WO2021035170A1 - Compositions et procédés de reprogrammation tcr à l'aide de protéines de fusion - Google Patents

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

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Publication number
WO2021035170A1
WO2021035170A1 PCT/US2020/047473 US2020047473W WO2021035170A1 WO 2021035170 A1 WO2021035170 A1 WO 2021035170A1 US 2020047473 W US2020047473 W US 2020047473W WO 2021035170 A1 WO2021035170 A1 WO 2021035170A1
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Prior art keywords
cell
tcr
nucleic acid
tfp
chain
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PCT/US2020/047473
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English (en)
Inventor
Aaron Martin
Patrick Alexander BAEUERLE
Robert Hofmeister
Daniel Getts
Philippe KIEFFER-KWON
Julie DONAGHEY
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Precision Biosciences, Inc.
TCR2 Therapeutics Inc.
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Application filed by Precision Biosciences, Inc., TCR2 Therapeutics Inc. filed Critical Precision Biosciences, Inc.
Publication of WO2021035170A1 publication Critical patent/WO2021035170A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • 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

Definitions

  • the invention relates to the field of oncology, cancer immunotherapy, molecular biology, and recombinant nucleic acid technology.
  • the invention relates to T cell receptor fusion proteins and their use for the treatment of diseases such as cancer.
  • 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
  • CD 19-specific CAR T cells 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 al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)).
  • TCR T cell receptor
  • TCR T cell receptor alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells. These 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.
  • modified T cells comprising fusion proteins of TCR complex subunits, including TCR alpha chains and TCR beta chains, with antigen binding domains specific for tumor associated antigens, that have the potential to overcome limitations of existing approaches. Additionally, these modified T cells may have functional disruption of an endogenous TCR subunit (e.g, a TCR alpha chain, TCR beta chain, 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. [0009] Provided herein are genetically modified T cells comprising one or more T cell receptor (TCR) fusion proteins (TFPs), methods of producing the modified T cells, and compositions and methods of use thereof for the treatment of diseases.
  • TCR T cell receptor
  • TFPs T cell receptors
  • a recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
  • T cell comprises a functional disruption of one or both of a TCR alpha and TCR beta subunit.
  • TCR complex subunit comprises a TCR alpha constant domain, a TCR beta constant domain, a CD3 epsilon chain, or fragments thereof.
  • the TCR complex subunit is encoded by a human, humanized, or murine sequence.
  • the antigen binding domain has specificity for a tumor associated antigen.
  • the tumor associated antigen is a cell surface antigen. In another embodiment, the tumor associated antigen is on the cell surface of a tumor cell. In some embodiments, the tumor associated antigen is CD 19. In one embodiment, the antigen binding domain comprises a single-chain variable fragment (scFv), a single-domain antibody (sdAb), or fragments thereof.
  • scFv single-chain variable fragment
  • sdAb single-domain antibody
  • the transmembrane domain is a transmembrane domain of a TCR alpha chain, a TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134,
  • CD137 or CD154.
  • the TCR complex subunit is a TCR alpha constant domain and the transmembrane domain is a TCR alpha chain transmembrane domain; the TCR complex subunit is a TCR beta constant domain and the transmembrane domain is a TCR beta chain transmembrane domain; or the TCR complex subunit is a CD3 epsilon chain and the transmembrane domain is a CD3 epsilon transmembrane domain.
  • the recombinant nucleic acid molecule comprises a promoter that drives expression of the TFP.
  • the TCR complex subunit of the TFP comprises a TCR alpha constant domain and the recombinant nucleic acid molecule further encodes a TCR beta chain lacking a variable domain.
  • the TCR alpha constant domain and the TCR beta chain are human, at least one of the TCR alpha constant domain and the TCR beta chain are murine, or the TCR alpha constant domain and the TCR beta chain are murine.
  • the TCR complex subunit of the TFP comprises a TCR beta constant domain and the recombinant nucleic acid molecule further encodes a TCR alpha chain lacking a variable domain.
  • the TCR beta constant domain and the TCR alpha chain are human, at least one of the TCR beta constant domain and the TCR alpha chain are murine, or the TCR beta constant domain and the TCR alpha chain are murine.
  • the TCR complex subunit of the TFP comprises a CD3 epsilon chain and the recombinant nucleic acid molecule further encodes a TCR alpha chain lacking a variable domain and/or a TCR beta chain lacking a variable domain.
  • the CD3 epsilon chain, the TCR alpha chain, and the TCR beta chain are human, at least one of the CD3 epsilon chain, the TCR alpha chain, and the TCR beta chain are murine, the CD3 epsilon chain is human and the TCR alpha chain and the TCR beta chain are murine, or the CD3 epsilon chain, the TCR alpha chain, and the TCR beta chain are murine.
  • the recombinant nucleic acid molecule encodes a first TFP and a second TFP, wherein the TCR complex subunit of the first TFP comprises a TCR alpha constant domain, and wherein the TCR complex subunit of the second TFP comprises a TCR beta constant domain.
  • the antigen binding domain of the first TFP and the antigen binding domain of the second TFP have specificity for the same tumor associated antigen.
  • the antigen binding domain of the first TFP and the antigen binding domain of the second TFP have specificity for different tumor associated antigens.
  • the recombinant nucleic acid molecule comprises a promoter that drives expression of the first TFP and the second TFP, and the sequences encoding the first TFP and the second TFP are separated by a 2A or IRES element.
  • the recombinant nucleic acid molecule comprises a first promoter which drives expression of the first TFP, and a second promoter which drives expression of the second TFP.
  • the TCR alpha constant domain and the TCR beta constant domain are human.
  • at least one of the TCR alpha constant domain and the TCR beta constant domain is murine.
  • the TCR alpha constant domain and the TCR beta constant domain are murine.
  • the TCR complex subunit of the TFP is a CD3 epsilon chain and the nucleic acid molecule further encodes a TCR alpha chain lacking a variable domain, a TCR beta chain lacking a variable domain, or both.
  • the recombinant nucleic acid molecule comprises a promoter that drives expression of the TFP, and further drives expression of the TCR alpha chain and/or the TCR beta chain, wherein the sequences encoding the TFP, the TCR alpha chain, and/or the TCR beta chain, are separated by 2A or IRES sequences.
  • the recombinant nucleic acid molecule comprises a first promoter that drives expression of the TFP and a second promoter that drives expression of the TCR alpha chain, the TCR beta chain, or both.
  • the CD3 epsilon chain, the TCR alpha chain, and the TCR beta chain are human, at least one of the CD3 epsilon chain, the TCR alpha chain, and the TCR beta chain are murine, the CD3 epsilon chain is human and the TCR alpha chain and the TCR beta chain are murine, or the CD3 epsilon chain, the TCR alpha chain, and the TCR beta chain are murine.
  • the recombinant nucleic acid molecule is a DNA or an RNA. In some embodiments, the nucleic acid molecule is an mRNA. In another embodiment, the recombinant nucleic acid molecule comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid molecule.
  • the vector comprising any of the recombinant nucleic acid molecules disclosed herein.
  • the vector is a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a Rous sarcoma viral (RSV) vector, a retroviral vector, a DNA vector, an RNA, or a plasmid.
  • the vector is an AAV vector having a serotype of AAV6.
  • the vector is an in vitro transcribed vector.
  • TFP polypeptide encoded by any recombinant nucleic acid molecule described herein.
  • a method of producing a genetically modified T cell expressing a TFP comprising introducing into a T cell: (a) a first nucleic acid encoding an engineered nuclease having specificity for a recognition sequence in an endogenous TCR alpha constant (TRAC) gene or an endogenous TCR beta constant (TRBC) gene, wherein the engineered nuclease is expressed in the T cell; and (b) a donor nucleic acid comprising any of the recombinant nucleic acid molecules disclosed herein that encode a TFP; wherein the engineered nuclease generates a cleavage site at the recognition sequence, and wherein the donor nucleic acid is inserted into the genome of the T cell at the cleavage site, and wherein expression of the endogenous TCR alpha chain or the endogenous TCR beta chain is disrupted.
  • TRAC endogenous TCR alpha constant
  • TRBC endogenous TCR beta constant
  • the donor nucleic acid is flanked by homology arms having homology to sequences flanking the recognition sequence, and wherein said donor template is inserted at the cleavage site by homologous recombination.
  • the genetically modified T cell does not have detectable cell-surface expression of an endogenous TCR.
  • the genetically modified T cell exhibits reduced binding to MHC-peptide complexes compared to a control cell.
  • the T cell is a human T cell.
  • the T cell is a CD4+ cell, a CD8+ cell, a CD4+/CD8+ T cell, a naive T cell, a memory stem T cell, a central memory T cell, a double-negative T cell, a effector memory T cell, a effector T cell, a Thl cell, a Tel cell, a Th2 cell, a Tc2 cell, a Thl7 cell, a Th22 cell, a gamma/delta T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.
  • the first nucleic acid is an mRNA.
  • the donor nucleic acid is delivered using an AAV vector.
  • the AAV vector has a serotype of AAV6.
  • the engineered nuclease is an engineered meganuclease, a zinc finger nuclease, a TALEN, a compact TALEN, a CRISPR system nuclease, or a megaTAL.
  • the engineered nuclease is an engineered meganuclease.
  • the recognition sequence consists of SEQ ID NO: 30.
  • the one or more TFPs are expressed in the genetically modified T cell and integrate into a cell-surface TCR complex.
  • the donor nucleic acid encodes a first TFP wherein the TCR complex subunit comprises a murine TCR alpha constant domain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP wherein the TCR complex subunit comprises a murine TCR beta constant domain, or wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a murine TCR beta chain lacking a variable domain.
  • the donor nucleic acid encodes a first TFP comprising a murine TCR beta constant domain
  • the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP comprising a murine TCR alpha constant domain
  • the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a murine TCR alpha chain lacking a variable domain.
  • the donor nucleic acid encodes a first TFP wherein the TCR complex subunit comprises a murine TCR alpha constant domain and a second TFP wherein the TCR complex subunit comprises a murine TCR beta constant domain.
  • the first TFP and the second TFP integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • the donor nucleic acid encodes a first TFP wherein the TCR complex subunit comprises a murine TCR alpha constant domain, and further encodes a murine TCR beta chain lacking a variable domain.
  • the first TFP and the murine TCR beta chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the donor nucleic acid encodes a first TFP wherein the TCR complex subunit comprises a murine TCR beta constant domain, and further encodes a murine TCR alpha chain lacking a variable domain.
  • the first TFP and the TCR alpha chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • the donor nucleic acid encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a murine TCR alpha chain lacking a variable domain and/or a nucleic acid sequence encoding a murine TCR beta chain lacking a variable domain.
  • the donor nucleic acid encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, and further encodes a murine TCR alpha chain lacking a variable domain and a murine TCR beta chain lacking a variable domain.
  • the TFP, the murine TCR alpha chain, and the murine TCR beta chain integrate into a cell-surface TCR complex with an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • the method further comprises introducing into the T cell a second nucleic acid encoding a second engineered nuclease, wherein the first engineered nuclease has specificity for a recognition sequence in the TRAC gene and the second engineered nuclease has specificity for a recognition sequence in the TRBC gene, and wherein the donor nucleic acid is inserted into a cleavage site generated in the TRAC gene or the TRBC gene, and wherein expression of the endogenous TCR alpha chain and the endogenous TCR beta chain are disrupted.
  • the donor nucleic acid encodes a TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain, or wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a human or murine TCR beta chain lacking a variable domain.
  • the donor nucleic acid encodes a TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain, or wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a human or murine TCR alpha chain lacking a variable domain.
  • the donor nucleic acid encodes a first TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain and a second TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain.
  • the first TFP and the second TFP integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the donor nucleic acid encodes a first TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain, and further encodes a human or murine TCR beta chain lacking a variable domain.
  • the first TFP and the human or murine TCR beta chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the donor nucleic acid encodes a first TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain, and further encodes a human or murine TCR alpha chain lacking a variable domain.
  • the first TFP and the human or murine TCR alpha chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • GVHD graft-versus-host disease
  • the donor nucleic acid encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a human or murine TCR alpha chain lacking a variable domain and/or a nucleic acid sequence encoding a human or murine TCR beta chain lacking a variable domain.
  • the donor nucleic acid encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, and further encodes a human or murine TCR alpha chain lacking a variable domain, and a human or murine TCR beta chain lacking a variable domain.
  • the TFP, the human or murine TCR alpha chain, and the human or murine TCR beta chain integrate into a cell-surface TCR complex with an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • the genetically modified T cell exhibits reduced binding to MHC-peptide complexes compared to a control cell.
  • a genetically modified T cell comprising in its genome any of the recombinant nucleic acid molecules disclosed herein, wherein: (a) the recombinant nucleic acid molecule is inserted into an endogenous TRAC gene or an endogenous TRBC gene; (b) expression of the endogenous TCR alpha chain or the endogenous TCR beta chain is disrupted; and (c) the TFP is expressed by the genetically modified T cell and integrates into a cell-surface TCR complex.
  • the genetically modified T cell does not have detectable cell-surface expression of an endogenous TCR. In another embodiment, the genetically modified T cell exhibits reduced binding to MHC-peptide complexes compared to a control cell.
  • the T cell is a human T cell.
  • the T cell is a CD4+ cell, a CD8+ cell, a CD4+/CD8+ T cell, a naive T cell, a memory stem T cell, a central memory T cell, a double-negative T cell, a effector memory T cell, a effector T cell, a Thl cell, a Tel cell, a Th2 cell, a Tc2 cell, a Thl7 cell, a Th22 cell, a gamma/delta T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.
  • NK natural killer
  • NKT natural killer T
  • the recombinant nucleic acid molecule is inserted into the TRAC gene at an engineered meganuclease recognition sequence comprising SEQ ID NO: 30.
  • the recombinant nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a murine TCR alpha constant domain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP wherein the TCR complex subunit comprises a murine TCR beta constant domain, or wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a murine TCR beta chain lacking a variable domain.
  • the recombinant nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a murine TCR beta constant domain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP wherein the TCR complex subunit comprises a murine TCR alpha constant domain, or wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a murine TCR alpha chain lacking a variable domain.
  • the nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a murine TCR alpha constant domain and a second TFP wherein the TCR complex subunit comprises a murine TCR beta constant domain.
  • the first TFP and the second TFP integrate into a cell- surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • the recombinant nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a murine TCR alpha constant domain, and further encodes a murine TCR beta chain lacking a variable domain.
  • the first TFP and the murine TCR beta chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains embodiments.
  • the recombinant nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a murine TCR beta constant domain, and further encodes a murine TCR alpha chain lacking a variable domain.
  • the first TFP and the TCR alpha chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • GVHD graft-versus-host disease
  • the recombinant nucleic acid molecule encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, and wherein the genetically-modified T cell further comprises in its genome a nucleic acid sequence encoding a human or murine TCR alpha chain lacking a variable domain, and/or a nucleic acid sequence encoding a human or murine TCR beta chain lacking a variable domain.
  • the nucleic acid molecule encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, a murine TCR alpha chain lacking a variable domain, and a murine TCR beta chain lacking a variable domain.
  • the TFP, the murine TCR alpha chain, and the murine TCR beta chain integrate into a cell-surface TCR complex with an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • the recombinant nucleic acid molecule encodes a TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain, wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a human or murine TCR beta chain lacking a variable domain.
  • the recombinant nucleic acid molecule encodes a TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain, and wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a second TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain, or wherein the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding a human or murine TCR alpha chain lacking a variable domain.
  • the recombinant nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain and a second TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain.
  • the first TFP and the second TFP integrate into a cell-surface TCR complex two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the recombinant nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a human or murine TCR alpha constant domain, and further encodes a human or murine TCR beta chain lacking a variable domain.
  • the first TFP and the human or murine TCR beta chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the recombinant nucleic acid molecule encodes a first TFP wherein the TCR complex subunit comprises a human or murine TCR beta constant domain, and further encodes a human or murine TCR alpha chain lacking a variable domain.
  • the first TFP and the human or murine TCR alpha chain integrate into a cell-surface TCR complex with two endogenous CD3 epsilon chains, an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • GVHD graft-versus-host disease
  • the recombinant nucleic acid molecule encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, and wherein the genetically-modified T cell further comprises in its genome a nucleic acid sequence encoding a human or murine TCR alpha chain lacking a variable domain and/or a nucleic acid sequence encoding a human or murine TCR beta chain lacking a variable domain.
  • the recombinant nucleic acid molecule encodes a TFP wherein the TCR complex subunit comprises a CD3 epsilon chain, and further encodes a human or murine TCR alpha chain lacking a variable domain and/or a human or murine TCR beta chain lacking a variable domain.
  • the TFP, the human or murine TCR alpha chain, and the human or murine TCR beta chain integrate into a cell-surface TCR complex with an endogenous CD3 gamma chain, an endogenous CD3 delta chain, and two endogenous CD3 zeta chains.
  • the genetically modified T cell does not induce graft-versus-host disease (GVHD) when administered to an allogeneic subject.
  • GVHD graft-versus-host disease
  • the genetically modified T cell further comprises in its genome a nucleic acid sequence encoding an inhibitory molecule, wherein the inhibitory molecule comprises a first polypeptide comprising at least a portion of an inhibitory molecule; and a second polypeptide comprising a positive signaling domain from an intracellular signaling domain.
  • the first polypeptide comprises at least a portion of PD-1
  • the second polypeptide comprises an optional co-stimulatory domain and a primary signaling domain.
  • a population of genetically modified T cells comprising a plurality of the genetically modified T cells disclosed herein. In one embodiment, at least about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to 100% of cells in the population are the genetically-modified T cells as disclosed herein. In one embodiment, up to about 90% of the genetically modified T cells that are CD8+ exhibit a naive phenotype. [0045] In another aspect is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the population of genetically modified T cells as disclosed herein.
  • a method for treating a disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as disclosed herein.
  • the method is an immunotherapy and the disease is a cancer.
  • the cancer is a leukemia or lymphoma.
  • the cancer cells express CD 19.
  • Figure 1 depicts TCR components used in the constructs disclosed herein (see, e.g ., Example 1). These constructs include truncated forms of aPTCR) and CD3e.
  • Figure 2 depicts constructs wherein the CD19-sepcific FMC63 scFv was attached via a Whitlow linker to either the human or murine versions of both the TCRa and b constant regions.
  • the scFv-TRAC and scFv-TRBC coding regions were separated by a P2A/furin site and the b chains were placed upstream of the a chains ( Figure 2A).
  • Figure 2B the order of the a and b chains was reversed
  • murine constant domains were encoded ( Figure 2C).
  • the fusion and TCR constant domains are separated by P2A/furin sites.
  • Figure 3 shows T cell subsets analyzed using antibodies specific for CD4, CD62L, and CD45RO.
  • Figure 3A shows 7069 TFP T cells and
  • Figure 3B shows 7070 CD3e-TFP T cells after magnetic depletion of non-edited cells; transgene + cells were identified on the basis of anti- FMC63 staining.
  • FIG 4 shows T cells with a CD19-specific CAR knocked into the TRAC locus.
  • non-edited T cells human o 3TCR +
  • Figure 4A shows residual human o 3TCR expression and scFv expression.
  • Figure 4B shows the frequency of transgene + cells identified on the basis of anti-FMC63 staining.
  • Figure 5 shows T cells with a CD 19-specific TCR TFP (encoded by construct 7069) knocked into the TRAC locus.
  • non-edited T cells human aPTCRf
  • Figure 5A shows residual human a.pTCR expression and scFv expression.
  • Figure 5B shows the frequency of transgene + cells identified on the basis of anti-FMC63 staining.
  • Figure 5C shows the frequency of transgene + cells identified on the basis of anti-TCR staining.
  • FIG. 6 shows T cells with a CD19-specific CD3e TFP (encoded by construct 7070) knocked into the TRAC locus.
  • non-edited T cells human a.pTCRT
  • Figure 6A shows residual human a.pTCR expression and scFv expression.
  • Figure 6B shows the frequency of transgene + cells identified on the basis of anti-FMC63 staining.
  • Figure 6C shows the frequency of transgene + cells identified on the basis of anti-TCR staining.
  • Figure 7 shows T cell subsets analyzed using antibodies specific for CD4, CD62L, and CD45RO.
  • Figure 7A shows 7206 CAR T cells after magnetic depletion of non-edited cells; transgene + cells were identified on the basis of anti-FMC63 staining.
  • the proportion of CD4+ and CD8+ cells for 7206 CAR T cells is shown in Figure 7B.
  • the proportion of CD4+ and CD8+ cells staining positive for CD26L is shown in Figures 7C and 7D, respectively.
  • Figure 8 shows T cell subsets analyzed using antibodies specific for CD4, CD62L, and CD45RO.
  • Figure 8A shows 7069 TFP T cells after magnetic depletion of non-edited cells; transgene + cells were identified on the basis of anti-FMC63 staining.
  • the proportion of CD4+ and CD8+ cells for 7069 TFP T cells is shown in Figure 8B.
  • the proportion of CD4+ and CD8+ cells staining positive for CD26L is shown in Figures 8C and 8D, respectively.
  • Figure 9 shows T cell subsets analyzed using antibodies specific for CD4, CD62L, and CD45RO.
  • Figure 9A shows 7070 TFP T cells after magnetic depletion of non-edited cells; transgene + cells were identified on the basis of anti-FMC63 staining.
  • the proportion of CD4+ and CD8+ cells for 7070 CAR T cells is shown in Figure 9B.
  • the proportion of CD4+ and CD8+ cells staining positive for CD26L is shown in Figures 9C and 9D, respectively.
  • Figure 10 shows a comparison of CAR T cell and TFP T cell expansion when cells were challenged with NALM/6 cells.
  • Figure 10A shows expansion of 7206 CAR T cells (circles) 7069 TCR- TFP T cells, (squares), and 7070 CD3e TFP T cells (triangles).
  • Figure 10B shows expansion of CD4+ cell (filled circles, squares, and triangles), and expansion of CD8+ cells (open circles, squares, and triangles).
  • Figure 11 is a graph showing a comparison of CAR T cell and TFP T cell expansion when cells were challenged with NALM/6 cells, including 7206 CAR T cells (circles) 7069 TCR-TFP T cells, (squares), and 7070 CD3e TFP T cells (triangles).
  • Figure 12 is a graph showing the total number of NALM/6 targets killed over the entire time course.
  • Figure 13 is a graph showing the results of challenge by Raji cells of CAR T and TFP T cells. Very similar kinetics were observed between 7206 CAR T cells and both TFP variants.
  • Figure 14 is a graph showing the kinetics of Raji cells killed by CAR T and TFP T cells over time.
  • Figure 15 is a graph showing the efficiency of Raji cells killed by CAR T and TFP T cells over time.
  • Figure 16 shows cell surface staining of T cells by flow cytometry.
  • Figure 16A shows FITC-conjugated CD 19 staining of non-transduced unedited T cells, non-transduced TCRocP-/- T cells, unedited T cells transduced with an anti-CD 19 epsilon-TFP fusion, or TCRocP-/- T cells transduced with an anti-CD 19 TCR-TFP fusion (AAV7069), an anti-CD 19 CD3 epsilon-TFP fusion (AAV7070), or a control TCR-TFP fusion.
  • Figure 16B shows cell-surface staining of the same cells with FITC-conjugated CD19 and anti-human CD3E.
  • Figure 16C shows staining of the same cells with anti -human CD3E and anti -human TCR.
  • Figure 16D shows staining of the same cells with anti -human CD3E and anti-murine TCR.
  • Figure 16E shows staining of the same cells with anti-human CD4 and anti-human CD8.
  • Figure 17 is a graph showing production of interferon gamma by the T cells shown (non- transduced unedited T cells, non-transduced TCRocP-/- T cells, unedited T cells transduced with an anti-CD 19 epsilon-TFP fusion, or TCRocP-/- T cells transduced with an anti-CD 19 TCR-TFP fusion (AAV7069), an anti-CD 19 CD3 epsilon-TFP fusion (AAV7070), or a control TCR-TFP fusion) when co-cultured with dendritic cells.
  • AAV7069 anti-CD 19 TCR-TFP fusion
  • AAV707070 anti-CD 19 CD3 epsilon-TFP fusion
  • AAV7070 anti-CD 19 CD3 epsilon-TFP fusion
  • Figure 18 is a series of graphs showing tumor burden in the Nalm6-luc tumor mouse model when treated with vehicle or the T cells shown (non-transduced unedited T cells, non- transduced TCRocP-/- T cells, unedited T cells transduced with an anti-CD 19 epsilon-TFP fusion, or TCRocP-/- T cells transduced with an anti-CD 19 TCR-TFP fusion (AAV7069), an anti-CD 19 CD3 epsilon-TFP fusion (AAV7070), or a control TCR-TFP fusion).
  • Figure 18A is an overlay of all of the plots.
  • Figure 18B shows individual plots for each treatment condition.
  • Figure 19 shows assessment of Graft-Versus Host Disease (GvHD) of the mice shown in Figure 18 at 49 days post-treatment.
  • Figure 19A shows immunohistochemistry staining of human CD7+ cells in mouse liver and skin tissue samples.
  • Figure 19B shows quantification of the immunohistochemistry data obtained as is described in Example 6. Tissues of two mice were examined for each treatment condition. The % of frames with CD7+ cells is shown on the Y- axis. Each bar is divided, from bottom to top, into the % of CD7+ frames with 1 CD7+ cell, with 2 CD7+ cells, and with 3+ CD7+ cells.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • a TCR complex subunit that does not comprise a variable domain
  • a transmembrane domain a transmembrane domain
  • an antigen binding domain comprising an antibody, or fragment thereof, a binding ligand or a fragment thereof that is capable of binding to an antibody or antibody fragment, or a ligand, or fragment thereof that binds to a receptor or polypeptide expressed on the surface of a cell; wherein the TCR complex subunit and the antibody are operatively linked.
  • vectors comprising the recombinant nucleic acid molecules disclosed herein.
  • modified T cells comprising the recombinant nucleic acid molecules 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 TFPs of the nucleic acid molecules disclosed herein or TFPs encoded by the sequence of the nucleic acid molecules disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified allogenic T cells comprising the sequence encoding the TFPs disclosed herein or TFPs encoded by the sequence of the nucleic acid molecules disclosed herein.
  • compositions comprising: (a) the modified T cells of the disclosure; and (b) a pharmaceutically acceptable carrier.
  • methods 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) introducing into the T cell containing a functional disruption of an endogenous TCR gene a recombinant nucleic acid molecule of the disclosure, or the vectors disclosed herein.
  • a method of producing the modified T cell of the disclosure comprising introducing into a T cell containing a functional disruption of an endogenous TCR gene a recombinant nucleic acid molecule 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.
  • compositions 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. [0081] As used herein, “treating” or “treatment” 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 tumor cell receptor
  • TCP T cell receptor
  • the TCR receptor complex is an octomeric complex of variable TCR receptor a and b chains with three dimeric signaling modules CD35/e, CD3y/e and CD247 z/z or z/h.
  • TCR alpha chain refers to these subunits in the TCR complex.
  • TCR beta chain refers to these subunits in the TCR complex.
  • CD3 epsilon chain refers to these subunits in the TCR complex.
  • CD3 delta chain refers to these subunits in the TCR complex.
  • the TCR alpha and beta subunits comprise both a variable domain and a constant, invariable domain.
  • 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
  • signal transduction event such as, but not limited to, signal transduction via the 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”.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.
  • 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 immobilised by, for example, coating on to a bead or plate.
  • HLA human leukocyte antigen system
  • MHC major histocompatibility complex
  • HLA class I antigens A, B & C
  • HLA class II antigens DP, DQ, & DR
  • HLA alleles A, B and C present peptides derived mainly from intracellular proteins, e.g ., proteins expressed within the cell.
  • 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 recognise 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 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.
  • an “intracellular signaling domain,” as the term is used herein, 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
  • examples of immune effector function, e.g, in a modified T-T cell include cytolytic activity and T helper cell activity, including the secretion of cytokines.
  • 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 ITAM (“immunoreceptor tyrosine-based activation motif’).
  • ITAM 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 DAP 10 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.
  • An intracellular 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 (CD 137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, 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- IBB 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 antigen binding domain 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 ak, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et ak, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et ak, 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 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. A skilled artisan will understand that 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. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that 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. Such 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.
  • CD 19 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.
  • 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.
  • the phrase 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.
  • ⁇ ективное 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.
  • 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 (GlyNer)
  • Also included within the scope of the present disclosure are linkers described in WO2012/138475 (incorporated herein by reference).
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • 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.
  • z 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. In the preferred embodiment of a construct for transient expression, 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.
  • poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • 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 associated antigen or “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.
  • the term “introduced” or “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 a plurality of TAL domain repeats fused to a nuclease domain or an active portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S 1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. See, for example, Christian et al. (2010) Genetics 186:757-761, which is incorporated by reference in its entirety.
  • Nuclease domains useful for the design of TALENs include those from a Type lis restriction endonuclease, including but not limited to Fokl, FoM, Stsl, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and AlwT Additional Type lis restriction endonucleases are described in International Publication No. WO 2007/014275.
  • the nuclease domain of the TALEN is a Fokl nuclease domain or an active portion thereof.
  • TAL domain repeats can be derived from the TALE (transcription activator-like effector) family of proteins used in the infection process by plant pathogens of the Xanthomonas genus.
  • TAL domain repeats are 33-34 amino acid sequences with divergent 12th and 13th amino acids. These two positions, referred to as the repeat variable dipeptide (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. Each base pair in the DNA target sequence is contacted by a single TAL repeat, with the specificity resulting from the RVD.
  • the TALEN comprises 16-22 TAL domain repeats. DNA cleavage by a TALEN requires two DNA recognition regions flanking a nonspecific central region ⁇ i.e., the "spacer").
  • spacer in reference to a TALEN refers to the nucleic acid sequence that separates the two nucleic acid sequences recognized and bound by each monomer constituting a TALEN.
  • the TAL domain repeats can be native sequences from a naturally- occurring TALE protein or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence (see, for example, Boch et al. (2009) Science 326(5959): 1509-1512 and Moscou and Bogdanove (2009) Science 326(5959): 1501, each of which is incorporated by reference in its entirety). See also, Ei.S. Publication No. 20110145940 and International Publication No.
  • each nuclease e.g ., Fokl
  • each nuclease monomer can be fused to a TAL effector sequence that recognizes a different DNA sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme.
  • TALEN can refer to a single TALEN protein or, alternatively, a pair of TALEN proteins (i.e., a left TALEN protein and a right TALEN protein) which bind to the upstream and downstream half-sites adjacent to the TALEN spacer sequence and work in concert to generate a cleavage site within the spacer sequence.
  • upstream and downstream half-sites can be identified using a number of programs known in the art (Kornel Labun; Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093/nar/gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen. (2014). CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42.
  • TALEN recognition sequence can be defined as the DNA binding sequence (i.e., half-site) of a single TALEN protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and the downstream half-site.
  • compact TALEN refers to an endonuclease comprising a DNA-binding domain with one or more TAL domain repeats fused in any orientation to any portion of the I-Tevl homing endonuclease or any of the endonucleases listed in Table 2 in U.S. Application No. 20130117869 (which is incorporated by reference in its entirety), including but not limited to Mmel, EndA, Endl, I-Basl, I-TevII, I-TevIII, I-Twol, Mspl, Mval, NucA, and NucM.
  • Compact TALENs do not require dimerization for DNA processing activity, alleviating the need for dual target sites with intervening DNA spacers.
  • the compact TALEN comprises 16-22 TAL domain repeats.
  • 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.
  • the caspase component of a CRISPR is an RNA- guided DNA endonuclease.
  • the caspase is a class II Cas enzyme.
  • the caspase is a class II, type II enzyme, such as Cas9.
  • the caspase is a class II, type V enzyme, such as Cpfl.
  • the guide RNA comprises a direct repeat and a guide sequence (often referred to as a spacer in the context of an endogenous CRISPR system), which is complementary to the target recognition site.
  • the CRISPR further comprises a tracrRNA (trans- activating CRISPR RNA) that is complementary (fully or partially) to a direct repeat sequence (sometimes referred to as a tracr- mate sequence) present on the guide RNA.
  • the caspase can be mutated with respect to a corresponding wild- type enzyme such that the enzyme lacks the ability to cleave one strand of a target polynucleotide, functioning as a nickase, cleaving only a single strand of the target DNA.
  • Non-limiting examples of caspase enzymes that function as a nickase include Cas9 enzymes with a D10A mutation within the RuvC I catalytic domain, or with a H840A, N854A, or N863 A mutation.
  • recognition sequences can be identified using a number of programs known in the art (Komel Labun; Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016).
  • CHOPCHOP v2 a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093/nar/gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen. (2014).
  • CHOPCHOP a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407).
  • 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
  • zinc finger nuclease or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, SI nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease.
  • Nuclease domains useful for the design of zinc finger nucleases include those from a Type IIs restriction endonuclease, including but not limited to Fokl, FoM, and Stsl restriction enzyme. Additional Type IIs restriction endonucleases are described in International Publication No. WO 2007/014275, which is incorporated by reference in its entirety. The structure of a zinc finger domain is stabilized through coordination of a zinc ion. DNA binding proteins comprising one or more zinc finger domains bind DNA in a sequence-specific manner.
  • the zinc finger domain can be a native sequence or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence ⁇ 18 basepairs in length, comprising a pair of nine basepair half-sites separated by 2-10 basepairs.
  • zinc finger nuclease can refer to a single zinc finger protein or, alternatively, a pair of zinc finger proteins (i.e., a left ZFN protein and a right ZFN protein) that bind to the upstream and downstream half-sites adjacent to the zinc finger nuclease spacer sequence and work in concert to generate a cleavage site within the spacer sequence.
  • upstream and downstream half-sites can be identified using a number of programs known in the art (Mandell JG, Barbas CF 3rd.
  • Zinc Finger Tools custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res.
  • a zinc finger nuclease recognition sequence can be defined as the DNA binding sequence (i.e., half-site) of a single zinc finger nuclease protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and the downstream half-site.
  • the term "recognition sequence” refers to a DNA sequence that is bound and cleaved by an endonuclease.
  • a recognition sequence comprises a pair of inverted, 9 basepair "half sites" which are separated by four basepairs.
  • the N-terminal domain of the protein contacts a first half site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3' "overhangs".
  • “Overhangs”, or “sticky ends” are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double- stranded DNA sequence.
  • the overhang comprises bases 10-13 of the 22 basepair recognition sequence.
  • the recognition sequence comprises a first CNNNGN sequence that is recognized by the I-Tevl domain, followed by a nonspecific spacer 4-16 basepairs in length, followed by a second sequence 16-22 bp in length that is recognized by the TAL-effector domain (this sequence typically has a 5' T base).
  • Cleavage by a Compact TALEN produces two basepair 3' overhangs.
  • the recognition sequence is the sequence, typically 16-24 basepairs, to which the guide RNA binds to direct cleavage. Full complementarity between the guide sequence and the recognition sequence is not necessarily required to effect cleavage.
  • Cleavage by a CRISPR can produce blunt ends (such as by a class II, type II caspase) or overhanging ends (such as by a class II, type V caspase), depending on the caspase.
  • cleavage by the CRISPR complex comprising the same will result in 5' overhangs and in certain embodiments, 5 nucleotide 5' overhangs.
  • Each caspase enzyme also requires the recognition of a PAM (protospacer adjacent motif) sequence that is near the recognition sequence complementary to the guide RNA.
  • PAMs are typically 2-5 base pair sequences adjacent to the target/recognition sequence.
  • PAM sequences for particular caspase enzymes are known in the art (see, for example, U.S. Patent Nos.
  • PAM sequences for novel or engineered caspase enzymes can be identified using methods known in the art, such as a PAM depletion assay (see, for example, Karvelis et al. (2017) Methods 121-122:3- 8, which is incorporated herein in its entirety).
  • the DNA binding domains typically recognize an 18- bp recognition sequence comprising a pair of nine basepair "half-sites" separated by 2-10 basepairs and cleavage by the nuclease creates a blunt end or a 5' overhang of variable length (frequently four basepairs).
  • target site refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease
  • homologous recombination or “HR” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11: 1958-1976).
  • the homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.
  • non-homologous end-joining refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11: 1958-1976).
  • NHEJ non-homologous end-joining
  • a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • 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 receptor (TCR) fusion protein (TFP), wherein the modified T cell also has a functionally disrupted endogenous TCR.
  • TCR T cell receptor
  • TCP T cell receptor
  • TCP T cell receptor
  • TCP T cell receptor
  • 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.
  • TFPs can integrate into a cell-surface TCR complex in a T cell to confer specificity of the T cell for specific targets, such as tumor associated antigens on cancer cells.
  • TFPs can be expressed in T cells having a functional disruption of a TCR alpha chain gene and/or a TCR beta chain gene, such that the resulting cell-surface TCRs lack variable domains and the genetically modified T cells are useful for allogeneic therapy.
  • the TCR complex subunit of a TFP can be a CD3 epsilon domain.
  • the T cell can be further modified to express TCR alpha chains and TCR beta chains lacking a variable domain (z.e., each chain comprises a constant domain and a transmembrane domain), which integrate into the same cell-surface TCR complex as the TFP.
  • the TCR complex subunit of a TFP can be a TCR alpha constant domain.
  • the T cell can be further modified to express a TCR beta chain lacking a variable domain (z.e., comprising a constant domain and a transmembrane domain), which integrates into the same cell-surface TCR complex as the TFP.
  • the TCR complex subunit of a TFP can be a TCR beta constant domain.
  • the T cell can be further modified to express a TCR alpha constant chain lacking a variable domain (z.e., comprising a constant domain and a transmembrane domain), which integrates into the same cell-surface TCR complex as the TFP.
  • a TCR alpha constant chain lacking a variable domain (z.e., comprising a constant domain and a transmembrane domain), which integrates into the same cell-surface TCR complex as the TFP.
  • the TCR complex subunit can be murine or human. In cases where only one of the endogenous TCR alpha or TCR beta chains is disrupted, and the TCR complex subunit is a TCR alpha constant domain or TCR beta constant domain, it is preferred that the TCR complex subunit is murine, and that it is co-expressed with another murine TCR alpha or beta chain lacking a variable domain, or with another TFP comprising a TCR complex subunit that is murine. In this manner, the two murine constant domains can co-integrate into a cell-surface TCR complex that lacks any variable domains.
  • the use of only murine constant domains can reduce the likelihood that a TFP will incorporate into a TCR complex with an endogenous human TCR alpha or beta chain, resulting in a TCR complex that comprises a variable domain and is no longer useful for allogeneic therapy.
  • the TCR complex subunit can be murine or human, and can be co-expressed with another murine or human TCR alpha or beta chain lacking a variable domain, or with another TFP comprising a TCR complex subunit that is murine or human. Because the endogenous TCR alpha and beta chains are disrupted, TCR complexes will assemble using the exogenous components and lack any variable domains on the alpha and beta constant domains.
  • both the endogenous TCR alpha and TCR beta chains can be disrupted, and the TFP can be co-expressed, for example, with a murine or human TCR alpha chain and a murine or human TCR beta chain that lack a variable domain.
  • the TFP can incorporate into a cell-surface TCR complex that lacks variable domains and is useful for allogeneic therapy.
  • 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 a scFv, a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as “an 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 (TFP) T cell receptor (TCR) fusion proteins (TFP)
  • the present disclosure encompasses recombinant nucleic acid molecules encoding one or more TFPs, wherein each TFP comprises an antigen binding domain that binds specifically to a tumor associated antigen, e.g ., human CD19wherein the sequence of the antigen binding domain is contiguous with, and is 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 of the TFP is specific for any antigen or epitope of interest, particularly any tumor associated antigen or epitope of interest.
  • the antigen of the target is a tumor associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD30, CD40, CLL-1, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M- CSF, prostase, prostase specific antigen (HER2/neu), carcinoembry
  • the antigen binding domain of the TFP has specificity for CD 19.
  • 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 murine 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
  • 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 of the TFP may comprise human, murine, or humanized residues for the antigen binding domain of an antibody or antibody fragment.
  • the antigen binding domain comprises a murine, humanized or human antibody or an antibody fragment.
  • the antigen binding domain is contiguous with, and in the same reading frame as, a leader sequence.
  • the antigen 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 an antibody, 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 an antibody, e.g, an antigen binding domain comprising one or more, e.g, all three, LC CDRs and one or more, e.g, all three, HC CDRs of an antibody.
  • the antigen 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 an antibody, e.g, the antigen binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
  • HC CDR1 heavy chain complementary determining region 1
  • HC CDR2 heavy chain complementary determining region 2
  • HC CDR3 heavy chain complementary determining region 3
  • the antigen binding domain comprises a murine, humanized, or human light chain variable region and/or a humanized or human heavy chain variable region.
  • the murine, humanized, or human antigen binding domain comprises a humanized heavy chain variable region, e.g, at least two humanized or human heavy chain variable regions described herein.
  • the antigen binding domain is a scFv, and a light chain variable region comprising an amino acid sequence, is attached to a heavy chain variable region of an antibody via a linker, e.g, a linker described herein.
  • the antigen 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 is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
  • the antigen binding domain (e.g ., a scFv) comprises: 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 light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy 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 with 95-99% identity to 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
  • the antigen 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)).
  • 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.
  • 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 ah, U.S. Pat. No. 5,585,089; and Riechmann et ah, 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.
  • Humanized antibodies and antibody fragments substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species.
  • Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.
  • 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 or 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.
  • 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. , the ability to bind the same tumor associated antigen.
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to a tumor associated antigen.
  • the antigen binding domain is characterized by particular functional features or properties of an antibody or antibody fragment, such as binding to a particular tumor associated antigen.
  • the antigen binding domain of a TFP composition of the present disclosure specifically binds human CD 19.
  • the antigen binding domain has the same or a similar binding specificity to human CD 19 as the FMC63 scFv described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).
  • a target antigen e.g ., CD 19 or any tumor associated antigen described elsewhere herein
  • VH 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 al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • scFv molecules can be produced by linking VH and VL 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 long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • a scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its VL and VH 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 (Gly4Ser) 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.
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • the stability of an antibody or antibody fragment can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control antibody or antibody fragment.
  • the antibody or antibody fragment 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 antibody or antibody fragment in the described assays.
  • the improved thermal stability of the antibody or antibody fragment is subsequently conferred to the entire TFP construct, leading to improved therapeutic properties of the TFP construct.
  • the thermal stability of the antibody or antibody fragment can be improved by at least about 2°C or 3°C as compared to a conventional antibody.
  • the antibody or antibody fragment e.g, scFv
  • the antibody or antibody fragment e.g, scFv
  • 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 antibody or antibody fragment (e.g ., scFv), comprises at least one mutation arising from the humanization process such that the mutated antibody or antibody fragment confers improved stability to the TFP construct.
  • the antibody or antibody fragment (e.g., scFv) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated antibody or antibody fragment confers improved stability to the 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 antigen binding domain described herein.
  • 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.
  • VH and/or VL variable regions
  • an 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, glutamic
  • 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 ak, (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et ak, (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 a starting antibody or antibody fragment (e.g ., scFv) amino acid sequence that generate functionally equivalent molecules.
  • VH or VL of an antibody or antibody fragment (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%,
  • 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%.
  • the extracellular domain of a TFP can comprise, for example, a human or murine constant domain of a TCR alpha chain or a TCR beta chain, fused to an antigen binding domain having specificity for a tumor associated antigen.
  • the extracellular domain of a TFP can comprise the extracellular domain of a CD3 epsilon chain fused to an antigen binding domain having specificity for a tumor associated antigen.
  • the extracellular domain is capable of associating with the transmembrane domain of the TFP.
  • the extracellular domain of a TFP does not comprise a variable domain, such as the endogenous TCR alpha chain variable domain or the endogenous TCR beta chain variable domain, in order to reduce the allogenicity of a TCR complex comprising one or more TFPs.
  • a TFP sequence comprises 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.
  • a transmembrane domain of particular use in this present disclosure may include at least the transmembrane region(s) of e.g, the alpha, beta, gamma, delta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can be attached to the extracellular domain of the TFP, e.g, TCR complex subunit 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 a linkage between the transmembrane domain and the cytoplasmic or extracellular domain of the TFP.
  • 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.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the linker has the sequence (G4S)n, wherein n is an integer between 1 and 4.
  • the cytoplasmic domain of a TCR complex comprising a TFP can include one or more intracellular signaling domains, such as the endogenous intracellular signaling domains of the CD3 gamma, delta, zeta, or epsilon polypeptides.
  • intracellular signaling domains such as the endogenous intracellular signaling domains of the CD3 gamma, delta, zeta, or epsilon polypeptides.
  • the TCR alpha chain subunit and TCR beta chain subunit generally lack an intracellular 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.
  • effector function refers to a specialized function of a cell.
  • 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 signaling domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that 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.
  • TCR T cell receptor
  • co-receptors that act in concert to initiate signal transduction following antigen receptor engagement
  • the TFP-expressing cell described herein can further comprise a second TFP, e.g ., a second TFP that includes a different antigen binding domain, e.g. , to the same target e.g. , CD19) or a different target (e.g, CD123) than the first TFP.
  • a second TFP e.g ., a second TFP that includes a different antigen binding domain, e.g. , to the same target e.g. , CD19) or a different target (e.g, CD123) than the first TFP.
  • 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 TFP and a 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. , 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, CD160, 2B4 and TGFR beta.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g.
  • the agent comprises a first polypeptide, e.g. , of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g, at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g, comprising a costimulatory domain (e.g, 4- IBB, CD27 or CD28, e.g, as described herein) and/or a primary signaling domain (e.g, a CD3 zeta signaling domain described herein).
  • a costimulatory domain e.g, 4- IBB, 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 Int. Immunol 8:765-75).
  • PD-L1 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 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43).
  • PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281- 7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). 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, 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).
  • ECD extracellular domain
  • PD1 TFP when used in combinations with a TFP described herein, improves the persistence of the T cell.
  • the present disclosure provides a population of TFP-expressing T cells, e.g ., TFP 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 an anti-CD 19 binding domain, and a second cell expressing a TFP having a different anti-CD 19 binding domain.
  • the population of TFP-expressing cells can include a first cell expressing a TFP that includes an anti-CD 19 binding domain and a second cell expressing a TFP that includes an antigen binding domain to a target other than CD 19 (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 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, CD160, 2B4 and TGFRbeta.
  • 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 TFP is encoded by a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA encoding the 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.
  • PCR polymerase chain reaction
  • 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.
  • 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 concatameric 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 polyT 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 al., RNA, 7:1468-95 (2001); Elango, et al., 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. Hum Gene Then, 12(8):861-70 (2001).
  • recombinant nucleic acid molecules encoding a TFP comprising: (a) a TCR complex subunit that does not comprise a variable domain; (b) a transmembrane domain; and (c) an antigen binding domain comprising an antibody, or fragment thereof, a binding ligand or a fragment thereof that is capable of binding to an antibody or antibody fragment, or a ligand, or fragment thereof that binds to a receptor or polypeptide expressed on the surface of a cell; wherein the TCR complex subunit and the antibody are operatively linked.
  • the TFP incorporates into a functional TCR complex when expressed in a T cell.
  • the TFP incorporates into a functional TCR complex with an exogenous TCR alpha chain and/or TCR beta chain that lacks a variable domain (i.e., only comprises the constant and transmembrane domains).
  • the lack of variable domains within the TCR complex allows the genetically modified T cell to be useful for allogeneic therapy.
  • the sequence encoding the TFP and the sequence(s) encoding exogenous TCR alpha chain and/or TCR beta chain are contained within a same nucleic acid molecule.
  • the sequence encoding the TFP and the sequence(s) encoding the exogenous TCR alpha chain and/or TCR beta chain are contained within different nucleic acid molecules.
  • the TCR complex subunit and the antigen binding domain are operatively linked by a linker sequence.
  • the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha or TCR beta.
  • the TCR complex into which the TFP integrates comprises intracellular domains of endogenous TCR complex components (e.g ., CD3 epsilon, CD3 gamma, CD3 delta, and/or CD3 zeta).
  • the TCR complex subunit comprises (i) at least a portion of a TCR subunit extracellular domain, and (ii) a TCR subunit transmembrane domain, wherein (i) and (ii) are from the same TCR subunit (e.g., a TCR alpha chain, a TCR beta chain, or a CD3 epsilon chain).
  • the TFP further comprises a CD3 epsilon intracellular domain.
  • the TCR complex subunit comprises an extracellular domain, or portion thereof, of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, or a CD3 epsilon chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR complex 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, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TFP is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR subunit polypeptide.
  • the TCR complex subunit is a TCR alpha constant chain lacking a variable domain, and the TFP functionally integrates into a TCR complex comprising an a TCR beta chain lacking a variable domain, two CD3 epsilon chains, a CD3 gamma chain , a CD3 delta chain, two CD3 zeta chains, or a combination thereof;
  • the TCR complex subunit is a TCR beta chain lacking a variable domain, and the TFP functionally integrates into a TCR complex comprising a TCR alpha chain lacking a variable domain, two CD3 epsilon chains, a CD3 gamma chain, a CD3 delta chain, two CD3 zeta chains, or a combination thereof; or
  • the TCR complex subunit is a CD3 e
  • the TCR alpha chain lacking a variable domain can comprise a constant domain and transmembrane domain only, or may be in the form of a TFP comprising a constant domain, a transmembrane domain, and an antigen binding domain.
  • the antigen binding domain is an antibody or antibody fragment.
  • the antibody fragment is a 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.
  • the recombinant nucleic acid molecule 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. [0244] In some instances, the recombinant nucleic acid molecule is selected from the group consisting of a DNA and an RNA.
  • the recombinant nucleic acid molecule is an mRNA. In some instances, the recombinant nucleic acid molecule 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-dimethylaminoethyloxy ethyl (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 G,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a
  • the antigen binding domain of a TFP 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 of a TFP 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.
  • vectors comprising the recombinant nucleic acid molecules 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 recombinant nucleic acid molecules of the present disclosure can be cloned into a number of types of vectors.
  • the recombinant nucleic acid molecules 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. 6,326,193).
  • the vector is an adenoviral vector.
  • 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 EFla 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 ak, 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 (MMTV), 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
  • MMTV 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.
  • Promoters capable of expressing a TFP can also include synthetic promoters, such as the JeT promoter (WO 2002/012514).
  • 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 [0260] 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.
  • modified T cells comprising the recombinant nucleic acid molecule disclosed herein, or the vectors disclosed herein; wherein the modified T cell comprises a functional disruption of an endogenous TCR. Also disclosed herein, in some embodiments, are modified T cells comprising a sequence encoding a TFP disclosed herein, or a TFP encoded by a sequence of a nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising a sequence encoding a TFP disclosed herein, or a TFP encoded by a sequence of a nucleic acid disclosed herein.
  • the T cell further comprises a nucleic acid sequence encoding a TCR alpha chain and/or TCR beta chain lacking a variable domain (; i.e ., comprising a TCR alpha or beta constant domain and transmembrane 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+ 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 Oncol ogyCytoMate, 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 Oncol ogyCytoMate, or the Haemonetics® Cell Saver® 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • buffers such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ® gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
  • 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.
  • the incubation time period is 24 hours.
  • 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, CDllb, 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
  • 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. [0276] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g ., particles such as beads), interactions between the particles and cells is minimized.
  • target antigens of interest such as CD28-negative T cells
  • CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations.
  • the concentration of cells used is 5xl0 6 /mL. In other aspects, the concentration used can be from about lxl0 5 /mL to lxl0 6 /mL, and any integer value in between.
  • 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 derived from induced pluripotent stem cells (iPSCs) that have been differentiated into T cells useful for the invention.
  • iPSCs induced pluripotent stem cells
  • 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 be used as can other methods commonly known in the art (Berg et ak, Transplant Proc. 30(8):3975-3977, 1998; Haanen et ak, J. Exp. Med. 190(9): 13191328, 1999; Garland et ak, J. Immunol. Meth. 227(1- 2):53-63, 1999).
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • 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+).
  • TH, CD4+ helper T cell population
  • TC cytotoxic or suppressor T cell population
  • 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.
  • infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
  • an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • TFP tissue plasminogen activator
  • 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. Assays to evaluate the effects of an anti-CD 19 are described in further detail below.
  • 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.
  • Cultures are re-stimulated with either CD 19+ K562 cells (K562-CD19), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1BBL in the presence of antiCD3 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 human CD19-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 ak, 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.
  • 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 NaCrtN, 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).
  • Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g. , in Barrett et al., Human Gene Therapy 22:1575-1586 (2011).
  • NOD/SCID/yc-/- (NSG) 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 CD 19 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, mannito
  • 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 ah, New Eng. J. of 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.
  • a potential issue that can arise in patients being treated using transiently expressing TFP T cells is anaphylaxis after multiple treatments.
  • 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, or both; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) introducing into the T cell a recombinant nucleic acid molecule of the disclosure encoding one or more TFPs, or the vectors disclosed herein.
  • disrupting comprises introducing into the T cell a nuclease protein, or a nucleic acid sequence encoding a nuclease protein, that targets an endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • methods of producing the modified T cell of the disclosure comprising introducing into a T cell containing a functional disruption of an endogenous TCR gene a recombinant nucleic acid molecule of the disclosure encoding one or more TFPs, or the vectors 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 compact TALEN, a CRISPR system nuclease, a CRISPR system nickase, or a megaTAL nuclease.
  • the sequence comprised by the recombinant nucleic acid molecule or the vector is inserted into the endogenous TCR subunit gene at the cleavage site generated by the nuclease, and insertion of the recombinant nucleic acid molecule into the endogenous TCR subunit gene functionally disrupts expression of 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.
  • 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 one or more TFPs 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 one or more TFPs 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.
  • 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 second subset of T cells express a second TFP.
  • 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 FK506, 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 JNeurosurg 108:963-971.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclospor
  • 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 IFN-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, CD160, 2B4 and TGFRbeta.
  • 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 expresses a TFP. In another embodiment, the fusion protein is expressed by a cell, e.g, a T cell that does not express a TFP.
  • Chimeric antigen receptors are molecules in which antibody fragments are linked to T cell receptor complex (CD3z) signaling domains. When delivered transgenically to T cells, this enables re-direction of the cell’s specificity. Tumor targets are engaged by the antibody fragment (e.g ., scFv), and TCR signaling is mimicked by the intracellular CD3zdomains of the CAR.
  • CD3z T cell receptor complex
  • TCR T cell receptor alpha constant region
  • the TCR components used in these constructs included truncated forms of aPTCR (i.e. only the constant domains of TCRa and TCRP) and CD3e ( Figure 1).
  • the CD19-sepcific FMC63 scFv was attached via a Whitlow linker to either the human or murine versions of both the TCRa and b constant regions.
  • the scFv-TRAC and scFv-TRBC coding regions were separated by a P2A/furin site and the b chains were placed upstream of the a chains (Figure 2A).
  • the order of the a and b chains was reversed ( Figure 2B).
  • TCRs without variable region domains were built into a set of constructs along with the CD3e fusion.
  • human constant domains were encoded, and in another construct murine domains were encoded ( Figure 2C).
  • the fusion and TCR constant domains were separated by P2A/furin sites.
  • Example 2 Production and characterization of T cell populations expressing TFPs.
  • T cells were enriched from an apheresis sample obtained from a healthy, informed, and compensated donor. Enrichment was accomplished using the CD3 positive selection Kit II (StemCell Technologies) and a manual magnet. T cells were stimulated for three days with ImmunoCult anti-CD2/3/28 (StemCell Technologies) in XVIVO-15 medium (Lonza) supplemented with 5% fetal bovine serum (HiClone/Gibco) and lOng/ml recombinant human IL-2 (Gibco).
  • T cells were electroporated with RNA encoding the TRC1-2L.1592 meganuclease (lpg/lxlO 6 cells) and transduced immediately thereafter with an AAV vector encoding an FMC63 -based CAR (AAV 7206), a TCR-TFP fusion (AAV 7069 - shown in Figure 1 A), or a CD3e-TFP fusion (AAV7070 - shown in Figure IB).
  • AAV 7206 FMC63 -based CAR
  • AAV 7069 TCR-TFP fusion
  • CD3e-TFP fusion AAV7070 - shown in Figure IB
  • Edited cells were cultured for an additional 5 days in X-VIVOTM 15 hematopoietic cell medium (Lonza Bioscience) supplemented with 30ng/ml IL-2 prior to magnetic depletion of non- edited cells, which continue to express endogenous TCR. This was accomplished using a biotinylated anti-human ajlTCR antibody and anti-biotin microbeads (both from Miltenyi Biotec).
  • TRAC knockout and CAR/TFP knock-in were carried out by staining samples with anti-human a.pTCR-biotin (Miltenyi Biotec) followed by streptavidin-BV421 (BioLegend), anti-mouse TCR-PE (H57-597 - BioLegend), and anti- FMC63-AlexaFluor® 647 (clone VM16, produced and conjugated in-house). Data were acquired on a Beckman-Coulter® CytoFLEX®-S.
  • CAR T cells CAR T cells, TCR-TFP T cells, and CD3e-TFP T cells were produced, analyzed, and enriched as described in Example 2.
  • T cell subsets were analyzed using antibodies specific for CD4 (BioLegend clone OKT4 conjugated to BV711), CD8 (BioLegend clone RPA-T8, BV421) CD62L (BD Biosciences clone DREG56, BB515), and CD45RO (BioLegend clone UCHL1, PE/Cy7).
  • transgene + cells were identified on the basis of anti-FMC63 staining Figure 7A for 7206 CAR T cells, Figure 3A for 7069 TCR-TFP T cells, and Figure 3B for 7070 CD3e-TFP T cells).
  • FMC63 + cells were found to have a CD4:CD8 ratio of nearly 1:1 for all three constructs analyzed Figures 7B, 8B, and 9B) and contained comparable frequencies of central memory (CD62L + CD45RO Lo ) transitional memory (CD62L + CD45RO Hl ) and effector memory T cells (CD62L CD45RO Hl ).
  • the 7206 CAR T cell product contained a lower frequency of central memory phenotype cells than the TFP variants, and this effect was more pronounced in the CD8 + fraction ( Figures 7-9, panels C and D).
  • CAR. T cells, TCR-TFP T cells, and CD3e-TFP T cells were prepared, enriched, and analyzed as described in Examples 2 and 3.
  • Co-cultures were assembled at a 1 : 1 ratio of targets to effectors on day 0. At each indicated time point, 10% of the culture volume was removed and the numbers of targets and effectors were enumerated by flow cytometry. Samples were stained with anti-CD8-FITC (clone RPA-T8, BD Biosciences), CD4-APC (clone OKT4, BioLegend), and anti-CD 19-PE (clone HIB19, BioLegend), and data were acquired on a Beckman Coulter® CytoFLEX®-S, equipped with cell counting function. Immediately following data acquisition, fresh target cells were added to each co-culture. The number of target cells added matched the number of T cells determined to remain in culture, such that a 1 : 1 T:E ratio was established. This was repeated until cultures failed to achieve approximately 90% clearance of tumor targets over each time interval.
  • FIG 10 shows CAR T cell and TFP T cell expansion when cells were challenged with NALM/6 cells. Both TFP variants exhibited a 3-4-fold expansion in the first 3 days, while the CAR T cells were not observed to expand until the d6 time point.
  • the CAR T cells were present in equal numbers to the TCR-TFP T cells, but the numbers of CD3e TFP T cells began to decline.
  • the TCR-TFP T cells began declining shortly thereafter (by the dl3 time point) while CAR T numbers were largely unchanged for the remainder if the experiment (through 17 days).
  • the maximum expansion of all three variants was approximately 10-fold, although this peak was reached at different times for each T cell variant.
  • Example 5 Characterization of TFP T cell activity and phenotype.
  • Human T cells were prepared as described in Example 2 to insert an anti-CD 19 TCR-TFP fusion (AAV7069), an anti-CD 19 CD3 epsilon-TFP fusion (AAV7070), or a control TCR-TFP fusion having a binder that should not bind the target cells into the TRAC site.
  • Untransduced TRA/TRB -/- T cells made according to the methods described above, were also used in the following experiments.
  • Unedited, non-transduced controls and unedited T cells transduced with an anti-CD 19 CD3 epsilon TFP were also used in the following experiments and were prepared according to the methods described in WO2016187349.
  • Results are shown in Figure 16. Post TFP transduction, edited TFP T cells are positive for FMC63 (Fig. 16A, bottom left and middle panel) and human CD3E (16B, bottom left and middle panel). Edited TFP T cells do not show surface expression of human TCRaP (Fig 16C bottom left and middle panel) but do show expression of murine TCRa.p (Fig. 16D bottom left and middle panel).
  • Interferon gamma levels are shown for T cells cultured with dendritic cells relative to T cells cultured in the absence of dendritic cells.
  • the edited cells show less of an increase in interferon gamma levels when cultured in the presence of interferon gamma cells relative to unedited cells.
  • Example 6 In vivo efficacy of edited TFP T cells.
  • a Nalm6-luc tumor mouse model was used to assess the in vivo efficacy of edited TFP T cells. 5 x 10 5 tumor cells were injected into NSG mice at Day -6. Mice received on injection of T cells on day 0 as is shown in Table 2 below.
  • mice were imaged to assess tumor load (Figure 18). Edited TFP T cells were able to slow or stop tumor growth for up to 80 days. Differences in efficacy between edited and unedited cells having the CDSsTFP may be due to the different production processes used to generate these cells.
  • liver and skin tissue was harvested and assessed for Graft-Versus Host Disease (GvHD) via tissue infiltration of human cells.
  • GvHD Graft-Versus Host Disease

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Abstract

L'invention concerne des acides nucléiquesrRecombinants codant pour le récepteur des lymphocytes T (TCR) des protéines de fusion (TFP), des lymphocytes T modifiés exprimant les molécules codées, et des procédés d'utilisation de ceux-ci pour le traitement de maladies, y compris le cancer.
PCT/US2020/047473 2019-08-21 2020-08-21 Compositions et procédés de reprogrammation tcr à l'aide de protéines de fusion WO2021035170A1 (fr)

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WO2023012584A2 (fr) 2021-08-03 2023-02-09 Genicity Limited Complexe tcr modifié et ses procédés d'utilisation
WO2023081767A1 (fr) 2021-11-05 2023-05-11 Precision Biosciences, Inc. Procédés d'immunothérapie
WO2023091910A1 (fr) 2021-11-16 2023-05-25 Precision Biosciences, Inc. Méthodes d'immunothérapie anticancéreuse
WO2023108150A1 (fr) 2021-12-10 2023-06-15 Precision Biosciences, Inc. Procédés d'immunothérapie anticancéreuse
WO2023215725A1 (fr) * 2022-05-02 2023-11-09 Fred Hutchinson Cancer Center Compositions et méthodes pour l'immunothérapie cellulaire
WO2024052389A1 (fr) * 2022-09-08 2024-03-14 F. Hoffmann-La Roche Ag Récepteurs de lymphocytes t recombinants
WO2024148167A1 (fr) 2023-01-05 2024-07-11 Precision Biosciences, Inc. Méganucléases modifiées optimisées ayant une spécificité pour le gène à région constante alpha du récepteur des lymphocytes t humain

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