDR3 of SEQ ID NO:90, or a CDR1 of SEQ ID NO:92, a CDR2 of SEQ ID NO:93, and a CDR3 of SEQ ID NO:94, or a CDR1 of SEQ ID NO:96, a CDR2 of SEQ ID NO:97, and a CDR3 of SEQ ID NO:98, or a CDR1 of SEQ ID NO: 100, a CDR2 of SEQ ID NO: 101, and a CDR3 of SEQ ID NO: 102, or a CDR1 of SEQ ID NO: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 106, or a CDR1 of SEQ ID NO:
  • a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof.
  • the antigen binding domain is humanized.
  • a humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991,
  • framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
  • a humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity.
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (see, e.g., Nicholson et al., Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).
  • the framework region e.g., all four framework regions, of the heavy chain variable region are derived from a VH4-4-59 germline sequence.
  • the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
  • the framework region e.g., all four framework regions of the light chain variable region are derived from a VK3-1.25 germline sequence.
  • the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
  • the portion of a TFP composition of the present disclosure that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties.
  • humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • a humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g., in the present disclosure, the ability to bind human a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to, e.g., human CD 19, human BCMA, or another tumor associated antigen.
  • the binding domain is characterized by particular functional features or properties of an antibody or antibody fragment.
  • the portion of a TFP composition of the present disclosure that comprises an antigen binding domain specifically binds human CD 19.
  • the antigen binding domain has the same or a similar binding specificity to human CD19 as the FMC63 scFv described in Nicholson et al., Mol. Immun. 34 (16-17): 1157-1165 (1997).
  • the present disclosure relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a CD 19 or BCMA protein or fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and/or a variable heavy chain that includes an amino acid sequence provided herein.
  • the scFv is contiguous with and in the same reading frame as a leader sequence.
  • the anti -tumor-associated antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv).
  • the anti-TAA binding domain is a Fv, a Fab, a (Fab’)2, or a bi-functional (e.g. bi-specific) hybrid antibody (e.g, Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).
  • the antibodies and fragments thereof of the present disclosure binds a CD 19 protein with wild-type or enhanced affinity.
  • the anti- TAA binding domain comprises a single domain antibody (sdAb or VHH).
  • a target antigen e.g., CD 19, BCMA or any target antigen described elsewhere herein for targets of fusion moiety binding domains
  • the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino
  • V H domains and scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et ah, (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • scFv molecules can be produced by linking V H and V L regions together using flexible polypeptide linkers.
  • the scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact.
  • the linker sequence comprises a linker sequence.
  • An scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its V L and V H regions.
  • the linker sequence may comprise any naturally occurring amino acid.
  • the linker sequence comprises amino acids glycine and serine.
  • the linker sequence comprises sets of glycine and serine repeats such as (Gly 4 Ser) n , where n is a positive integer equal to or greater than 1.
  • the linker can be (Gly4Ser)4 or (Gly4Ser)3. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • a tumor associated antigen binding domain e.g., scFv molecules (e.g., soluble scFv)
  • scFv molecules e.g., soluble scFv
  • biophysical properties e.g, thermal stability
  • the humanized or human scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a parent scFv in the described assays.
  • the improved thermal stability of the anti-TAA binding domain e.g., scFv is subsequently conferred to the entire TAA-TFP construct, leading to improved therapeutic properties of the anti-TAA TFP construct.
  • the thermal stability of the binding domain e.g., scFv or sdAb, can be improved by at least about 2 °C or 3 °C as compared to a conventional antibody.
  • the binding domain has a 1 °C improved thermal stability as compared to a conventional antibody.
  • the binding domain has a 2 °C improved thermal stability as compared to a conventional antibody.
  • the scFv has a 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, or 15 °C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described in more detail below.
  • the anti-TAA binding domain e.g., scFv or sdAb
  • the anti-TAA binding domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv or sdAb confers improved stability to the TAA-TFP construct.
  • the antigen binding domain of the TFP comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-tumor-associated antigen antibody fragments described herein.
  • the TFP composition of the present disclosure comprises an antibody fragment.
  • that antibody fragment comprises a scFv.
  • the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g, VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions.
  • the TFP composition of the present disclosure comprises an antibody fragment.
  • that antibody fragment comprises a scFv.
  • the antibody or antibody fragment of the present disclosure may further be modified such that they vary in amino acid sequence (e.g ., from wild-type), but not in desired activity.
  • additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein.
  • a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g, aspartic acid, gluta
  • Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g, 60% identity, optionally 70%, 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ah, (1977 )Nuc. Acids Res. 25:3389-3402; and Altschul et ah, (1990) J. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • the present disclosure contemplates modifications of the starting antibody or fragment (e.g., scFv) amino acid sequence that generate functionally equivalent molecules.
  • the VH or VL of a binding domain, e.g., scFv, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the present disclosure contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules.
  • the TFP construct can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain.
  • An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
  • the extracellular domain is a TCR extracellular domain.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the TCR extracellular domain comprises an IgC domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the extracellular domain comprises a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma.
  • the extracellular domain comprises a sequence encoding an IgC domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain can be a TCR extracellular domain.
  • the TCR extracellular domain can be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit or a CD3 delta TCR subunit.
  • the extracellular domain can be a full-length TCR extracellular domain or fragment (e.g., functional fragment) thereof.
  • the extracellular domain can comprise a variable domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a variable domain and a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. In some cases, the extracellular domain may not comprise a variable domain.
  • the extracellular domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a fragment (e.g., functional fragment) of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
  • the TCR chain can be a murine or human TCR chain.
  • the extracellular domain can comprise a constant domain of a murine TCR alpha chain, a murine TCR beta chain, a human TCR gamma chain or a human TCR delta chain.
  • a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence.
  • a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3,
  • the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another TFP on the TFP- T cell surface.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target.
  • the TCR- integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the transmembrane domain comprises, or comprises at least 5, 6,
  • TCR alpha chain 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more consecutive amino acid residues of the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the transmembrane domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.
  • the transmembrane domain can be attached to the extracellular region of the TTP, e.g., the antigen binding domain of the TFP, via a hinge, e.g., a hinge from a human protein.
  • a hinge e.g., a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • a short oligo- or polypeptide linker may form the linkage between the binding element and the TCR extracellular domain of the TFP.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length.
  • the linker comprises the amino acid sequence of GGGGSGGGGS or a sequence (GGGGS)x wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more.
  • X is 2. In some embodiments, X is 4. In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC. Cytoplasmic Domain
  • the cytoplasmic domain of the TFP can include an intracellular domain.
  • the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain.
  • An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more consecutive amino acid residues of the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain.
  • the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain.
  • the transmembrane domain comprises a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the intracellular domain comprises, or comprises at least 5, 6, 7, 8,
  • the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).
  • primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine- based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine- based activation motifs
  • ITAMs containing primary intracellular signaling domains include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • a TFP of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 -epsilon.
  • a primary signaling domain comprises a modified IT AM domain, e.g., a mutated IT AM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
  • a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g ., an optimized and/or truncated ITAM-containing primary intracellular signaling domain.
  • a primary signaling domain comprises one, two, three, four or more ITAM motifs.
  • the intracellular signaling domain of the TFP can comprise a CD3 signaling domain, e.g., CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure.
  • the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain.
  • the costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al., Blood. 2012;
  • the intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8,
  • the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another.
  • a cell expressing a first and second TFP can have an antigen binding domain of the first TFP, e.g., as a fragment, e.g., a scFv, that does not form an association with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a VHH.
  • the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a modified T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., PD1
  • inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, LAG3, CTLA4,
  • a costimulatory domain e.g, 4- 1BB, CD27 or CD28, e.g., as described herein
  • a primary signaling domain e.g., a CD3 zeta signaling domain described herein.
  • the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g, at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g, a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
  • PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.
  • PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al., 1996, Ini. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al., 2000./. Exp. Med.
  • PD-L1 is abundant in human cancers (Dong et al., 2003 J. Mol. Med. 81:281-7;
  • Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.
  • the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP).
  • the PD1 TFP when used in combinations with an anti-TAA TFP described herein, improves the persistence of the T cell.
  • the TFP is a PD1 TFP comprising the extracellular domain of PD 1.
  • TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death-Ligand 1 (PD-L1) or Programmed Death-Ligand 2 (PD-L2).
  • the present disclosure provides a population of TFP-expressing T cells, e.g., FFP-T cells.
  • the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs.
  • the population of TFP-T cells can include a first cell expressing a TFP having a binding domain described herein, and a second cell expressing a TFP having a different anti-TAA binding domain, e.g., a binding domain described herein that differs from the binding domain in the TFP expressed by the first cell.
  • the population of TFP-expressing cells can include a first cell expressing a TFP that includes a first binding domain binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than the binding domain of the first cell (e.g., another tumor-associated antigen).
  • the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a modified T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD- L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta.
  • the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • RNA encoding TFPs include methods for producing in vitro transcribed RNA encoding TFPs.
  • the present disclosure also includes a TFP encoding RNA construct that can be directly transfected into a cell.
  • a method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”), a 5’ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
  • RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP.
  • the anti-TAA TFP is encoded by a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA encoding the anti-TAA TFP is introduced into a T cell for production of a TFP-T cell.
  • the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
  • the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • PCR polymerase chain reaction
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired template for in vitro transcription is a TFP of the present disclosure.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs).
  • the nucleic acid can include exons and introns.
  • the DNA to be used for PCR is a human nucleic acid sequence.
  • the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and 3’ UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein.
  • the portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection.
  • Methods for performing PCR are well known in the art.
  • Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
  • “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
  • the primers can be designed to be substantially complementary to any portion of the DNA template.
  • the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs.
  • the primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest.
  • the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs.
  • Primers useful for PCR can be generated by synthetic methods that are well known in the art.
  • “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
  • Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
  • reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
  • Downstream is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.
  • Any DNA polymerase useful for PCR can be used in the methods disclosed herein.
  • the reagents and polymerase are commercially available from a number of sources.
  • the RNA preferably has 5’ and 3’ UTRs.
  • the 5’ UTR is between one and 3000 nucleotides in length.
  • the length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest.
  • UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3’UTR sequences can decrease the stability of mRNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid.
  • a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts but do not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • a circular DNA template for instance, plasmid DNA
  • RNA polymerase produces a long concatemeric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3 ’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res. , 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem ., 270:1485-65 (2003).
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a poly-T tail, such as 100 T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP).
  • E-PAP E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods disclosed herein include a 5’ cap.
  • the 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et ah, RNA, 7:1468-95 (2001); Elango, et ah, Biochim. Biophys. Res. Commun ., 330:958-966 (2005)).
  • RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector®-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser® II (BioRad, Denver, Colo.), Multiporator® (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al.
  • TFP T cell receptor
  • TCR T cell receptor
  • TCR subunit comprising at least a portion of a TCR extracellular domain.
  • TCR subunit can further comprise a transmembrane domain.
  • the TCR subunit can further comprise an intracellular domain of TCR gamma, TCR delta, TCR alpha or TCR beta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta.
  • the TFP can further comprise an antibody (e.g., a human, humanized, or murine antibody) comprising an antigen binding domain.
  • the recombinant nucleic acid molecule can further comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR subunit and the antibody can be operatively linked.
  • the TFP can functionally incorporate into a TCR complex (e g., an endogenous TCR complex) when expressed in a T cell.
  • the constant domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain
  • the constant domain can comprise a full- length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise a fragment (e.g., functional fragment) of the full- length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can further encode the transmembrane domain and/or intracellular region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can encode a full-length constant region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant region of a TCR chain can comprise a constant domain, a transmembrane domain, and a intracellular region.
  • the constant region of a TCR chain can also exclude the transmembrane domain and the intracellular region of the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
  • the TCR chain can be a murine or human TCR chain.
  • the constant domain can comprise a constant domain of a murine or human TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain.
  • the constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO:143, SEQ ID NO: 146, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO:155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO:155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:
  • SEQ ID NO: 20 SEQ ID NO: 21, or SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:243 or SEQ ID NO:265.
  • the murine TCR alpha constant domain can comprise positions 2-137 of SEQ ID NO: 146.
  • the murine TCR alpha constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of positions 2-137 of SEQ ID NO: 146
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise a sequence or fragment thereof of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of positions 2-137 of SEQ ID NO: 146.
  • the murine TCR beta constant domain can comprise positions 2-173 of SEQ ID NO:152.
  • the murine TCR beta constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of positions 2-173 of SEQ ID NO: 152.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of positions 2-173 of SEQ ID NO: 152.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of positions 2-173 of SEQ ID NO:152.
  • the constant domain can comprise a sequence or fragment thereof of positions 22-173 of SEQ ID NO: 152.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6,
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of positions 2-173 of SEQ ID NO:152.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of positions 2-173 of SEQ ID NO: 152.
  • the TCR constant domain is a TCR delta constant domain.
  • the TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID N0 265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modification.
  • the TCR delta constant domain can comprise SEQ ID NO:243.
  • the TCR delta constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:243.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:243.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:243.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:243.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:243.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:243.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:243.
  • the TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding a TCR delta constant domain further encodes a TCR delta variable domain, thereby encoding a full TCR delta domain.
  • the full TCR delta domain can be delta 2 or delta 1.
  • the full TCR delta constant domain can comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the full TCR delta domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the delta domain can comprise a truncated version of a delta domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:256.
  • the delta domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:256.
  • the delta domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:256.
  • the delta domain can comprise a sequence or fragment thereof of SEQ ID NO:256.
  • the delta domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:256.
  • the delta domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
  • the delta domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID N0 256 [0269]
  • the TCR gamma constant domain can comprise SEQ ID NO:21.
  • the TCR gamma constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:21.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:21.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:21.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:21.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:21.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:21.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 243.
  • the TCR gamma constant domain can comprise SEQ ID NO:21 or SEQ ID NO:155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR gamma constant domain further encodes a TCR gamma variable domain, thereby encoding a full TCR gamma domain.
  • the full TCR gamma domain can be gamma 9 or gamma 4.
  • the full TCR gamma domain can comprise SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the full TCR gamma domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the gamma domain can comprise a truncated version of a gamma domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:255.
  • the gamma domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:255.
  • the gamma domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:255.
  • the gamma domain can comprise a sequence or fragment thereof of SEQ ID NO:255.
  • the gamma domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or gamma of the sequence of SEQ ED NO:255.
  • the gamma domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ED NO:255.
  • the gamma domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 255.
  • TCR beta chain (Homo sapiens):
  • the murine TCR beta chain constant region canonical sequence is:
  • TCR alpha constant region (Mus musculus) (or [mm]TRAC(82-137)): ATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 17).
  • the murine TCR alpha chain constant (mTRAC) region canonical sequence is: XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSN GAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILL LKVAGFNLLMTLRLW S S (SEQ ID NO: 146).
  • TCR beta constant region (Mus musculus) (or [mm]TRBCl(123-173)):
  • GRADCGITS AS YQQGVLSATILYEILLGKATLY AVLV STLVVMAMVKRKN S (SEQ ID NO: 18).
  • the murine TCR beta chain constant region canonical sequence is:
  • TCR beta chain (Homo sapiens):
  • TCR delta constant region version 1 (Homo sapiens):
  • TCR gamma constant region (Homo sapiens) (or [hs]TRGC(l-173)): DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN C SKD ANDTLLLQLTNT S A YYM YLLLLLK S V VYF AIITC CLLRRT AF CCN GEK S (SEQ ID NO: 21).
  • the TCR constant domain is a TCR delta constant domain.
  • the sequence encoding the TCR delta constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR delta constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the TCR constant domain is a TCR gamma constant domain.
  • the sequence encoding the TCR gamma constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR gamma constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the recombinant nucleic acid comprise a sequence encoding a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR gamma constant domain can comprise SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR gamma constant domain can further encode a TCR gamma variable domain, thereby encoding a full TCR gamma domain
  • the TCR gamma domain can be gamma 9 or gamma 4.
  • the full TCR gamma domain comprises SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR delta constant domain can further encode a TCR delta variable domain, thereby encoding a full TCR delta domain.
  • the TCR delta domain can be delta 2 or delta 1.
  • the full TCR delta domain can comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell.
  • the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within different nucleic acid molecules.
  • the sequence can further encode a cleavage site (e.g., a protease cleavage site) between the encoded TFP and the TCR constant domain.
  • the cleavage site can be a protease cleavage site.
  • the cleavage site can be a self cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.
  • the cleavage site can comprise a sequence of SEQ ID NO: 23.
  • T2A cleavage site EGRGSLLT C GD VEENPGP (SEQ ID NO: 23).
  • the TCR subunit of the TFP and the constant domain can comprise a sequence derived from a same TCR chain or a different TCR chain. In some cases, the TCR subunit of the TFP and the constant domain are derived from different TCR chains.
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR alpha chain, and the constant domain can comprise a constant domain of a TCR beta chain
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR beta chain, and the constant domain can comprise a constant domain of a TCR alpha chain.
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR gamma chain, and the constant domain can comprise a constant domain of a TCR delta chain.
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR delta chain, and the constant domain can comprise a constant domain of a TCR gamma chain.
  • the TCR subunit and the antibody domain, the antigen domain or the binding ligand or fragment thereof are operatively linked by a linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR gamma, TCR delta, TCR alpha or TCR beta.
  • the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR gamma, only TCR delta, only TCR alpha or only TCR beta.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two or all of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD 134, CD 137, CD 154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a TCR intracellular domain of TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or a fragment thereof.
  • the TCR subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit can comprise (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain of a TCR gamma chain or a TCR delta chain.
  • the TCR extracellular domain can comprise the extracellular portion of a constant domain of a TCR gamma chain or a TCR delta chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a delta constant domain, or a fragment thereof, e.g., a delta constant domain described herein.
  • the delta constant domain can have the sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • thhe TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a gamma constant domain, e.g., a gamma constant domain described herein.
  • the gamma constant domain can have the sequence of SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the extracellular domain of the TFP may not comprise the variable domain of a gamma chain or a delta chain.
  • the TCR subunit of the TFP can comprise the extracellular, transmembrane and intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta.
  • recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon, , CD3 gamma, or CD3 delta and the constant domains of TCR beta and TCR alpha.
  • recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon and the constant domains of TCR gamma and TCR delta.
  • recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon and full length TCF gamma and full length TCR delta.
  • the TCR subunit of the TFP comprises CD3 epsilon.
  • the TCR subunit of CD3 epsilon can comprise the sequence of SEQ ID NO:258 functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprising at least a portion of a murine TCR alpha or murine TCR beta extracellular domain and a murine TCR alpha or murine TCR beta transmembrane domain is or comprises a TCR alpha constant domain or a TCR beta constant domain.
  • the TCR subunit can comprise an intracellular domain of murine TCR alpha or murine TCR beta.
  • the TCR constant domain can be a TCR alpha constant domain, e.g., a TCR alpha constant domain described herein.
  • the TCR alpha constant domain can comprise SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO:207, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR alpha constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR alpha constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the TCR alpha constant domain can comprise a murine TCR alpha constant domain.
  • the murine TCR alpha constant domain can comprise amino acids 2-137 of the murine TCR alpha constant domain.
  • the murine TCR alpha constant domain can comprise amino acids 2-137 of SEQ ID NO: 146.
  • the murine TCR alpha constant domain can comprise a sequence of SEQ ID NO:207.
  • the murine TCR alpha constant domain can comprise amino acids 82-137 of SEQ ID NO: 146.
  • the murine TCR alpha constant domain comprises a sequence of SEQ ID NO: 17.
  • the TCR constant domain can be a TCR beta constant domain, e.g., a TCR beta constant domain described herein.
  • the TCR beta constant domain can comprise SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR beta constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR beta constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • TCR beta constant domain can comprise a murine TCR beta constant domain.
  • the murine TCR beta constant domain can comprise amino acids 2-173 of the murine TCR beta constant domain.
  • the murine TCR beta constant domain can comprise amino acids 2- 173 of SEQ ID NO: 152.
  • the murine TCR beta constant domain can comprise SEQ ID NO:209.
  • the TCR beta constant domain can comprise amino acids 123-173 of SEQ ID NO: 152.
  • the TCR beta constant domain can comprise SEQ ID NO: 18
  • the recombinant nucleic acid can comprise sequence encoding a TCR alpha constant domain and a TCR beta constant domain.
  • the TCR alpha constant domain can comprise SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO:207, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR beta constant domain can comprise SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the intracellular signaling domain can be CD3 epsilon, CD3 gamma, or CD3 delta
  • the intracellular signaling domain can be CD3 epsilon.
  • the sequence encoding the TCR constant domain can comprise from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5 ’-3’, a first leader sequence, a TRAC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRBC gene sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence.
  • the at least one but not more than 20 modifications thereto of a sequence described herein can comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the TCR subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, IC AM- 1 , LFA-1 (CDlla/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
  • the TCR subunit comprises an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3 a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD
  • IT AM immunorecept
  • the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the TFP, the TCR gamma constant domain, the TCR delta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR delta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof
  • the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof
  • the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the antibody or antigen binding domain can be an antibody fragment.
  • the antibody or antigen binding domain can be murine, human or humanized.
  • the human or humanized antibody is an antibody fragment.
  • the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain.
  • human or humanized antibody comprising an antigen binding domain is selected from a group consisting of an anti-CD 19 binding domain, anti-B-cell maturation antigen (BCMA) binding domain, anti- mesothelin (MSLN) binding domain, anti-CD22 binding domain, anti -PD- 1 binding domain, anti-BAFF orBAFF receptor binding domain, and anti-ROR-1 binding domain.
  • An antigen binding domain described herein can be selected from a group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain, an anti-HER2 binding domain, an anti-PMSA binding domain, an anti-MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti BAFF or BAFF receptor binding domain, an anti-Nectin-4 binding domain, an anti-TROP-2 binding domain, an anti-GPC3 binding domain, and anti-ROR-1 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some instances, the nucleic acid is an mRNA. In some instances, the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2’-0-methyl, 2’-0-methoxyethyl (2’-0-MOE), 2’-0- aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-0-aminopropyl (2’-0-AP), 2-0- dimethylaminoethyl (2’-0-DMAOE), 2’-0-dimethylaminopropyl (2’-0-DMAP), T-O- dimethylaminoethyloxyethyl (2’-0-DMAEOE), 2’-0-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thio
  • the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR beta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain.
  • TCR T cell receptor
  • TFP T cell receptor
  • TCR subunit comprising at least a portion of a TCR extracellular domain.
  • the TCR subunit can further comprise a transmembrane domain.
  • the TCR subunit can further comprise an intracellular domain of TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or a fragment thereof.
  • the TCR subunit comprises an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or comprises an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta.
  • the TFP can further comprise a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof.
  • the recombinant nucleic acid molecule can comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the binding ligand or fragment thereof are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a T cell.
  • the binding ligand is capable of binding an Fc domain of the antibody.
  • the binding ligand is capable of selectively binding an IgGl antibody. In some instances, the binding ligand is capable of specifically binding an IgGl antibody. In some instances, the antibody or fragment thereof binds to a cell surface antigen. In some instances, the antibody or fragment thereof binds to a cell surface antigen on the surface of a tumor cell. In some instances, the binding ligand comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer. In some instances, the binding ligand does not comprise an antibody or fragment thereof. In some instances, the binding ligand comprises a CD 16 polypeptide or fragment thereof. In some instances, the binding ligand comprises a CD 16-binding polypeptide.
  • the binding ligand is human or humanized.
  • the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof capable of being bound by the binding ligand.
  • the antibody or fragment thereof is capable of being secreted from a cell.
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some instances, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell. In some instances, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule. In some instances, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within different nucleic acid molecules.
  • the TCR subunit and the antibody domain, the antigen domain or the binding ligand or fragment thereof are operatively linked by a linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR alpha or only TCR beta.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD 134, CD 137, CD 154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, IC AM- 1 , LFA-1 (CDlla/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
  • the TCR subunit comprises an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66
  • IT AM immunorecept
  • the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the TFP, the TCR gamma constant domain, the TCR delta constant domain, the TCR alpha constant domain, the TCR beta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR beta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR alpha, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR alpha constant domain and the TFP functionally integrates into a TCR complex comprising
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the human or humanized antibody is an antibody fragment.
  • the antibody fragment is a scFv, a single domain antibody domain (sdAb), a VH domain or a VL domain.
  • human or humanized antibody comprising an antigen binding domain is selected from a group consisting of an anti-CD 19 binding domain, anti-B-cell maturation antigen (BCMA) binding domain, anti-mesothelin (MSLN) binding domain, anti- CD22 binding domain, anti-PD-1 binding domain, anti PD-L1 binding domain, anti IL13Ra2 binding domain, anti-BAFF or BAFFR binding domain, and anti-ROR-1 binding domain.
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some instances, the nucleic acid is an mRNA. In some instances, the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2’-0-methyl, 2’-0-methoxyethyl (2’-0-M0E), 2’-0- aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-0-aminopropyl (2’-0-AP), 2-0- dimethylaminoethyl (2’-0-DMAOE), 2’-0-dimethylaminopropyl (2’-0-DMAP), T-O- dimethylaminoethyloxyethyl (2’-0-DMAEOE), 2’-0-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiol,
  • the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR beta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain. Alternatively, the recombinant nucleic acid comprises a sequence encoding a TCR gamma or TCR delta domain, e.g., a transmembrane domain.
  • TCR T cell receptor
  • TFP T cell receptor
  • TCR subunit TCR subunit
  • TCR extracellular domain TCR extracellular domain
  • TCR subunit can further comprise a transmembrane domain.
  • the TCR subunit can further comprise an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta
  • the TFP can comprise an antigen domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell.
  • the recombinant nucleic acid molecule can comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antigen domain are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a T cell.
  • the antigen domain comprises a ligand.
  • the ligand binds to the receptor of a cell.
  • the ligand binds to the polypeptide expressed on a surface of a cell.
  • the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide.
  • the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4 and combinations thereof.
  • the antigen domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the antigen domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the ligand or fragment thereof is a monomer or a dimer. In some instances, the antigen domain does not comprise an antibody or fragment thereof. In some instances, the antigen domain does not comprise a variable region. In some instances, the antigen domain does not comprise a CDR. In some instances, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • NVG2D Natural Killer Group 2D
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some instances, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell. In some instances, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule. In some instances, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within different nucleic acid molecules.
  • the TCR subunit and the antibody domain, the antigen domain or the binding ligand or fragment thereof are operatively linked by a linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR delta, or TCR gamma.
  • the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR alpha, only TCR beta, only TCR delta, or only TCR gamma.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR delta chain, a TCR gamma chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR delta chain, a TCR gamma chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a TCR intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta. In some instances, the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, IC AM- 1 , LFA-1 (CDlla/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
  • the TCR subunit comprises an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66
  • IT AM immunorecept
  • the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the TFP, the TCR gamma constant domain, the TCR delta constant domain, the TCR alpha constant domain, the TCR beta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR beta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR alpha constant domain and the TFP functionally integrates into a TCR complex
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the human or humanized antibody is an antibody fragment.
  • the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain.
  • human or humanized antibody comprising an antigen binding domain is selected from a group consisting of an anti-CD 19 binding domain, anti-CD20 binding domain, anti-mesothelin binding domain, anti-PMSA binding domain, anti-CD70 binding domain, anti-CD79b binding domain, anti-MUC16 binding domain, anti-anti -B-cell maturation antigen (BCMA) binding domain, anti-mesothelin (MSLN) binding domain, anti-IL13Ra2 binding domain, anti-CD22 binding domain, anti-BAFF or anti-BAFFR binding domain, anti- PD-1 binding domain, anti-PD-Ll binding domain, and anti-ROR-1 binding domain.
  • BCMA anti-mesothelin
  • MSLN anti-mesothelin
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some instances, the nucleic acid is an mRNA. In some instances, the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2’-0-methyl, 2’-0-methoxyethyl (2’-0-MOE), 2’-0- aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-0-aminopropyl (2’-0-AP), 2-0- dimethylaminoethyl (2’-0-DMAOE), 2’-0-dimethylaminopropyl (2’-0-DMAP), T-O- dimethylaminoethyloxyethyl (2’-0-DMAEOE), 2’-0-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thio
  • the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR beta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR gamma transmembrane domain.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR delta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR gamma transmembrane domain and a sequence encoding a TCR delta transmembrane domain.
  • vectors comprising the recombinant nucleic acid disclosed herein.
  • the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector is an AAV6 vector.
  • the vector further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • the gene of interest can be produced synthetically, rather than cloned.
  • the present disclosure also provides vectors in which a DNA of the present disclosure is inserted.
  • Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35).
  • the expression of nucleic acids encoding TFPs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below lune et al. 2009 Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference.
  • the expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties).
  • the present disclosure provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, ( e.g ., WO 01/96584; WO 01/29058; and U.S. Pat. No.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EF la promoter.
  • the native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009)).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTY), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTY mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • inducible promoters are also contemplated as part of the present disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic- resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82) Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection [0357] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine- nucleic acid complexes are also contemplated.
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELIS As and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
  • moleukin assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELIS As and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
  • the present disclosure further provides a vector comprising a TFP encoding nucleic acid molecule.
  • a TFP vector can be directly transduced into a cell, e.g., a T cell
  • the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, mini circles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs.
  • the vector is capable of expressing the TFP construct in mammalian T cells.
  • the mammalian T cell is a human T cell.
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and (ii) an antibody comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TCR subunit and the antibody
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to a sequence encoding a delta constant domain.
  • the intracellular domain is an intracellular domain of TCR gamma.
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to a sequence encoding a gamma constant domain.
  • the intracellular domain is an intracellular domain of TCR delta.
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to both a sequence encoding a TCR delta constant domain or fragment thereof and a TCR gamma constant domain or fragment thereof.
  • the intracellular signaling domain is CD3 epsilon, CD3 gamma, or CD3 delta. In some embodiments, the intracellular signaling domain is CD3 epsilon. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, the recombinant nucleic acid further comprises a portion of a TCR alpha constant domain, a portion of a TCR beta domain, or both.
  • the sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRGC gene sequence. In some embodiments, the sequence comprises, from 5’ -3’, a first leader sequence, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker sequence, and a TRGC gene sequence.
  • the sequence comprises, from 5 ’-3’, a first leader sequence, an antigen binding domain sequence, a first linker sequence, a TRDC gene sequence, a cleavable linker, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, and a TRGC gene sequence.
  • the sequence comprises, from 5 ’-3’, a first leader sequence, a TRDC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRGC gene sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence.
  • the sequence comprises, from 5 ’-3’, a first leader sequence, a first antigen binding domain sequence, a first linker sequence, a TRDC gene sequence or fragment thereof, a TRAC gene sequence or fragment thereof, a cleavable linker sequence, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, a TRGC gene sequence or fragment thereof, and a TRBC gene sequence or fragment thereof.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:l.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:2.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:3.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO:4. In some embodiments, the sequence encodes the polypeptide as set forth in SEQ ID NO:5. In some embodiments, the binding ligand is capable of binding an Fc domain of the antibody. In some embodiments, the binding ligand is capable of selectively binding an IgGl antibody. In some embodiments, the binding ligand is capable of specifically binding an IgG4 antibody. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen In some embodiments, the antibody or fragment thereof is murine, human or humanized. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen on the surface of a tumor cell.
  • the binding ligand comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer. In some embodiments, the binding ligand does not comprise an antibody or fragment thereof. In some embodiments, the binding ligand comprises a CD 16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD 16-binding polypeptide. In some embodiments, the binding ligand is human or humanized.
  • the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof capable of being bound by the binding ligand.
  • the antibody or fragment thereof is capable of being secreted from a cell.
  • a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and (ii) an antigen binding domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and
  • the recombinant nucleic acid further comprises at least a partial sequence encoding a TCR alpha constant domain, a TCR beta constant domain, or a partial sequence of both a TCR alpha constant domain and a TCR beta constant domain.
  • the antigen binding domain comprises a ligand.
  • the ligand binds to the receptor of a cell.
  • the ligand binds to the polypeptide expressed on a surface of a cell.
  • the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide.
  • the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4 and combinations thereof.
  • the antigen binding domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the antigen binding domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the ligand or fragment thereof is a monomer or a dimer.
  • the antigen binding domain does not comprise an antibody or fragment thereof.
  • the antigen binding domain does not comprise a variable region.
  • the antigen binding domain does not comprise a CDR.
  • the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell.
  • the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within different nucleic acid molecules.
  • the TCR subunit and the antibody domain, the antigen binding domain or the binding ligand or fragment thereof are operatively linked by a linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR delta, or TCR gamma.
  • the encoded intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR alpha, TCR beta, TCR gamma, or TCR delta
  • the encoded TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR extracellular domain comprises a constant domain or a portion thereof of a TCR gamma chain or a TCR delta chain.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137,
  • the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit further comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
  • the TCR subunit comprises an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional
  • IT AM
  • the IT AM replaces an IT AM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the IT AM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the TFP, the TCR gamma constant domain, the TCR delta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR delta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the antibody is an antibody fragment.
  • the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain.
  • an antigen binding domain is selected from a group consisting of an anti-CD 19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti- 79b binding domain, an anti-HER2 binding domain, an anti-PMSA binding domain, an anti- MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti BAFF or BAFF receptor binding domain, and anti-ROR-1 binding domain.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR beta transmembrane domain. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain.
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and (ii) an antibody or fragment thereof comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain or a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TFP
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain or a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain; wherein the TCR subunit and the binding lig
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to a sequence encoding an alpha constant domain.
  • the intracellular domain is an intracellular domain of TCR beta.
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to a sequence encoding a beta constant domain.
  • the intracellular domain is an intracellular domain of TCR alpha.
  • the sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO: 10.
  • the recombinant nucleic acid further comprises at least one leader sequence and at least one linker.
  • the binding ligand is capable of binding an Fc domain of the antibody.
  • the binding ligand is capable of selectively binding an IgGl antibody.
  • the binding ligand is capable of specifically binding an IgG4 antibody.
  • the antibody or fragment thereof binds to a cell surface antigen.
  • the antibody or fragment thereof is murine, human or humanized.
  • the antibody or fragment thereof binds to a cell surface antigen on the surface of a tumor cell.
  • the binding ligand comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer. In some embodiments, the binding ligand does not comprise an antibody or fragment thereof. In some embodiments, the binding ligand comprises a CD 16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD 16-binding polypeptide. In some embodiments, the binding ligand is human or humanized.
  • the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof capable of being bound by the binding ligand.
  • the antibody or fragment thereof is capable of being secreted from a cell.
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and (ii) an antigen binding domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain or a TCR beta constant domain or a TCR alpha constant domain and a TCR
  • the TCR constant domain is a murine TCR constant domain, e.g., a murine TCR alpha constant domain or a murine TCR beta constant domain or a murine TCR alpha constant domain and a murine TCR beta constant domain.
  • the extracellular domain comprises at least a portion of a TCR alpha extracellular domain or TCR beta extracellular domain.
  • the TCR alpha extracellular domain or TCR beta extracellular domain is a murine TCR alpha extracellular domain or a murine TCR beta extracellular domain.
  • the extracellular domain comprises at least a portion of a TCR alpha constant domain or TCR beta constant domain.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain. In some embodiments, the recombinant nucleic acid further comprises at least a partial sequence encoding a TCR gamma constant domain, a TCR delta constant domain, or a partial sequence of both a TCR gamma constant domain and a TCR delta constant domain.
  • the antigen binding domain comprises a ligand. In some embodiments, the ligand binds to the receptor of a cell. In some embodiments, the ligand binds to the polypeptide expressed on a surface of a cell.
  • the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide.
  • the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4 and combinations thereof.
  • the antigen binding domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer. In some embodiments, the antigen binding domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer. In some embodiments, the ligand or fragment thereof is a monomer or a dimer. In some embodiments, the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not comprise a variable region. In some embodiments, the antigen binding domain does not comprise a CDR. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • NSG2D Natural Killer Group 2D
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some embodiments, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within different nucleic acid molecules. In some embodiments, the TCR subunit and the antibody domain, the antigen binding domain or the binding ligand or fragment thereof are operatively linked by a linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR delta, or TCR gamma.
  • the encoded intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR alpha, TCR beta, TCR gamma, or TCR delta
  • the encoded TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit further comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2,
  • the TCR subunit comprises an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b
  • IT AM immunoreceptor tyrosine-based activation motif
  • the IT AM replaces an IT AM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the IT AM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the TFP, the TCR alpha constant domain, the TCR beta domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR alpha constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR alpha constant domain and a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, (2) a murine TCR alpha or murine TCR beta transmembrane domain, and (3) an intracellular domain of murine TCR alpha or murine TCR beta, and (ii) an antibody or fragment thereof comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain or a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the antibody are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a modified T cell
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, (2) a murine TCR alpha or murine TCR beta transmembrane domain, and (3) an intracellular domain of murine TCR alpha or murine TCR beta, and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain or a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the binding ligand or fragment thereof are operatively linked, and wherein the TFP functionally
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to a sequence encoding an alpha constant domain.
  • the intracellular domain is an intracellular domain of TCR beta.
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to a sequence encoding a beta constant domain.
  • the intracellular domain is an intracellular domain of TCR alpha.
  • the sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence encodes the polypeptide as set forth in SEQ ID NO: 10.
  • the recombinant nucleic acid further comprises at least one leader sequence and at least one linker.
  • the binding ligand is capable of binding an Fc domain of the antibody.
  • the binding ligand is capable of selectively binding an IgGl antibody.
  • the binding ligand is capable of specifically binding an IgG4 antibody.
  • the antibody or fragment thereof binds to a cell surface antigen.
  • the antibody or fragment thereof is murine, human or humanized.
  • the antibody or fragment thereof binds to a cell surface antigen on the surface of a tumor cell.
  • the binding ligand comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer. In some embodiments, the binding ligand does not comprise an antibody or fragment thereof. In some embodiments, the binding ligand comprises a CD 16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD 16-binding polypeptide. In some embodiments, the binding ligand is human or humanized.
  • the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof capable of being bound by the binding ligand.
  • the antibody or fragment thereof is capable of being secreted from a cell.
  • the present disclosure provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a murine TCR alpha or murine TCR beta extracellular domain, (2) a murine TCR alpha or murine TCR beta transmembrane domain, and (3) an intracellular domain of murine TCR alpha or murine TCR beta, and (ii) an antigen binding domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a murine TCR alpha constant domain or a murine TCR beta constant domain or a murine TCR alpha constant domain and a murine TCR beta constant domain; wherein the TCR subunit and the antigen binding domain are
  • the extracellular domain comprises at least a portion of a TCR alpha constant domain or TCR beta constant domain.
  • the recombinant nucleic acid further comprises at least a partial sequence encoding a TCR gamma constant domain, a TCR delta constant domain, or a partial sequence of both a TCR gamma constant domain and a TCR delta constant domain.
  • the antigen binding domain comprises a ligand.
  • the ligand binds to the receptor of a cell.
  • the ligand binds to the polypeptide expressed on a surface of a cell.
  • the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide.
  • the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4 and combinations thereof.
  • the antigen binding domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the antigen binding domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the ligand or fragment thereof is a monomer or a dimer. In some embodiments, the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not comprise a variable region. In some embodiments, the antigen binding domain does not comprise a CDR. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some embodiments, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell.
  • the encoded TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain or a TCR beta chain, e.g., a murine TCR alpha chain or a TCR beta chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a TCR alpha chain or a TCR beta chain, e.g., a murine TCR alpha chain or a TCR beta chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a TCR intracellular domain of a TCR alpha chain or a TCR beta chain, e.g., a murine TCR alpha chain or a TCR beta chain, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit further comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2,
  • the TCR subunit comprises an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b
  • IT AM immunoreceptor tyrosine-based activation motif
  • the IT AM replaces an IT AM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the IT AM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the TFP, the TCR alpha constant domain, the TCR beta domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR alpha constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR alpha constant domain and a TCR beta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a circRNA. In some embodiments, the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2 ’-O-methyl, 2 ’-O-m ethoxy ethyl (2’-0-M0E), 2’-0-aminopropyl, 2’-deoxy, T- deoxy-2’-fluoro, 2’-0-aminopropyl (2’-0-AP), 2'-0-dimethylaminoethyl (2’-0-DMA0E), 2’-0- dimethylaminopropyl (2’-0-DMAP), T-O-dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2’-0- N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a morpholino,
  • the recombinant nucleic acid further comprises a leader sequence. In some embodiments, the recombinant nucleic acid further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3’UTR sequence. In some embodiments, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid.
  • the present disclosure provides a vector comprising the recombinant nucleic acid described herein.
  • the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector is an AAV6 vector.
  • the vector further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • the present disclosure provides a modified T cell comprising the recombinant nucleic acid described herein, or the vector described herein, and the modified T cell comprises a functional disruption of an endogenous TCR.
  • the present disclosure provides modified T cell comprising the sequence encoding the TFP of the nucleic acid described herein or a TFP encoded by the sequence of the nucleic acid described herein encoding the TFP, and the modified T cell comprises a functional disruption of an endogenous TCR.
  • the present disclosure provides a modified allogenic T cell comprising the sequence encoding the TFP described herein or a TFP encoded by the sequence of the nucleic acid described herein encoding the TFP.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain.
  • the TCR constant domain e g., the TCR alpha constant domain, the TCR beta constant domain or the TCR alpha constant domain and the TCR beta constant domain
  • is a murine TCR constant domain e.g., a murine TCR alpha constant domain, a murine TCR beta constant domain or a murine TCR alpha constant domain and a murine TCR beta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain.
  • the endogenous TCR that is functionally disrupted has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell.
  • the functional disruption is a disruption of a gene encoding the endogenous TCR.
  • the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
  • the T cell is a human T cell selected from CD4 cells, CD8 cells, naive T-cells, memory stem T-cells, central memory T- cells, double negative T-cells, effector memory T-cells, effector T-cells, ThO cells, TcO cells, Thl cells, Tel cells, Th2 cells, Tc2 cells, Thl7 cells, Th22 cells, alpha/beta T cells, gamma/delta T cells, natural killer (NK) cells, natural killer T ( KT) cells, hematopoietic stem cells and pluripotent stem cells.
  • the T cell is a CD8+ or CD4+ T cell
  • the T cell is an allogenic T cell.
  • the modified T cell comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • the present disclosure provides a pharmaceutical composition comprising: the modified T cells described herein; and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of producing the modified T cell described herein, the method comprising disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid described herein, or the vector described herein.
  • disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • the present disclosure provides a method of producing the modified T cell described herein, the method comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid described herein, or the vector described herein.
  • the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • the T cell is a human T cell.
  • the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
  • the nuclease is a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
  • the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit.
  • the nuclease is a meganuclease.
  • the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence.
  • the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified T cell produced according to the method described herein; and (b) a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a modified T cell produced according to the method described herein; and (b) a pharmaceutically acceptable carrier.
  • the modified T cell is an allogeneic T cell. In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell. In some embodiments, less cytokines are released in the subject compared a subject administered an effective amount of a modified T cell comprising the recombinant nucleic acid described herein, or the vector described herein. In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition. In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition.
  • the cancer is a solid cancer, a lymphoma or a leukemia.
  • the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer.
  • less cytokines are released in the subject compared to a subject administered an effective amount of an autologous T cell expressing the TFP described herein.
  • the method does not induce graft versus host disease.
  • the subject has a reduced risk of developing graft versus host disease compared to a subject administered an effective amount of an autologous T cell expressing the TFP described herein.
  • the present disclosure provides the recombinant nucleic acid described herein, the vector described herein, the modified T cell described herein, or the pharmaceutical composition described herein, for use as a medicament or in the preparation of a medicament.
  • recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, TCR gamma, TCR delta, TCR alpha or TCR beta, and (ii) a human or humanized antibody comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain, wherein the TCR
  • recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR gamma or TCR delta and (ii) a binding ligand or a fragment thereof that is capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain; wherein the TCR subunit and the binding ligand
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to a sequence encoding a delta constant domain.
  • a sequence encoding the antigen binding domain or ligand binding domain is operatively linked to both a sequence encoding a TCR delta constant domain or fragment thereof and a TCR gamma constant domain or fragment thereof.
  • the intracellular signaling domain is CD3s. In another embodiment, the intracellular signaling domain is CD3y.
  • the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In another embodiment, the recombinant nucleic acid further comprises a portion of a TCR alpha constant domain, a portion of a TCR beta domain, or both. [0407] In another embodiment, the recombinant nucleic acid sequence comprises, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRGC gene sequence.
  • the recombinant nucleic acid sequence comprises, from 5’-3’, a first leader sequence, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker sequence, and a TRGC gene sequence.
  • the recombinant nucleic acid sequence comprises, from 5’-3’, a first leader sequence, an antigen binding domain sequence, a first linker sequence, a TRDC gene sequence, a cleavable linker, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, and a TRGC gene sequence.
  • the recombinant nucleic acid sequence comprises, from 5’-3’, a first leader sequence, a TRDC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRGC gene sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence.
  • the recombinant nucleic acid sequence comprises, from 5’-3’, a first leader sequence, a first antigen binding domain sequence, a first linker sequence, a TRDC gene sequence or fragment thereof, a TRAC gene sequence or fragment thereof, a cleavable linker sequence, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, a TRGC gene sequence or fragment thereof, and a TRBC gene sequence or fragment thereof.
  • sequence encodes the polypeptide as set forth in SEQ ID NO: 1.
  • sequence encodes the polypeptide as set forth in SEQ ID NO:2. In another embodiment, the sequence encodes the polypeptide as set forth in SEQ ID NO:3. In another embodiment, the sequence encodes the polypeptide as set forth in SEQ ID NO:4. In another embodiment, the sequence encodes the polypeptide as set forth in SEQ ID NO: 5. In one embodiment, the recombinant nucleic acid sequence further comprises at least a partial sequence encoding a TCR alpha constant domain, a TCR beta constant domain, or a partial sequence of both a TCR alpha constant domain and a TCR beta constant domain.
  • the binding ligand is capable of binding an Fc domain of the antibody. In some instances, the binding ligand is capable of selectively binding an IgGl antibody. In some instances, the binding ligand is capable of specifically binding an IgGl antibody. In some instances, the antibody or fragment thereof binds to a cell surface antigen. In some instances, the antibody or fragment thereof binds to a cell surface antigen on the surface of a tumor cell. In some instances, the binding ligand comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the binding ligand does not comprise an antibody or fragment thereof. In some instances, the binding ligand comprises a CD 16 polypeptide or fragment thereof. In some instances, the binding ligand comprises a CD16-binding polypeptide. In some instances, the binding ligand is human or humanized. In some instances, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof capable of being bound by the binding ligand. In some instances, the antibody or fragment thereof is capable of being secreted from a cell.
  • recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR gamma or TCR delta and (ii) an antigen domain comprising a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on a surface of a cell; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain
  • the antigen domain comprises a ligand.
  • the ligand binds to the receptor of a cell.
  • the ligand binds to the polypeptide expressed on a surface of a cell.
  • the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide.
  • the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4 and combinations thereof.
  • the antigen domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the antigen domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the ligand or fragment thereof is a monomer or a dimer. In some instances, the antigen domain does not comprise an antibody or fragment thereof In some instances, the antigen domain does not comprise a variable region. In some instances, the antigen domain does not comprise a CDR. In some instances, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some instances, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR gamma or TCR delta
  • the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, only CD3 delta, only TCR alpha, only TCR beta, only TCR gamma or only TCR delta.
  • the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4- 1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-lBB (CD 137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
  • the TCR subunit comprises an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof, and
  • the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the TFP, the TCR gamma constant domain, the TCR delta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR delta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof
  • the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof
  • the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the antibody is an antibody fragment.
  • the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain.
  • human or humanized antibody comprising an antigen binding domain is selected from a group comprising an anti-CD 19 binding domain, anti-B-cell maturation antigen (BCMA) binding domain, anti-mesothelin (MSLN) binding domain, anti-MUC16 binding domain, anti-IL13Ra2 binding domain, anti-CD22 binding domain, anti-PD-1 binding domain, anti-PD-Ll binding domain, anti-BAFF or BAFF receptor binding domain, and anti-ROR-1 binding domain.
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some instances, the nucleic acid is an mRNA.
  • the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2 ’-O-methyl, 2 ’-O-m ethoxy ethyl (2’-0-M0E), 2’-0-aminopropyl, 2’-deoxy, T- deoxy-2’-fluoro, 2’-0-aminopropyl (2’-0-AP), 2'-0-dimethylaminoethyl (2’-0-DMA0E), 2’-0- dimethylaminopropyl (2’-0-DMAP), T-O-dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2’-0- N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (EN
  • the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some instances, the nucleic acid is an in vitro transcribed nucleic acid. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain.
  • the recombinant nucleic acid further comprises a sequence encoding a TCR beta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha transmembrane domain and a sequence encoding a TCR beta transmembrane domain.
  • vectors comprising the recombinant nucleic acid disclosed herein.
  • the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector is an AAV6 vector.
  • the vector further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • modified T cell comprising the recombinant nucleic acid disclosed above, or the vector disclosed above; wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed above or a TFP encoded by the sequence of the nucleic acid disclosed above encoding the TFP, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified allogenic T cell comprising the sequence encoding the TFP disclosed above or a TFP encoded by the sequence of the nucleic acid disclosed above encoding the TFP.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain.
  • the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
  • the functional disruption is a disruption of a gene encoding the endogenous TCR.
  • the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
  • the T cell is a human T cell.
  • the T cell is a CD8+ T cell, a CD4+ T cell, a naive T cell, a memory stem T cell, a central memory T cell, a double negative T cell, an effector memory T cell, an effector T cell, a ThO cell, a TcO cell, a Thl cell, a Tel cell, a Th2 cell, a Tc2 cell, a Thl 7 cell, a Th22 cell, a gamma delta T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a hematopoietic stem cell, or a pluripotent stem cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the T cell is a CD4+CD8+ T cell. In some instances, the T cell is an allogenic T cell. In some instances, the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • compositions comprising: (a) the modified T cells of the disclosure; and (b) a pharmaceutically acceptable carrier.
  • a method of producing the modified T cell of the disclosure comprising (a) disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid, or the vector disclosed herein.
  • disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • method of producing the modified T cell of the disclosure comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid, or the vector disclosed herein.
  • the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • the T cell is a human T cell.
  • the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell.
  • the nuclease is a meganuclease, a zinc- finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
  • the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit.
  • the nuclease is a meganuclease.
  • the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence.
  • the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein.
  • method of treating cancer in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising (a) a modified T cell produced according to the methods disclosed herein; and (b) a pharmaceutically acceptable carrier.
  • the modified T cell is an allogeneic T cell.
  • less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell.
  • the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition. In some instances, the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition.
  • the cancer is a solid cancer, a lymphoma or a leukemia. In some instances, the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer.
  • nucleic acid for use as a medicament or in the preparation of a medicament.
  • modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein; wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta constant domain, an endogenous TCR alpha constant domain and an endogenous TCR beta constant domain, an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta chain.
  • the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
  • the functional disruption is a disruption of a gene encoding the endogenous TCR.
  • the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
  • the T cell is a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the T cell is an allogenic T cell.
  • the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • 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). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • 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. In certain aspects of the present disclosure, any number of T cell lines available in the art, may be used.
  • 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 may be 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. Initial activation steps in the absence of calcium can lead to magnified activation.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow- through” centrifuge (for example, the Cobe® 2991 cell processor, the Baxter Oncology CytoMateTM, or the Haemonetics® Cell Saver® 5) according to the manufacturer’s instructions.
  • a semi-automated “flow- through” centrifuge for example, the Cobe® 2991 cell processor, the Baxter Oncology CytoMateTM, or the Haemonetics® Cell Saver® 5
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ® gradient or by counterflow centrifugal elutriation.
  • T cells can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS ® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • multiple rounds of selection can also be used in the context of this present disclosure. In certain aspects, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • a T cell population can be selected that expresses one or more of IFN-g TNF-alpha, 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.
  • Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013/126712.
  • the concentration of cells and surface e.g., particles such as beads
  • the concentration of cells and surface can be varied.
  • a concentration of 2 billion cells/mL is used.
  • a concentration of 1 billion cells/mL is used.
  • greater than 100 million cells/mL is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used.
  • concentrations of 125 or 150 million cells/mL can be used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • the concentration of cells used is 5xl0 6 /mL. In other aspects, the concentration used can be from about lxl0 5 /mL to lxlOVmL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature.
  • 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.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • cryopreserved cells are thawed and washed as described herein 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.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3
  • T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631.
  • the T cells of the present 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 costimulatory molecule on the surface of the T cells.
  • 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 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 can stimulate proliferation of either CD4+ T cells or CD8+ T cells.
  • an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth.
  • T cells may additionally be activated and expanded in the presence of a cytokine with or without an anti-CD3 and/or CD28 antibody.
  • cytokines include IL-2, IL-7, IL-15, and IL-21.
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+).
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • CD4 and CD8 markers vary significantly, but in large part, reproducibly during the course of the cell expansion process.
  • anti-CD 19 anti-BCMA, anti-CD22, anti-RORl, anti-PD-1, or anti-BAFF, anti- MUC16, anti-mesothelin, anti-HER2, anti-PMSA, anti-CD20, anti-CD70, anti-GPC3, anti- Nectin-4, anti-Trop2, or antiCD79b TFP is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate in vitro and animal models.
  • TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • T cells (1:1 mixture of CD4 + and CD8 + T cells) expressing the TFPs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions.
  • TFPs are detected by western blotting using an antibody to a TCR chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.
  • TFP + T cells following antigen stimulation can be measured by flow cytometry.
  • a mixture of CD4 + and CD8 + T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed.
  • exemplary promoters include the CMV IE gene, EF-lalpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters.
  • GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g ., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP along with eGFP using a 2A ribosomal skipping sequence.
  • TAA+ K562 cells K562-TAA
  • wild-type K562 cells K562 wild type
  • K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibody (K562-BBL-3/28) following washing.
  • Exogenous IL-2 is added to the cultures every other day at 100 IU/mL.
  • GFP+ T cells are enumerated by flow cytometry using bead-based counting (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • Sustained TFP+ T cell expansion in the absence of re-stimulation can also be measured (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP on day 1.
  • mice can also be used to measure a TFP-T activity.
  • xenograft model using, e.g., human CD 19-specific TFP+ T cells to treat a primary human pre-B ALL in immunodeficient mice can be used (see, e.g., Milone et al, Molecular Therapy 17(8): 1453-1464 (2009)).
  • mice are randomized as to treatment groups. Different numbers of engineered T cells are coinjected at a 1:1 ratio into NOD/SC I D/y-/- mice bearing B- ALL. The number of copies of each vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for leukemia at weekly intervals.
  • Peripheral blood CD 19+ B-ALL blast cell counts are measured in mice that are injected with alphaCD19-zeta TFP+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log-rank test.
  • absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD/SCID/y-/- mice can also be analyzed. Mice are injected with leukemic cells and 3 weeks later are injected with T cells engineered to express TFP by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are normalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are assessed for leukemia at 1-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.
  • Dose dependent TFP treatment response can be evaluated (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • peripheral blood is obtained 35-70 days after establishing leukemia in mice injected on day 21 with TFP T cells, an equivalent number of mock-transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood CD 19+ ALL blast counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.
  • TFP-mediated proliferation is performed in microtiter plates by mixing washed T cells with K562 cells expressing the tumor associated antigen (TAA, e.g., CD19) CD19 (K19) or CD32 and CD137 (KT32-BBL) for a final T cell:K562 ratio of 2: 1.
  • TAA tumor associated antigen
  • K562 cells are irradiated with gamma- radiation prior to use.
  • Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo.
  • T cells are enumerated in cultures using CountBrightTM fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer.
  • TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP-expressing lentiviral vectors. For TFP+ T cells not expressing GFP, the TFP+ T cells are detected with biotinylated recombinant CD 19 protein and a secondary avidin-PE conjugate.
  • CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re-stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer’s instructions. Fluorescence is assessed using a FACScaliburTM flow cytometer (BD Biosciences), and data are analyzed according to the manufacturer’s instructions.
  • Cytotoxicity can be assessed by a standard 51 Cr-release assay (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • Target cells K562 lines and primary pro-B-ALL cells
  • 51 Cr as NaCrCU, New England Nuclear
  • Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector cell Target cell (E:T).
  • NOD/SCID/yc-/- mice are injected IV with Nalm-6 cells (ATCC® CRL-3273TM) followed 7 days later with T cells 4 hour after electroporation with the TFP constructs.
  • the T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence.
  • therapeutic efficacy and specificity of a single injection of TFP+ T cells in Nalm-6 xenograft model can be measured as the following: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail-vein injection of T cells electroporated with a TAA-TFP 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive leukemia in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.
  • compositions comprising: (a) the modified T cells of the disclosure; and (b) a pharmaceutically acceptable carrier.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
  • the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
  • an immunologically effective amount When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g ., Rosenberg et al., New Eng. J. Med. 319:1676, 1988).
  • T cells can be activated from blood draws of from 10 cc to 400 cc.
  • T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
  • compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection.
  • the T cell compositions of the present disclosure are administered by i.v. injection.
  • the compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
  • subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells.
  • T cell isolates may be expanded by methods known in the art and treated such that one or more TFP constructs of the present disclosure may be introduced, thereby creating a modified T-T cell of the present disclosure.
  • Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded modified T cells of the present disclosure.
  • expanded cells are administered before or following surgery.
  • the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the scaling of dosages for human administration can be performed according to art-accepted practices.
  • the dose for alemtuzumab will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days.
  • the preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Pat. No. 6,120,766).
  • the TFP is introduced into T cells, e.g ., using in vitro transcription, and the subject ⁇ e.g., human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.
  • more than one administration of the TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the present disclosure are administered per week.
  • the subject receives more than one administration of the TFP T cells per week (e.g, 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g, more than one administration of the TFP T cells per week) is administered to the subject.
  • the subject receives more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
  • the TFP T cells are administered every other day for 3 administrations per week
  • the TFP T cells of the present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.
  • CD 19 TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP-T cells generated that way will have stable TFP expression.
  • TFP T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction.
  • Transient expression of TFPs can be affected by RNA TFP vector delivery.
  • the TFP RNA is transduced into the T cell by electroporation.
  • anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype. It is thought that a patient’s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen day break in exposure to antigen.
  • TFP T cell infusion breaks should not last more than ten to fourteen days.
  • a method of producing the modified T cell of the disclosure comprising (a) disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of the disclosure, or the vectors disclosed herein.
  • disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • the T cell is a human T cell.
  • the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
  • the nuclease is a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, CRISPR/Cas nickase, or a megaTAL nuclease.
  • the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit.
  • the nuclease is a meganuclease.
  • the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence.
  • the meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucl eases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Urnov et ah, Nat. Rev.
  • CRISPR® clustered regularly interspaced short palindromic repeats
  • TALE transcription activator-like effector
  • TALENs transcription activator-like effector
  • meganucleases endodeoxyribonucl eases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs
  • a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et ah, 2009 Nature Reviews Immunol. 9.10: 704-716, each incorporated herein by reference.
  • one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).
  • the endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain can be inactivated in the modified cell (e.g., modified T cell) described herein.
  • the inactivation can include disruption of genomic gene locus, gene silencing, inhibition or reduction of transcription, or inhibition or reduction of translation.
  • the endogenous TCR gene can be silenced, for example, by inhibitory nucleic acids such as siRNA and shRNA.
  • the translation of the endogenous TCR gene can be inhibited by inhibitory nucleic acids such as microRNA.
  • gene editing techniques are employed to disrupt an endogenous TCR gene.
  • mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene.
  • multiplex genomic editing techniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD1), and/or other genes.
  • HLAs human leukocyte antigens
  • PD1 programmed cell death protein 1
  • nickase nucleases generate single-stranded DNA breaks (SSB).
  • DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.
  • ssDI single strand DNA incorporation
  • ssTR single strand template repair
  • Genome DNA can be performed using site-specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest.
  • Methods for producing engineered, site-specific endonucleases are known in the art.
  • ZFNs zinc- finger nucleases
  • ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme.
  • the zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence -18 basepairs in length.
  • TAL-effector nucleases can be generated to cleave specific sites in genomic DNA.
  • a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol 23:93-9).
  • the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair.
  • Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762).
  • a Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-Tevl homing endonuclease.
  • I-Tevl does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
  • Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63).
  • the CRISPR gene-editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA.
  • a CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short “guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • a caspase effector nuclease typically microbial Cas9
  • a short “guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • CRISPR systems There are two classes of CRISPR systems known in the art (Adli (2016) Nat. Commun. 9:1911), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class P contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR/Cas system is the type II CRISPR- Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli (2016) Nat. Commun. 9: 1911).
  • Homing endonucleases are a group of naturally-occurring nucleases that recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins.
  • MN Meganucleases
  • meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.
  • chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy 24: 430-446).
  • a megaTAL is a single chimeric protein, which is the combination of the easy-to- tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.
  • the nucleases and in the case of the CRISPR/ Cas9 system, a gRNA, may need to be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
  • the endogenous TCR gene (e.g., a TRAC locus or a TRBC locus) encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain can be inactivated by CRISPR/Cas9 system.
  • the gRNA used to inactivate (e.g., disrupt) the TRAC locus can comprise a sequence of SEQ ID: 196.
  • the gRNA used to disrupt the TRBC locus can comprise a sequence of SEQ ID: 197.
  • CTCGACCAGCTTGACATCAC (SEQ ID NO: 196).
  • compositions comprising (a) a modified T cell produced according to the methods disclosed herein; and (b) a pharmaceutically acceptable carrier.
  • the modified T cell is an allogeneic T cell. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of a modified T cell comprising the recombinant nucleic acid disclosed herein, or the vector disclosed herein.
  • the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition. In some instances, the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition.
  • the cancer is a solid cancer, a lymphoma or a leukemia.
  • the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney and stomach cancer.
  • the present disclosure includes a type of cellular therapy where T cells are genetically modified to express a TFP and a TCR gamma and/or delta constant domain and the modified T cell is infused to a recipient in need thereof.
  • the infused cell is able to kill tumor cells in the recipient.
  • modified T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
  • the T cells administered to the patient, or their progeny persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
  • the present disclosure also includes a type of cellular therapy where T cells are modified, e.g ., by in vitro transcribed RNA, to transiently express a TFP and a TCR gamma and/or delta constant domain and the modified T cell is infused to a recipient in need thereof.
  • the infused cell is able to kill tumor cells in the recipient.
  • the T cells administered to the patient is present for less than one month, e.g., three weeks, two weeks, or one week, after administration of the T cell to the patient.
  • the anti-tumor immunity response elicited by the modified T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response.
  • the human modified T cells of the disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal
  • the mammal is a human.
  • ex vivo immunization at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP and a TCR gamma and/or delta constant domain to the cells or iii) cryopreservation of the cells.
  • cells are isolated from a mammal (e.g ., a human) and genetically modified (i.e., transduced or transfected in vitro ) with a vector disclosed herein.
  • the modified T cell can be administered to a mammalian recipient to provide a therapeutic benefit.
  • the mammalian recipient may be a human and the modified cell can be autologous with respect to the recipient.
  • the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo.
  • other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
  • compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient are also provided.
  • the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
  • the modified T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
  • a modified T cell described herein may be used in combination with other known agents and therapies.
  • Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”.
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • the “at least one additional therapeutic agent” includes a modified T cell.
  • T cells that express multiple TFPs, which bind to the same or different target antigens, or same or different epitopes on the same target antigen.
  • populations of T cells in which a first subset of T cells express a first TFP and a TCR gamma and/or delta constant domain and a second subset of T cells express a second TFP and a TCR gamma and/or delta constant domain.
  • a modified T cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially.
  • the modified T cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
  • a modified T cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, cytokines, and irradiation peptide vaccine, such as that described in Izumoto et al., 2008 J. Neurosurg. 108:963-971.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus
  • antibodies or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine,
  • the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a modified T cell.
  • Side effects associated with the administration of a modified T cell include but are not limited to cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS).
  • CRS cytokine release syndrome
  • HHL hemophagocytic lymphohistiocytosis
  • MAS Macrophage Activation Syndrome
  • Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
  • the methods disclosed herein can comprise administering a modified T cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a modified T cell.
  • the soluble factor elevated in the subject is one or more of PTM-g,
  • an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors.
  • agents include, but are not limited to a steroid, an inhibitor of TNFa, and an inhibitor of IL-6.
  • An example of a TNFa inhibitor is entanercept.
  • An example of an IL-6 inhibitor is tocilizumab (toe).
  • the subject can be administered an agent which enhances the activity of a modified T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., Programmed Death 1 (PD1)
  • PD1 can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response.
  • inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta.
  • Inhibition of an inhibitory molecule e.g., by inhibition at the DNA, RNA or protein level, can optimize a modified T cell performance.
  • an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
  • an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
  • the inhibitor is a shRNA.
  • the inhibitory molecule is inhibited within a modified T cell.
  • a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the TFP.
  • the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule.
  • the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as Yervoy ® ; Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206)).
  • the agent is an antibody or antibody fragment that binds to TIM3.
  • the agent is an antibody or antibody fragment that binds to LAG3.
  • the agent which enhances the activity of a modified T cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein.
  • the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein.
  • the fusion protein is expressed by the same cell that expressed the TFP. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-TAA TFP.
  • T-Cell Receptor is formed by a complex of dimers TCRoc/b, CD3y/s, CD36/s and the homodimer ⁇ 3z/z.
  • TCRy/d are expressed instead of TCRa/b to form a functional TCR.
  • TCRa/b/g/d have a constant domain common to all T-cells and a variable domain specific to an antigen.
  • TRAC, TRBC, TRGC and TRDC genes encode for the constant C-terminal region of TCRa, TCRb, TCRy and TCRb respectively.
  • TCRa only pairs with TCRb and TCRy only pairs with TCRb.
  • a TCR complex is formed with TCRa/b in a/b T cells or with TCRy/d in g/d T cells.
  • TCRa/b/g/d constant region(s) blocks the translocation of TCR protein(s) to the cell surface. Thus, inhibiting assembly of the TCR receptor complex. Impairing the translocation of a TCRa or TCRb is enough to inhibit the assembling of entire TCR receptor in TCRa/b T cells. Similarly, impairing the translocation of a TCRy or TCRb is enough to inhibit assembly of the entire TCR receptor in TCRy/d T cells.
  • Inactivation of the TCR complex may therefore be done by targeting the TRAC or TRBC genes with a gene editing method using clustered regularly interspaced short palindromic repeat (CRISPR) method, transcription activator-like effector nucleases (TALENs), zinc finger nucleases or meganucleases.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • TALENs transcription activator-like effector nucleases
  • TFP T cells based on CD3s or CD3y or CD35 fusion proteins require surface expression of TCRot/b or TCRy/d to incorporate into a functional TCR complex.
  • Activation of the TCR complex on the surface of alloreactive donor T cells by mismatched HLA molecules or cognate antigens i.e., recognition of antigens presented by the major histocompatibility complex (MHC) on antigen presenting cells
  • MHC major histocompatibility complex
  • CRS cytokine release syndrome
  • the following Examples describe methods of introducing a transgene in TCRa or TCR knock out cells encoding for a truncated version of TCRa (murine) and TCRP (murine) having a binder on one or both, or on a CD3 TFP, or a truncated or full length TCRy or TCR5, having a binder on one or both truncated TCRs, or on a CD3 TFP with the fusion protein itself separated by a self cleavage signal (e.g., T2A).
  • the truncated version of TCRy or TCR5 includes the transmembrane domain and the connecting peptide domain (CP), and the constant domain of TCRy or TCR6.
  • the truncated version of TCRy or TCR5 includes the transmembrane domain and the connecting peptide domain (CP), and the constant domain of TCRa or TCR .
  • the TFP’s antigen binding domain is fused at the N- terminal end of either or both the truncated TCRy and/or TCR5.
  • crRNAs to inactivate TRA were designed with “Dunne 2017” algorithm accessible on DeskGenTM CRISPR library website (www.deskgen.com). Any crRNAs binding the TRA locus are able to efficiently generate double strand breaks in the TRA gene.
  • the crRNAs used have an off-target score of >90%, comprising at least 3 mismatches with the closest homolog sequence in the Genome Reference Consortium Human genome build 38 (GRCh38/hg38) genome.
  • one mismatch is located in the 8 bp upstream to the protospacer adjacent motif (PAM).
  • Tables 1-2 show exemplary crRNA sequences selected to inactivate the TRA gene (Table 1) and predicted off target activity (Table 2).
  • crRNAs to inactivate the TRBC genes were designed with Dunne 2017 algorithm as described above. As the constant region of TCRJ3 is encoded by two genes, TRBC1 and TRBC2, crRNAs are directed against sequences identical in both TRBC1 and TRBC2. Consequently, the off-target score generated by DeskGenTM is lower than 94%. However, aside from targeting TRBC1 and TRBC2, other homolog sequences between crRNAs and the GRCh38/hg38 genome carry at least 3 mismatches. In a preferred embodiment, one of those mismatches is localized in the 8 bp upstream to the Protospacer adjacent motif (PAM). Tables 3-4 show exemplary crRNA sequences selected to inactivate the TRB gene (Table 3) and predicted off target activity (Table
  • Inactivation of the TRAC or TRBC genes in Jurkat cells was done by electroporation of SpCas9 ribonucleoproteins (RNPs) directed against TRAC or TRBC genes.
  • RNPs SpCas9 ribonucleoproteins
  • Cells were maintained at 0.2xl0 6 cells per mL in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 300mg/L L-Glutamine until electroporation.
  • SpCas9 ribonucleoproteins targeting TRA or TRB genes were prepared by annealing crRNA targeting either TRAC (TRAC2-4598) or TRBC (TRBC-44345) with tracrRNA at a molecular ratio of 1 : 1.
  • Annealed duplexes were mixed with SpCas9 protein at a molecular ratio of 1.5:1. 0.61 mM of RNPs were mixed with 2.5xl0 6 T cells and electroporated according to the manufacturer’s protocol for the Neon Transfection System (Thermo Fisher Scientific). Electroporation was set at 1600V, 10ms, 3 pulses. After pulse the cells were immediately transferred to warm medium and incubated at 37°C for three days.
  • TCRa and TCRp knock out cells are herein called TRA-/- or TRB-/- Jurkat cells.
  • TFP transgenes were introduced in Jurkat cells using lentiviruses as described, e.g., in co pending U.S. Patent Publication No. 2017-0166622.
  • Jurkat cells were incubated with virus at a multiplicity of infection (MO I) of five. Medium was replaced twenty-four-hours post incubation.
  • Transduction efficacy and TFP expression was assessed with flow cytometry using a ligand specific to the TFP binder of interest and/or surface expression of TCRaJ3 and CD3s.
  • TRAC-/- and TRBC-/- Jurkat cells were transduced with TCR/d TFPs and restoration of surface TCR was indicated by highly positive CD3e staining ( Figure 2).
  • TRBC-/- Jurkat cells were also transduced with anti-CD 19 TCRfi TFP or anti-CD 19 TCRfl TFP having only the constant domain of TCRP (Figure 12B).
  • the results show that, while the TFP having the entire human TCRP is expressed on the cell surface, the TFP having only the constant domain of human TCRP is not expressed on the cell surface ( Figure 13). This is because the constant domain of TCRP is not able to localize to the cell surface, whereas it is known that the constant domain of murine TCRP and murine human chimeras of the TCRP are able to localize to the cell surface.
  • TFP transgenes were introduced into T cells using lentiviruses as described, e.g., in copending U.S. Patent Publication No. 2017-0166622. T cells were mixed together with viruses at a multiplicity of infection (MOI) of five plus lOOng/mL of LentiBOOSTTM (Sirion Biotech). Transduction efficacy and TFP expression was assessed with flow cytometry using a ligand specific to the TFP binder of interest and/or surface expression of TCRaP and CD3e.
  • MOI multiplicity of infection
  • TRAC or TRBC blocks the translocation to the cell surface of all TCR subunits.
  • TCRa or TCRP cannot pair with TCRy or TCR5. Consequently, an exogenous TRGC and TRDC transgenes or TRAC and TRBC transgenes are expressed in TRAC 7 or TRBC 7 cells to restore a functional TFP T cell.
  • TCRa negative cells still express TCRP and, reciprocally, TCRa is expressed in TCRP negative cells; However, TCRa or TCRp cannot pair with TCRv or TCR6. Therefore, TCRy TFP and TCR5 TFPs were expressed together in TRAC 7 cells or in TRB 7 cells. Multiple formats of TCRy/d TFPs were tested in TCR negative cells to determine the optimal construction to restore translocation of the entire TCR complex (see Figure 3 schematic). In one embodiment, TCRy/d TFPs were generated by assembling the constant domains of TCRy or/and TCR5 with an antigen binder (e.g., scFv or sdAb).
  • an antigen binder e.g., scFv or sdAb
  • TCRv/d constant domains are expressed together with a CD3c TFP ( Figure 4)
  • TRGC1 and TRDC residues are numerated according to the the sequences provided herein and according to international ImMunoGeneTics information system (IMGT).
  • IMGT international ImMunoGeneTics information system
  • a TCR complex contains the CD3-epsilon polypeptide, the CD3-gamma poly peptide, the CD3-delta polypeptide, and the TCR alpha chain polypeptide and the TCR beta chain polypeptide or the TCR delta chain polypeptide and the TCR gamma chain polypeptide.
  • TCR alpha, TCR beta, TCR gamma, and TCR delta recruit the CD3 zeta polypeptide.
  • the human CD3-epsilon polypeptide canonical sequence is Uniprot Accession No. P07766.
  • the human CD3-gamma polypeptide canonical sequence is Uniprot Accession No. P09693.
  • the human CD3-delta polypeptide canonical sequence is Uniprot Accession No. P043234.
  • the human CD3- zeta polypeptide canonical sequence is Uniprot Accession No. P20963.
  • the human TCR alpha chain canonical sequence is Uniprot Accession No. Q6ISU1.
  • the murine TCR alpha chain canonical sequence is Uniprot Accession No. A0A075B662.
  • the human TCR beta chain constant region canonical sequence is Uniprot Accession No. P01850.
  • the murine TCR beta chain constant region canonical sequence is Uniprot Accession No. P01852.
  • the human CD3-epsilon polypeptide canonical sequence is:
  • the mature human CD3-epsilon polypeptide sequence is:
  • the signal peptide of human CD3e is:
  • VMS VATIVIVDICIT GGLLLLVYYW S (SEQ ID NO: 127).
  • the intracellular domain of human CD3 is:
  • KNRR AK AKP VTRGAGAGGRQRGQNKERPPP VPNPD YEPIRKGQRDL Y S GLN QRRI (SEQ ID NO: 128).
  • the human CD3-gamma polypeptide canonical sequence is:
  • the mature human CD3-gamma polypeptide sequence is:
  • transmembrane domain of human CD3 g is:
  • the mature human CD3 -delta polypeptide sequence is:
  • the signal peptide of human CD35 is:
  • the transmembrane domain of human CD36 is:
  • the intracellular domain of human CD35 is:
  • the human CD3-zeta polypeptide canonical sequence is:
  • the human TCR alpha chain constant region canonical sequence is:
  • the human TCR alpha chain human IgC sequence is:
  • the transmembrane domain of the human TCR alpha chain is:
  • VIGFRILLLK V AGFNLLMTLRLW (SEQ ID NO: 144).
  • the intracellular domain of the human TCR alpha chain is: SS (SEQ ID NO: 145)
  • the murine TCR alpha chain constant (mTRAC) region canonical sequence is: XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSN GAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILL LKV AGFNLLMTLRLW S S (SEQ ID NO: 146)
  • the transmembrane domain of the murine TCR alpha chain is:
  • MGLRILLLKV AGFNLLMTLRLW (SEQ ID NO: 147).
  • the intracellular domain of the murine TCR alpha chain is: SS
  • the human TCR beta chain constant region (mTRBC) canonical sequence is:
  • the human TCR beta chain human IgC sequence is:
  • the transmembrane domain of the human TCR beta chain is:
  • the intracellular domain of the human TCR beta chain is:
  • VKRKDF (SEQ ID NO: 151)
  • the murine TCR beta chain constant region canonical sequence is:
  • the transmembrane domain of the murine TCR beta chain is:
  • the intracellular domain of the murine TCR beta chain is:
  • the human TCR gamma chain constant region canonical sequence is: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN C SKD ANDTLLLQLTNT S A YYM YLLLLLK S V VYF AIITC CLLRRT AF CCN GEK S (SEQ ID NO:21).
  • the human TCR gamma human IgC sequence is:
  • transmembrane domain of the human TCR gamma chain is:
  • the intracellular domain of the human TCR gamma chain is:
  • the human TCR delta chain C region canonical sequence is: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG K YEDSNS VT C S VQHDNKT VHS TDFEVKTD S TDHVKPKETENTKQP SKS CHKPK AIVHTE KVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO:243).
  • the human TCR delta human IgC sequence is:
  • the transmembrane domain of the human TCR delta chain is:
  • LGLRMLF AKT V A VNFLLTAKLFF (SEQ ID NO: 158).
  • the intracellular domain of the human TCR delta chain is: L
  • Nt-FMC63-TRDC(1-153)-T2A-FMC63-TRGC1(1-173)-Ct Nt-FMC63-TRDC(-6,1 89)-2A- FMC63-TRGC1(1.8, 189)-Ct according to IMGT numbering:
  • Nt-TRDC(l-153)-T2A-TRGCl(l-173)-T2A-FMC63-CD3s(l-185)-Ct Nt-TRDC(-6, 189)-2A- TRGC1(1.8, 189)-2A-FMC63-CD3s(l, 186)-Ct according to IMGT numbering:
  • Nt-FMC63-TRDC(1-153)-T2A-TRGC1(1-173)-Ct without signal peptide (amino acid sequence) Nt-FMC63-TRDC(-6, 189)-2A-TRGC1(1.8, 189)-Ct according to IMGT numbering:
  • Nt-FMC63-TRDC(1-153)-T2A-TRGC1(1-173)-Ct without signal peptide (nucleic acid sequence) Nt-FMC63-TRDC(-6, 189)-2A-TRGC1(1.8, 189)-Ct according to IMGT numbering:

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Abstract

L'invention concerne des acides nucléiques recombinés codant pour des protéines de fusion (TFP) de récepteurs de lymphocytes T (TCR) et un domaine constant de TCR, des lymphocytes T modifiés exprimant les molécules codées, et leurs méthodes d'utilisation pour le traitement de maladies, dont le cancer.
EP20862242.3A 2019-09-12 2020-09-11 Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion Pending EP4028033A4 (fr)

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