EP3519561A1 - Gedächtnis-t-zellen mit modifizierten stammzellen, verfahren zur herstellung und verfahren zur verwendung davon - Google Patents

Gedächtnis-t-zellen mit modifizierten stammzellen, verfahren zur herstellung und verfahren zur verwendung davon

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Publication number
EP3519561A1
EP3519561A1 EP17794119.2A EP17794119A EP3519561A1 EP 3519561 A1 EP3519561 A1 EP 3519561A1 EP 17794119 A EP17794119 A EP 17794119A EP 3519561 A1 EP3519561 A1 EP 3519561A1
Authority
EP
European Patent Office
Prior art keywords
cell
modified
cells
composition
tscm
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17794119.2A
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English (en)
French (fr)
Inventor
Eric Ostertag
Devon SHEDLOCK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Poseida Therapeutics Inc
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Poseida Therapeutics Inc
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Filing date
Publication date
Application filed by Poseida Therapeutics Inc filed Critical Poseida Therapeutics Inc
Publication of EP3519561A1 publication Critical patent/EP3519561A1/de
Pending legal-status Critical Current

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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • B82—NANOTECHNOLOGY
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    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
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    • C12N2800/00—Nucleic acids vectors
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Definitions

  • the disclosure is directed to molecular biology, and more, specifically, to methods of making and using modified stem-cell memory T cells.
  • modified-T cells of the disclosure possess the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments.
  • modified-T cells of the disclosure must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses.
  • intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory cells, especially stem cell memory (TSCM).
  • TSCM stem cell memory
  • Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM), effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term modified-T cell engraftment.
  • Modified-T cells of the disclosure include, but are not limited to, those cells that express an antigen receptor comprising a protein scaffold of the disclosure.
  • Modified-T cells of the disclosure include, but are not limited to, those cells that express a chimeric antigen receptor (CAR) (i.e. CAR-T cells of the disclosure).
  • CAR chimeric antigen receptor
  • Chimeric antigen receptors (CARs) of the disclosure may comprise one or more sequences that each specifically bind an antigen, including, but not limited to, a single chain antibody (e.g. a scFv), a sequence comprising one or more fragments of an antibody (e.g. a VHH, referred to in the context of a CAR as a VCAR), an antibody mimic, and a Centyrin (referred to in the context of a CAR as a CARTyrin).
  • a single chain antibody e.g. a scFv
  • a sequence comprising one or more fragments of an antibody e.g. a VHH, referred to in the context of a CAR as a VCAR
  • an antibody mimic e.g. a Centyrin (referred to in the context of a CAR as a CARTyrin).
  • Modified cells of the disclosure may be further subjected to genomic editing.
  • a genomic editing construct may be introduced into the modified cells of the disclosure in a transposon or other means of delivery through electroporation or
  • modified T cell with an edited genome that retains a stem-like phenotype is a modified T cell with an edited genome that retains a stem-like phenotype.
  • This modified T cell with an edited genome that retains a stem-like phenotype may be used as a cellular therapy.
  • modified cells of the disclosure may be subject to a first electroporation or nucleofection and a subsequent electroporation or nucleofection to introduce a genomic editing construct.
  • the disclosure provides a method of producing a modified stem memory T cell (TSCM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a modified stem memory T cell (TSCM).
  • the disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising introducing into a plurality of primary' human T cell
  • transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%,
  • the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • TSCM modified stem memory T cells
  • the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality' of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell- surface markers of the CAR-TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD 127, CD45RO, CD95, CD95 and IL-2R ⁇ , In certain embodiments, the cell-surtace markers of the CAR-TSCM comprise one or more of CD45RA, CD95, IL-2R ⁇ , CR7, and CD62L.
  • the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggy Bac transposon.
  • the transposase is a piggy BacTM or a Super piggyBacTM (SPB) transposase.
  • the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements.
  • the transposon is a piggyBac transposon.
  • the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase.
  • the sequence encoding the transposase is an mRNA sequence.
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme.
  • PB piggyBac
  • the piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:
  • PB piggyBacTM
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4.
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4.
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 4.
  • the amino acid substitution at position 30 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I).
  • the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G).
  • the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M).
  • the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N).
  • the transposase enzyme is a Super piggyBacTM (SPB) transposase enzyme.
  • the Super piggyBacTM (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 4 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N).
  • the Super piggyBacTM (SPB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%,
  • the piggy BacTM or Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119,
  • transposase comprises the above-described mutations at positions 30, 165, 282 and/or
  • the piggyBacTM or Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200,
  • amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for a serine (S).
  • amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A).
  • amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (I).
  • amino acid substitution at position 103 of SEQ ID NO: 5 is a substitution of a serine (S) for an alanine (A).
  • amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A).
  • amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (I).
  • amino acid substitution at position 103 of SEQ ID NO: 5 is a substitution of a serine (S) for an alan
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S).
  • the amino acid substitution at position 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for an arginine (R).
  • the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine
  • the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ
  • the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for a tyrosine (Y).
  • the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a phenylalanine (F).
  • amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 4 is amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5
  • the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A).
  • the amino acid substitution at position 200 of SEQ ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F).
  • the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A).
  • the amino acid substitution at position 200 of SEQ ID NO: 5 is a substitution of a valine (V)
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenylalanine (F).
  • W tryptophan
  • F phenylalanine
  • the amino acid substitution at position 209 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a valine (V).
  • the amino acid substitution at position 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a methionine (M).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L).
  • the amino acid substitution at position 240 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V).
  • the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine
  • SEQ ID NO: 4 is a substitution of a lysine (K) for a proline (P).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for an asparagine (N).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a leucine (L).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L).
  • the amino acid substitution at position 298 of SEQ YD NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a proline (P).
  • the amino acid substitution at position 31 1 of SEQ YD NO: 4 or SEQ ID NO: 5 is a substitution of a valine for a proline (P).
  • the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T).
  • G glycine
  • T threonine
  • amino acid substitution at position 327 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T).
  • the amino acid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a tyrosine (Y).
  • the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C).
  • the amino acid substitution at position 340 of SEQ ID NO: 5 is a substitution of an arginine (R) for a tyrosine (Y).
  • the amino acid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a tyrosine (Y).
  • the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C).
  • the amino acid substitution at position 340 of SEQ ID NO: 5 is a substitution of an arginine
  • amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D).
  • amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a valine (V).
  • amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine (M).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L).
  • F phenylalanine
  • L leucine
  • the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a methionine (M).
  • the amino acid substitution at position 552 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V).
  • the amino acid substitution at position 570 of SEQ YD NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A).
  • the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline
  • the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a glutamine (Q).
  • the piggyBacTM transposase enzyme may comprise or the Super piggy BacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or
  • the piggyBacTM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the transposase comprises the above-described mutations at positions
  • the piggyBacTM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at positions
  • the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M).
  • the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R).
  • the amino acid substitution at position 375 of SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S).
  • the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M).
  • the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R).
  • the amino acid substitution at position 375 of SEQ ID NO: 5 is a substitution of a proline (P) for a serine (
  • amino acid substitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for an aspartic acid (D).
  • amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S).
  • amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for an asparagine (N).
  • the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4.
  • the piggyBacTM transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, and a substitution of an alanine (A) for a lysine ( ) at position 375 of SEQ ID NO: 4.
  • the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 4 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 4.
  • the disclosure provides a method of producing a modified stem memory T cell
  • TSCM stem memory T cell
  • TSCM TSCM
  • T cells comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%,
  • the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell-surface markers of the CAR-TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R ⁇
  • the cell-surface markers of the CAR-TSCM comprise one or more of CD45RA, CD95, lL-2R ⁇ , CR7, and CD62L.
  • the transposon is a Sleeping Beauty transposon.
  • the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
  • the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%,
  • the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:
  • the disclosure provides a method of producing a modified stem memory T cell
  • TSCM stem memory T cell
  • the disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality' of modified stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • TSCM stem memory T cell
  • the method produces a plurality of modified T cells, wherein at least 80% of the plurality' of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • TSCM stem memory T cell
  • the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell-surface markers of the CAR-TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL- 2R ⁇ , In certain embodiments, the cell-surface markers of the CAR-TSCM comprise one or more of CD45RA, CD95, IL-2R ⁇ , CR7, and CD62L.
  • the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase.
  • the transposase is a Helitron transposase.
  • Helitron transposases mobilize the Helraiser transposon, an ancient element from the bat genome that was active about 30 to 36 million years ago.
  • An exemplary Helraiser transposon of the disclosure includes Helibatl , which comprises a nucleic acid sequence comprising:
  • the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a RepHel motif made up of a replication initiator domain (Rep) and a DNA helicase domain.
  • the Rep domain is a nuclease domain of the HUH superfamily of nucleases.
  • An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:
  • a hairpin close to the 3' end of the transposon functions as a terminator.
  • this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences.
  • Helraiser transposition generates covalently closed circular intermediates.
  • Helitron transpositions can lack target site duplications.
  • the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5'-TC/CTAG- 3' motif.
  • a 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence
  • the disclosure provides a method of producing a modified stem memory T cell (TSCM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a modified stem memory T cell (TSCM).
  • TSCM stem memory T cell
  • the disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSC ), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • TSC stem memory T cell
  • TSCM stem memory T cell
  • TSCM stem memory T cell
  • the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • TSCM stem memory T cell
  • the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM).
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell-surface markers of the CAR-TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL- 2RB. In certain embodiments, the cell-surface markers of the CAR-TSCM comprise one or more of CD45RA, CD95, IL-2R ⁇ , CR7, and CD62L.
  • the transposon is a Tol2 transposon. In certain embodiments, including those embodiments wherein the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.
  • the transposase is a Tol2 transposase.
  • Tol2 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family.
  • Exemplary Tol2 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the Tol2 transposase, which contains four exons.
  • An exemplary Tol2 transposase of the disclosure comprises an amino acid sequence comprising the following:
  • An exemplary Tol2 transposon of the disclosure including inverted repeats, subterminal sequences and the Tol2 transposase, is encoded by a nucleic acid sequence comprising the following:
  • the disclosure provides a method of producing a modified central memory T-cell (TCM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface markers) of a central memory T-cell (TCM), thereby producing a modified central memory T- cell (TCM).
  • TCM central memory T-cell
  • the disclosure provides a method of producing a plurality of modified central memory T-cells (TCM), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surtace marker(s) of a central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM).
  • TCM central memory T-cells
  • the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell- surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM).
  • the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM).
  • the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell-surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM).
  • the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T- cells (TCM).
  • the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface markers) of central memory T-cell (TCM), thereby producing a plurality of modified central memory T-cells (TCM).
  • the cell-surface markers comprise one or more of CD45RO, CD95, IL-2Rp, CCR7, and CD62L.
  • the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements.
  • the transposon is a piggy Bac transposon.
  • the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase.
  • the transposon is a Sleeping Beauty transposon.
  • the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty' transposase (SB100X).
  • the transposon is a Helraiser transposon.
  • the transposase is a Helitron transposase.
  • the transposon is a Tol2 transposon.
  • the transposase is a Tol2 transposase.
  • the disclosure provides a method of producing a composition comprising a plurality of modified stem memory T-cells (TSCM) and a plurality of modified central memory T-cells
  • TC comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a composition comprising a plurality of modified TSCM and a plurality of modified TCM, wherein the plurality of modified TSCM expresses one or more
  • the modified stem memory thereby producing a composition comprising a plurality of modified TSCM and a plurality of modified TCM.
  • the modified stem memory thereby producing a composition comprising a plurality of modified TSCM and a plurality of modified TCM.
  • T-cells comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
  • the modified central memory T-cells comprise at least 1%, 2%, 5%, 7%,
  • the modified stem memory T-cells are 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition.
  • TSCM comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 90% of the total number of cells of the composition.
  • TCM modified central memory T-cells
  • TSCM comprise at least 90% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 10% of the total number of cells of the composition.
  • TCM central memory T-cells
  • TSCM comprise at least 20% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 80% of the total number of cells of the composition.
  • TCM central memory T-cells
  • TSCM comprise at least 80% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 20% of the total number of cells of the composition.
  • TCM central memory T-cells
  • TSCM comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 70% of the total number of cells of the composition.
  • the modified stem memory T-cells (TSCM) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 30% of the total number of cells of the composition.
  • the modified stem memory T-cells (TSCM) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 60% of the total number of cells of the composition.
  • the modified stem memory T-cells comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 40% of the total number of cells of the composition.
  • the modified stem memory T-cells comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 50% of the total number of cells of the composition.
  • the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements.
  • the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
  • the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, including those embodiments wherein the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.
  • the transposon may be derived or recombined from any species. Alternatively, or in addition, the transposon may be synthetic.
  • the antigen receptor is a T- cell receptor.
  • the T-cell receptor is naturally-occurring .
  • the T-cell receptor is not naturally-occurring.
  • the T- cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
  • the T-cell receptor is a recombinant T-cell receptor.
  • the antigen receptor is a Chimeric Antigen Receptor (CAR).
  • the CAR is a CARTyrin.
  • the CAR comprises one or more VHH sequences).
  • the CAR is a VCAR.
  • the methods further comprise introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase, the methods further comprise introducing into a primary human T cell (c) a second transposon composition comprising a transposon comprising a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein.
  • the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein.
  • the transposase composition of (b) transposes the transposon of (a) and the transposon of (c).
  • this methods further comprises introducing into the primary human T cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase.
  • the second transposase composition transposes the transposon of (c).
  • the transposase composition of (b) transposes the transposon of (a) and the transposase composition of (d) transposes the transposon of (c).
  • the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis- regulatory insulator elements.
  • the transposon is a piggyBac transposon.
  • the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase.
  • the transposon is a Sleeping Beauty transposon.
  • the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
  • the transposon is a Helraiser transposon.
  • the transposase is a Helitron transposase.
  • the transposon is a Tol2 transposon.
  • the transposase is a Tol2 transposase.
  • the disclosure provides a method of producing a modified stem memory T cell
  • TSCM stem memory T cell
  • TSCM stem memory T cell
  • the disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising: (a) introducing into a plurality of primary' human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%,
  • the method produces a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a stem memory T cell
  • the method produces a plurality of activated modified T cells, wherein at least 50% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stem memory T cells (TSCM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a stem memory T cell
  • the method produces a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stem memory T cells (TSCM).
  • the method produces a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stem memory T cells (TSCM).
  • the method produces a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stem memory T cells (TSCM).
  • the method produces a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stem memory T cells (TSCM).
  • the method produces a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated modified stem memory T cells (TSCM).
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell-surface markers of the activated modified TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD 127, CD45RO, CD95, CD95 and IL-2R ⁇ , In certain emboiiiments, the cell-surface markers of the activated modified TSCM comprise one or more of CD45RA, CD95, IL-2R ⁇ ,, CR7, and CD62L.
  • a modified stem memory T cell comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti- human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
  • this method further comprises the step of (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM).
  • TSCM stem memory T cell
  • TSCM stem memory T cell
  • this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%,
  • this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface markers) of a stem memory T cell (TSCM).
  • the enriching step comprises isolating modified T-cells that express one or more cell-surface markers) of a stem memory T cell (TSCM) from the plurality of enriched modified T-cells.
  • the enriching step further comprises contacting the isolated modified TSCM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol,
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-
  • the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
  • the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
  • T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints.
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg.
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 umol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 umol/kg and 75 umol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 umol/kg and 25 umol/kg, inclusive of the endpoints.
  • octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints
  • palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 ⁇ /kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the disclosure provides a method of producing a modified central memory T-cell
  • TCM comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified T-cell expresses one or more cell-surface markers) of a central memory
  • T-cell T-cell
  • TCM central memory T-cell
  • the disclosure provides a method of producing a plurality of modified central memory T-cell (TCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human
  • CD3 monospecific tetrameric antibody complex an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
  • the method produces a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a central memory T cell (TCM), thereby producing a plurality of activated modified central memory T cell (TCM).
  • the method produces a plurality of activated modified T cells, wherein at least
  • the method produces a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a central memory T cell
  • the method produces a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a central memory T cell (TCM), thereby producing a plurality of activated modified central memory T cell (TCM). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a central memory T cell
  • the method produces a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a central memory T cell (TCM), thereby producing a plurality of activated modified central memory T cell (TCM).
  • the method produces a plurality of activated modified T cells, wherein at least 90% of the plurality' of activated modified T cells expresses one or more cell-surface markers) of a central memory T cell
  • the method produces a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modified T cells expresses one or more cell-surface markers) of a central memory T cell (TCM), thereby producing a plurality of activated modified central memory T cell (TCM).
  • the cell-surface markers of the activated modified TCM comprise one or more of CD45RO, CD95, IL-2R ⁇ , CCR7, and CD62L.
  • a modified central memory T cell comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti- human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
  • this method further comprises the step of (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove ' s MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface markers) of a central memory T cell (TCM).
  • TCM central memory T cell
  • this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface markers) of a central memory T cell (TCM).
  • this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell- surface markers) of a central memory T cell (TCM).
  • the enriching step comprises isolating modified T-cells that express one or more cell-surface markers) of a central memory T cell (TCM) from the plurality of enriched modified T-cells.
  • the enriching step further comprises contacting the isolated modified TCM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TCM.
  • a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TCM.
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
  • TMDD 2,4,7,9-tetramethyl-5-decyn-4,7-diol
  • DIPA diisopropyl adipate
  • n-butyl-benzenesulfonamide 1,2-benzenedicarboxylic acid
  • palmitic acid palmitic acid
  • linoleic acid oleic acid
  • the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints.
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg.
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 ⁇ /kg and 75 ⁇ mol/kg, inclusive of the endpoints; oleic acid at a
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 ⁇ /kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the disclosure provides a method of producing a composition comprising a plurality of modified stem memory T-cells (TSCM) and a plurality of modified central memory T-cells (TCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (TSCM) and a plurality of activated modified central memory T-cells (TCM), wherein the plurality of activated modified TSCM expresses one or more CD62L, CD45RA, CD28, CCR7, CD 127,
  • the modified stem memory T-cells comprise at least 1%
  • the modified central memory T-cells comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%,
  • the modified stem memory T-cells comprise at least 10% of the total number of cells of the composition and the modified central memory T- cells (TCM) comprise at least 90% of the total number of cells of the composition.
  • the modified stem memory T-cells comprise at least
  • TCM modified stem memory T-cells
  • TCM modified stem memory T-cells
  • TCM modified stem memory T-cells
  • the modified central memory T-cells comprise at least 70% of the total number of cells of the composition.
  • the modified stem memory T-cells comprise at least
  • TCM modified stem memory T-cells
  • TCM comprise at least 60% of the total number of cells of the composition.
  • TSCM modified stem memory T-cells
  • TCM comprise at least 40% of the total number of cells of the composition.
  • TSCM modified stem memory T-cells
  • TCM comprise at least 50% of the total number of cells of the composition.
  • composition comprising a plurality of modified stem memory T-cells (TSCM) and a plurality of modified central memory T-cells (TCM), comprising: (a) introducing into a plurality of primary human
  • T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (TSCM) and a plurality of activated modified central memory T-cells (TCM), the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
  • this method further comprises the step of (c) contacting the composition the plurality of activated modified stem memory T-cells
  • TSCM and the plurality of activated modified central memory T-cells (TCM) with a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein the plurality of expanded modified TSCM expresses one or more CD62L, CD45RA, CD28, CCR7, CD127,
  • CD45RO, CD95, CD95 and YL- ⁇ and the plurality of expanded modified TCM expresses one or more CD45RO, CD95, IL-2R ⁇ , CCR7, and CD62L, thereby producing a composition comprising a plurality of expanded modified TSCM and a plurality of expanded modified TCM.
  • the enriching step comprises isolating modified T- cells that express one or more cell-surface markers) of a stem memory T cell (TSCM) from the plurality of enriched modified T-cells or isolating modified T-cells that express one or more cell-surface markers) of a central memory T cell (TCM) from the plurality of enriched modified T-cells.
  • the enriching step comprises isolating modified T-cells that express one or more cell-surface markers) of a stem memory
  • the enriching step further comprises contacting the composition comprising the isolated modified TSCM and the isolated modified TCM with a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol,
  • Iscove's MDM and an expansion supplement to produce a composition comprising a plurality of expanded enriched modified TSCM and a plurality of expanded enriched modified
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol
  • the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg.
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 ⁇ mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 ⁇ /kg and 70 ⁇ mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 ⁇ /kg and 75 ⁇ /kg, inclusive of the endpoints; oleic acid at a
  • the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 ⁇ /kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the modified stem memory T-cells (TSCM) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
  • the modified central memory T-cells comprise at least 1%, 2%, 5%, 7%, 10%,
  • the modified stem memory T-cells 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition.
  • TSCM comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 90% of the total number of cells of the composition.
  • TCM modified central memory T-cells
  • TSCM comprise at least 90% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 10% of the total number of cells of the composition.
  • TCM central memory T-cells
  • TSCM comprise at least 20% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 80% of the total number of cells of the composition.
  • TCM central memory T-cells
  • TSCM comprise at least 80% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 20% of the total number of cells of the composition.
  • TCM central memory T-cells
  • TSCM comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 70% of the total number of cells of the composition.
  • TCM modified central memory T-cells
  • TSCM comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 30% of the total number of cells of the composition.
  • the modified stem memory T-cells (TSCM) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 60% of the total number of cells of the composition.
  • the modified stem memory T-cells (TSCM) comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 40% of the total number of cells of the composition.
  • the modified stem memory T-cells (TSCM) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 50% of the total number of cells of the composition.
  • TCM of the disclosure including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, the introducing step comprises a homologous recombination.
  • a genomic editing composition contacts a genomic sequence of at least one primary T cell of the plurality of T cells.
  • a genomic editing composition contacts a genomic sequence of a portion of primary T cells of the plurality of T cells.
  • the portion of primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
  • a genomic editing composition contacts a genomic sequence of each primary T cell of the plurality of T cells.
  • a genomic editing composition induces a single strand break.
  • a genomic editing composition induces a double strand break.
  • the introduction step further comprises a donor sequence composition.
  • the donor sequence composition comprises a sequence encoding the antigen receptor.
  • the donor sequence composition comprises a sequence encoding the antigen receptor, a 5' genomic sequence and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 5' to the break point induced by the genomic editing composition and the 3' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3' to the break point induced by the genomic editing
  • the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence sequentially, and the genomic editing composition is provided first. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA binding domain and a nuclease domain.
  • the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA -binding domain of a TALEN. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genomic editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof.
  • the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic
  • the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32, comprising a substitution of an
  • the nuclease domain comprises or further comprises a type IIS endonuclease.
  • the type IIS endonuclease comprises Acil, Mnll, Alwl, Bbvl, Bccl, BceAI, BsmAI, BsmFI, BspCNI, Bsrl, BtsCI, Hgal, Hphl, HpyAV, Mboll, My II, Plel, SfaNI, Acul, BciVI, BfuAI, BmgBI, Bmrl, Bpml, BpuEI, Bsal, BseRI, Bsgl, Bsml, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Btsl, Earl, Ecil, Mmel, NmeAIII, Bb
  • the type IIS endonuclease comprises Clo051.
  • the nuclease domain comprises or further comprises a TALEN or a nuclease domain thereof.
  • the nuclease domain comprises or further comprises a ZFN or a nuclease domain thereof.
  • the introduction step comprising a homologous recombination the genomic editing composition induces a break in a genomic sequence and the donor sequence composition is inserted using the endogenous DNA repair mechanisms of the primary T cell.
  • the introduction step comprising a homologous recombination the insertion of the donor sequence composition eliminates a DNA binding site of the genomic editing composition, thereby preventing further activity of the genomic editing composition.
  • a viral vector comprises the antigen receptor.
  • the viral vector comprises one or more sequences isolated, derived, or recombined from an RNA virus.
  • the RNA virus is a single-stranded or a double-stranded virus.
  • the viral vector comprises one or more sequences isolated, derived, or recombined from a DNA virus.
  • the DNA virus is a single-stranded or a double-stranded virus.
  • the virus is replication- defective.
  • TCM of the disclosure including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor.
  • the viral vector comprises a sequence isolated or derived from a retrovirus.
  • the viral vector comprises a sequence isolated or derived from a lentivirus.
  • a viral vector comprises the antigen receptor.
  • the viral vector comprises a sequence isolated or derived from a retrovirus.
  • the viral vector comprises a sequence isolated or derived from a gamma retrovirus.
  • TCM of the disclosure including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor.
  • the viral vector comprises a sequence isolated or derived from an adeno-associated virus
  • the AAV is a serotype AAVl, AAV2, AAV3, AAV4,
  • AAV comprises a sequence from one or more of AAVl, AAV2, AAV3, AAV4, AAV5,
  • the AAV comprises a sequence isolated, derived, or recombined from one or more of AAVl, AAV2,
  • the AAV comprises a sequence isolated, derived, or recombined from AAV2.
  • the AAV comprises a sequence isolated, derived, or recombined from AAV9.
  • Exemplarj' adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g.
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03, rAAV-NP59 and rAAV-NP84.
  • a nucleic acid vector comprises the antigen receptor.
  • a DNA vector comprises the antigen receptor.
  • an mRNA vector comprises the antigen receptor.
  • the nucleic acid vector is a plasmid or a minicircle vector.
  • a nanoparticle vector comprises the antigen receptor.
  • Nanoparticles may be comprised of polymers disclosed in, for example, International Patent Publication No. WO 2012/094679, International Patent Publication No. WO 2016/022805, International Patent Publication No. WO/2011/133635, International Patent Publication No.
  • the antigen receptor is a T-cell receptor.
  • the T-cell receptor is naturally-occurring.
  • the T-cell receptor is not naturally-occurring.
  • the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
  • the T- cell receptor is a recombinant T-cell receptor.
  • the antigen receptor is a Chimeric Antigen Receptor (CAR).
  • the CAR is a CARTyrin.
  • the CAR comprises one or more VHH sequence(s).
  • the CAR is a VCAR.
  • TCM of the disclosure including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, the method further comprises introducing into the primary human T cell, a composition comprising a therapeutic protein to produce a modified T cell capable of expressing the therapeutic protein.
  • the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein.
  • the introducing step comprises a homologous recombination and a donor sequence comprises a sequence encoding the therapeutic protein.
  • the donor sequence that comprises the antigen receptor further comprises the therapeutic protein.
  • a first donor sequence comprises the antigen receptor and a second donor sequence comprises the therapeutic protein.
  • a vector comprises a sequence encoding the therapeutic protein.
  • the vector is a viral vector.
  • the vector is a nanoparticle.
  • the vector that comprises the antigen receptor further comprises the therapeutic protein.
  • a first vector comprises the antigen receptor and a second vector template comprises the therapeutic protein.
  • the disclosure provides a method of producing a modified stem memory T cell
  • TSCM stem memory T cell
  • TSCM stem memory T cell
  • the disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%,
  • the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
  • T cell comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-T cell and (b) contacting the CAR-T cell and a T-cell activator composition comprising one or more of an anti-human T cell (TSCM), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-T cell and (b) contacting the CAR-T cell and a T-cell activator composition comprising one or more of an anti-human
  • CAR chimeric antigen receptor
  • CD3 monospecific tetrameric antibody complex an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce an activated CAR-T cell, wherein the activated
  • CAR-T cell expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a CAR-expressing stem memory T cell (TSCM) (CAR-TSCM).
  • TSCM stem memory T cell
  • CAR-TSCM CAR-TSCM
  • TSCM chimeric antigen receptor
  • CAR chimeric antigen receptor
  • T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,
  • the methods further comprises the step of: (c) contacting the activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM).
  • the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide,
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 ⁇ /kg, inclusive of the endpoints; oleic acid at a concentration of between
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 umol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ mol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 ⁇ mol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%,
  • the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%,
  • the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface markers) of a stem memory T cell (TSCM).
  • the enriching step further comprises isolating modified T-cells that express one or more cell-surface markers) of a stem memory T cell (TSCM) from the plurality of enriched modified T-cells.
  • the enriching step further comprises contacting the isolated modified TSCM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TSCM.
  • a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TSCM.
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfbnamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
  • TMDD 2,4,7,9-tetramethyl-5-decyn-4,7-diol
  • DIPA diisopropyl adipate
  • n-butyl-benzenesulfbnamide 1,2-benzenedicarboxylic acid
  • palmitic acid palmitic acid
  • linoleic acid oleic acid
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol).
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 umol/kg, inclusive of the endpoints; oleic acid at a concentration of between
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 ⁇ /kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 umol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 ⁇ /kg.
  • the disclosure provides a method of producing a modified central memory T cell
  • TCM central memory T cell
  • the disclosure provides a method of producing a plurality of modified central memory T cells (TCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%,
  • the T- cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
  • the methods further comprises the step of: (c) contacting the activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface markerfs) of a central memory T cell
  • the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about
  • the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 ⁇ /kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 umol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 umol/kg and 75 umol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 ⁇ /kg and 25 ⁇ /kg, inclusive of the endpoints.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ /kg, oleic acid at a concentration of about 7.56 ⁇ mol/kg and a sterol at a concentration of about 2.61 umol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ /kg, oleic acid at a concentration of 7.56 ⁇ /kg and a sterol at a concentration of 2.61 umol/kg.
  • At least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (TCM).
  • at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (TCM).
  • the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface markers) of a central memory T cell (TCM).
  • the method further comprises the step of: (d) enriching the plurality of expanded modified T- cells to produce a composition comprising at least 60% of modified T-cells that express cell- surface markers) of a central memory T cell (TCM).
  • the enriching step further comprises isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (TCM) from the plurality of enriched modified T-cells.
  • the enriching step further comprises contacting the isolated modified TCM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified TCM.
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfbnamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
  • TMDD 2,4,7,9-tetramethyl-5-decyn-4,7-diol
  • DIPA diisopropyl adipate
  • n-butyl-benzenesulfbnamide 1,2-benzenedicarboxylic acid
  • palmitic acid palmitic acid
  • linoleic acid oleic acid
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol).
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 umol/kg, inclusive of the endpoints; oleic acid at a concentration of between
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 ⁇ /kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 umol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 umol/kg.
  • the disclosure provides a method of producing a composition comprising a plurality of modified stem memory T-cells (TSCM) and a plurality of modified central memory T-cells
  • TCM modified stem memory T-cells
  • TCM modified central memory T-cells
  • a transposon comprises the antigen receptor
  • TCM modified central memory T-cells
  • a transposon comprises the antigen receptor
  • TCM T-cell activator composition
  • the plurality of activated modified TSCM expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RP and the plurality of activated modified TCM expresses one or more CD45
  • the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
  • the methods further comprises the step of: (c) contacting the composition and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the composition comprising a plurality of expanded modified T-cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM).
  • TSCM stem memory T cell
  • the methods further comprises the step of: (c) contacting the composition and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove ' s MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the composition comprising a plurality of expanded modified T-cells expresses one or more cell- surface marker(s) of a central memory T cell (TCM).
  • TCM central memory T cell
  • the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n- butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
  • TMDD 2,4,7,9-tetramethyl-5-decyn-4,7-diol
  • DIPA diisopropyl adipate
  • n- butyl-benzenesulfonamide 1,2-benzenedicarboxylic acid
  • palmitic acid palmitic acid
  • linoleic acid oleic acid
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol).
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 ⁇ /kg, inclusive of the endpoints; oleic acid at a concentration of between
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ /kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 umol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 ⁇ mol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%,
  • composition comprising a plurality of expanded modified
  • TSCM and a plurality of expanded modified TCM expresses cell-surface markers) of a stem memory T cell (TSCM).
  • TSCM stem memory T cell
  • the method further comprises the step of: (d) enriching the composition to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%,
  • the method further comprises the step of: (d) enriching the composition to produce a composition comprising at least 2%, 5%, 10%, 15%,
  • the enriching step further comprises isolating modified T-cells that express one or more cell-surface markers) of a stem memory
  • T cell (TSCM) from the composition or isolating modified T-cells mat express one or more cell-surface markers) of a central memory T cell (TCM) from the composition.
  • the enriching step further comprises isolating modified T-cells that express one or more cell-surface markers) of a stem memory T cell (TSCM) from the composition and isolating modified T-cells that express one or more cell-surface markers) of a central memory T cell (TCM) from the composition.
  • the enriching step further comprises contacting the isolated modified TSCM and/or TCM and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a composition comprising a plurality of expanded enriched modified TSCM and/or
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about
  • the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 ⁇ /kg and 640 ⁇ mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 ⁇ mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 ⁇ /kg and 75 umol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 umol/kg and 75 umol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 ⁇ /kg and 25 umol/kg, inclusive of the endpoints.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 ⁇ /kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ /kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 ⁇ mol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ /kg, oleic acid at a concentration of 7.56 ⁇ /kg and a sterol at a concentration of 2.61 ⁇ /kg.
  • the modified stem memory T-cells (TSCM) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%,
  • the modified central memory T-cells comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
  • the modified stem memory T-cells comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 90% of the total number of cells of the composition.
  • the modified stem memory T-cells comprise at least 90% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 10% of the total number of cells of the composition.
  • the modified stem memory T-cells comprise at least 20% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 80% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TSCM) comprise at least 80% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 20% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TSCM) comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 70% of the total number of cells of the composition.
  • the modified stem memory T-cells (TSCM) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 30% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TSCM) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TSCM) comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 40% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TSCM) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (TCM) comprise at least 50% of the total number of cells of the composition.
  • the method comprises introducing into a primary human T cell (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the method further comprises introducing into the primary human T cell (c) a second transposon composition comprising a transposon comprising a therapeutic protein, to produce a modified
  • the modified T cell wherein the modified T cell is capable of expressing the therapeutic protein.
  • the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein.
  • the method further comprises introducing atransposase composition.
  • the transposase composition transposes the transposon of (a) and the second transposon.
  • the method comprises introducing a first transposase composition and a second transposase composition.
  • the method comprises introducing a first transposase composition and a second transposase composition, the first transposase composition transposes the transposon of (a) and the second transposase composition transposes the second transposon.
  • the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements.
  • the transposon is a piggyBac transposon.
  • the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase.
  • the transposon is a Sleeping Beauty transposon.
  • the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
  • the transposon is a Helraiser transposon.
  • the transposase is a Helitron transposase.
  • the transposon is a Tol2 transposon.
  • the transposase is a Tol2 transposase.
  • the method comprises introducing into a primary human T cell (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-ccll activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the method further comprises introducing into the primary human T cell a sequence encoding a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein.
  • the introducing step comprises a homologous recombination.
  • a vector comprises the sequence encoding the therapeutic protein.
  • the vector is a viral vector.
  • the vector is a nanoparticle.
  • the introducing step further comprises a composition comprising a genomic editing construct.
  • the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease. In certain embodiments, the genomic editing construct encodes a fusion protein. In certain embodiments, the genomic editing construct encodes the DNA binding domain and the type IIS endonuclease and wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a Cas9 endonuclease.
  • the sequence derived from a Cas9 endonuclease is the DNA binding domain.
  • the sequence derived from a Cas9 endonuclease encodes an inactive Cas9.
  • the sequence derived from a Cas9 endonuclease encodes an inactive Cas9.
  • sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
  • the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D10A).
  • the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
  • sequence derived from a Cas9 endonuclease comprises dCas9 (SEQ ID
  • sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
  • sequence derived from a Cas9 endonuclease comprises dSaCas9
  • the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
  • TALEN transcription activator-like effector nuclease
  • the sequence derived from a TALEN is the DNA binding domain.
  • the genomic editing construct comprises a TALEN.
  • the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
  • ZFN zinc-finger nuclease
  • the sequence derived from a ZFN is the DNA binding domain.
  • the genomic editing construct comprises a zinc-finger nuclease (ZFN).
  • the transposon is a plasmid
  • the introducing step further comprises a composition comprising an mRNA sequence encoding a transposase.
  • the transposon is a piggyBac transposon.
  • the transposase is a Super piggyBacTM (SPB) transposase.
  • SPB Super piggyBacTM
  • the sequence encoding the transposase is an mRNA sequence.
  • the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4.
  • the piggyBac transposase is a hyperactive variant and the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4.
  • the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I) (I30V).
  • SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G) (G165S).
  • the amino acid substitution at position 282 of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V).
  • the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K).
  • the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5.
  • the transposon is a Sleeping Beauty transposon.
  • the transposase is a Sleeping Beauty transposon.
  • the transposon is a Helraiser transposon. In certain embodiments, in particular those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments, the transposon is a Tol2 transposon. In certain embodiments, in particular those embodiments wherein the transposon is a Tol2 transposon, the transposase is a Tol2 transposase. In certain embodiments, the sequence encoding the transposase is an mRNA sequence. In certain embodiments, the transposon may be derived or recombined from any species. Alternatively, or in addition, the transposon may be synthetic.
  • the transposon further comprises a selection gene.
  • the T-cell expansion composition further comprises a selection agent.
  • the antigen receptor is a T- cell receptor.
  • the T-cell receptor is naturally-occurring .
  • the T-cell receptor is not naturally-occurring.
  • the T- cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
  • the T-cell receptor is a recombinant T-cell receptor.
  • the antigen receptor is a Chimeric Antigen Receptor (CAR).
  • the CAR is a CARTyrin.
  • the CAR comprises one or more VHH sequence(s).
  • the CAR is a VCAR.
  • the cell-surface markers of the modified TSCM comprise CD62L and CD45RA.
  • the cell-surface markers of the modified TSCM comprise one or more of CD62L, CD4SRA, CD28, CCR7, CD 127, CD45RO, CD95, CD95 and IL-2R ⁇ ,
  • the cell-surface markers of the modified TSCM comprise one or more of CD45RA, CD95, IL-2RfJ, CR7, and CD62L.
  • the plurality of expanded modified T-cells comprises a naive T-cell (modified TN) and the cell-surface markers of the
  • CAR-TN comprise one or more of CD45RA, CCR7 and CD62L.
  • the plurality of expanded modified T-cells comprises a central memory T-cell (modified TCM) and the cell-surface markers of the CAR-TCM comprise one or more of CD45RO, CD95, IL-
  • the plurality of expanded modified T-cells comprises an effector memory T-cell (modified TEM) and the cell-surface markers of the
  • CAR-TEM comprise one or more of CD45RO, CD95, and lL-2R ⁇
  • plurality of expanded modified T-cells comprises an effector T-ccll (modified TEFF) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2R ⁇ ,
  • the plurality of expanded modified T-cells comprises a central memory T-cell (modified TCM) and the cell-surface markers of the CAR-TCM comprise one or more of CD45RO, CD95, IL-2R ⁇ , CCR7, and
  • the most abundant cell in the plurality of expanded modified T-cells is a central memory T-cell (modified TCM) and the cell-surface markers of the CAR-TCM comprise one or more of CD45RO, CD95, IL-2R ⁇ , CCR7, and CD62L. In certain embodiments, wherein the most abundant cell in the plurality of expanded modified
  • T-cells is a central memory T-cell (modified TCM), the plurality of expanded modified T-cells comprises a TSCM cell and the cell-surface markers of the TSCM cell comprise one or more of
  • the disclosure provides a method of producing a modified stem memory T cell
  • TSCM stem memory T cell
  • CAR-TSCM CAR-expressing stem memory T cell
  • the disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,
  • CAR chimeric antigen receptor
  • the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality' of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell- surface markers of the activated CAR TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD 127, CD45RO, CD95, CD95 and IL-2Rp * . In certain embodiments, the cell- surface markers of the activated CAR TSCM comprise one or more of CD45RA, CD95, IL- 2R ⁇ , CR7, and CD62L.
  • the disclosure provides a method of producing a modified stem memory T cell (TSCM), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-T cell and (b) contacting the CAR-T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated CAR-T cell, wherein the activated CAR-T cell expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a CAR-expressing stem memory T cell (TSCM) (CAR-TSCM).
  • CAR chimeric antigen receptor
  • the disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality
  • the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell-surface markers of the activated CAR TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp. In certain embodiments, the cell-surface markers of the activated CAR TSCM comprise one or more of CD45RA, CD95, IL-2R ⁇ , CR7, and CD62L.
  • this method may further comprise the step of: (c) contacting the activated CAR-T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol,
  • Iscove's MDM and an expansion supplement to produce a plurality of expanded CAR-T cells, wherein at least 2% of the plurality of expanded CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM) (CAR-TSCM).
  • TSCM stem memory T cell
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIP A), n- butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
  • TMDD 2,4,7,9-tetramethyl-5-decyn-4,7-diol
  • DIP A diisopropyl adipate
  • n- butyl-benzenesulfonamide 1,2-benzenedicarboxylic acid
  • palmitic acid palmitic acid
  • linoleic acid oleic acid
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol).
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 ⁇ /kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 umol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 umol/kg.
  • CAR-TSCM stem memorj' T cell
  • the plurality of expanded CAR-T cells may be enriched for CAR-T cells that express cell-surface markers) of a stem memory T cell (TSCM) (CAR-TSCM), and, therefore, following an enrichment step, the method may produce an enriched composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of CAR-T cells that express cell-surface markers) of a stem memory T cell (TSCM) (CAR-TSCM).
  • the cell- surface markers comprise CD62L and CD45RA.
  • the cell-surface markers of the CAR-TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R ⁇ , In certain embodiments, the cell-surface markers of the CAR-TSCM comprise one or more of CD45RA, CD95, lL-2R ⁇ , CR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises a naive T- cell (CAR-TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L.
  • CAR-TN naive T- cell
  • the plurality of expanded CAR-T cells comprises a central memory T-cell (CAR-TCM) and the cell-surface markers of the CAR-TCM comprise one or more of CD45RO, CD95, IL-2R ⁇ , CCR7, and CD62L.
  • CAR-TCM central memory T-cell
  • the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-TEM) and the cell-surface markers of the CAR-TEM comprise one or more of CD45RO, CD95, and IL-2R ⁇
  • the plurality of expanded CAR-T cells comprises an effector T-cell (CAR-TEFF) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2R ⁇
  • CAR-TEFF effector T-cell
  • Additional cell-surface markers are described in Gattinoni et al. (Nat Med. 2011 Sep 18; 17(10): 1290-7; the contents of which are incorporated herein by reference in their entirety).
  • the disclosure provides a method of producing a modified stem memory T cell
  • TSCM stem memory T cell
  • CAR-TSCM CAR-expressing stem memory T cell
  • the disclosure provides a method of producing a plurality of modified stem memory T cells
  • TSCM chimeric antigen receptor
  • CAR chimeric antigen receptor
  • T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • TSCM stem memory T cell
  • the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality' of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell- surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell-surface markers) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of activated CAR stem memory T cells (TSCM).
  • the cell-surface markers comprise CD62L and CD45RA.
  • the cell-surface markers of the activated CAR TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R ⁇ , In certain embodiments, the cell-surface markers of the activated CAR TSCM comprise one or more of CD45RA, CD95, IL-2R ⁇ , CR7, and CD62L.
  • the plurality of expanded CAR-T cells comprises a naive T-cell (CAR-TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L.
  • the plurality of expanded CAR-T cells comprises a central memory T-cell (CAR-TCM) and the cell-surface markers of the CAR-TCM comprise one or more of CD45RO, CD95, IL-2R ⁇ , CCR7, and CD62L.
  • the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-TEM) and the cell-surface markers of the CAR-TEM comprise one or more of CD45RO, CD9S, and IL-2R ⁇ , In certain embodiments, the plurality of expanded CAR-T cells comprises an effector T-cell (CAR-TKFF) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2R ⁇ ,
  • a transposon comprises a chimeric antigen receptor (CAR) of the disclosure.
  • the transposon may be a plasmid DNA transposon with a sequence encoding the CAR flanked by two cis-regulatory insulator elements.
  • the transposon is a piggyBac transposon.
  • a step introducing a composition comprising a chimeric antigen receptor (CAR) of the disclosure may further a composition comprising an mRNA sequence encoding a transposase.
  • the transposase is a Super piggyBacTM (SPB) transposase.
  • a transposon of the disclosure may further comprise a selection gene.
  • the T-cell expansion composition of the methods of the disclosure may further comprise a selection agent to simultaneously select and expand an activated or modified T cell of the disclosure.
  • a CAR of the disclosure may be a CARTyrin.
  • the CAR comprises one or more VHH sequence(s).
  • the CAR is aVCAR.
  • the introducing step may comprise an electroporation or a nucleofection.
  • the nucleofection may comprise the steps of:
  • composition comprising a plurality of primary human T cells in a cuvette; (b) applying one or more electrical pulses to the cuvette, and (c) incubating the composition comprising the plurality of primary human T cells in a composition comprising a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37°C.
  • the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfbnamide, 1,2-benzenedicarboxylic acid, bis(2- methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane.
  • the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 ⁇ /kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 ⁇ /kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 ⁇ /kg and 75 umol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 ⁇ /kg and 75 umol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 ⁇ /kg and 25 umol/kg, inclusive of the endpoints.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/kg.
  • the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 umol/kg.
  • the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 umol/kg.
  • the transposon composition is a 0.5 ug/ ⁇ solution comprising nuclease free water and the cuvette comprises 2 ul of the transposon composition to yield 1 Mg of transposon.
  • the transposon composition may comprise a piggyBac transposon.
  • the transposon composition may comprise a Sleeping Beauty transposon.
  • the transposase composition comprises 5 ug of transposase.
  • the transposase composition may comprise a hyperactive piggyBacTM or Super piggyBacTM (SPB) transposase.
  • the transposase composition may comprise a hyperactive Sleeping Beauty (SB100X) transposase.
  • the transposon may comprise a Hel raiser transposon and the transposase composition may comprise a Helitron transposase.
  • the transposon may comprise a Tol2 transposon and the transposase composition comprises a Tol2 transposase.
  • the nucleofection comprises contacting a first transposon composition and a first transposase composition and a composition comprising a plurality of primary human T cells in a cuvette.
  • the nucleofection comprises contacting a first transposon composition, a second transposon composition, a first transposase composition and a composition comprising a plurality of primary human T cells in a cuvette.
  • the nucleofection comprises contacting a first transposon composition, a second transposon composition, a first transposase composition, a second transposase composition and a composition comprising a plurality of primary human T cells in a cuvette.
  • the first transposon comprises a sequence encoding an antigen receptor.
  • the second transposon comprises a sequence encoding a therapeutic protein.
  • the first transposon composition and the second transposon composition are identical. In certain embodiments, the first transposon composition and the second transposon composition are not identical.
  • the first transposase mobilizes the first transposon composition and the second transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition but not the second transposon composition. In certain embodiments, the second transposase mobilizes the second transposon composition but not the first transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition and the second transposase mobilizes the second transposon composition. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding an antigen receptor. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding a therapeutic protein.
  • the first transposon composition comprises a sequence encoding an antigen receptor and the second transposon composition comprises a sequence encoding a therapeutic protein.
  • the therapeutic protein is a secreted or secretable protein.
  • the nucleofection comprises contacting a transposon composition, a first transposase composition, a second transposase composition and a composition comprising a plurality of primary human T cells in a cuvette.
  • the transposon composition comprises a sequence encoding the antigen receptor. In certain embodiments, the transposon composition comprises a sequence encoding the therapeutic protein. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection further comprises contacting a composition capable of inducing homologous recombination at a specific site in the genome with a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments, the composition capable of inducing homologous recombination comprises an exogenous donor molecule.
  • the exogenous donor molecule comprises a sequence encoding the antigen receptor and the transposon comprises a sequence encoding the therapeutic protein. In certain embodiments, the exogenous donor molecule comprises a sequence encoding the therapeutic protein and the transposon comprises a sequence encoding the antigen receptor.
  • the composition comprising the transposon, the composition comprising the transposase and the composition capable of inducing homologous recombination at a specific site in the genome are contacted with the composition comprising a plurality of primary human T cells simultaneously. In certain embodiments, the composition comprising the transposon and the composition comprising the transposase are contacted with the composition comprising a plurality of primary human
  • composition capable of inducing homologous recombination at a specific site in the genome is contacted with the composition comprising a plurality of primary human
  • the composition capable of inducing homologous recombination at a specific site in the genome is contacted with the composition comprising a plurality- of primary human T cells first and the composition comprising the transposon and the composition comprising the transposase are contacted with the composition comprising a plurality of primary human T cells second.
  • the composition comprising primary human T cells comprises a buffer that maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells.
  • the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells prior to the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells during the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells following the nucleofection. In certain embodiments, the buffer comprises a P3 primary cell solution (Lonza).
  • the buffer comprises one or more of KC1, MgCh, ClNa, Glucose and Ca(N03)2 in any absolute or relative abundance or concentration, and, optionally, the buffer further comprises a supplement selected from the group consisting of HEPES, Tris/HCl, and a phosphate buffer.
  • the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(N03)2.
  • the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(NO.i)2 and a supplement comprising 20 mM HEPES and 75 mM Tris/HCl. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(N03)2 and a supplement comprising 40 mM
  • composition comprising primary human T cells comprises 100 ul of the buffer and between 5x10 6 and 25x10 6 cells.
  • the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and/or CD19.
  • the composition comprising primary human T cells comprises 100 ul of the buffer and between 5x10 6 and 25x10 6 cells.
  • the terms "supplemented T-ccll expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37°C.
  • the terms "supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of phosphorus, an octanoic fatty acid, a palmitic fatty acid, a linoleic fatty acid and an oleic acid.
  • the media comprises an amount of phosphorus that is 10-fold higher than may be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available at ThermoFisher Scientific as Catalog number 12440053).
  • IMDM Iscove's Modified Dulbecco's Medium
  • the terms "supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol,
  • supplied T-cell expansion composition or 'T-cell expansion composition
  • media comprising one or more of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium.
  • boron boron, sodium, magnesium, phosphorus, potassium, and calcium.
  • supplied T-cell expansion composition or 'T-cell expansion composition
  • media comprising one or more of the following elements present in the corresponding average concentrations: boron at 3.7 mg/L, sodium at 3000 mg/L, magnesium at 18 mg/L, phosphorus at 29 mg/L, potassium at 15 mg/L and calcium at 4 mg/L.
  • the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37°C.
  • the terms “supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No.
  • TMDD 2,4,7,9-tetramethyl-5- decyn-4,7-diol
  • DIPA diisopropyl adipate
  • n-butyl-benzenesulfonamide CAS No. 3622-84-2
  • 1,2-benzenedicarboxylic acid bis(2-methylpropyl) ester
  • palmitic acid CAS No. 57-10-3
  • linoleic acid CAS No. 60-33-3
  • oleic acid CAS No. 112-80-1
  • stearic acid hydrazide CAS No.
  • oleamide CAS No. 3322-62-1
  • sterol e.g., cholesterol
  • alkanes e.g., nonadecane
  • supplied T-cell expansion composition ' ' or 'T-cell expansion composition may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5- decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938- 94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No.
  • palmitic acid CAS No. 57-10-3
  • linoleic acid CAS No. 60-33-3
  • oleic acid CAS No. 112-80-1
  • stearic acid hydrazide CAS No. 4130- 54-5
  • oleamide CAS No. 3322-62-1
  • sterol e.g., cholesterol
  • alkanes e.g., nonadecane
  • phenol red CAS No. 143-74-8
  • the terms "supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-(iiol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2- benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-(iiol (TMDD) (CAS No
  • the terms "supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol,
  • supplied T-cell expansion composition or 'T-cell expansion composition
  • media comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate and phosphate.
  • the terms "supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol,
  • supplied T-cell expansion composition or 'T-cell expansion composition
  • a media comprising one or more of the following free amino acids: histidine, asparagine, serine, glutamate, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine and tryptophan.
  • the terms "supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about 1%), asparagine (about 0.5%), serine (about
  • valine about 3.5%
  • isoleucine about 3%
  • leucine about 3.5%
  • T-cell expansion composition or 'T-cell expansion composition
  • a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about .78%), asparagine (about
  • the terms "supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol).
  • a media comprising one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol).
  • the terms “supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90
  • 'T-cell expansion composition may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of
  • 'T-cell expansion composition may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 6.4 umol/kg and 640 umol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 umol/kg and 70 umol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 ⁇ /kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 ⁇ /kg and 75 ⁇ mol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 ⁇ /kg and 25 umol/kg, inclusive of the endpoints.
  • the terms "supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 64 umol/kg, palmitic acid at a concentration of about 7 ⁇ /kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 ⁇ /kg.
  • the terms "supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ /kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 ⁇ mol/kg.
  • the terms "supplemented T-cell expansion composition” or 'T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 umol/kg, linoleic acid at a concentration of about 7.57 ⁇ mol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 ⁇ /kg.
  • P3 buffer may be used interchangeably with a buffer comprising one or more of KC1, MgCh, CINa, Glucose and Ca(N03)2 in any absolute or relative abundance or concentration, and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/HCl, and a phosphate buffer.
  • P3 buffer may be used interchangeably with a buffer comprising 5 raM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(N03>2 , and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/HCl, and a phosphate buffer.
  • P3 buffer may be used interchangeably with a buffer comprising 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(N03)2 and a supplement comprising 20 mM HEPES and 75 mM Tris/HCl.
  • P3 buffer may be used interchangeably with a buffer comprising 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(N03)2 and a supplement comprising 40 mM
  • the terms "supplemented RPMI-1640 media” or 'T-cell conditioned media (TCCM)” may be used interchangeably with a media comprising one or more of water, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HC1, L- Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine
  • supplied RPMI-1640 media or “T-cell conditioned media (TCCM)” may be used interchangeably with a media comprising water, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine
  • L-Aspartic acid L-Cysteine 2HC1, L-Glutamic acid
  • Glycine L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HQ, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2H2O, L- Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D- Panthothenic acid (hemicalcium), pyridoxine HC1, riboflavin, thiamine HC1, vitamin B12, D- Glucose, Glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration.
  • the terms "supplemented AIM-V” or “supplemented AIMV” media may be used interchangeably with a media comprising one or more of water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic
  • supplied AIM-V or “supplemented AIMV” media may be used interchangeably with a media comprising water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L- Isoleucine, L-Leucine, L-Lysine HQ, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Thre
  • ImmunoCultTM medium may be used interchangeably with a medium comprising one or more of water, human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine,
  • L-Arginine hydrochloride L-Asparagine, L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, L-Glutamine, L-Histidine hydrochloride H20, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-
  • ImmunoCultTM medium may be used interchangeably with a medium comprising water, human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cysteine
  • Threonine L-Tryptophan, L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-
  • Modified T-cells of the disclosure may be incubated, cultured, grown, stored, or otherwise, combined at any step in the methods of the procedure with a growth medium comprising one or more inhibitors a component of a PI3K pathway.
  • exemplary inhibitors a component of a PI3K pathway include, but are not limited to, an inhibitor of GSK3P such as TWS119 (also known as GSK 3B inhibitor ⁇ ; CAS Number 601514-19-6 having a chemical formula C18H14N4O2).
  • Exemplary inhibitors a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIOTM).
  • the terms “electroporation” and “nucleofection” are meant to describe alternative means to deliver a nucleic acid, transposon, vector or composition of the disclosure to a cell by providing an electric pulse that induces a cell membrane (the cell membrane, nuclear membrane, or both) to become permeable or to become more permeable to the nucleic acid, transposon, vector or composition of the disclosure.
  • the method is performed one or more cuvette(s) simultaneously. In certain embodiments of the nucleofection, the method is performed in two cuvettes simultaneously. For a process performed on a larger scale for clinical or commercial applications, for example, the nucleofections may be performed in a large-volume cassette with many procedures ongoing simultaneously.
  • the incubating step comprises incubating the composition comprising the plurality of primary human T cells in a pre-warmed T-cell expansion composition. The incubation step may have a period of at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11,
  • the incubation step may have a period of at least 1 , 2, 3, 4, 5, 6 or 7 days or any number/portion of days in between.
  • the incubation step may have a period of at least 1 week.
  • the incubation step has a period of two days.
  • the applying step may comprise applying one or more of the following program(s) EI-115, EI-151, EI-156, EI-158, EG-115, EG- 142,
  • the applying step may comprise applying one or more of the following program(s) EI-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, EO- 151, EO-148, EO-156, EO-210, EO-213, and FI-156, or a program that provides the same number of electrical pulses, each pulse having the same duration and intensity, and a substantially similar interpulse duration of time.
  • the applying step may be performed using a known clectroporation/nucleofection device, including, but not limited to, Lonza Amaxa, MaxCyte technology, BTX PulseAgile, and BioRad GenePulser.
  • the applying step may comprise applying at least one electrical pulse.
  • the applying step may comprise applying at least one electrical pulse sufficient to induce the cell membrane and/or nuclear membrane of a cell to become permeable to a composition of the disclosure.
  • the activation supplement comprises one or more cytokine(s).
  • the one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines.
  • Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFy).
  • the one or more cytokine(s) may comprise IL-2.
  • the expansion supplement comprises one or more cytokine(s).
  • the one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines.
  • Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interieukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFy).
  • the one or more cytokine(s) may comprise IL-2.
  • the primary human T cell is a naive T cell.
  • the naive T cell may express CD45RA, CCR7 and CD62L.
  • the method is applied to a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between of naive T cells.
  • the efficiency of production of modified TSCM and/or TCM of the disclosure may be increased by increasing a proportion or percentage of naive T cells in a cell population to which the methods of the disclosure are applied.
  • the primary human T cell is a memory T cell.
  • the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R ⁇ ,
  • the primary human T cell is a naive T-cell (modified TN) and the modified TN expresses one or more of CD45RA, CCR7 and CD62L.
  • the primary human T cell is a modified TSCM a T memory stem cell (modified TSCM) and the modified TSCM expresses one or more of CD45RA, CD95, IL-2RfJ, CR7, and CD62L.
  • the primary human T cell is a central memory T-cell (modified TCM) and the modified TCM expresses one or more of CD45RO, CD95, IL-2R ⁇ , CCR7, and CD62L.
  • the primary human T cell is an effector memory T-cell (modified TEM) and the modified TEM expresses one or more of CD45RO, CD95, and IL-2R ⁇
  • the primary human T cell is an effector T-cell (modified TKFF) and the modified TEFF expresses one or more of CD45RA, CD95, and IL-2R ⁇
  • the primary human T cell may express CD4 and/or CD8. In certain embodiments, the primary human T cell may express CD4 and/or CD8 at various ratios. In certain embodiments, the primary human T cell may express CD4 and/or CD8 at various ratios that are not naturally-occurring. In certain embodiments, the primary human T cells that express CD4 and/or CD8 at various ratios, that may be not naturally occurring, are a heterologous cell population.
  • the primary human T cell may be isolated, prepared or derived from for example, whole blood, peripheral blood, umbilical cord blood, lymph fluid, lymph node tissue, bone marrow, and cerebral spinal fluid (CSF).
  • peripheral blood refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow.
  • Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver or bone marrow.
  • cord blood refers to blood that remains in the placenta and in the attached umbilical cord after child birth.
  • Cord blood often contains stem cells including hematopoietic cells.
  • pan T-cells include all T lymphocytes isolated from a biological sample, without sorting by subtype, activation status, maturation state, or cell-surface marker expression.
  • the method further comprises introducing into a modified TSCM or TCM cell a composition comprising a genomic editing construct or composition.
  • the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats
  • the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease.
  • the genomic editing construct encodes a fusion protein.
  • the genomic editing construct encodes the DNA binding domain and the type
  • the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease
  • the genomic editing construct comprises a sequence derived from a
  • Cas9 endonuclease In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a Cas9 endonuclease is the DNA binding domain. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes an inactive Cas9. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
  • sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D 1 OA).
  • sequence derived from a Cas9 endonuclease comprises or further comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
  • sequence derived from a Cas9 endonuclease comprises an inactivated Cas9 (dCas9) (SEQ ID NO: 33).
  • the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an alanine (A) for an Asparagine (N) at position 580 (N580A).
  • the sequence derived from a Cas9 endonuclease comprises a truncated and inactivated Cas9 (dSaCas9) (SEQ ID NO: 32).
  • the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
  • the sequence derived from a TALEN is the DNA binding domain.
  • the genomic editing construct comprises a TALEN.
  • the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
  • ZFN zinc-finger nuclease
  • the sequence derived from a ZFN is the DNA binding domain.
  • the genomic editing construct comprises a zinc-finger nuclease (ZFN).
  • modified TSCM and/or TCM cells of the disclosure may be optimized to produce a greater number or greater proportion of modified TSCM and/or TCM cells.
  • the population of cells subjected to the methods of the disclosure may be enriched to contain an increased number or greater proportion of naive T cells.
  • the number and/or proportion of naive T cells increases in the population of T cells subjected to the methods of the disclosure, the number and/or proportion of modified TSCM and/or TCM cells of the disclosure produced also increases.
  • the length of time or duration required for a method of disclosure to precede decreases, the number and/or proportion of modified TSCM and/or TCM cells of the disclosure produced by the method increases.
  • the length of time or duration required for a method of disclosure to precede, or the "manufacturing period" may also be referred to as the "out-of-life period" of the T cells subjected to the methods of the disclosure.
  • the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD 127, CD4SRO, CD95, CD9S and IL-2R ⁇
  • the primary human T cell is a naive T-cell (TN) and the TN expresses one or more of CD45RA, CCR7 and CD62L.
  • the primary human T cell is a T memory stem cell (TSCM) and the TSCM expresses one or more of CD45RA, CD95, IL-2R ⁇ , CR7, and CD62L.
  • TSCM T memory stem cell
  • the primary human T cell is a central memory T-cell (TCM) and wherein the TCM expresses one or more of CD45RO, CD95, IL-2R ⁇ , CCR7, and CD62L.
  • TCM central memory T-cell
  • the primary human T cell is an effector memory T-cell (TEM) and the EM expresses one or more of CD45RO, CD9S, and IL-2Rp.
  • the primary human T cell is an effector T-cell (TEFF) and the TEFF expresses one or more of CD45RA, CD95, and IL-2R ⁇ ,
  • the primary human T cell expresses CD4 and/or CD8.
  • the disclosure provides a composition comprising a modified TSCM produced a method of the disclosure.
  • the disclosure provides a composition comprising a modified TCM produced a method of the disclosure.
  • the disclosure provides a composition comprising a modified TSCM and a modified TCM produced a method of the disclosure.
  • a plurality of TSCM may comprise at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% or the composition. .
  • a plurality of TCM may comprise at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% or the composition.
  • composition comprising a modified TSCM and/or
  • TCM produced a method of the disclosure for the manufacture of a medicament to treat a subject in need thereof.
  • the modified TSCM and/or TCM is autologous.
  • the modified TSCM and/or TCM is allogeneic.
  • the antigen receptor is a T-cell receptor. In certain embodiments, the
  • the T-cell receptor is naturdly-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring . In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-c ⁇ xurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-occurring, the T- cell receptor is a recombinant T-cell receptor. In certain embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
  • CAR Chimeric Antigen Receptor
  • the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a modified TSCM and/or TCM produced a method of the disclosure.
  • the modified TSCM and/or TCM is autologous.
  • the modified TSCM and/or TCM is allogeneic.
  • the antigen receptor is a T-cell receptor.
  • the T-cell receptor is naturally-occurring .
  • the T-cell receptor is not naturally- occurring.
  • the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor.
  • the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
  • the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen.
  • the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antigen.
  • the disease or disorder is a disease or disorder caused by a lack of an activity or an insufficient amount of a secretory protein.
  • the disease or disorder is a disease or disorder treated by a replacement of an activity of a therapeutic protein or by an increase in an amount of the therapeutic protein.
  • the therapeutic protein is a secreted protein.
  • the secretory protein is lacking an activity or a sufficient amount within a local area of a body.
  • the local area of a body is accessible by a native T-cell or a modified T-cell.
  • the modified T-cell is produced in vivo, ex vivo, in vitro or in situ.
  • Figure 1 is a series of plots depicting the emergence of the CAR-TSCM phenotype at Day 11 of the method of Example 1.
  • Cells were nucleofected with a surrogate CARTyrin plasmid.
  • CAR-TSCM cells express CD62L and CD45RA as shown in the bottom two plots.
  • Figure 2 is a series of plots depicting the purity of the CAR-TSCM produced by the method of Example 1 at day 19.
  • the population of CAR-TSCM cells produced by the method described in Example 1 at day 19 contained no B cells or lymphocytes. The majority' of the cells are CD3+ T-cells. Only 1.1% are Natural Killer cells and 1.7% are Natural Killer T- cells.
  • Figure 3 is a plot showing that at Day 11 of the method described in Example 1, the majority of the T-cells produced express the CARTyrin.
  • Figure 4 is a series of plots depicting an enrichment of the CAR-TSCM phenotype at Day 19 of the method described in Example 1.
  • Cells were nucleofected with a surrogate CARTyrin plasmid.
  • CAR-TSCM cells express CD62L and CD4SRA as shown in the bottom two plots.
  • Figure 5 is a series of plots depicting the absence of T-cell exhaustion at Day 19 of the method described in Example 1.
  • the cell population produced by this method does not express PD1, which is a marker for T cell activation and exhaustion.
  • PD1 is a marker for T cell activation and exhaustion.
  • These cells expressing the CARTyrin have almost successfully reached a resting state post-manufacture. They do not exhibit signs of antigen-independent (tonic) signaling which would otherwise drive higher levels of PD1 expression.
  • Tonic signaling is hypothesized to be caused by some CAR molecules that lead to early exhaustion and reduced efficacy of a CAR T-cell therapy.
  • FIG. 6A is a series of plots depicting T cells transposed with a plasmid containing a sequence encoding atransposon comprising a sequence encoding an inducible caspase polypeptide (a safety switch, "iC9"), a CARTyrin (anti-BCMA), and a selectable marker.
  • a safety switch "iC9”
  • anti-BCMA anti-BCMA
  • Left-hand plots depict live T cells exposed to transposase in the absence of the plasmid.
  • Right-hand plots depict live T cells exposed to transposase in the presence of the plasmid.
  • Cells were exposed to either a hyperactive transposase (the "Super piggyBac”) or a wild type piggy Bac transposase.
  • Figure 6B is a series of plots depicting T cells transposed with a plasmid containing a sequence encoding a green fluorescent protein (GFP).
  • Left-hand plots depict live T cells exposed to transposase in the absence of the plasmid.
  • Right-hand plots depict live T cells exposed to transposase in the presence of the plasmid.
  • Cells were exposed to either a hyperactive transposase (the "Super piggy Bac") or a wild type piggyBac transposase.
  • FIG. 6C is a table depicting the percent of transformed T cells resulting from transposition with WT versus hyperactive piggyBac transposase.
  • T cells contacted with the hyperactive piggyBac transposase (the Super piggyBac transposase) were transformed at a rate 4-fold greater than WT transposase.
  • Figure 6D is a table depicting the percent of transformed T cells resulting from transposition with WT versus hyperactive piggyBac transposase 5 days after nucleofection.
  • T cells contacted with the hyperactive piggyBac transposase (the Super piggyBac transposase) were transformed at a rate far greater than WT transposase.
  • FIG. 7 is a graph showing a phenotypic difference between piggyBacTM- and lentivirus-produced CAR+ T cells.
  • CAR+ T cells were produced using either piggyBac transposition or lentivirus transduction.
  • Human pan T cells were transposed with piggyBac encoding CAR, stimulated with anti-CD3/CD28 beads at day 2 post-transposition, expanded, and examined on day 19 post-transposition.
  • pan T cells were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding CAR (MOI 5), expanded, and examined on day 18 post-stimulation.
  • each population of CAR+ T cells was characterized based on their expression of the standard memory markers CD62L, CD45RA and CD95.
  • the percentage of each CAR+ T cell subset was defined as naive (CD62L+CD45RA+), Tcm (CD62L+CD45RA-), Tem (CD62L-CD45RA-) and Teff (CD62L-CD45RA+). All CAR+ T cells were CD95+.
  • Figure 8A-B is a pair of graphs showing that piggyBacTM preferentially transposes naive T cells.
  • Human pan T cells were sorted (using a BD FACSAria ⁇ flow cytometer) into naive (CD62L+CD45RA+), Tcm (CD62L+CD45RA-), Tem (CD62L-CD45RA-), and Teff
  • FIG. 9 is a pair of graphs showing that the piggyBacTM manufacturing process yields high levels of TSCM in samples from multiple myeloma (MM) patients even when naive T cells are rare.
  • T cells from MM patients were characterized for memory marker expression by flow cytometry before (left) and after (right) the Poseida manufacturing process. Expression of CD45RA and CD62L was assessed by FACS and plots are shown for the MM patients and a healthy donor. It is known that T cells from MM patients generally have lower frequencies of naive and TSCM cells, but higher frequencies of Teff, unlike those from healthy normal donors which are the opposite.
  • Figure 10 is a series of Fluorescence Activated Cell Sorting (FACs) plots characterizing T and TSCM cell markers in human pan T cells transformed with the Sleeping
  • Tscm Stem cell memory phenotype
  • the x-axis in order from left to right, in the first and second columns shows Forward Scatter (FSC), units from 0 to 250 thousand (abbreviated "k"), in increments of 50k.
  • FSC Forward Scatter
  • k 250 thousand
  • the x axis of the third column from the left shows CD8 expression, with markings reading from 0 to 10 s incrementing by powers of 10.
  • the final right hand column shows CD62L expression, with markings reading from 0 to 10 s incrementing by powers of 10.
  • the y-axis, in the first column shows Side Scatter (SSC), in units from 0 to 250k in increments of 50k.
  • the y-axis in the second column from the left shows expression of the cell viability marker 7 aminoactinomycin D (7AAD), from 0 to 10 s incrementing by powers of 10.
  • the y-axis of the third column from the left shows the expression of the marker CD4, from 0 to 10 s incrementing by powers of 10.
  • the y-axis in the right hand column show expression of the marker CD45RA, from 0 to 10 3 incrementing by powers of 10.
  • FIG. 11 is a schematic diagram showing the human coagulation pathway leading to blood clotting.
  • Contact activation for example by damaging an endothelium, activates an intrinsic clotting pathway.
  • Tissue factors activate an extrinsic clotting pathway, for example following trauma. Both pathways converge onto the conversion of Prothrombin into
  • Thrombin which catalyzes the conversion of fibrinogen into fibrin. Polymerized fibrin together with platelets forms a clot. In the absence of Factor IX (circled), clotting is defective. Factor VHI (FVm) deficiency leads to development of Hemophilia A. Factor IX
  • Hemophilia B is a rare disease, occurring with a frequency of about one in between 25,000 and 30,000. Sixty percent of hemophilia B cases are severe. Fewer than one percent of individuals with Hemophilia B have normal FIX levels.
  • the standard treatment for hemophilia B involved an infusion of recombinant FIX every 2 to 3 days, at an expense of approximately $250,000 per year.
  • TSCM cells of the disclosure are maintained in humans for several decades.
  • Figure 12 is a series of Fluorescence-Activated Cell Sorting (FACS plots) depicting
  • T cells encoding a human Factor IX transgene showed a TSCM
  • FSC Forward scatter
  • SSC side scatter
  • k 250 thousand
  • APC conjugated to a Cy7 dye (CDC56-APC-Cy7), units from 0 to 10 s incrementing in powers of 10.
  • On the y-axis is shown anti-CD3 conjugated to phycoerythrin (PE), units from
  • Figure 13A is a graph showing human Factor IX secretion during production of modified T cells of the disclosure.
  • On the x-axis are shown 9 day and 12 day T cells.
  • Figure 13B is a graph showing the clotting activity of the secreted Factor IX produced by the T cells. On the y-axis is shown percent Factor IX activity relative to human plasma, from 0 to 8 in increments of 2. On the x-axis are 9 and 12 day T cells.
  • Figures 14A-E are a series of plasmid maps for site-specific integration into the AAVS1 site using either HR or MMEJ and corresponding sequences.
  • HR site-specific
  • AAVSl homologous recombination
  • MMEJ microhomology-mediated end- joining
  • FIG. 15 is a graph showing transgene (GFP) expression in primary human pan T cells 3 days post-nucleofection.
  • HR or MMEJ donor plasmids were co-delivered with or without CRISPR ribonucleoprotein (RNP) targeting reagents into pan T cells via
  • RNP CRISPR ribonucleoprotein
  • T cells receiving donor plasmids alone were included as controls.
  • Pan T cells were also modified using the piggyBacTM transposon delivery system.
  • T cells were activated via TCR stimulation on Day 0 and GFP+ T cell percentage was accessed at day 3 post- nucleofection by flow cytometry and data are summarized in bar graph.
  • FIG. 16 is a graph showing transgene (GFP) expression in primary human pan T cells 11 days post-nucleofection and selection.
  • Activated T cells with stably integrated transgenes were selected by methotrexate addition using the DHFR selection gene encoded in the bi-cistronic GFP-2A-DHFR integration cassettes.
  • GFP+ cell percentage was assessed at Day 11 post-nucleofection by flow cytometry and data are summarized in bar graph.
  • GFP+ cells were highly enriched via selection in pan T cells receiving transposition reagents, RNP plus HR or MMEJ donor plasmids, but not in T cells receiving donor plasmids alone.
  • Figure 17A-C is a series of graphs showing the phenotype of primary human pan T cells modified by HR and MMEJ at the AAVS1 site.
  • the phenotype of GFP+ CD8+ pan T cells was analyzed at Day 11 post-nucleofection by flow cytometry.
  • A) Cells were stained with 7AAD (cell viability), CD4, CD8, CD45RA and CD62L, and FACS plots show gating strategy.
  • CD8+ T cell subsets were defined by expression of CD45RA+CD62L+ (stem cell memory T cells (Tscm)), CD45RA-CD62L+ (central memory T cells (Tcm)), CD45RA- CD62L- (effector memory T cells (Tem)), and CD45RA+CD62L- (T effectors (Teff)).
  • Tscm stem cell memory T cells
  • Tcm central memory T cells
  • CD45RA- CD62L- effector memory T cells
  • Tefff CD45RA+CD62L-
  • Figure 18A-B is a pair of photographs of gel electrophoresis results showing site- specific integration into the AAVSl site. Selected cells from each group were harvested and genomic DNA was extracted and used as template for PCRto confirm site-specific integration into the AAVSl site for A) HR and B) MMEJ. Two pairs of primers individually amplify the 5 '-end junction (with one primer priming the promoter region of the insertion EFla-2r CACCGGAGCCAATTCCCACT (SEQ ID NO: 36) and the other priming the AAVSl region beyond the 500 bp homologue arm at the 5 '-end AAVS-3r
  • CTGCACCACGTGATGTCCTC (SEQ ID NO: 37), yielding a 0.73 kb DNA fragment for both HR or MMEJ) and 3 '-end junction (with one primer priming the polyA signaling region SV40pA-lr GTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38) and the other priming the AAVSl region beyond the 500 bp 5'-homologue arm AAVS-2f
  • CTGGGGACTCTTTAAGGAAAGAAG SEQ ID NO: 39
  • the disclosure provides a method for producing human chimeric antigen receptor (CAR) expressing-T cells using the piggy BacTM Transposon System under conditions that preserve or induce stem-cell memory T cells (TSCM) with potent CAR activity (referred to herein as a CAR-TSCM.
  • Compositions comprising CAR-TSCM produced using the methods of the disclosure comprise > 60% CAR-TSCM and exhibit a distinct functional profile that is consistent with this T cell subset.
  • T cell subsets found in the compositions of the disclosure include, but are not limited to, central memory CAR-T cells (CAR-TCM), effector memory CAR-T cells (CAR-TEM), effector CAR-T cells (CAR-TE), and terminally- differentiated effector CAR-T cells (CAR-TIE).
  • CAR-TCM central memory CAR-T cells
  • CAR-TEM effector memory CAR-T cells
  • CAR-TE effector CAR-T cells
  • CAR-TIE terminally- differentiated effector CAR-T cells
  • a linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN) > TSCM > TCM > TEM > TE > TTE, whereby TN is the parent precursor cell that directly gives rise to TSCM, which then, in turn, directly gives rise to TCM, etc.
  • compositions comprising CAR-TSCM, CARTyrin-TscM and/or VCAR-TSCM of the disclosure may comprise one or more of each parental CAR-T cell subset with CAR-TSCM being the most abundant (e.g. TSCM > TCM > TEM > TE > TTE). While, the absolute quantities/abundances and relative proportions of each parental T cell subset may vary among samples of patient blood and naturally-occurring cell populations, and naturally- occurring cell populations may have a high abundance and/or proportion of TSCM, compositions of the disclosure comprising non-naturally occurring CAR-TSCM are more potent and efficacious in treating patients against diseases and cancers.
  • CAR-T cells chimeric-antigen receptor (CAR)-T cells
  • Ag tumor-associated antigen
  • CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments.
  • CAR-T cells must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses.
  • a stem cell-like CAR-T would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM), effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term CAR-T engraftment.
  • TCM central memory
  • TEM effector memory
  • TE effector T cells
  • CAR-TSCM of the disclosure may comprise a Centyrin-based CAR, referred to as a CARTyrin (and hence, the cell may be referred to as a CARTyrin-Tso-i).
  • Centyrins are alternative scaffold molecules based on human consensus tenascin FN3 domain, are smaller than scFv molecules, and can be selected for monomelic properties that favor stability and decrease the likelihood of tonic signaling in CAR molecules.
  • CARTyrins of the disclosure may be introduced to T cells using a plasmid DNA transposon encoding the CARTyrin that is flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking improper gene activation or silencing.
  • CAR-TSCM of the disclosure may comprise a VHH-based CAR, referred to as a VCAR (and hence, the cell may be referred to as a VCAR-TSCM).
  • VCARS of the disclosure may be introduced to T cells using a plasmid DNA transposon encoding the VHH that is flanked by two cis-regulatory insulator elements to help stabilize VHH expression by blocking improper gene activation or silencing.
  • the piggyBacTM (PB) Transposon System may be used for stable integration of antigen-specific (including cancer antigen-specific) CARTyrin or VCAR into resting pan T cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme (although the transposon and transposase would be comprised in separate compositions until they were introduced into a cell), called Super piggyBacTM (SPB), in a single electroporation reaction. Delivery of piggyBacTM transposon into untouched, resting primary human pan T cells resulted in 20- 30% of cells with stable integration and expression of PB-delivered genes.
  • SPB Super piggyBacTM
  • modified CARTyrin-expressing T cells were positive for expression of CD62L and CD45RA, markers commonly associated with stem memory T-cells (TSCM cells).
  • TSCM cells stem memory T-cells
  • the modified CARTyrin-expressing T cells positive for expression of CD62L and CD45RA were activated via stimulation of CD3 and CD28.
  • > 60% of CARTyrin+ T cells exhibited a stem-cell memory phenotype.
  • these cells which expressed a CARTyrin specific for a cancer antigen, were fully capable of expressing potent anti-tumor effector function.
  • phenotypic and functional characteristics were measured including kinetic analysis of memory and exhaustion-associated markers, secondary proliferation in response to homeostatic cytokine or tumor-associated Ag, cytokine production, and lytic capability in response to target tumor cells.
  • the LV-transduced CARTyrin + T cells did not exhibit an augmented memory phenotype.
  • PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of target tumor cells.
  • CARTyrin + T cells exhibit strong anti-tumor activity and may give rise to cells that persist longer in vivo due to the use of a Centyrin-based CAR, which may be less prone to tonic signaling and functional exhaustion.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises one or more sequences that each specifically bind an antigen; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • the antigen recognition region may comprise two sequences that each specifically bind an antigen to produce a bi-specific or tandem CAR
  • the antigen recognition region may comprise three sequences that each specifically bind an antigen to produce a tri-specific CAR
  • the ectodomain may further comprise a signal peptide.
  • the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
  • Sequences that each specifically bind an antigen may include, but not limited to, a single chain antibody (e.g. a scFv), a sequence comprising one or more fragments of an antibody (e.g. a VHH, referred to in the context of a CAR as a VCAR), an antibody mimic, and a Centyrin (referred to in the context of a CAR as a CARTyrin).
  • the signal peptide may comprise a sequence encoding a human CD2, CD35, CD3e, CD3y, CD3£, CD4, CD8a,
  • the signal peptide may comprise a sequence encoding a human CD8a signal peptide.
  • the human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8).
  • the human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8).
  • the human CD8a signal peptide may be encoded by a nucleic acid sequence comprising
  • the transmembrane domain may comprise a sequence encoding a human CD2, CD36, CD3e, CD3y, CD3 ⁇ CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain.
  • the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain.
  • the CD8a transmembrane domain may comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10).
  • transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatttgggoiccactggccgggacctgtgg (SEQ ID NO:
  • the endodomain may comprise a human ⁇ 3 ⁇ endodomain.
  • the at least one costimulatory domain may comprise a human 4- IBB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.
  • the at least one costimulatory domain may comprise a CD28 and/or a 4- IBB costimulatory domain.
  • the CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • the CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising
  • the 4- 1BB costimulatory domain may be encoded by the nucleic acid sequence comprising
  • domain may be located between the transmembrane domain and the CD28 costimulatory domain.
  • the hinge may comprise a sequence derived from a human CD8a, IgG4, and/or CD4 sequence. In certain embodiments of the CARs of the disclosure, the hinge may comprise a sequence derived from a human CD8a sequence.
  • the hinge may comprise a human CD8a amino acid sequence comprising TTTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising TTTPAPRPPTTAPTTASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16).
  • the human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising
  • the disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
  • the disclosure provides a transposon comprising the CAR of the disclosure.
  • Transposons of the disclosure be episomally maintained or integrated into the genome of the recombinant/modified cell.
  • the transposon may be part of a two component piggyBac system that utilizes a transposon and transposase for enhanced non-viral gene transfer.
  • Transposons of the disclosure may comprise a selection gene for identification, enrichment and/or isolation of cells that express the transposon.
  • Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for cell viability and survival.
  • Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for conferring resistance to a drug challenge against which the cell is sensitive (or which could be lethal to the cell) in the absence of the gene product encoded by the selection gene.
  • Exemplar ⁇ ' selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for viability and/or survival in a cell media lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene.
  • Exemplar ⁇ ' selection genes include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to
  • Methotrexate TYMS (encoding Thymidylate Synthetase), MGMT (encoding 0(6)- methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member A 1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).
  • ALDH1 encoding Aldehyde dehydrogenase 1 family, member A 1
  • FRANCF RAD51C (encoding RAD51 Paralog C)
  • GCS encoding glucosylceramide synthase
  • NKX2.2 encoding NK2 Homeobox 2.
  • Transposons of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between one or more of a sequence that specifically binds an antigen and a selection gene of the disclosure.
  • the at least one self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18).
  • a GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%,
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).
  • a GSG- F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
  • Transposons of the disclosure may comprise a first and a second self-cleaving peptide, the first self-cleaving peptide located, for example, upstream of one or more of a sequence that specifically binds an antigen of the disclosure the second self-cleaving peptide located, for example, downstream of the one or more of a sequence that specifically binds an antigen of the disclosure.
  • the first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18).
  • a GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%,
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 023).
  • a GSG- F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
  • composition comprising the transposon the disclosure.
  • a method introducing the composition may further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme.
  • the sequence encoding a transposase enzyme may be an mRNA sequence.
  • Transposons of the disclosure may comprise piggyBac transposons.
  • Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
  • the disclosure provides a vector comprising the CAR of the disclosure.
  • the vector is a viral vector.
  • the vector may be a recombinant vector.
  • Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lenti virus, an adenovirus, an adeno-associated virus or any combination thereof.
  • the viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV).
  • the viral vector may comprise a recombinant AAV (rAAV).
  • Exemplary adeno- associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (ITR) sequences located in cis next to one or more of a sequence that specifically binds an antigen.
  • ITR inverted terminal repeat
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to all serotypes (e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).
  • Exemplar ⁇ ' adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g. AAV2/5, AAV-DJ and AAV-DJ8).
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.
  • Viral vectors of the disclosure may comprise a selection gene.
  • the selection gene may encode a gene product essential for cell viability and survival.
  • the selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions.
  • Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.
  • Exemplary selection genes of the disclosure may include, but are not limited to, neo
  • DHFR encoding Dihydrofolate Reductase and conferring resistance to Methotrexate
  • TYMS encoding Thymidylate Synthetase
  • MGMT encoding 0(6)-methylguanine-DNA methyltransferase
  • MDR1 multidrug resistance gene
  • ALDH1 encoding Aldehyde dehydrogenase 1 family, member Al
  • FRANCF RAD51C
  • RADS1 Paralog C encoding RADS1 Paralog C
  • GCS encoding glucosylceramide synthase
  • NKX2.2 encoding NK2 Homeobox 2 or any combination thereof.
  • Viral vectors of the disclosure may comprise at least one self-cleaving peptide.
  • the vector may comprise at least one self-cleaving peptide and wherein a self-cleaving peptide is located between a CAR and a selection gene.
  • the vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR.
  • the self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).
  • a GSG- F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
  • the disclosure provides a vector comprising the CAR of the disclosure.
  • the vector is an mRNA vector.
  • the vector may be a recombinant mRNA vector.
  • T cells of the disclosure may be expanded prior to contacting the T-cell and the mRNA vector comprising the CAR of the disclosure.
  • the T cell comprising the mRNA vector, the modified T cell may then be administered to a subject.
  • the disclosure provides a vector comprising the CAR of the disclosure.
  • the vector is a nanoparticle.
  • Exemplary nanoparticle vectors of the disclosure include, but are not limited to, nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof), amino acids (L-amino acids, D -amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g. polymersomes), micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any combination thereof.
  • a nanoparticlc vector may be passively or actively transported across a cell membrane.
  • Nanoparticle vectors of the disclosure may comprise a selection gene.
  • the selection gene may encode a gene product essential for cell viability and survival.
  • the selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions.
  • Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.
  • Exemplar ⁇ ' selection genes of the disclosure may include, but are not limited to, mo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT (encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.
  • mo conferring resistance to neomycin
  • DHFR encoding Dihydrofolate Reductase and conferring resistance to Methotrexate
  • TYMS encoding Thymidylate Synthetase
  • MGMT encoding
  • Nanoparticle vectors of the disclosure may comprise at least one self-cleaving peptide.
  • the nanoparticle vector may comprise at least one self- cleaving peptide and wherein a self-cleaving peptide is located between a CAR and the nanoparticle.
  • the nanoparticle vector may comprise at least one self- cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR.
  • the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self- cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR.
  • the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and a selection gene.
  • the self- cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG- P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising
  • EGRGSLLTCGDVEENPGP SEQ ID NO: 18 or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).
  • a GSG- F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
  • the disclosure provides a composition comprising a vector of the disclosure.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one Centyrin; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • a CAR comprising a Centyrin is referred to as a CARTyrin.
  • the antigen recognition region may comprise two Centyrins to produce abi-specific or tandem CAR.
  • the antigen recognition region may comprise three Centyrins to produce a tri-specific CAR.
  • the ectodomain may further comprise a signal peptide.
  • the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one protein scaffold or antibody mimetic; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • CAR chimeric antigen receptor
  • the antigen recognition region may comprise two scaffold proteins or antibody mimetics to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three protein scaffolds or antibody mimetics to produce a tri-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
  • the signal peptide may comprise a sequence encoding a human CD2, CD36, CD3e, CD3y, CD3£, CD4, CD8a,
  • the signal peptide may comprise a sequence encoding a human CD8a signal peptide.
  • the human CD8a signal peptide may comprise an amino acid sequence comprising
  • the human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8).
  • CD8a signal peptide may be encoded by a nucleic acid sequence comprising
  • the transmembrane domain may comprise a sequence encoding a human CD2, CD36, CD3e, CD3y, CD3£ CD4, CD8a,
  • CD19, CD28, 4- IBB or GM-CSFR transmembrane domain In certain embodiments of the
  • the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain.
  • the CD8a transmembrane domain may comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVrTLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10).
  • the CD8a transmembrane domain may be encoded by the nucleic acid sequence comprising
  • the endodomain may comprise a human ⁇ 3 ⁇ endodomain.
  • the at least one costimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.
  • the at least one costimulatory domain may comprise a CD28 and/or a 4- IBB costimulatory domain.
  • the CD28 costimulatory domain may comprise an amino acid sequence comprising
  • T e 4- IBB cost mu atory oma n may comp se an am no ac sequence comprising KRGRKKLLYTFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • 1BB costimulatory domain may be encoded by the nucleic acid sequence comprising
  • the hinge may comprise a sequence derived from a human CD8ct, IgG4, and/or CD4 sequence. In certain embodiments of the CARs of the disclosure, the hinge may comprise a sequence derived from a human CD8a sequence.
  • the hinge may comprise a human CD8a amino acid sequence comprising TTTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16).
  • the human CD8a hinge amino acid sequence may be encoded by the nucleic acid
  • Centyrins of the disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding an antigen.
  • Centyrins of the disclosure may comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type ⁇ (FN3) domain, wherein the scaffold is capable of specifically binding an antigen.
  • the at least one fibronectin type ⁇ (FN3) domain may be derived from a human protein.
  • the human protein may be Tenascin-C.
  • the consensus sequence may comprise
  • sequence may encoded by a nucleic acid sequence comprising
  • Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type ⁇ (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type III (FN3) domains.
  • the scaffold may bind an antigen with at least one affinity selected from a KD of less than or equal to 10 ⁇ M, less than or equal to 10 "10 M, less than or equal to 10 "1 'M, less than or equal to 10 "12 M, less than or equal to 10 "13 M, less than or equal to 10 "14 M, and less than or equal to 10 ⁇ 15 M.
  • the KD may be determined by surface plasmon resonance.
  • the disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
  • the disclosure provides a transposon comprising the CAR of the disclosure.
  • Transposons of the disclosure be episomally maintained or integrated into the genome of the recombinant/modified cell.
  • the transposon may be part of a two component piggyBac system that utilizes a transposon and transposase for enhanced non-viral gene transfer.
  • Transposons of the disclosure may comprise a selection gene for identification, enrichment and/or isolation of cells that express the transposon.
  • Exemplar ⁇ ' selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for cell viability and survival.
  • Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for conferring resistance to a drug challenge against which the cell is sensitive (or which could be lethal to the cell) in the absence of the gene product encoded by the selection gene.
  • Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for viability and/or survival in a cell media lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene.
  • Exemplar ⁇ ' selection genes include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to
  • Methotrexate TYMS (encoding Thymidylate Synthetase), MGMT (encoding 0(6)- methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member A 1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), andNKX2.2 (encoding NK2 Homeobox 2).
  • Transposons of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between on or more of a protein scaffold, Centyrin or C ARTyrin of the disclosure and a selection gene of the disclosure.
  • the at least one self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18).
  • a GSG- T2A peptide may comprise an amino acid sequence comprising
  • GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).
  • a GSG-F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:
  • Transposons of the disclosure may comprise a first and a second self-cleaving peptide, the first self-cleaving peptide located, for example, upstream of one or more of a protein scaffold, Centyrin or CARTyrin of the disclosure the second self-cleaving peptide located, for example, downstream of the one or more of a protein scaffold, Centyrin or CARTyrin of the disclosure.
  • the first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18).
  • a GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).
  • a GSG- F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:
  • composition comprising the transposon the disclosure.
  • a method introducing the composition may further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme.
  • the sequence encoding a transposase enzyme may be an mRNA sequence.
  • Transposons of the disclosure may comprise piggyBac transposons.
  • Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
  • the disclosure provides a vector comprising the CAR of the disclosure.
  • the vector is a viral vector.
  • the vector may be a recombinant vector.
  • Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lenti virus, an adenovirus, an adeno-associated virus or any combination thereof.
  • the viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV).
  • the viral vector may comprise a recombinant AAV (rAAV).
  • Exemplary adeno- associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (ITR) sequences located in cis next to a sequence encoding a protein scaffold, Centyrin or CARTyrin of the disclosure.
  • ITR inverted terminal repeat
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to all serotypes (e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g. AAV2/5, AAV-DJ and AAV-DJ8).
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.
  • Viral vectors of the disclosure may comprise a selection gene.
  • the selection gene may encode a gene product essential for cell viability and survival.
  • the selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions.
  • Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.
  • Exemplary selection genes of the disclosure may include, but are not limited to, neo
  • DHFR encoding Dihydrofolate Reductase and conferring resistance to Methotrexate
  • TYMS encoding Thymidylate Synthetase
  • MGMT encoding 0(6)-methylguanine-DNA methyltransferase
  • MDR1 multidrug resistance gene
  • ALDHl encoding Aldehyde dehydrogenase 1 family, member Al
  • FRANCF RAD51C
  • RADS1 Paialog C GCS
  • GCS encoding glucosylceramide synthase
  • NKX2.2 encoding NK2 Homeobox 2 or any combination thereof.
  • Viral vectors of the disclosure may comprise at least one self-cleaving peptide.
  • the vector may comprise at least one self-cleaving peptide and wherein a self-cleaving peptide is located between a CAR and a selection gene.
  • the vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR
  • the self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).
  • a GSG- F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
  • the disclosure provides a vector comprising the CAR of the disclosure.
  • the vector is an mRNA vector.
  • the vector may be a recombinant mRNA vector.
  • T cells of the disclosure may be expanded prior to contacting the T-cell and the mRNA vector comprising the CAR of the disclosure.
  • the T cell comprising the mRNA vector, the modified T cell may then be administered to a subject.
  • the disclosure provides a vector comprising the CAR of the disclosure.
  • the vector is a nanoparticle.
  • Exemplary nanoparticle vectors of the disclosure include, but are not limited to, nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof), amino acids (L-amino acids, D -amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g. polymersomes), micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any combination thereof.
  • a nanoparticle vector may be passively or actively transported across a cell membrane.
  • Nanoparticle vectors of the disclosure may comprise a selection gene.
  • the selection gene may encode a gene product essential for cell viability and survival.
  • the selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions.
  • Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.
  • Exemplary selection genes of the disclosure may include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT (encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDHl (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.
  • neo conferring resistance to neomycin
  • DHFR encoding Dihydrofolate Reductase and conferring resistance to Methotrexate
  • TYMS encoding Thymidylate Synthetase
  • Nanoparticle vectors of the disclosure may comprise at least one self-cleaving peptide.
  • the nanoparticle vector may comprise at least one self- cleaving peptide and wherein a self-cleaving peptide is located between a CAR and the nanoparticle.
  • the nanoparticle vector may comprise at least one self- cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR.
  • the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self- cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR.
  • the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and a selection gene.
  • the self- cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG- P2A peptide.
  • a T2A peptide may comprise an amino acid sequence comprising
  • EGRGSLLTCGDVEENPGP SEQ ID NO: 18 or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19).
  • a GSG-T2A peptide may comprise a nucleic acid sequence comprising
  • An E2A peptide may comprise an amino acid sequence comprising
  • QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).
  • An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).
  • a GSG- F2A peptide may comprise an amino acid sequence comprising
  • GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising
  • a P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).
  • a GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
  • the disclosure provides a composition comprising a vector of the disclosure.
  • a Centyrin is one example of a protein scaffold of the disclosure.
  • An antigen recognition region of a CAR of the disclosure may comprise at least one protein scaffold.
  • Protein scaffolds of the disclosure may be derived from a fibronectin type III (FN3) repeat protein, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods of making and using them.
  • the protein scaffold is comprised of a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter 'Tenascin").
  • the protein scaffold of the present invention is a consensus sequence of 15 FN3 domains.
  • the protein scaffolds of the disclosure can be designed to bind various molecules, for example, a cellular target protein.
  • the protein scaffolds of the disclosure can be designed to bind an epitope of a wild type and/or variant form of an antigen.
  • Protein scaffolds of the disclosure may include additional molecules or moieties, for example, the Fc region of an antibody, albumin binding domain, or other moiety influencing half-life.
  • the protein scaffolds of the disclosure may be bound to a nucleic acid molecule that may encode the protein scaffold.
  • the disclosure provides at least one method for expressing at least one protein scaffold based on a consensus sequence of multiple FN3 domains, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one protein scaffold is expressed in detectable and/or recoverable amounts.
  • the disclosure provides at least one composition comprising (a) a protein scaffold based on a consensus sequence of multiple FN3 domains and/or encoding nucleic acid as described herein; and (b) a suitable and/or pharmaceutically acceptable carrier or diluent. [0177]
  • the disclosure provides a method of generating libraries of a protein scaffold based on a fibronectin type in (FN3) repeat protein, preferably, a consensus sequence of multiple FN 3 domains and, more preferably, a consensus sequence of multiple FN3 domains from human Tenascin.
  • the library is formed by making successive generations of scaffolds by altering (by mutation) the amino acids or the number of amino acids in the molecules in particular positions in portions of the scaffold, e.g., loop regions.
  • Libraries can be generated by altering the amino acid composition of a single loop or the simultaneous alteration of multiple loops or additional positions of the scaffold molecule. The loops that are altered can be lengthened or shortened accordingly.
  • Such libraries can be generated to include all possible amino acids at each position, or a designed subset of amino acids.
  • the library members can be used for screening by display, such as in vitro or CIS display (DNA, RNA, ribosome display, etc.), yeast, bacterial, and phage display.
  • Protein scaffolds of the disclosure provide enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations; they may be expressed and folded in prokaryotic systems, such as E. coli, in eukaryotic systems, such as yeast, and in in vitro transcription/translation systems, such as the rabbit reticulocyte lysate system.
  • prokaryotic systems such as E. coli
  • eukaryotic systems such as yeast
  • in vitro transcription/translation systems such as the rabbit reticulocyte lysate system.
  • the disclosure provides an isolated, recombinant and/or synthetic protein scaffold based on a consensus sequence of fibronectin type III (FN3) repeat protein, including, without limitation, mammalian-derived scaffold, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding protein scaffold based on the consensus FN3 sequence.
  • the disclosure further includes, but is not limited to, methods of making and using such nucleic acids and protein scaffolds, including diagnostic and therapeutic compositions, methods and devices.
  • the protein scaffolds of the disclosure offer advantages over conventional therapeutics, such as ability to administer locally, orally, or cross the blood-brain barrier, ability to express in E. Coli allowing for increased expression of protein as a function of resources versus mammalian cell expression ability to be engineered into bispecific or tandem molecules that bind to multiple targets or multiple epitopes of the same target, ability to be conjugated to drugs, polymers, and probes, ability to be formulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.
  • the protein scaffolds possess many of the properties of antibodies in relation to their fold that mimics the variable region of an antibody. This orientation enables the FN3 loops to be exposed similar to antibody complementarity determining regions
  • CDRs CDRs
  • CDRs 1-3 complementarity determining regions
  • the remaining three loops are surface exposed in a manner similar to antibody CDRs.
  • These loops span at or about residues 13-16, 22-28, 38-43, 51-54,
  • the loop regions at or about residues 22-28,
  • loop regions 51-54, and 75-81 are altered for binding specificity and affinity.
  • One or more of these loop regions are randomized with other loop regions and/or other strands maintaining their sequence as backbone portions to populate a library and potent binders can be selected from the library having high affinity for a particular protein target.
  • One or more of the loop regions can interact with a target protein similar to an antibody CDR interaction with the protein.
  • Scaffolds of the disclosure may comprise a single chain antibody (e.g. a scFv).
  • Single chain antibodies of the disclosure may comprise three light chain and three heavy chain
  • the single chain antibodies of the disclosure comprise three light chain and three heavy chain CDRs of an antibody, wherein the complementarity-determining regions (CDRs) of the single chain antibody are human sequences.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one single chain antibody (e.g. a scFv); (b) a transmembrane domain, and
  • the antigen recognition region may comprise two single chain antibodies (e.g. two scFvs) to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three single chain antibodies (e.g. three scFvs) to produce atri-specific CAR.
  • the ectodomain may further comprise a signal peptide.
  • the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
  • Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a VHH). Sequence comprising one or more fragments of an antibody of the disclosure may comprise two heavy chain variable regions of an antibody. In certain embodiments, the sequence comprises two heavy chain variable regions of an antibody, wherein the complementarity-determining regions (CDRs) of the VHH are human sequences. Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a VHH). The disclosure provides a chimeric antigen receptor
  • CAR comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one a sequence comprising one or more fragments of an antibody (e.g. a VHH); (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • the antigen recognition region may comprise two sequences comprising one or more fragments of an antibody (e.g. two VHHs) to produce a bi-specific or tandem CAR.
  • the antigen recognition region may comprise three sequences comprising one or more fragments of an antibody (e.g.
  • the ectodomain may further comprise a signal peptide.
  • the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
  • Scaffolds of the disclosure may comprise an antibody mimetic.
  • antibody mimetic is intended to describe an organic compound that specifically binds a target sequence and has a structure distinct from a naturally-occurring antibody.
  • Antibody mimetics may comprise a protein, a nucleic acid, or a small molecule.
  • the target sequence to which an antibody mimetic of the disclosure specifically binds may be an antigen.
  • Antibody mimetics may provide superior properties over antibodies including, but not limited to, superior solubility, tissue penetration, stability towards heat and enzymes
  • antibody mimetics include, but are not limited to, an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, and avimer (also known as avidity multimer), a DARPin (Designed
  • Ankyrin Repeat Protein a Fynomer, a Kunitz domain peptide, and a monobody.
  • Affibody molecules of the disclosure comprise a protein scaffold comprising or consisting of one or more alpha helix without any disulfide bridges.
  • affibody molecules of the disclosure comprise or consist of three alpha helices.
  • an affibody molecule of the disclosure may comprise an immunoglobulin binding domain.
  • An affibody molecule of the disclosure may comprise the Z domain of protein A.
  • Affilin molecules of the disclosure comprise a protein scaffold produced by modification of exposed amino acids of, for example, either gamma-B crystallin or ubiquitin. Affilin molecules functionally mimic an antibody's affinity to antigen, but do not structurally mimic an antibody. In any protein scaffold used to make an affilin, those amino acids mat are accessible to solvent or possible binding partners in a properly-folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.
  • Affimer molecules of the disclosure comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide a high affinity binding site for a specific target sequence.
  • Exemplary affimer molecules of the disclosure comprise a protein scaffold based upon a cystatin protein or tertiary structure thereof.
  • Exemplary affimer molecules of the disclosure may share a common tertiary structure of comprising an alpha- helix lying on top of an anti-parallel beta-sheet.
  • Affitin molecules of the disclosure comprise an artificial protein scaffold, the structure of which may be derived, for example, from a DNA binding protein (e.g. the DNA binding protein Sac7d).
  • Affitins of the disclosure selectively bind a target sequence, which may be the entirety or part of an antigen.
  • Exemplary affitins of the disclosure are manufactured by randomizing one or more amino acid sequences on the binding surface of a DNA binding protein and subjecting the resultant protein to ribosome display and selection.
  • Target sequences of affitins of the disclosure may be found, for example, in the genome or on the surface of a peptide, protein, virus, or bacteria.
  • an affitin molecule may be used as a specific inhibitor of an enzyme.
  • Affitin molecules of the disclosure may include heat-resistant proteins or derivatives thereof.
  • Alphabody molecules of the disclosure may also be referred to as Cell-Penetrating Alphabodies (CPAB).
  • CPAB Cell-Penetrating Alphabodies
  • Alphabody molecules of the disclosure comprise small proteins (typically of less than 10 kDa) that bind to a variety of target sequences (including antigens). Alphabody molecules are capable of reaching and binding to intracellular target sequences.
  • alphabody molecules of the disclosure comprise an artificial sequence forming single chain alpha helix (similar to naturally occurring coiled-coil structures).
  • Alphabody molecules of the disclosure may comprise a protein scaffold comprising one or more amino acids that are modified to specifically bind target proteins. Regardless of the binding specificity of the molecule, alphabody molecules of the disclosure maintain correct folding and thermostability.
  • Anticalin molecules of the disclosure comprise artificial proteins that bind to target sequences or sites in either proteins or small molecules.
  • Anticalin molecules of the disclosure may comprise an artificial protein derived from a human lipocalin.
  • Anticalin molecules of the disclosure may be used in place of, for example, monoclonal antibodies or fragments thereof.
  • Anticalin molecules may demonstrate superior tissue penetration and thermostability than monoclonal antibodies or fragments thereof.
  • Exemplary anticalin molecules of the disclosure may comprise about 180 amino acids, having a mass of approximately 20 kDa.
  • anticalin molecules of the disclosure comprise a barrel structure comprising antiparallel beta-strands pairwise connected by loops and an attached alpha helix.
  • anticalin molecules of the disclosure comprise a barrel structure comprising eight antiparallel beta-strands pairwise connected by loops and an attached alpha helix.
  • Avimer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen).
  • Avimcrs of the disclosure may recognize multiple binding sites within the same target or within distinct targets. When an avimer of the disclosure recognize more than one target, the avimer mimics function of a bi- specific antibody.
  • the artificial protein avimer may comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. Amino acid sequences of one or more of the peptides of the avimer may be derived from an A domain of a membrane receptor.
  • Avimers have a rigid structure that may optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure may demonstrate greater heat stability compared to an antibody.
  • DARPins Designed Ankyrin Repeat Proteins
  • DARPins of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high affinity for a target sequence.
  • DARPins of the disclosure arc derived from ankyrin proteins and, optionally, comprise at least three repeat motifs (also referred to as repetitive structural units) of the ankyrin protein.
  • Ankyrin proteins mediate high-affinity protein-protein interactions.
  • DARPins of the disclosure comprise a large target interaction surface.
  • Fynomers of the disclosure comprise small binding proteins (about 7 kDa) derived from the human Fyn SH3 domain and engineered to bind to target sequences and molecules with equal affinity and equal specificity as an antibody.
  • Kunitz domain peptides of the disclosure comprise a protein scaffold comprising a
  • Kunitz domains comprise an active site for inhibiting protease activity.
  • Kunitz domains of the disclosure comprise a disulfide-rich alpha+beta fold. This structure is exemplified by the bovine pancreatic trypsin inhibitor. Kunitz domain peptides recognize specific protein structures and serve as competitive protease inhibitors. Kunitz domains of the disclosure may comprise Ecallantide (derived from a human lipoprote in- associated coagulation inhibitor (LACI)).
  • LACI human lipoprote in- associated coagulation inhibitor
  • Monobodies of the disclosure are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable in size to a single chain antibody. These genetically engineered proteins specifically bind target sequences including antigens.
  • Monobodies of the disclosure may specifically target one or more distinct proteins or target sequences.
  • monobodies of the disclosure comprise a protein scaffold mimicking the structure of human fibronectin, and more preferably, mimicking the structure of the tenth extracellular type III domain of fibronectin.
  • the tenth extracellular type III domain of fibronectin, as well as a monobody mimetic thereof, contains seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three
  • CDRs complementarity determining regions
  • a monobody In contrast to the structure of the variable domain of an antibody, a monobody lacks any binding site for metal ions as well as a central disulfide bond. Multispecific monobodies may be optimized by modifying the loops BC and FG. Monobodies of the disclosure may comprise an adnectin.
  • At least one scaffold protein of the disclosure can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y.
  • Amino acids from a scaffold protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.
  • scaffold proteins can be engineered with retention of high affinity for the antigen and other favorable biological properties.
  • the scaffold proteins can be optionally prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional 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 sequences and can measure possible immunogenicity (e.g., Immunofiltcr program of Xencor, Inc. of Monrovia, Calif).
  • the methods of the disclosure produce a modified TSCM of the disclosure regardless of the method used for introducing an antigen receptor into a primary human T cell of the disclosure.
  • the methods of the disclosure produce a modified TSCM of the disclosure with greater efficacy and/or a greater abundance, proportion, yield of modified -TSCM of the disclosure when the antigen receptor or the therapeutic protein of the disclosure is introduced to the primary human T cell using the piggyBac transposon system.
  • a piggyBac transposon system of the disclosure may comprise a piggyBac transposon comprising an antigen receptor of the disclosure.
  • the primary human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a transposase of the disclosure simultaneously (or in very close temporal proximity, e.g. the primary human T cell, the transposon and the transposase are contained in the same container (such as a cuvette) prior to introduction of the transposon and transposase into the cell - however they would not be permitted to interact in the absence of the cell.
  • the primary' human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a Super piggyBacTM (SPB) transposase of the disclosure simultaneously prior to introduction of the transposon and transposase into the cell.
  • the Super piggyBacTM (SPB) transposase is an mRNA sequence encoding the Super piggyBacTM (SPB) transposase.
  • the disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell.
  • the introducing steps are mediated by the piggy Bac transposon system.
  • the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements.
  • the transposon is a piggyBac transposon.
  • the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase.
  • the sequence encoding the transposase is an mRNA sequence.
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme.
  • PB piggyBac
  • the piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:
  • PB piggyBacTM
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4.
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4.
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 4.
  • the amino acid substitution at position 30 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I).
  • the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G).
  • the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M).
  • the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N).
  • the transposase enzyme is a Super piggyBacTM (SPB) transposase enzyme.
  • the Super piggyBacTM (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 4 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N).
  • the Super piggyBacTM (SPB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%,
  • the piggyBacTM or Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119,
  • transposase comprises the above-described mutations at positions 30, 165, 282 and/or
  • the piggyBacTM or Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200,
  • amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for a serine (S).
  • amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A).
  • amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (I).
  • amino acid substitution at position 103 of SEQ ID NO: 5 is a substitution of a serine (S) for an alanine (A).
  • amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A).
  • amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (I).
  • amino acid substitution at position 103 of SEQ ID NO: 5 is a substitution of a serine (S) for an alan
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S).
  • the amino acid substitution at position 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (?) for an arginine (R).
  • the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C).
  • L leucine
  • C cysteine
  • the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for a tyrosine (Y).
  • the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a phenylalanine (F).
  • amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 4 is amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5
  • the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A).
  • the amino acid substitution at position 200 of SEQ ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F).
  • the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A).
  • the amino acid substitution at position 200 of SEQ ID NO: 5 is a substitution of a valine (V)
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenylalanine (F).
  • W tryptophan
  • F phenylalanine
  • the amino acid substitution at position 209 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a valine (V).
  • the amino acid substitution at position 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a methionine (M).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L).
  • the amino acid substitution at position 240 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V).
  • the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine
  • SEQ ID NO: 4 is a substitution of a lysine (K) for a proline (P).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for an asparagine (N).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a leucine (L).
  • the amino acid substitution at position 296 of SEQ ID NO: 5 is a substitution of a serine (S) for an asparagine (N).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L).
  • the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a
  • amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L).
  • amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a methionine (M).
  • amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a proline (P).
  • the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine for a proline (P).
  • the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T).
  • G glycine
  • T threonine
  • amino acid substitution at position 327 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T).
  • the amino acid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a tyrosine (Y).
  • the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C).
  • the amino acid substitution at position 340 of SEQ ID NO: 5 is a substitution of an arginine (R) for a tyrosine (Y).
  • the amino acid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a tyrosine (Y).
  • the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C).
  • the amino acid substitution at position 340 of SEQ ID NO: 5 is a substitution of an arginine
  • amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D).
  • amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a valine (V).
  • amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine (M).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L).
  • F phenylalanine
  • L leucine
  • the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a methionine (M).
  • the amino acid substitution at position 552 of SEQ ID NO: 5 is a substitution of a lysine (K) for a serine (S).
  • the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M).
  • the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a methionine (M).
  • SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V).
  • the amino acid substitution at position 570 of SEQ ⁇ ) NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A).
  • the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline
  • the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a glutamine (Q).
  • the piggyBacTM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or
  • the piggyBacTM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the transposase comprises the above-described mutations at positions
  • the piggyBacTM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at positions
  • the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M).
  • the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R).
  • the amino acid substitution at position 375 of SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S).
  • the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M).
  • the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R).
  • the amino acid substitution at position 375 of SEQ ID NO: 5 is a substitution of a proline (P) for a serine (
  • amino acid substitution at position 450 of SEQ YD NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for an aspartic acid (D).
  • amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S).
  • amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for an asparagine (N).
  • the piggy BacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4.
  • the piggyBacTM transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, and a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 4.
  • the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 4 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 4.
  • introducing is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell.
  • the methods of the invention do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host.
  • Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • endogenous refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.
  • stable transformation is intended that the polynucleotide construct introduced into a plant integrates into the genome of the host and is capable of being inherited by progeny thereof.
  • transient transformation is intended that a polynucleotide construct introduced into the host does not integrate into the genome of the host.
  • the piggyBac transposon system is used to introduce exogenous sequences into a primary human T cell by stable transformation to generate a modified TscMor TCM.
  • the transposon is a Sleeping Beauty transposon.
  • the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
  • the disclosure provides a method of producing a modified stem memory T-cell (TSCM) or a modified central memory T-cell (TCM) , comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface markers) of a modified stem memory T-cell (TSCM) or a modified central memory T-cell (TCM), thereby producing a modified stem memory T- cell (TSCM) or a modified central memory T-cell (TCM).
  • TSCM modified stem memory T-cell
  • TCM central memory T-cell
  • the disclosure provides a method of producing a plurality of modified stem memory T-cells (TSCM) or a plurality of modified central memory T-cells (TCM), comprising introducing into a plurality of primary human T cells (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell- surface markers) of a stem memory T-cell (TSCM) or a central memory T-cell (TCM), thereby producing a plurality of modified stem memory T-cells (TSCM) or a plurality of modified central memory T-
  • the transposon is a Sleeping
  • the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
  • the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%,
  • n certan em o ments o t e met o s o t e scosure, t e yperactve eepng Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:
  • the transposase is a Helitron transposase.
  • Helitron transposases mobilize the Helraisertransposon, an ancient element from the bat genome that was active about 30 to 36 million years ago.
  • Helraiser transposon of the disclosure includes Helibatl, which comprises a nucleic acid sequence comprising:
  • the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a RepHel motif made up of a replication initiator domain (Rep) and a DNA helicase domain.
  • the Rep domain is a nuclease domain of the HUH superfamily of nucleases.
  • An exemplar ⁇ ' Helitron transposase of the disclosure comprises an amino acid sequence comprising:
  • a hairpin close to the 3' end of the transposon functions as a terminator.
  • this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences.
  • Helraiser transposition generates covalently closed circular intermediates.
  • Helitron transpositions can lack target site duplications.
  • the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5'-TC/CTAG- 3' motif.
  • a 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence
  • the transposase is a Tol2 transposase.
  • Tol2 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family.
  • Exemplary Tol2 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the Tol2 transposase, which contains four exons.
  • An exemplary Tol2 transposase of the disclosure comprises an amino acid sequence comprising the following:
  • An exemplary Tol2 transposon of the disclosure including inverted repeats, subterminal sequences and the Tol2 transposase, is encoded by a nucleic acid sequence comprising the following:
  • a modified CAR-TSCM or CAR-TCM of the disclosure is produced by introducing an antigen receptor into a primary human T cell of the disclosure by homologous recombination.
  • the homologous recombination is induced by a single or double strand break induced by a genomic editing composition or construct of the disclosure.
  • Homologous recombination methods of the disclosure comprise contacting a genomic editing composition or construct of the disclosure to a genomic sequence to induce at least one break in the sequence and to provide an entry point in the genomic sequence for an exogenous donor sequence composition.
  • Donor sequence compositions of the disclosure are integrated into the genomic sequence at the induced entry point by the cell's native DNA repair machinery.
  • homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a "genomic safe harbor" site.
  • a mammalian genomic sequence comprises the genomic safe harbor site.
  • a primate genomic sequence comprises the genomic safe harbor site.
  • a human genomic sequence comprises the genomic safe harbor site.
  • Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism.
  • Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ottholog of the mouse Rosa26 locus.
  • AAVS1 adeno-associated virus site 1
  • CCR5 chemokine receptor 5
  • homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a sequence encoding one or more components of an endogenous T-cell receptor or a major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MHC disrupts the endogenous gene, and optionally, replaces part of the coding sequence of the endogenous gene with a donor sequence composition of the disclosure.
  • inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MHC disrupts the endogenous gene, and optionally, replaces the entire coding sequence of the endogenous gene with a donor sequence composition of the disclosure.
  • introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor to an endogenous T cell promoter.
  • introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional or translational regulatory element.
  • introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a
  • the transcriptional regulatory element comprises an endogenous T cell 5' UTR.
  • a genomic editing composition contacts a genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of a portion of primary T cells of the plurality of T cells. In certain embodiments, the portion of primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%,
  • a genomic editing composition contacts a genomic sequence of each primary T cell of the plurality of T cells.
  • a genomic editing composition induces a single strand break.
  • a genomic editing composition induces a double strand break.
  • the introduction step further comprises a donor sequence composition.
  • the donor sequence composition comprises a sequence encoding the antigen receptor.
  • the donor sequence composition comprises a sequence encoding the antigen receptor, a 5' genomic sequence and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 5' to the break point induced by the genomic editing composition and the 3' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3' to the break point induced by the genomic editing composition.
  • the 5" genomic sequence and/or the 3' genomic sequence comprises at least 50 bp, 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 1100 bp, at least 1200 bp, at least 1300 bp, at least
  • the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially.
  • the genomic editing composition and donor sequence composition are contacted with the genomic sequence sequentially, and the genomic editing composition is provided first.
  • the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA binding domain and a nuclease domain. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a TALEN. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genomic editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof.
  • the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic
  • the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32, comprising a substitution of an
  • the nuclease domain comprises or further comprises a type IIS endonuclease.
  • the type IIS endonuclease comprises Acil, Mnll, Alwl, Bbvl, Bccl, BceAI, BsmAI, BsmFI, BspCNI,
  • the type US endonuclease comprises
  • the nuclease domain comprises or further comprises a TALEN or a nuclease domain thereof. In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a ZFN or a nuclease domain thereof.
  • the introduction step comprising a homologous recombination the genomic editing composition induces a break in a genomic sequence and the donor sequence composition is inserted using the endogenous DNA repair mechanisms of the primary T cell. In certain embodiments of the introduction step comprising a homologous recombination, the insertion of the donor sequence composition eliminates a DNA binding site of the genomic editing composition, thereby preventing further activity of the genomic editing composition.
  • the nuclease domain of a genomic editing composition or construct is capable of introducing a break at a defined location in a genomic sequence of the primary human T cell, and, furthermore, may comprise, consist essentially of or consist of, a homodimcr or a heterodimer.
  • the nuclease is an endonuclease. Effector molecules, including those effector molecules comprising a homodimer or a heterodimer, may comprise, consist essentially of or consist of, a Cas9, a Cas9 nuclease domain or a fragment thereof.
  • the Cas9 is a catalytically inactive or "inactivated” Cas9 (dCas9).
  • the Cas9 is a catalytically inactive or ''inactivated” nuclease domain of Cas9.
  • the dCas9 is encoded by a shorter sequence that is derived from a full length, catalytically inactivated, Cas9, referred to herein as a "small" dCas9 or dSaCas9.
  • the inactivated, small, Cas9 operatively-linked to an active nuclease.
  • the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA binding domain and molecule nuclease, wherein the nuclease comprises a small, inactivated Cas9 (dSaCas9).
  • the dSaCas9 of the disclosure comprises the mutations D10A and N580A (underlined and bolded) which inactivate the catalytic site.
  • the dSaCas9 of the disclosure comprises the amino acid sequence of:
  • the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphyloccocus pyogenes.
  • the dCas9 comprises a dCas9 with substitutions at positions 10 and 840 of the amino acid sequence of the dCas9 which inactivate the catalytic site. In certain embodiments, these substitutions are D10A and H840A.
  • the amino acid sequence of the dCas9 comprises the sequence of:
  • the nuclease domain may comprise, consist essentially of or consist of a dCas9 or a dSaCas9 and a type IIS endonuclease. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and a type IIS endonuclease, including, but not limited to, Acil,
  • nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and Clo05 l.
  • exemplary CloOS 1 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:
  • An exemplary dCas9-Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of (Clo051 sequence underlined, linker bold italics, dCas9 sequence in italics):
  • the nuclease capable of introducing a break at a defined location in the genomic DNA of the primary human T cell may comprise, consist essentially of or consist of, a homodimer or a heterodimer.
  • Nuclease domains of the genomic editing compositions or constructs of the disclosure may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a transcription-activator-like effector nuclease (TALEN).
  • TALENs are transcription factors with programmable DNA binding domains that provide a means to create designer proteins that bind to pre-determined DNA sequences or individual nucleic acids.
  • Modular DNA binding domains have been identified in transcriptional activator-like (TAL) proteins, or, more specifically, transcriptional activator-like effector nucleases (TALENs), thereby allowing for the de novo creation of synthetic transcription factors that bind to DNA sequences of interest and, if desirable, also allowing a second domain present on the protein or polypeptide to perform an activity related to DNA.
  • TAL proteins have been derived from the organisms Xanthomonas and Ralstonia.
  • the nuclease domain of the genomic editing composition or construct may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a TALEN and a type IIS endonuclease.
  • the type IIS endonuclease may comprise, consist essentially of or consist of Acil, Mnll, Alwl, Bbvl, Bccl, BceAI, BsmAI, BsmFI, BspCNI,
  • the type IIS endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID NO: 34).
  • the nuclease domain of the genomic editing composition or construct may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a zinc finger nuclease (ZFN) and a type IIS endonuclease.
  • ZFN zinc finger nuclease
  • the type IIS endonuclease may comprise, consist essentially of or consist of Acil, Mnll, Alwl, Bbvl, Bccl, BceAI, BsmAI,
  • the type IIS endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID NO: 34).
  • the DNA binding domain and the nuclease domain may be covalently linked.
  • a fusion protein may comprise the DNA binding domain and the nuclease domain.
  • the DNA binding domain and the nuclease domain may be operably linked through a non- covalent linkage.
  • modified T-cclls express therapeutic proteins.
  • Therapeutic proteins of the disclosure include secreted proteins.
  • the therapeutic protein is a human protein, including a secreted human protein.
  • the combination comprising the CAR-T cell and the therapeutic protein secreted therefrom may be considered a monotherapy.
  • the CAR-T cells of the disclosure may be administered as a combination therapy with a second agent.
  • a database of human secreted proteins that may be expressed or secreted by modified T-cell of the disclosure can be found at proteinatlas.Org/search/protein_class:Predicted%20secreted%20p the contents of which are incorporated herein by reference.
  • Exemplary human secreted proteins are provided, but are not limited to the human secreted proteins, in Table 1.
  • T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins.
  • compositions comprising CAR-T cells modified to express or to secrete a human protein are used to treat a clotting disorder. Blood clotting occurs through a multistep process known as the coagulation cascade. In the extrinsic pathway, Tissue Factor (also known as factor ⁇ or thromboplastin) comes into contact with factor VII to form an activated Vila complex.
  • Tissue Factor also known as factor ⁇ or thromboplastin
  • Factor XII into Factor Xlla.
  • Factor Xlla converts Factor XI into Factor XIa, and Factor XIa activates Factor IX to produce Factor IXa, which, together with FVIIIa form the tenase complex, which activates Factor X, which helps convert Prothrombin (II) into Thrombin
  • Fibrinogen (I) Thrombin in turn leads to the conversion of Fibrinogen (I) into Fibrin, which together with Factor Xllla forms a cross-linked fibrin clot.
  • Many clotting disorders are the result of low levels of secreted proteins in the blood that are involved in the coagulation cascade.
  • Clotting disorders can drastically increase the amount of blood leaving the body upon injury, or cause bleeding to occur under the skin or in vital organs. These disorders are frequently genetic. Exemplary, but non-limiting diseases caused by deficiencies in clotting factors include Hemophilias, von Willebrand disease and deficiencies in Antithrombin ⁇ , protein C or protein S. Hemophila A and B are X-linked, and are caused by insufficient levels of clotting factor VIII and factor IX (FIX) respectively. Hemophila C is caused by insufficient factor XI. Factor II, VII, X or XII deficiencies can also cause bleeding disorders. Von
  • Willebrand disease is due to a low level of the von Willebrand clotting factor in the blood.
  • deficiencies in blood proteins that regulate clotting lead can lead to too much clotting.
  • Factor V Leiden is a genetic disorder, where the factor V Leiden protein overreacts, causing the blood to clot too often or too much.
  • Deficiencies in Antithrombin ⁇ , protein C or protein S which help regulate bleeding, can also cause excessive clotting.
  • clotting disorders such as Hemophilia are treated with blood transfusions or infusions of the missing clotting factor (replacement therapy).
  • replacement therapy complications of replacement therapy include developing antibodies to the clotting factor, contracting viral infections from blood derived products and damage to joints. There thus exists a need for additional therapies.
  • T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins.
  • compositions comprising CAR-T cells modified to express or to secrete a human protein are used for enzyme replacement therapy.
  • Enzyme replacement therapy typically involves intravenous infusions of therapeutically effective amounts of compositions comprising enzymes that balance underlying enzyme deficiencies that cause the symptoms of the disease. The missing enzyme activity is thus supplied exogenously in this manner.
  • Exemplary diseases that can be treated by modified T cells of the disclosure include, but are not limited to, lysosomal storage diseases Gaucher' s disease (glucocerebrosidase enzyme), Fabry disease, mucopolysaccharidosis I (MPS I), mucopolysaccharidosis I (MPS ⁇ , or Hunter syndrome, caused by iduronate-2-sulfatase deficiency), mucopolysaccharidosis VI (MPS VI, caused by arylsulfatase B deficiency) and Pompe disease (or glycogen stoarage disease type II, caused by a deficiency in acid alpha-glucosidase).
  • Gaucher' s disease glucocerebrosidase enzyme
  • Fabry disease mucopolysaccharidosis I
  • MPS ⁇ mucopolysaccharidosis I
  • MPS ⁇ or Hunter syndrome, caused by iduronate-2-sulfatase defici
  • Additional diseases treatable with enzyme replacement therapy include but are not limited to Adenosine deaminase (ADA) deficiency, Hyperammonemia due to the deficiency of the hepatic enzyme N-acetylglutamate synthetase (NAGS), Hypophosphatasia, Lysosomal acid lipase deficiency, Morquio Syndrome A, Wolman LAL Lysosomal Acid Lipase deficiency, A1AT (Alpha 1- Antitrypsin) deficiency and Urea cycle disorder.
  • ADA Adenosine deaminase
  • NAGS N-acetylglutamate synthetase
  • Hypophosphatasia Lysosomal acid lipase deficiency
  • Morquio Syndrome A Wolman LAL Lysosomal Acid Lipase deficiency
  • A1AT Alpha 1- Antitrypsin
  • Enzymes supplied to patients during enzyme replacement therapy include, but are not limited to Alphal -Antitrypsin, ⁇ - Glucocerebrosidase, Adenosine Deaminase, Alpha-Galactosidase A, a-L-Iduromdase, Iduronate-2-Sulfatase, N-Acetylgalactosamine-6 Sulfatase, -Acetylgalactosamine-4 Sulfatase and Lysosomal Acid Lipase.
  • T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins.
  • compositions comprising CAR-T cells modified to express or to secrete a human protein are used to produce human antibodies.
  • the disease to be treated by modified T cells expressing secreted proteins is a disease that can be treated through the intravenous infusion or injection of an antibody or an antibody fragment.

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EP17794119.2A 2016-09-30 2017-10-02 Gedächtnis-t-zellen mit modifizierten stammzellen, verfahren zur herstellung und verfahren zur verwendung davon Pending EP3519561A1 (de)

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WO2018064681A1 (en) 2018-04-05
JP2021191312A (ja) 2021-12-16
AU2017337147A1 (en) 2019-03-21
CA3036926A1 (en) 2018-04-05
IL265157A (en) 2019-05-30
KR20200108927A (ko) 2020-09-21
IL276907A (en) 2020-10-29
CA3105873A1 (en) 2018-04-05
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IL276907B (en) 2021-12-01
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