CA3036926A1 - Modified stem cell memory t cells, methods of making and methods of using same - Google Patents

Modified stem cell memory t cells, methods of making and methods of using same Download PDF

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CA3036926A1
CA3036926A1 CA3036926A CA3036926A CA3036926A1 CA 3036926 A1 CA3036926 A1 CA 3036926A1 CA 3036926 A CA3036926 A CA 3036926A CA 3036926 A CA3036926 A CA 3036926A CA 3036926 A1 CA3036926 A1 CA 3036926A1
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cell
modified
cells
composition
transposon
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CA3036926C (en
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Eric Ostertag
Devon SHEDLOCK
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Poseida Therapeutics Inc
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Poseida Therapeutics Inc
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Abstract

The disclosure provides a method of producing modified stem memory T cells (e.g. CAR-T cells) for administration to a subject as, for example an adoptive cell therapy.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

MODIFIED STEM CELL MEMORY T CELLS, METHODS OF MAKING AND
METHODS OF USING SAME
RELATED APPLICATIONS
[01] This application claims the benefit of provisional applications USSN
62/402,707 filed September 30, 2016, USSN 62/502,508 filed May 5, 2017, USSN 62/553,058 filed August 31, 2017 and USSN 62/556,309 filed September 8, 2017, the contents of each of which are herein incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[02] The contents of the text filed named "POTH-012_001WO_SeqList.txt", which was created on 2 October 2017 and is 110 KB in size, are hereby incorporated by reference in their entirety.
FIELD OF THE DISCLOSURE
[03] The disclosure is directed to molecular biology, and more, specifically, to methods of making and using modified stem-cell memory T cells.
BACKGROUND
[04] There has been a long-felt but unmet need in the art for a method of producing modified stem-cell memory T cells for administration to a subject as, for example, an adoptive cell therapy. The disclosure provides a solution to this long-felt but unmet need.
SUMMARY
[05] Unlike traditional biologics and chemotherapeutics, modified-T cells of the disclosure possess the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, 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. Thus, 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).
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 (TErm) and effector T cells (TB), 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). 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).
[06] Modified cells of the disclosure may be further subjected to genomic editing. For example, 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 nucleofection and allowed to integrate into the genome of the cell during the following incubation phase. The resultant cell 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. Alternatively, or in addition, 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.
[071 Specifically, 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 marker(s) 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 (Tscb,), 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%, 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 marker(s) 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 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). 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 marker(s) 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 600/0 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). 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). In certain embodiments, 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 marker(s) 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscb,). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, 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-2120, CR7, and CD62L. In certain embodiments of this method, 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 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.
[081 In certain embodiments of the methods of the disclosure, 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 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, and, in particular, those embodiments wherein the transposase is a Super piggyBac"' (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.
P91 In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBacTM (PB) transposase enzyme. 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:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
4).
[010] In certain embodiments of the methods of the disclosure, 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:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
4).
[0111 In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, 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).
[012] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBacTm (SPB) transposase enzyme. In certain embodiments, 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). In certain embodiments, the Super piggyBacTM (SPB) 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:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
5).
[013] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ
ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the 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). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO:
4 or SEQ ID
NO: 5 is a substitution of a serine (5) for an alanine (A). In certain embodiments, the 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). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tiyptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, 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
ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 180 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a phenylalanine (F).
In certain embodiments, the amino acid substitution at position 180 of SEQ ID
NO: 4 or SEQ
ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 187 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenylalanine (F),In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a valine (V). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 226 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) fora leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID
- 7 -NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ TD 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 TD 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
ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for an arginine (R),In certain embodiments, the amino acid substitution at position 319 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 340 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 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the 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). In certain embodiments, the 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). In certain embodiments, the amino acid substitution at position 470 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a lysine (K) for a serine (5). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 552 of SEQ
- 8 -ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V).
In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, 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).
[014) In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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 SEQ ID NO: 5. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID
NO: 5. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 375 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the 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). In certain embodiments, the 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). In certain embodiments, the 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
- 9 -asparagine (N). In certain embodiments, the piggyBacTm transposase enzyme may comprise a substitution of a valine (V) fora methionine (M) at position 194 of SEQ ID NO:
4. In certain embodiments, including those embodiments wherein 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.
In certain embodiments, 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. In certain embodiments, 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.
10151 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 marker(s) of a stem memory T cell (Tscm), thereby producing a modified stem memory T cell (TKO. 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 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%,
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 marker(s) 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 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). 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 marker(s) 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 60% 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 (TKO. 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 (Tscts,t). In certain embodiments, 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 marker(s) 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 marker(s) 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 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsai), 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 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). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, 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-2RD, CR7, and CD62L. 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).
[016] In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:
- 11 -301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 6).
[0171 In certain embodiments of the methods of the disclosure, 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:

301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7).
[018] 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 marker(s) 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 stein 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 marker(s) 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 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). 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified
- 12 -stem memory T cells (Tscm). In certain embodiments, 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 (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 marker(s) 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 80% 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).
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 marker(s) 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 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). In certain embodiments, 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). In certain embodiments, the cell-surface markers comprise CD62L
and CD45RA. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RD. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2110, CR7, and CD62L. In certain embodiments of this method, 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.
[019) In certain embodiments of the methods of the disclosure, 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:
1 TCCTATATAA TAAAAGAGAA ACATGC.AAA.T TGACCATCCC TCCGCTACGC TCAA.GCCACG
- 13 -421 GTCCCACCCC CAC.AGAATCA GCCAGAA.TCA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG

1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGAC.AGTAk AAATTTCATG

1321 CTCATGATCA ACATCAACAA CCTCATGCAT GAAATAAATG AATTAACAAA. ATCGTACAAG
1381 ATGCTACATG AGGTAGAAAA. GGAAGCCCAA TCTGAAGCAG CAGCAAAAGG TATTGCTCCC

1681 GGTGAAAAAG GCTGGGGAAC AGATATTGCA TTAAGACTC.A GAGAC.AACAG TGTAATCGA.0 1981 AATGAC,AkTG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA GGGTAGTCCC

2281 CATGTCATTG AATTTCAGAA ACGCGGA.CTG CCTCACGCTC ACATATTATT GATATTAGA.T
- 14 -2461 TGTGGAATAC AAAATCCAAA. TAGTCCATGT ATGGAAAATG GAAAA.TGTTC AAAGGGATAT
2521 CCAAAAGAAT TTCAAAATGC GACCATTGGA AATATTGATG GATATCCCAA. ATACAAACGA

2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA. TGCGAA.TGCA TGACCAATCT

3421 CATTTTATTG AAGA.TTTCTG TTGGAAA.TTA CACCGAAGAG AAGGTGCCTG TGTGAACTGT

4081 CAATGTCTCA GTATTGTACC ACATGCTATG CGA.TCGGCC.A TAGTACAAAC GAGTTTAAA.G

4381 ATTCTTTGTC CAA.kAAATGA GCATGTTCAA AAATTAAATG AAGAAATTTT GGATATACTT
4441 GATGGAGATT TTCACACATA TTTGAGTGAT GATTCCATTG ATTCAACAGA. TGATGCTGAA

4561 AAATTAA.AAT TGAAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA TAGTAAATGG

4681 GAAGTATTAA CAGGATCTGC AGAGGGA.GAG GTTGTTCTGA. TTCCAA.GAAT TGATTTGTCC
- 15 -4921 TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCACAAG GGAAA.TTAGT CAAGCACTCT

5281 TGCACCGGGC CACTAG (SEQ ID NO: 27).
10201 Unlike other transposases, 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.
10211 An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:
- 16 -1441 LPEPVTAHGQ LYVAFSRVRR ACDVKVKVVN TSSQGKLVEH SESVFTLNVV YREILE (SMID
NO: 28).
[022) In Helitron transpositions, a hairpin close to the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, 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 GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 29).
[023) 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 marker(s) 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 (TKO, 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 marker(s) 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 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). 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 marker(s) 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 60% of the plurality of modified T cells expresses one or
- 17 -more cell-surface marker(s) 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 marker(s) 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 80% 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).
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 marker(s) 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 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). In certain embodiments, 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). In certain embodiments, the cell-surface markers comprise CD62L
and CD45RA. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CDI27, CD45RO, CD95, CD95 and IL-2143. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2110, CR7, and CD62L. In certain embodiments of this method, the transposon is a To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
[024] In certain embodiments of the methods of the disclosure, the transposase is a To12 transposase. To12 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family. Exemplary To12 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the To12 transposase, which contains four exons. An exemplary To12 transposase of the disclosure comprises an amino acid sequence comprising the following:
- 18 -601 NTPLRASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SE:QM/if/30).
10251 An exemplary To12 transposon of the disclosure, including inverted repeats, subtenuinal sequences and the To12 transposase, is encoded by a nucleic acid sequence comprising the following:

301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA. GTGACGTCAT GTCACATCTA

841 CCACGGGAAA. TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT

1441 GGTTAGTTCA CCC.AAAAATG AAAATAA.TGT CATTAATGAC TCGCCCTCAT GTCGTTCC.AA
- 19 -1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA. TGCTGAATAA

2641 TTTTGAGGTA CTGGCCAGTG CCATGAA.TGA TATCCACTC.A GAGTATGAAA TACGTGAC.AA

2821 TGAAGGCTGT GGTGAGGGAA GTGATGGTGT GGAATTCCAA. GATGCCTCAC GAGTCCTGGA

2941 TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAAA.CT

3301 CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA. GTGGGCCAAC ACAA.TGCGTC

3601 ATGATGA.kAC CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC

3901 GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAA.TT TGTTCCCAGT
-20 -4441 AACCTTGTAT GCATTTCATT TAATGTTTTT TGA.GATTAAA. AGCTTAAACA AGAATCTCTA

4681 TG (SEQ ID NO: 31).
10261 The disclosure provides a method of producing a modified central memory T-cell (Tem), 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 marker(s) of a central memory T-cell (Tem), thereby producing a modified central memory T-cell (TcbA). The disclosure provides a method of producing a plurality of modified central memory T-cells (Tem), 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 marker(s) of a 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 25% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tcm), thereby producing a plurality of modified central memory T-cells (Tem). 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 marker(s) 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 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tem), thereby producing a plurality of
- 21 -modified central memoiy 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 marker(s) 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 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) 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 marker(s) 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 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) 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 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tem), thereby producing a plurality of modified central memory T-cells (Tcm). In certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, CCR7, and CD62L. In certain embodiments of this method, 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 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 piggyBac' m (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). In certain embodiments of this method, 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 To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
- 22 -10271 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 (Tem), 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 Tsem and a plurality of modified Tcm, wherein the plurality of modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-21(13 and the plurality of modified Tem expresses one or more CD45RO, CD95, IL-2113, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified Tscm and a plurality of modified Tcm. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified central memory T-cells (Tcm) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (TKO comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (Tom) comprise at least 90% 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 90% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 10% 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 20% of the total number of cells of the composition and the modified central memory T-cells (Tem) 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 (Tsem) 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 (Tem) comprise at least 70% of the total number of cells of the composition. In certain embodiments of this method. the modified stem memory T-cells
- 23 -(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 munber of cells of the composition and the modified central memory T-cells (Tem) 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 memoiy 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 (Tem) comprise at least 50% of the total number of cells of the composition. In certain embodiments of this method, 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 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 piggyBac' m (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). In certain embodiments of this method, 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 To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
10281 In certain embodiments of the methods of the disclosure, the transposon may be derived or recombined from any species. Alternatively, or in addition, the transposon may be synthetic.
[029] In certain embodiments of the methods of the disclosure, the antigen receptor is a T-ull receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein 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
-24 -certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, 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.
[030J In certain embodiments of the methods of the disclosure, including those wherein the method comprises 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.
In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the transposase composition of (b) transposes the transposon of (a) and the transposon of (c). In certain embodiments, 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. In certain embodiments, the second transposase composition transposes the transposon of (c). In certain embodiments, the transposase composition of (b) transposes the transposon of (a) and the transposase composition of (d) transposes the transposon of (c). In certain embodiments of this method, 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. In certain embodiments, 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). In certain embodiments of this method, 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
- 25 -is a To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
[031] 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 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 marker(s) of a stem memory T
cell (Taw), thereby producing a modified stein memory T cell (Tscm). 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%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between 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). In certain embodiments, 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 marker(s) 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 50% 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). 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T
cells (Tscm). In
-26 -certain embodiments, 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 marker(s) 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 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). In certain embodiments, 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 marker(s) 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 90% 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). In certain embodiments, 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 marker(s) of a stem memory T cell (Tsci4), thereby producing a plurality of activated modified stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated modified Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-21113. In certain embodiments, the cell-surface markers of the activated modified Tsc.m comprise one or more of CD45RA, CD95, IL-2R. CR7, and CD62L.
[032] In certain embodiments of the methods of the disclosure of producing a modified stem memory T cell (Tscm), 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. In certain embodiments, this method further comprises the step of (c) contacting the activated modified T-cell and a T-cell expansion
- 27 -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 marker(s) of a stem memory T
cell (Tscm). In certain embodiments of this method, 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 stem memory T cell (Tscm). In certain embodiments of this method, at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, 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 marker(s) of a stem memory T cell (Tscm). In certain embodiments, 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 marker(s) of a stem memory T cell (Tsci4). In certain embodiments of this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tscm) from the plurality of enriched modified T-cells. In certain embodiments of this method, 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. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-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. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, 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
- 28 -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. In certain embodiments of this method, 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. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 gmol/kg and 640 gmol/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.7 gmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 gmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 gmol/kg and 25 gmol/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 pmol/kg, palmitic acid at a concentration of about 7 gmol/kg, linoleic acid at a concentration of about 7.5 gmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 gmol/kg.
[033] The disclosure provides a method of producing a modified central memmy 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 marker(s) of a central memory T-cell (Tcm), thereby producing a central memory T-cell (Tcm). 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
-29 -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%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated modified T cells expresses one or more cell-surface marker(s) 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 25% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tem), thereby producing a plurality of activated modified central memory T cell (Tem). In certain embodiments, 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 marker(s) of a central memory T cell (Tem), thereby producing a plurality of activated modified central memory T cell (Tem). 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 marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T
cell (Tem). In certain embodiments, 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 marker(s) of a central memory T cell (Tem), 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 marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T
cell (Tem). In certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 85 /0 of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tem), 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 90% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T
cell (Tem). In certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 95 /0 of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tem), thereby producing a plurality of activated
-30-modified central memoiy T cell (Tcm). In certain embodiments, the cell-surface markers of the activated modified Tcm comprise one or more of CD45RO, CD95, IL-2R13, CCR7, and CD621,.
10341 In certain embodiments of the methods of the disclosure 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, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, 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 marker(s) of a central memory T cell (Tcm). In certain embodiments of this method, 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). In certain embodiments of this method, at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (Tcm). In certain embodiments, 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 marker(s) of a central memory T cell (Tcm). In certain embodiments, 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 marker(s) of a central memory T cell (Tcm). In certain embodiments of this method, the enriching step 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.
In certain embodiments of this method, the enriching step further comprises contacting the
- 31 -isolated modified Tem 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 Tem. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-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. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, 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. In certain embodiments of this method, 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. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 punol/kg and 640 pmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 mmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 Limol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 Amol/kg and 75 ptmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 punol/kg and 25 punol/kg, inclusive of the endpoints.
In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 tunol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 Ltmol/kg, oleic acid at a concentration of about 7.5 innol/kg and a sterol at a concentration of about 2.5 gmol/kg.
[035] 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
- 32 -(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 (Tem), wherein the plurality of activated modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2120 and the plurality of activated modified Tcm expresses one or more CD45RO, CD95, IL-2R13, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified Tscm and a plurality of modified Tem. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified central memory T-cells (Tcm) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%

or any percentage of cells in between 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 10% of the total number of cells of the composition and the modified central memory T-eens (Tem) comprise at least 90% of the total number of cells of the composition. In certain embodiments of this method, the modified stein memory T-cells (TKO comprise at least 90% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 10% 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 20% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 80% of the total number of cells of the composition.
In certain embodiments of this method, the modified stein memory T-cells (Tsai) comprise at least 80% of the total number of cells of the composition and the modified central memory T-cells (Tem) 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
-33-(Tcm) comprise at least 70% 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 70% of the total number of cells of the composition and the modified central memory T-cells (Teri) 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 (Tem) comprise at least 60% of the total number of cells of the composition.
In certain embodiments of this method, the modified stein memory T-cells (Tsem) comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (Tem) 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 (Tem) comprise at least 50% of the total number of cells of the composition.
[0361 In certain embodiments of methods of the disclosure of producing a composition comprising a plurality of modified stem memory T-cells (Taw) and a plurality of modified central memory T-cells (Tem), 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 (Tsem) and a plurality of activated modified central memory T-cells (Tem), the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, 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 (Tem) 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 IL-2R( and the plurality of expanded modified Tcm expresses one or more CD45RO, CD95, CCR7, and CD62L, thereby producing a composition
-34 -comprising a plurality of expanded modified Tsem and a plurality of expanded modified Tem.
In certain embodiments of this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tsem) from the plurality of enriched modified T-cells or isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tem) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tsem) from the plurality of enriched modified T-cells and isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tem) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step further comprises contacting the composition comprising the isolated modified TSCM
and the isolated modified Tat 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 Tsem and a plurality of expanded enriched modified Tem. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicaiboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, 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. In certain embodiments of this method, 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. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.41.tmol/kg
-35-and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 gmol/kg and 25 gmol/kg, inclusive of the endpoints.
In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 gmol/kg, oleic acid at a concentration of about 7.5 gmol/kg and a sterol at a concentration of about 2.5 gmol/kg. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified central memory T-cells (Tcm) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between 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 10% of the total munber 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. In certain embodiments of this method, the modified stem 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. In certain embodiments of this method, the modified stem 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. 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. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 70% of the total munber of cells of the composition and the
-36-modified central memmy T-cells (Tem) 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 (Tsem) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (Tem) 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 (TKO comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (Tem) 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 (Tsem) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 50% of the total number of cells of the composition.
[037] In certain embodiments of the methods of producing an activated modified Tsem or Tem 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.
In certain embodiments of the introduction step comprising a homologous recombination, 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%, 71)/0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of the total number of primary T cells in 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 each primary T cell of the plurality of T
cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a single strand break. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a double strand break. In certain embodiments of the introduction step comprising a homologous recombination, the introduction step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments, 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. 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 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. 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.
In certain embodiments of the genomic editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic Acid (D) at position 10 (DIOA) and a substitution of Alanine (A) for Histidine (H) at position 840 (H840A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32, comprising a substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D10A) and a substitution of an Alanine (A) for an Asparagine (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a type IIS
endonuclease. In certain embodiments of the genomic editing composition, the type IIS
endonuclease comprises AciI, Mn11, AlwI, Bbvi, BccI, BceAI, BsmAI, BsmF1, BspCNI,
-38-BsrI, BtsCI, Hgal, HphI, HpyAV, Mbo II, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, Bird, BpmI, BpuEl, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsT, Earl, EciI, Mmel, NmeAIII, BbvCI, Bpul0I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, Fokl or Clo051. In certain embodiments, the type IIS endonuclease comprises Clo051. In certain embodiments of the genomic editing composition, 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. In certain embodiments of 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.
[038] In certain embodiments of the methods of producing an activated modified Tscm or 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. In certain embodiments, the viral vector comprises one or more sequences isolated, derived, or recombined from an RNA virus. In certain embodiments, the RNA virus is a single-stranded or a double-stranded virus. In certain embodiments, the viral vector comprises one or more sequences isolated, derived, or recombined from a DNA virus. In certain embodiments, the DNA virus is a single-stranded or a double-stranded virus. In certain embodiments, the virus is replication-defective.
[039] In certain embodiments of the methods of producing an activated modified Tscm or 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. In certain embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus.
In certain embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus.
[040J In certain embodiments of the methods of producing an activated modified Tscm or Tem 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. In certain embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus.
In certain embodiments, the viral vector comprises a sequence isolated or derived from a gamma retrovirus.
[0411 In certain embodiments of the methods of producing an activated modified Tscm or Tem 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. In certain embodiments, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In certain embodiments, the AAV is a serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO or AAV I 1. In certain embodiments, the AAV comprises a sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11. In certain embodiments, the AAV
comprises a sequence isolated, derived, or recombined from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11. In certain embodiments, the AAV comprises a sequence isolated, derived, or recombined from AAV2.
In certain embodiments, including those in which the vector crosses the blood brain barrier
-40 -(BBB), the AAV comprises a sequence isolated, derived, or recombined from 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, rAAV-NP59 and rAAV-NP84.
[042] In certain embodiments of the methods of producing an activated modified Tscm or Tas,i of the disclosure, a nucleic acid vector comprises the antigen receptor.
In certain embodiments, a DNA vector comprises the antigen receptor. In certain embodiments, an mRNA vector comprises the antigen receptor. In certain embodiments, the nucleic acid vector is a plasmid or a minicircle vector.
[043) In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, 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.
WO/2016/090111, International Patent Publication No. WO/2017/004498, WO/2017/004509, International Patent Application No. PCT/US2017/030271, US Patent No.
6,835,394, US
Patent No. 7,217,427, and US Patent No. 7,867,512.
[044] In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments. the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein 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, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, 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.
-41 -[045] In certain embodiments of the methods of producing an activated modified TSCM or 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. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the introducing step comprises a homologous recombination and a donor sequence comprises a sequence encoding the therapeutic protein.
In certain embodiments, the donor sequence that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first donor sequence comprises the antigen receptor and a second donor sequence comprises the therapeutic protein. In certain embodiments, a vector comprises a sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle. In certain embodiments, the vector that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first vector comprises the antigen receptor and a second vector template comprises the therapeutic protein.
[046] 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 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-htunan 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 marker(s) of a stem memory T cell (Tscm), thereby producing a modified stein memory T cell (Tscm). 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,
-42 -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%, 60%, 75%, 80%, 85%, 90%, 95% or 99% 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 modified stem memory T cell (Tscm). In certain embodiments of this method, at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments of this method, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. The disclosure provides a method of producing a modified stem memory T cell (Tscm), comprising: (a) introducing into a primaiy 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, 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 marker(s) of a stem memory T
cell (Taw), thereby producing a CAR-expressing stem memory T cell (TKO (CAR-Tscm). 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%, 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 of activated CAR stein memory T cells (Tscm). In certain embodiments, 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,
-43 -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 marker(s) of a stem memory T cell (Tscm). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonarnide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, 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 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 (wherein mg/kg =
parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 Ltmol/kg and 640 mmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 Rmol/kg and 70 ttmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 ttmol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 Ltmol/kg and 75 Ltmol/kg, inclusive of the endpoints; and a sterol at a concentration of
-44 -between 0.25 mol/kg and 25 tunol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 punol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 prnol/kg and a sterol at a concentration of about 2.5 tunol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 prnol/kg, linoleic acid at a concentration of about 7.57 p.mol/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 gmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 prnol/kg, palmitic acid at a concentration of about 7.27 p.mol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 tunol/kg and a sterol at a concentration of 2.61 mol/kg. In certain embodiments, 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 stem memory T cell (Tscm). In certain embodiments, at least 60%
of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T
cell (Tscm). In certain embodiments, 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 /0, 35%, 40%, 45 /o, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, 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 marker(s) of a stem memory T cell (Tscm). In certain embodiments, the enriching step further comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tscm) from the plurality of enriched modified T-cells. In certain embodiments, 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. In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD). diisopropyl adipate
-45 -(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. In certain embodiments, 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 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 (wherein mg/kg =
parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 gmol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints: oleic acid at a concentration of between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pawl/kg and 25 gmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 gmol/kg, linoleic acid at a concentration of about 7.5 gmol/kg, oleic acid at a concentration of about 7.5 gmol/kg and a sterol at a concentration of about 2.5 gmol/kg. In certain embodiments, the T-cell expansion
-46 -composition comprises one or more of octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 tunol/kg, linoleic acid at a concentration of about 7.57 p.mol/kg, oleic acid at a concentration of about 7.56 ttmol/kg and a sterol at a concentration of about 2.61 ttmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27 tunol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of 7.56 Innol/kg and a sterol at a concentration of 2.61 pmol/kg.
[047] 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 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, wherein the activated modified-T cell expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a modified central memory T cell (Tcm). The disclosure provides a method of producing a plurality of modified central memory T cells (Tem), 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%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T
cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a modified central memory T cell (Tcm). In certain embodiments of this method, at least 60 A of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm). In certain embodiments of this method, the T-ull activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, 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, htunan transferrin, 2-Mercaptoethanol, Iscoves 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 marker(s) of a central memory T cell (Tcm). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-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. In certain embodiments, 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 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 (wherein mg/kg = parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 mmol/kg and 640 mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 Amol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 mol/kg and 75 mol/kg, inclusive of the endpoints; and a
-48 -sterol at a concentration of between 0.25 mol/kg and 25 mmol/kg, inclusive of the endpoints.
In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 punol/kg, palmitic acid at a concentration of about 7 punol/kg, linoleic acid at a concentration of about 7.5 pimolVkg, oleic acid at a concentration of about 7.5 pimol/kg and a sterol at a concentration of about 2.5 mmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 tnnol/kg, palmitic acid at a concentration of about 7.27 mmol/kg, linoleic acid at a concentration of about 7.57 Rmol/kg, oleic acid at a concentration of about 7.56 gmol/kg and a sterol at a concentration of about 2.61 punol/kg.
In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 mmol/kg, palmitic acid at a concentration of about 7.27 mol/kg, linoleic acid at a concentration of about 7.57 Rmol/kg, oleic acid at a concentration of 7.56 I.imol/kg and a sterol at a concentration of 2.61 punol/kg. In certain embodiments, 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 memoiy T cell (Teri). In certain embodiments, at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, 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%, 700/0, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, 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 marker(s) of a central memory T cell (Tem). In certain embodiments, the enriching step further comprises isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tem) from the plurality of enriched modified T-cells.
In certain embodiments, the enriching step further comprises contacting the isolated modified Tem and a T-cell expansion composition comprising one or more of hiunan serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified Tem.
In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD). diisopropyl adipate
-49 -(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. In certain embodiments, 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 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 (wherein mg/kg =
parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 gmol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 grnol/kg, inclusive of the endpoints: oleic acid at a concentration of between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 grnol/kg and 25 gmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 gmol/kg, linoleic acid at a concentration of about 7.5 gmol/kg, oleic acid at a concentration of about 7.5 gmol/kg and a sterol at a concentration of about 2.5 gmol/kg. In certain embodiments, the T-cell expansion
-50-composition comprises one or more of octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 gmol/kg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of about 7.56 gmol/kg and a sterol at a concentration of about 2.61 gmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of 7.56 gmol/kg and a sterol at a concentration of 2.61 ttmol/kg.
[048] 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 composition comprising a plurality of modified stem memory T-cells (Tscm) and a plurality of modified central memory T-cells (Tcm), wherein a transposon comprises the antigen receptor, and (b) contacting the composition 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, CD127, CD45RO, CD95, CD95 and IL-2R[ and the plurality of activated modified Tcm expresses one or more CD45RO, CD95, IL-2120, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified Tscm and a plurality of modified Tcm. In certain embodiments of this method, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, 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 stem memory T cell (Tscm). In certain embodiments, 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
- 51 -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). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (Dl PA). li-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. In certain embodiments, 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 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 (wherein mg/kg =
parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 Ltmol/kg and 640 mmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 tunol/kg and 70 ttmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 umol/kg and 75 uniol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 Lunol/kg and 75 Limol/kg, inclusive of the endpoints; and a sterol at a concentration of
- 52 -between 0.25 gmol/kg and 25 tunol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 ptmol/kg, palmitic acid at a concentration of about 7 Amol/kg, linoleic acid at a concentration of about 7.5 mmol/kg, oleic acid at a concentration of about 7.5 1=01/kg and a sterol at a concentration of about 2.5 1=01/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 ptmol/kg, palmitic acid at a concentration of about 7.27 Lunol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of about 7.56 innol/kg and a sterol at a concentration of about 2.61 1=01/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 1=01/kg, palmitic acid at a concentration of about 7.27 ptmol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 Rmol/kg and a sterol at a concentration of 2.61 pnol/kg. In certain embodiments, 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 cells the composition comprising a plurality of expanded modified Tscm and a plurality of expanded modified Tcm expresses cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, 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 cells the composition comprising a plurality of expanded modified Tscm and a plurality of expanded modified Tcm expresses cell-surface marker(s) of a central memory T
cell (Tcm). In certain embodiments, the method further comprises the step of:
(d) enriching the composition 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 marker(s) of a stem memory T cell (Tscm). In certain embodiments, the method further comprises the step of: (d) enriching the composition 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 marker(s) of a central memory T cell (Tcm). In certain embodiments, the enriching step further comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tscm) from the composition or isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the composition.
In certain embodiments, the enriching step further comprises isolating modified T-cells that express one
- 53 -or more cell-surface marker(s) of a stem memory, T cell (Tscm) from the composition and isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the composition. In certain embodiments, 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 Tcm. In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-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. In certain embodiments, 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 concentration of between 0.2 ing/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 (wherein mg/kg = parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4
-54 -gmol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 gmol/kg and 25 gmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 gmol/kg, linoleic acid at a concentration of about 7.5 mol/kg, oleic acid at a concentration of about 7.5 gmol/kg and a sterol at a concentration of about 2.5 1=01/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 gmol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of about 7.56 gmol/kg and a sterol at a concentration of about 2.61 gmol/kg.
In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 gmol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of 7.56 gmol/kg and a sterol at a concentration of 2.61 gmol/kg. In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified central memory T-cells (Tem) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 90% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 90% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 10% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 20% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 80% of the total number of cells of the composition. In certain embodiments of this method, the modified
-55-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 munber 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. In certain embodiments of this method, the modified stem memory T-cells (TKO 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.
[049] In certain embodiments of the methods of the disclosure, including those wherein 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 T cell, wherein the modified T cell is capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the method further comprises introducing a transposase composition. In certain embodiments, the transposase composition transposes the transposon of (a) and the second transposon. In certain embodiments, the method comprises introducing a first transposase composition and a
-56-second transposase composition. In certain embodiments, including those wherein 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. In certain embodiments of this method, 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.
In certain embodiments, 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 piggyBac' m or a Super piggyBac' m (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). In certain embodiments of this method, 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 To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
[050] In certain embodiments of the methods of the disclosure, including those wherein 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 a sequence encoding a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein. In certain embodiments of introducing a sequence encoding a therapeutic protein, the introducing step comprises a homologous recombination.
In certain embodiments of introducing a sequence encoding a therapeutic protein, a vector comprises the sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle.
-57-[051] In certain embodiments of the methods of the disclosure, the introducing step further comprises a composition comprising a genomic editing construct. In certain embodiments, the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA
endonuclease. In certain embodiments, the genomic editing construct comprises a DNA binding domain and a type ITS 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 ITS 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. 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. In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (DIOA).
In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dCas9 (SEQ ID
NO: 33). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dSaCas9 (SEQ ID NO: 32). 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 transcription activator-like effector nuclease (TALEN). 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 TALEN is the DNA binding domain. In certain embodiments,
-58-the genomic editing construct comprises a TALEN. 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 zinc-finger nuclease (ZFN). 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 ZFN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a zinc-finger nuclease (ZFN).
10521 In certain embodiments of the methods of the disclosure, 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. In certain embodiments of this method, the introducing step further comprises a composition comprising an mRNA sequence encoding a transposase. In certain embodiments, 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 Super piggyBacTm (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBac Tm (SPB) transposase, the sequence encoding the transposase is an mRNA
sequence. In certain embodiments, the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4. In certain embodiments, 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. In certain embodiments, the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I) (I30V). In certain embodiments, the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G) (G1655). In certain embodiments, the amino acid substitution at position 282 of SEQ TD NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V). In certain embodiments, the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K). In certain embodiments, the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5. In certain embodiments, 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). In certain embodiments, the transposon is a Helraiser transposon. In certain embodiments, in particular those embodiments wherein the transposon is a Helraiser transposon, the
- 59 -transposase is a Helitron transposase. In certain embodiments, the transposon is a To12 transposon. In certain embodiments, in particular those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 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.
[053) In certain embodiments of the methods of the disclosure, the transposon further comprises a selection gene. In certain embodiments, the T-cell expansion composition further comprises a selection agent.
[054] In certain embodiments of the methods of the disclosure, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein 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, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, 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.
[0551 In certain embodiments of the methods of the disclosure, the cell-surface markers of the modified Tscm comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the modified 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 modified Tscm comprise one or more of CD45RA, CD95, IL-2R(, CR7, and CD62L.
[056] In certain embodiments of the methods of the disclosure, the plurality of expanded modified T-cells comprises a naïve T-cell (modified TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L. In certain embodiments, the plurality of expanded modified T-cells comprises a central memory T-cell (modified Tcm) and the cell-surface markers of the CAR-Tem comprise one or more of CD45RO, CD95, IL-21213, CCR7, and CD62L. In certain embodiments, 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 IL-2RD. In certain embodiments,
-60 -plurality of expanded modified T-cells comprises an effector T-cell (modified TEFF) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2R[.
[0571 In certain embodiments of the methods of the disclosure, 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, CCR7, and CD62L. In certain embodiments, 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 CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RP.
[0581 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, 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 marker(s) of a stem memory T cell (Tscm), thereby producing a CAR-expressing stem memory T cell (Tscm) (CAR-Tscm). 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%, 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 of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at
- 61 -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). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, 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 marker(s) of a stein memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, 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). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R13. In certain embodiments. the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-21213, 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)
-62 -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 marker(s) of a stem memory, T cell (Tscm), thereby producing a CAR-expressing stem memory T cell (Tscm) (CAR-Tscm).
[059) 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-htunan 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 of activated CAR stem memory T cells (Tscm). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60 /0 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). In certain embodiments, 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, 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
-63 -(Tscm). In certain embodiments, 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 stein memory T cells (Tscm). In certain embodiments, 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, 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). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R13. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-2110, CR7, and CD62L.
[060] In certain embodiments, 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 senun 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 marker(s) of a stem memory T cell (Tscm) (CAR-Tscm). In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-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. In certain embodiments, 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;
pahnitic 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 (wherein mg/kg =
-64 -parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 mol/kg and 640 mmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 timol/kg and 70 mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/kg and 75 timol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 punol/kg and 75 timol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 mol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 tunol/kg, palmitic acid at a concentration of about 71=01/kg, linoleic acid at a concentration of about 7.5 mol/kg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 pmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 tunol/kg, palmitic acid at a concentration of about 7.27 punol/kg, linoleic acid at a concentration of about 7.57 punol/kg, oleic acid at a concentration of about 7.56 mol/kg and a sterol at a concentration of about 2.61 punol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 tunol/kg, palmitic acid at a concentration of about 7.27 mmol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of 7.56 timol/kg and a sterol at a concentration of 2.61 larnol/kg. In certain embodiments, 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 CAR-T cells expresses cell-surface
-65 -marker(s) of a stem memory T cell (Tscm) (CAR-Tscm). In certain embodiments, the plurality of expanded CAR-T cells may be enriched for CAR-T cells that express cell-surface marker(s) 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 marker(s) of a stem memory T cell (Tscm) (CAR-Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, 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, CD95,11,-2RO, CR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises a naïve T-cell (CAR-TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L. In certain embodiments, 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, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-Tovi) and the cell-surface markers of the CAR-Tim comprise one or more of CD45RO, CD95, and IL-2RP. In certain embodiments, 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[. 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).
[061] 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 marker(s) of a stem memory T
cell (Tscm), thereby producing a CAR-expressing stem memory T cell (Tscm) (CAR-Tscm).
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
-66 -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 of activated CAR
stem memory T cells (Tscm). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, 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 stein memory T cells (Tscm). In certain embodiments, 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, 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 marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (TKO. In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95%
of the
-67 -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). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, 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-21113, CR7, and CD62L.
[062] In certain embodiments of the methods of the disclosure, the plurality of expanded CAR-T cells comprises a naïve T-cell (CAR-TN) and the cell-surface markers of the CAR-TN
comprise one or more of CD45RA, CCR7 and CD62L. In certain embodiments, 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, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-Tad) and the cell-surface markers of the CAR-TEm comprise one or more of CD45RO, CD95, and IL-2R13. In certain embodiments, 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(.
[063] In certain embodiments of the methods of the disclosure, 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-regulatoiy insulator elements. In certain preferred embodiments, the transposon is a piggyBac transposon. In certain embodiments, 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. In certain preferred embodiments, the transposase is a Super piggyBacTm (SPB) transposase.
[064] In certain embodiments, a transposon of the disclosure may further comprise a selection gene. When a transposon of the disclosure comprises 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.
[065] In certain embodiments a CAR of the disclosure may be a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
-68 -[066] In certain embodiments of the methods of producing a modified Tscm of the disclosure, the introducing step may comprise an electroporation or a nucleofection. When the introducing step comprises a nucleofection, the nucleofection may comprise the steps of:
(a) contacting a transposon composition, a transposase composition, and a 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. In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-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. In certain embodiments, 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 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 (wherein mg/kg = parts per million). In certain embodiments, 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 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, 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 (wherein mg/kg = parts per million). In certain embodiments, 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
-69 -(wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 gmol/kg and 640 Amol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 mol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 iimol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 mol/kg and 75 mol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 mol/kg and 25 mol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 mol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 mol/kg, oleic acid at a concentration of about 7.5 Lunol/kg and a sterol at a concentration of about 2.5 mol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27 mol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of about 7.56 mol/kg and a sterol at a concentration of about 2.61 I.Lmol/kg.
In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27 mol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of 7.56 mol/kg and a sterol at a concentration of 2.61 mol/kg. In certain embodiments of the nucleofection, the transposon composition is a 0.5 g/ 1 solution comprising nuclease free water and the cuvette comprises 2 I of the transposon composition to yield I lig of transposon. The transposon composition may comprise a piggyBac transposon. The transposon composition may comprise a Sleeping Beauty transposon. In certain embodiments of the nucleofection, the transposase composition comprises 5 Lig 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.
In certain embodiments, the transposon may comprise a Helraiser transposon and the transposase composition may comprise a Helitron transposase. In certain embodiments, the transposon may comprise a To12 transposon and the transposase composition comprises a To12 transposase.
[067] In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition and a first transposase
-70 -composition and a composition comprising a plurality of primary human T cells in a cuvette.
In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, 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. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an cicctroporation, 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.
In certain embodiments, the first transposon comprises a sequence encoding an antigen receptor. In certain embodiments, the second transposon comprises a sequence encoding a therapeutic protein. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, the first transposon composition comprises a sequence encoding an antigen receptor and the second transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein is a secreted or secretable 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 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.
In certain embodiments, the transposon composition comprises a sequence encoding the
- 71 -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. In certain embodiments, 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. In certain embodiments, 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 T cells first, and 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 second. In certain embodiments, 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. In certain embodiments of the methods of producing a modified Tscm of the disclosure, 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. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stern-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
-72 -(Lonza). In certain embodiments, the buffer comprises one or more of KC1, MgCl2, ClNa, Glucose and Ca(NO3)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/HC1, and a phosphate buffer. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCl2, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(NO3)2. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCl2, 90 mM ClNa, 10 mM
Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 20 inIVI HEPES and 75 mM
Tris/HC1. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCl2, 90 mM
ClNa, 10 mM Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 40 mM
Na2HPO4/NaH2PO4 at pH 7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 ill of the buffer and between 5x106 and 25x106 cells.
1068j In certain embodiments of the methods of producing a modified Tscm of the disclosure, the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and/or CD19. In certain embodiments, the composition comprising primary human T cells comprises 100 td of the buffer and between 5x106 and 25x106 cells.
[069] As used herein, 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. Alternatively, or in addition, 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. In certain embodiments, 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 ((TMDM); available at ThermoFisher Scientific as Catalog number 12440053).
[070] As used herein, 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. Alternatively, or in addition, 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 elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. In certain embodiments, the terms "supplemented T-cell expansion composition" or `7-cell expansion composition"
may be
-73 -used interchangeably with a 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.
[071] As used herein, 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, Iscoves MDM, and an expansion supplement at 37 C. Alternatively, or in addition, 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-tetramethy1-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No.

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. 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) (CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5). In certain embodiments, the terms "supplemented T-cell expansion composition" or `7-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-tetramethy1-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No.

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. 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) (CAS No. 57-88-5), alkanes (e.g., nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). In certain embodiments, 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-tetramethy1-5-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. 84-69-5), palmitic acid (CAS
No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid
-74 -hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS
No. 143-74-8) and lanolin alcohol.
[072] As used herein, 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. Alternatively, or in addition, 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 ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate and phosphate.
[073] As used herein, 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. Alternatively, or in addition, 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:
histidine, asparagine, serine, glutamate, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine and tryptophan. In certain embodiments, 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 1.5%), glutamine (about 67%), arginine (about 1.5%), glycine (about 1.5%), aspartic acid (about 1%), glutamic acid (about 2%), threonine (about 2%), alanine (about 1%), proline (about 1.5%), cysteine (about 1.5%), lysine (about 3%), tyrosine (about 1.5%), methionine (about lq, valine (about 3.5%), isoleucine (about 3%), leucine (about 3.5%), phenylalanine (about 1.5%) and tryptophan (about 0.5%). In certain embodiments, 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 .78%), asparagine (about 0.4%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine
-75 -(about 3.14%), leucine (about 3.3%), phenylalanine (about 1.64%) and tryptophan (about 0.37%).
[074] As used herein, 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). In certain embodiments, 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 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 (wherein mg/kg = parts per million). In certain embodiments, 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 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 (wherein mg/kg = parts per million). ). In certain embodiments, 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 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 (wherein mg/kg =
parts per million). In certain embodiments, 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 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 (wherein mg/kg = parts per million). In certain embodiments, 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 6.4 p.mol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 p.mol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/kg
-76 -and 75 gmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 timol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 1=01/kg and 25 timol/kg, inclusive of the endpoints. In certain embodiments, 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 mol/kg, palmitic acid at a concentration of about 7p.mol/kg, linoleic acid at a concentration of about 7.5 tunol/kg, oleic acid at a concentration of about 7.5 1=01/kg and a sterol at a concentration of about 2.5 timol/kg. In certain embodiments, the terms "supplemented T-cell expansion composition" or `7-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 tunol/kg, palmitic acid at a concentration of about 7.27 1=01/kg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of about 7.56 p.mol/kg and a sterol at a concentration of about 2.61 timol/kg.
In certain embodiments, 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 1.1mol/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of 7.561=01/kg and a sterol at a concentration of 2.61 mol/kg.
[075] As used herein, the term "P3 buffer" may be used interchangeably with a buffer comprising one or more of KCI, MgCl2, ClNa, Glucose and Ca(NO3)2 in any absolute or relative abundance or concentration, and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/HC1, and a phosphate buffer.
The term "P3 buffer" may be used interchangeably with a buffer comprising 5 mM KCl, 15 mM
MgC12, 90 mM ClNa, 10 mM Glucose and 0.4 mM Ca(NO3)2, and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/HC1, and a phosphate buffer. The term "P3 buffer" may be used interchangeably with a buffer comprising 5 mM KC1, 15 mM MgCl2, 90 mM ClNa, 10 mM Glucose and 0.4 mM
Ca(NO3)2 and a supplement comprising 20 mM HEPES and 75 mM Tiis/HC1. The term "P3 buffer"
may be used interchangeably with a buffer comprising 5 mM KCl, 15 mM MgCl2, 90 mM
ClNa, 10 mM Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 40 mM
Na2HPO4/NaH2PO4 at pH 7.2.
[076] As used herein, the terms "supplemented RPMI-1640 media" or '7-cell conditioned media (TCCM)" may be used interchangeably with a media comprising one or more of
-77 -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 HCI, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-'Threonine, L-Tryptophan, L-Tyrosine 2Na 2H20, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid. D-Panthothenic acid (hemicalcium), pyridoxine HCI, riboflavin, thiamine HCI, vitamin B12, D-Glucose, Glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration. The terms "supplemented 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 (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HCI, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2H20, 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.
10771 As used herein, the terms "supplemented AIM-V" or "supplemented AIMN"' 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 (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 HCI, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 21-120, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12, D-Glucose,
-78 -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 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 HCl, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 21-120, 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.
[078] As used herein, the term ImmunoCultrm 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-Tryptophan, L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-Calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-Inositol, calcium chloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in any absolute or relative abundance or concentration. The term "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 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-Tryptophan, L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-Calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine
-79 -hydrochloride, vitamin B12, i-Inositol, calcium chloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in any absolute or relative abundance or concentration.
[079] Modified T-cells of the disclosure, including modified Tsem and/or Tem 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 GSK3f3 such as TWS119 (also known as (]SK
3B inhibitor XII; CAS Number 601514-19-6 having a chemical formula C18H14N402).
Exemplary inhibitors a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIOTm).
[0801 As used herein, 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 penneable to the nucleic acid, transposon, vector or composition of the disclosure.
[ON] In certain embodiments of the nucleofection, 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. In certain embodiments of the nucleofection, 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, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or any number/portion of hours in between. The incubation step may have a period of at least I, 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. In certain embodiments of the nucleofection, the incubation step has a period of two days. In certain embodiments of the nucleofection, the applying step may comprise applying one or more of the following program(s) ET-115, El-151, El-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. In certain embodiments, the applying step may comprise applying one or more of the following
- 80 -
81 program(s) EI-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, E0-151, EO-148, EO-156, EO-210, E0-213, 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. In certain embodiments, the applying step may be performed using a known electroporation/nucleofection device, including, but not limited to, Lonza Amaxa, MaxCyte technology, B'TX PulseAgile, and BioRad GenePulser. In certain embodiments of the nucleofection, the applying step may comprise applying at least one electrical pulse. In certain embodiments of the nucleofection, 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.
10821 'While the amounts provided herein are exemplary and non-limiting, the relationship between these amounts (e.g. ratios or relative abundances) may be used to modify the methods exemplified herein for larger-scale processes and manufacturing.
[083] In certain embodiments of the methods of producing a modified T cell (e.g. a Tscm and/or Tcm) 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. Exemplay 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.
[084] In certain embodiments of the methods of producing a modified T cell (e.g. a Tscm and/or Tcm) of the disclosure, 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), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFO. The one or more cytokine(s) may comprise IL-2.
10851 In certain embodiments of the methods of producing a modified T cell (e.g. a Tscm and/or Tcm) of the disclosure, the primary human T cell is a naive T cell. The naive T cell may express CD45RA, CCR7 and CD62L. In certain embodiments, 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. In certain embodiments, the efficiency of production of modified Tsem and/or Tem of the disclosure may be increased by increasing a proportion or percentage of naïve T
cells in a cell population to which the methods of the disclosure are applied.
[086] In certain embodiments of the methods of producing a modified Tsem and/or Tem of the disclosure, the primary human T cell is a memory T cell.
[087) In certain embodiments of the methods of producing a modified Tscm and/or Tem of the disclosure, the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2110.
[088] In certain embodiments of the methods of producing a modified Tscm and/or Tcm of the disclosure, the primary human T cell is a naïve T-cell (modified TN) and the modified TN
expresses one or more of CD45RA, CCR7 and CD62L. In certain embodiments of the methods of producing a modified Tsem and/or Tcm of the disclosure, the primary human T
cell is a modified Tscm a T memory stem cell (modified Tsem) and the modified Tscm expresses one or more of CD45RA, CD95, IL-2R3, CR7, and CD62L. In certain embodiments of the methods of producing a modified Tscm and/or Tem of the disclosure, the primary human T cell is a central memory T-cell (modified Tcm) and the modified Tem expresses one or more of CD45RO, CD95, IL-2R3, CCR7, and CD62L. In certain embodiments of the methods of producing a modified Tscm and/or Tcm of the disclosure, the primary human T cell is an effector memory T-cell (modified TEO and the modified Ti expresses one or more of CD45RO, CD95, and IL-2R(. In certain embodiments of the methods of producing a modified Tscm and/or Tcm of the disclosure, the primary human T
cell is an effector T-cell (modified TEFF) and the modified TEFF expresses one or more of CD45RA. CD95, and IL-2113.
[089] In certain embodiments of the methods of producing a modified Tscm and/or Tem of the disclosure, 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.
[0901 In certain embodiments of the methods of producing a modified Tscm and/or Tem of the disclosure, the primary human T cell may be isolated, prepared or derived from for
- 82 -example, whole blood, peripheral blood, umbilical cord blood, lymph fluid, lymph node tissue, bone marrow, and cerebral spinal fluid (CSF). The term "peripheral blood" as used herein, 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. The terms "umbilical cord blood", "umbilical blood" or "cord blood", which can be used interchangeably, 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.
[091] Primary human T cells of the disclosure may comprise pan T cells. As used herein, 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.
1092) In certain embodiments of the methods of the disclosure, the method further comprises introducing into a modified Tscm or Tcm cell a composition comprising a genomic editing construct or composition. In certain embodiments, the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease. In certain embodiments, 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 11S
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. 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. In certain embodiments, the
- 83 -sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (Dl OA). In certain embodiments, the 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). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an inactivated Cas9 (dCas9) (SEQ ID NO: 33). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an alanine (A) for an Asparagine (N) at position 580 (N580A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises a truncated and inactivated Cas9 (dSaCas9) (SEQ ID NO:
32). 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 transcription activator-like effector nuclease (TALEN).
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 TALEN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a TALEN. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type HS
endonuclease, the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
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 ZFN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a zinc-finger nuclease (ZFN).
[093] The methods of making modified Tsem and/or Tem cells of the disclosure may be optimized to produce a greater number or greater proportion of modified Tsem and/or Tcm cells. For example, the population of cells subjected to the methods of the disclosure may be enriched to contain an increased number or greater proportion of naïve T
cells. As 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 Tem cells of the disclosure produced also increases. Alternatively, or in addition, as 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 Tem cells of the disclosure produced by the method increases. The length of time or duration required for a method of disclosure to precede, or
- 84 -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.
[094] In certain embodiments of the methods of making modified T-cells of the disclosure, the primary human Tee!! expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-21(13. In certain embodiments, the primary human T
cell is a naïve T-cell (TN) and the TN expresses one or more of CD45RA, CCR7 and CD62L.
In certain embodiments, the primary human T cell is a T memory stem cell (Tscm) and the Tscm expresses one or more of CD45RA, CD95, IL-2R13, CR7, and CD62L. In certain embodiments, the primary human T cell is a central memory T-cell (Tcm) and wherein the Teri expresses one or more of CD45RO, CD95, IL-2R3, CCR7, and CD62L. In certain embodiments, the primary human T cell is an effector memory T-cell (TEm) and the EM
expresses one or more of CD45RO, CD95, and IL-2R13. In certain embodiments, the primary human T cell is an effector T-cell (Ten) and the TEFF expresses one or more of CD45RA, CD95, and IL-2120. In certain embodiments, the primary human T cell expresses CD4 and/or CD8.
[095] The disclosure provides a composition comprising a modified Tsem 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 Tem produced a method of the disclosure. In certain embodiments of the composition comprising a modified Tsem and a modified Tcm produced a method of the disclosure, a plurality of Tsem 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. . In certain embodiments of the composition comprising a modified Tsem and a modified Tcm produced a method of the disclosure, a plurality of Tem 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.
[096) The disclosure provides a use of a composition comprising a modified Tscm and/or Tem produced a method of the disclosure for the manufacture of a medicament to treat a subject in need thereof. In certain embodiments of this use, the modified Tsem and/or Tem is autologous. In certain embodiments of this use, the modified Tscm and/or Tem is allogeneic.
In certain embodiments, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not
- 85 -naturally-occurring. In certain embodiments, and, in particular, in those embodiments wherein 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. 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.
[097] 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 Tem produced a method of the disclosure. In certain embodiments of this method, the modified Tscm and/or Tcm is autologous. In certain embodiments of this method, the modified Tscm and/or Tcm is allogeneic. In certain embodiments, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-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-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. 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. In certain embodiments of this method, the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen. In certain embodiments of this method, the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antigen. In certain embodiments, the disease or disorder is a disease or disorder caused by a lack of an activity or an insufficient amount of a secretory protein. In certain embodiments, 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. In certain embodiments, the therapeutic protein is a secreted protein. In certain embodiments, the secretory protein is lacking an activity or a sufficient amount within a local area of a body. In certain embodiments, the local area of a
- 86 -body is accessible by a native T-cell or a modified T-cell. In certain embodiments, the modified T-cell is produced in vivo, ex vivo, in vitro or in situ.
BRIEF DESCRIPTION OF THE DRAWINGS
[098] 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.
[099] 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.
[0100] 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.
[0101] Figure 4 is a series of plots depicting an enrichment of the CAR-Tscm phenotype at Day 19 of the method described in Example I. Cells were nucleofected with a surrogate CARTyrin plasmid. CAR-Tscm cells express CD62L and CD45RA as shown in the bottom two plots.
[0102] Figure 5 is a series of plots depicting the absence of T-cell exhaustion at Day 19 of the method described in Example 1. At Day 19, the cell population produced by this method does not express PD1, which 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 PD! 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.
[0103] Figure 6A is a series of plots depicting T cells transposed with a plasmid containing a sequence encoding a transposon comprising a sequence encoding an inducible caspase polypeptide (a safety switch, "iC9"), a CARTyrin (anti-BCIvIA), and a selectable marker.
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.
- 87 -[0104] 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 piggyBac") or a wild type piggyBac transposase.
[0105] Figure 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.
[0106] 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.
[0107] Figure 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. For production using lentivirus, pan T cells were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding CAR (MOI
5), expanded, and examined on day 18 post-stimulation. Then, 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+), Tem (CD62L+CD45RA-), Tem (CD62L-CD45RA-) and Teff (CD62L-CD45RA+). All CAR+ T cells were CD95+.
[0108] Figure 8A-B is a pair of graphs showing that piggyBac Tm preferentially transposes naive T cells. Human pan T cells were sorted (using a BD FACSAria II flow cytometer) into naive (CD62L+CD45RA+), Tem (CD62L+CD45RA-), Tem (CD62L-CD45RA-), and Teff (CD62L-CD45RA+) subsets. The sorted subsets were each either transposed with piggyBac-GFP or transduced with lentivirus-GFP. For the former, each sorted subset was transposed with PiggyBac-GFP, stimulated with anti-CD3/CD28 beads at day 2 post-transposition, expanded, and examined on day 19 post-transposition. For the latter, the sorted subsets were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding GFP (MOI
5), expanded, and examined on day 19 post-stimulation. n=3 donors.
- 88 -[0109] Figure 9 is a pair of graphs showing that the piggyBac rm 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 (triangles) and healthy donors (circles) were characterized for memory marker expression by flow cytometiy 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 naïve and Tscm cells, but higher frequencies of Teff, unlike those from healthy normal donors which are the opposite.
Regardless of the input frequency of naïve and Tscm from different MM
patients, production of P-BCMA-101 using the Poseida manufacturing process resulted in a product that exhibited a high level of CD8+ Tscm (E). This was also true for a MM patient who was actively receiving treatment (red triangle).
[0110] 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 Beauty (SB100x) transposition system and the methods of the disclosure.
Sleeping Beauty (SB100x) Transposition yields predominately Tscm phenotype using Poseida manufacture process. Human pan T cells were transposed using In of either a Sleeping Beauty or piggyBac transposon plasmid and SB100x or SPB mRNA, respectively as shown.
Following transposition, cells were expanded ex vivo and all non-transposed cells were depleted using the Poseida manufacture drug selection system. Following 18 days in culture, cells were stained with the phenotypic markers CD4, CD8, CD45RA, and CD62L. Stem cell memory phenotype (Tscm) is defined by CD45RA and CD62L double positive cells and make up >65% of the cells in all of samples. All panels in a column share common x-axis and y-axis parameters. In each row, from top to bottom, are shown data from T cells transposed with (top), 2.5 microgram (Ltg) of the Sleeping Beauty transposon SB100x, (second from top) 5 Lig of SB100x, (3KI from top) 10 flg of SB100x, (second from bottom) 5 tg of the piggyBac transposon P-BCMA-101 and at bottom, an unstained control. 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. The x axis of the third column from the left shows CD8 expression, with markings reading from 0 to 105 incrementing by powers of 10.
The final right hand column shows CD62L expression, with markings reading from 0 to 105 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
- 89 -shows expression of the cell viability marker 7 aminoactinomycin D (7AAD), from 0 to 105 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 105 incrementing by powers of 10. The y-axis in the right hand column show expression of the marker CD45RA, from 0 to 105 incrementing by powers of 10.
[01.111 Figure 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 VIII (FVIII) deficiency leads to development of Hemophilia A. Factor IX
(FIX) deficiency leads to development of Hemophilia B. 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. Prior to the compositions and methods of the disclosure, 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. In sharp contrast to this standard treatment option, Tscm cells of the disclosure are maintained in humans for several decades.
[01.12] Figure 12 is a series of Fluorescence-Activated Cell Sorting (FACS
plots) depicting FIX-secreting T cells. T cells encoding a human Factor IX transgene showed a Tscm phenotype in approximately 80% of cells. The 6 panels are described in order from left to right. (1) Forward scatter (FSC) on the x-axis versus side scatter (SSC) on the y-axis. The x-axis is from 0 to 250 thousand (abbreviated k) in increments of 50k, the y-axis is for 0 to 250k, in increments of 50k. (2) FSC on the x-axis versus the cell viability marker 7 aminoactinomycin D (7AAD). The x-axis is labeled from 0 to 250k in increments of 50k.The y-axis reads, from top to bottom, -103, 0, 103, 104, 105. (3) On the x-axis is shown anti-CD56-APC conjugated to a Cy7 dye (CDC56-APC-Cy7), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown anti-CD3 conjugated to phycoerythrin (PE), units from 0 to 105 incrementing in powers of 10. (4) On the x-axis is shown anti-CD8 conjugated to f1uoresceii isothiocyanate (FITC), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown anti-CD4 conjugated to Brilliant Violet 650 dye (BV650), units from 0 to 105 incrementing in powers of 10. (5) On the x-axis is shown an anti CD62L
antibody conjugated
-90 -to a Brilliant Violet 421 dye (BV421), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown an anti-CD45RA antibody conjugated to PE and Cy7, units from 0 to 105 incrementing in powers of 10. This panel is boxed. (6) On the x-axis is shown an anti-CCR7 antibody conjugated to Brilliant Violet 786 (BV786), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown anti-CD45RA conjugated to PE and Cy7, units from 0 to 105 incrementing in powers of 10.
[0113] Figure 13A is a graph showing human Factor IX secretion during production of modified T cells of the disclosure. On the y-axis, Factor IX concentration in nanograms (ng) per milliliter (mL) from 0 to 80 in increments of 20. On the x-axis are shown 9 day and 12 day T
[01141 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.
[0115] 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. Donor plasmids for testing stable integration into the genome of human pan T cells via A) site-specific (AAVS1) homologous recombination (HR), B) site-specific (AAVS1) microhomology-mediated end-joining (MMEJ) recombination and C) 'MA-specific piggyBacTM transposition. For HR and MMEJ donor plasmids, GFP-2A-DHFR gene expression cassettes were flanked by CRISPR/Cas9 targeting sites and homology arms for AAVS1 site integration; for piggyBacTM donor plasmid, GFP-2A-DHFR gene expression cassette is flanked by piggyBacTm transposon elements. The homology arms for the HR and MMEJ plasmids are 500 bp and 25 bp, respectively. Panels D and E, and F depict SEQ ID NOs 41 and respectively.
[0116] Figure 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 nucleofection. 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.
[0117] Figure 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
- 91 -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 cytometiy 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 atone.
[01181 Figure 17A-C is a series of graphs showing the phenotype of primary human pan T
cells modified by HER 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 (Tern)), and CD45RA+CD62L- (T effectors (Teft)). B) Percentage of total GFP+ CD8+ T cells in each T cell subset is summarized in bar graph. An enriched population of GFP+ Tscm was achieved in all cases using either the piggyBacTm transposon system, or HR and MMEJ in combination with Cas9 RNP. C) The total number of pan T cells was analyzed at day 13 post-nucleofection and data are summarized in bar graph.
[0119] Figure 18A-B is a pair of photographs of gel electrophoresis results showing site-specific integration into the AAVS I site. Selected cells from each group were harvested and genomic DNA was extracted and used as template for PCR to confirm site-specific integration into the AAVS1 site for A) HR and B) MMEJ. Two pairs of primers individually amplify the 5s-end junction (with one primer priming the promoter region of the insertion EF la-2r CACCGGAGCCAATTCCCACT (SEQ ID NO: 36) and the other priming the AAVSI 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 3s-end junction (with one primer priming the polyA
signaling region SV40pA-Ir GTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38) and the other priming the AAVS1 region beyond the 500 bp 5'-homologue arm AAVS-2f CTGGGGACTCTTTAAGGAAAGAAG (SEQ ID NO: 39), yielding a 0.76 kb DNA
fragment for HR or MMEJ) of the AAVSI target site. PCR products were displayed on Agarose gel. Non-specific bands in HR samples are the result of only a single round of PCR
and would likely have been resolved given additional rounds.
DETAILED DESCRIPTION
-92 -[01201 The disclosure provides a method for producing human chimeric antigen receptor (CAR) expressing-T cells using the piggyBacTM Transposon System under conditions that preserve or induce stem-cell memory T cells (Tsai) 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. Other T cell subsets found in the compositions of the disclosure include, but are not limited to, central memory CAR-T cells (CAR-Tcm), effector memoiy CAR-T cells (CAR-TEm), effector CAR-T cells (CAR-TB), and terminally-differentiated effector CAR-T cells (CAR-TTE). A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN) > Tscm > Tcm > Tat > TB > TIE, 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. Tscrs,i > Tcm > T1> TB > 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.
[0121] Tmmunotherapy using chimeric-antigen receptor (CAR)-T cells is emerging as an exciting therapeutic approach for cancer therapies. Autologous CAR-modified T
cells targeting a tumor-associated antigen (Ag) can result in robust tumor killing, in some cases resulting in complete remission of CD19+ hematological malignancies. Unlike traditional biologics and chemotherapeutics, CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments. To achieve this, 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.
Thus, intensive efforts have been focused on the development of CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as of a CAR-T
product containing early memory cells, especially stem cell memory (Tscm). A
stein cell-like CAR-T would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (Tcm), effector memory (Tat) and effector T cells (TB), thereby producing better minor eradication and long-term CAR-T engraftment.
-93 -[0122] 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-Tscm).
Centyrins are alternative scaffold molecules based on human consensus tenascin FN3 domain, are smaller than scFv molecules, and can be selected for monomeric 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.
[0123] 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.
[0124] In certain embodiments of the methods of the disclosure, 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.
Unexpectedly, a majority of these modified CARTyrin-expressing T cells were positive for expression of CD62L and CD45RA, markers commonly associated with stem memory T-cells (Tscm cells).
To confirm that this phenotype was retained upon CAR-T cell stimulation and expansion, the modified CARTyrin-expressing T cells positive for expression of CD62L and CD45RA were activated via stimulation of CD3 and CD28. As a result of stimulation of CD3 and CD28, >
60% of CARTyrin+ T cells exhibited a stem-cell memory phenotype. Furthermore, these cells, which expressed a CARTyrin specific for a cancer antigen, were fully capable of expressing potent anti-tumor effector function.
[0125] To determine whether or not the PB system directly contributed to enhancing the expression of stem-like markers, the phenotype of CAR-T cells generated either by PB
transposition or lentiviral (LV) transduction was compared. To do this, a new vector was constructed by subcloning the CARTyrin transgene into a common LV construct for
-94 -production of virus. Following introduction of the CARTyrin to untouched resting T cells either by PB-transposition or LV-transduction, the CARTyrin + cells were expanded and then allowed to return to a resting state. A variety' of 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, cy-tokine production, and lytic capability in response to target tumor cells. Unlike the PB-transposed CARTyrin + T cells, the LV-transduced CARTyrin + T cells did not exhibit an augmented memoiy phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of target tumor cells.
Together, these data demonstrate that CAR-T cells produced by PB transposition are predominantly Tscm cells, a highly desirable product phenotype in the CAR-T field. Furthermore, these 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.
Chimeric Antigen Receptors [01261 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. In certain embodiments, the antigen recognition region may comprise two sequences that each specifically bind an antigen to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three sequences that each specifically bind an antigen 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. 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).
[0127] In certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD3.5, CD3e, CD3y, CD3c, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD8a signal
-95 -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 atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO:
9).
[0128] In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD38, CD3E, CD3y, CD3C, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. The CD8a transmembrane domain may comprise an amino acid sequence comprising IYTWAPLAGTCGVLLLSLVITLYC (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 atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ
ID NO:
11).
[0129] In certain embodiments of the CARS of the disclosure, the endodomain may comprise a human CD31; endodomain.
[0130] In certain embodiments of the CARS of the disclosure, the at least one costimulatory domain may comprise a htunan 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof In certain embodiments of the CARS of the disclosure, the at least one costimulatory domain may comprise a CD28 and/or a 4-1BB
costimulatory domain. The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDICRRGRDPEMGGKPRRICNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATICD'TYDALHMQALP
PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRICNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising
-96 -cgcgtgaagtttagtegatcagcagatgccccagatacaaacagggacagaaccagctgtataacgagctgaatctggg ccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcagg aagg cctglataacgagclgcagnaggacannatggcagaagcctattetgagatcggcatgaagggggagegacggagaggc aaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctcca agg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID
NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYTEKQPFMRPVQTTQEEDGCSCREPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgetgtatattttcaaacagccettcatgcgccccgtgcagactacccaggaggaagacg ggtgctcc tgtcgattccctgaegaagageaaggegggtgtgagctg (SEQ ID NO: 15). The 4-1BB
costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.
101311 In certain embodiments of the CARS of the disclosure, 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 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (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 sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagccectgagtctgagacctgaggcct gcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).
101321 The disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
[0133] 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.
[0134] Transposons of the disclosure may comprise a selection gene for identification, enrichment and/or isolation of cells that express the transposon. Exemplary selection genes
-97 -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. Exemplaiy 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.
Exemplaiy 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 Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).
101351 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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLICLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). 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
-98 -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 GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). 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).
101361 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 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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). 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 QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an
-99 -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 GSGQCTNYALLICLAGDVESNPGP (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 GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). 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).
[0137] The disclosure provides a composition comprising the transposon the disclosure. In certain embodiments, 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.
[0138] Transposons of the disclosure may comprise piggyBac transposons.
Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
[0139] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.
[0140] Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovinis, a lentivirus, 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. 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).
- 100 -Exemplaq 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.
[0141] 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 (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding 'Thymidylate Synthetase), MGN1T ( 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.
[0142] Viral vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, 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. In some embodiments, 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 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 ggatctggagagggaaggggaagectgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An
- 101 -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 QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). 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 GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). 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).
[0143] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, 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.
101441 The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, 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. poly-mersomes), micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any
- 102 -combination thereof A nanoparticle vector may be passively or actively transported across a cell membrane.
[0145] 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, nen (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thytnidylate 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.
101461 Nanoparticle vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 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
- 103 -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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). 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 QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). 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 GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). 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).
[0147] The disclosure provides a composition comprising a vector of the disclosure.
CAR7'yrins [0148] 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. As used throughout the disclosure, a CAR
comprising a Centyrin is referred to as a CARTyrin. In certain embodiments, the antigen recognition region may comprise two Centyrins to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three Centyrins to
- 104 -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.
[0149] 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. In certain embodiments, 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.
101501 in certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD38, CD3e, CD3y, CD3C CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments of the CARs of the disclosure, 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 atggcactgccagtcaccgccctgctgctgcctctggctctgetgctgcacgcagctagacca (SEQ ID NO:
9).
[0151] In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CDT& CD3e, CD3y, CD3t, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments of the CARS of the disclosure, 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). The CD8a
- 105 -transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ
ID NO:
11).
101521 In certain embodiments of the CARS of the disclosure, the endodomain may comprise a human CD3 endodomain.
101.531 In certain embodiments of the CARS of the disclosure, the at least one costimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof In certain embodiments of the CARS of the disclosure, the at least one costimulatory domain may comprise a CD28 and/or a 4-1BB
costimulatory domain. The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDICRRGRDPEMGGKPRRICNPQ
EGLYNELQICDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRICNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATICDTYDALHMQALP
PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagatacaaacagggacagaaccagctgtataacgagagaatctgggc cgccga gaggaatatgacgtgctggataageggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcagg aagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggc aaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgactgcatatgcaggcactgcctccaa gg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID
NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgctg,tatattttcaaacagcccticatgegccccgtgcagactacccaggaggaagac gggtgacc tgtegattccctgaggaagaggaaggcgggtg,tgagctg (SEQ ID NO: 15). The 4-1BB
costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.
- 106 -[0154] In certain embodiments of the CARS of the disclosure, 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 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (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 sequence comprising actaccacaccagcacctagaccaccaactccagaccaaccatcgcgagtcagccectgagtctgagacctgaggcctg caggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).
[0155] 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 III (FN3) domain, wherein the scaffold is capable of specifically binding an antigen.
The at least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be Tenascin-C. The consensus sequence may comprise LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDL
TGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) or MLPAPKNLVVSEV'TEDSLRLSWTAPDAAFDSFLIQYQESEKVGEATNLTVPGSERSYD
LTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). The consensus sequence may encoded by a nucleic acid sequence comprising atgctgectgcaccaaagaacctggtggtgtacatgtgacagaggatagtgccagactgtcatggactgctcccgacgc agccttcg atagattatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtectgacagtgccagggtecgaacgctet tatgacctg acagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagatccctctgtccgc aatcttcac caca (SEQ ID NO: 3). The consensus sequence may be modified at one or more positions within (a) a A-B loop comprising or consisting of the amino acid residues TEDS
at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of the amino acid residues TAPDAAF at positions 22-28 of the consensus sequence; (c) a C-D loop comprising or consisting of the amino acid residues SEKVGE at positions 38-43 of the consensus sequence; (d) a D-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence; (e) a E-F loop comprising or consisting of the amino acid residues GLKPG at positions 60-64 of the consensus sequence; (f) a F-G loop
- 107 -comprising or consisting of the amino acid residues KGGHRSN at positions 75-81 of the consensus sequence: or (g) any combination of (a)-(f). Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type III (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 Kt of less than or equal to 10-91, less than or equal to 10-1 M, less than or equal to 10-"M, less than or equal to 10-12M, less than or equal to 10-13M, less than or equal to 10-'4M, and less than or equal to 10-15M. The KD may be determined by surface plasmon resonance.
[0156] The disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
[0157] 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.
101581 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. 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.
Exemplary 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 Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).
[0159] 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 CARTyrin of the disclosure and a selection gene of the disclosure. The at least one self-cleaving peptide may
- 108 -comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A
peptide. A 12A
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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). 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 QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). 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 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). 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).
[0160] 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
- 109 -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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). 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 QCTNYALLKLAGDVESNPGP (SEQ TD NO:21). 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 GSGVKQTLNFDLLKLAGDVESNPGP (SEQ TD NO: 24). 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).
- 110 -[0161] The disclosure provides a composition comprising the transposon the disclosure. In certain embodiments, 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.
[0162] Transposons of the disclosure may comprise piggyBac transposons.
Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
[0163] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.
[0164] Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lentivirus, 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. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to all seroty, pes (e.g. AAV I, 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.
[0165] 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 (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 (MDR I), 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.
[0166] Viral vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, 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. In some embodiments, 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 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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90 A, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLICLAGDVESNPGP (SEQ ID NO: 21). 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 GSGQCTNYALLICLAGDVESNPGP (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 GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). 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 ATNIFSLLKQAGDVEENPGP (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).
[0167] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, 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.
[0168] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, 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. poly-mersomes), 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.
[0169] 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), MGN1T
(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.
[0170] Nanoparticle vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggnaaacccaggacca (SEQ ID NO:
20). 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 QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). 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 GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). 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).
[0171.1 The disclosure provides a composition comprising a vector of the disclosure.
Scaffold Proteins [01721 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.
[0173] Protein scaffolds of the disclosure may be derived from a ftbronectin type ITT (FN3) repeat protein, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods of making and using them. In a preferred embodiment, the protein scaffold is comprised of a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter "Tenascin"). In a further preferred embodiment, 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. In a preferred embodiment, the protein scaffolds of the disclosure can be designed to bind an epitope of a wild type and/or variant form of an antigen.
[0174] Protein scaffolds of the disclosure may include additional molecules or moieties, for example, the Fe region of an antibody, albumin binding domain, or other moiety influencing half-life. In further embodiments, the protein scaffolds of the disclosure may be bound to a nucleic acid molecule that may encode the protein scaffold.
[0175] 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.
[0176] 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 ITT (FN3) repeat protein, preferably, a consensus sequence of multiple FN3 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.
[0178] 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. coil, in eukaiyotic systems, such as yeast, and in in vitro transcription/translation systems, such as the rabbit reticulocy-te lysate system.
[0179] 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.
[0180] 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 fonnulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.

[0181] Moreover, 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). They should be able to bind to cellular targets and the loops can be altered, e.g., affinity matured, to improve certain binding or related properties.
[0182] Three of the six loops of the protein scaffold of the disclosure correspond topologically to the complementarity determining regions (CDRs 1-3), i.e., antigen-binding regions, of an antibody, while 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, 60-64, and 75-81 of SEQ ID NO: 1. Preferably, the loop regions at or about residues 22-28, 51-54, and 75-81 are altered for binding specificity and affmity. 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.
[0183] 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 CDRs of an antibody. In certain embodiments, 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 (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, 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 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.
[0184] Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a 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. In certain embodiments, 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. In certain embodiments, the antigen recognition region may comprise three sequences comprising one or more fragments of an antibody (e.g. three VHHs) 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.
[0185] Scaffolds of the disclosure may comprise an antibody mimetic.
[0186] The term "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 (e.g. resistance to enzymatic degradation), and lower production costs.
Exemplary 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.
[0187] Affibody molecules of the disclosure comprise a protein scaffold comprising or consisting of one or more alpha helix without any disulfide bridges.
Preferably, affibody molecules of the disclosure comprise or consist of three alpha helices. For example, 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.
[01881 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 that 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.
[0189] 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.
[0190] 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. In certain embodiments of the disclosure, 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.
101911 Alphabody molecules of the disclosure may also be referred to as Cell-Penetrating Alphabodies (CPAB). 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.
Structurally, 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.
[0192] 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.
Structurally, anticalin molecules of the disclosure comprise a barrel structure comprising antiparallel beta-strands pairwise connected by loops and an attached alpha helix. In preferred embodiments, anticalin molecules of the disclosure comprise a barrel structure comprising eight antiparallel beta-strands pairwise connected by loops and an attached alpha helix.
101931 Avimer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). Avimers 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.
[0194] DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high affmity for a target sequence. In certain embodiments, DARPins of the disclosure are 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.
[0195] 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.
[0196] Kunitz domain peptides of the disclosure comprise a protein scaffold comprising a Kunitz domain. Kunitz domains comprise an active site for inhibiting protease activity.
Structurally. 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 lipoprotein-associated coagulation inhibitor (LAC)).
[0197] 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. In preferred embodiments, 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 complementarity determining regions (CDRs) of an antibody. 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 FO. Monobodies of the disclosure may comprise an adnectin.
Production and Generation of Scaffold Proteins [0198] 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. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);
Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).
[0199] 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.
[0200] Optionally, scaffold proteins can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, 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., Immunofilter program of Xencor, Inc. of Monrovia, Calif.). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability of the candidate scaffold protein to bind its antigen.
In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved.
Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used.
piggyBac Transposon System [0201] 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. Preferably, 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. Preferably, 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. In certain preferred embodiments, the Super piggyBacTm (SPB) transposase is an mRNA sequence encoding the Super piggyBacTm (SPB) transposase.
[0202] Additional disclosure regarding piggyBac transposons and Super piggyBac Tm (SPB) transposases may be found in International Patent Publication WO 2010/099296, US Patent No. 8,399,643, US Patent No. 9,546,382, US Patent No. 6,218,185, US Patent No.
6,551,825, US Patent No. 6,962,810, and US Patent No. 7,105,343, the contents of which are each herein incorporated by reference in their entireties.
[0203] The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. Preferably, the introducing steps are mediated by the piggyBac transposon system.
[0204] In certain embodiments of the methods of the disclosure, 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. In certain embodiments, 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 piggyBac Tm or a Super piggyBacTM (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBac Tm (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.
[0205] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBacTM (PB) transposase enzyme. 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:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
4).
[0206] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac' (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:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
4).
[0207] In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, the transposase enzyme is a piggyBacrm (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. In certain embodiments, 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 (0. In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of a serine (5) for a glycine (G). In certain embodiments, 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). In certain embodiments, 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).
[0208] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBacTM (SPB) transposase enzyme. In certain embodiments, 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). In certain embodiments, the Super piggyBacTm (SPB) 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:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
5).
[0209] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ
ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the 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). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO:
4 or SEQ ID
NO: 5 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a thmonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a ttyptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, 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
ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a phenylalanine (F).
In certain embodiments, the amino acid substitution at position 180 of SEQ ID
NO: 4 or SEQ
ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 200 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenylalanine (F),In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a valine (V). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 235 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a ty, rosine (Y) for a leucine (L). In certain embodiments, 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). 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 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 saline (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a proline (P). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for an arginine (R),In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 340 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 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 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the 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). In certain embodiments, the amino acid substitution at position 456 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a lysine (K) for a serine (S). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, 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).
In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, 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).
[0210] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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 SEQ ID NO: 5. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, 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. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super piggyBacTm transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID
NO: 5. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a serine (5). In certain embodiments, 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). In certain embodiments, 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). In certain embodiments, the amino acid substitution at position 375 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the 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). In certain embodiments, the 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). In certain embodiments, the 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). In certain embodiments, the piggyBacTm transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID
NO: 4. In certain embodiments, including those embodiments wherein 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.
In certain embodiments, 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. In certain embodiments, 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.
102111 By "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.
[0212] As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.
[0213] By "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.
[0214] By "transient transformation" is intended that a poly-nucleotide construct introduced into the host does not integrate into the genome of the host.

[02151 In preferred embodiments, the piggyBac transposon system is used to introduce exogenous sequences into a primary human T cell by stable transformation to generate a modified Tscm or Tem.
Additional 7'ransposon Systems [0216] In certain embodiments of the methods of the disclosure, 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).
[0217] 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 marker(s) of a modified stem memory T-cell (Tsai) or a modified central memory T-cell (Tem), thereby producing a modified stem memory T-ull (Tscm) or a modified central memory T-cell (Tem). 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 (Tem), 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 marker(s) of a stem memory T-cell (Tscm) or a central memory T-cell (Teri), thereby producing a plurality of modified stem memory T-cells (Tscm) or a plurality of modified central memory T-cells (Tem).
[0218] In certain embodiments of the methods of the disclosure, 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).
[0219] In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 6).
[0220] In certain embodiments of the methods of the disclosure, 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:

301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7).
[0221] In certain embodiments of the methods of the disclosure, the transposase is a Helitron transposase. Helitron tmnsposases 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:

301 GAGCAGGAGG CCGCTGGACA TAGAGC.AGAG CGA.GAGAGAG GGTGGCTTGG AGGGCGTGGC
361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA. CTGAC.AGCAG GGAGGAGGAA

661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA. ACAGCGGCGT

1081 GAAAATATTC GTTCCATAAA. TAGTTCTTTT GCTTTTGCTT CCATGGGTGC AAATATTGCA

2041 AGAAATATGC AGC.AGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC

2221 AATGCTCTTT TAAA.TGATAT ATGTAAA.TTC CATTTATTTG GCAAAGTAAT AGCTAAAATT

2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA ATTGTAAAA.T CAAATATGGT ACATGGACCA

2701 ATTAAAAGTG TCAAATATTT ATTTAAA.TAC ATCTATAAAG GGCACGATTG TGCAAATATT

2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA. TGCGAA.TGCA TGACCAATCT

3301 GATACGATTG ACGA.TGCAAT CATCCTTAAT ATGCCCAAAC AACTACGGCA ACTTTTTGCA

3541 CATTTCA.kAC TTCCGGACTA TCCTTTATTA ATGAATGCAA ATACAT GT GA TCAATTGTAC

3841 GGAAGAACCT TTC.ATTCCCA ATATAAA.TTA CC.AATTCCAT TAAATGAAAC TTCAATTTCT

4021 ATTATGAATT T GAA.T GTT GC ATTTGGTGGG AAA.GTTCTCC TTCTCGGAGG GGATTTTCGA

4201 GATTCTGCTT ATAGTGAATG GTTAGTAAAA CTTGGAGATG GCAAACTT GA. TAGCAGTTTT

4441 GAT GGAGATT TTC.ACACATA TTT GAGT GAT GATTCCATTG ATTCAA.CAGA T GAT GCT GAA

5281 TGCACCGGGC CACTAG ( SEQ ID NO: 27 ) .
[0222] Unlike other transposases, the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a Reptlel 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.
[0223] An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:

1441 LPEPVFAHGQ LYVAFSRVRR ACDVFVKVVN TSSQGKLVFH SESVFTLNVV YREILE (SEQUJ
NO: 28).
[0224] In Helitron transpositions, a hairpin close to the 3 end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, 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 GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 29).
[0225] In certain embodiments of the methods of the disclosure, the transposase is a To12 transposase. To12 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family. Exemplary To12 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the To12 transposase, which contains four exons. An exemplary To12 transposase of the disclosure comprises an amino acid sequence comprising the following:

601 NTPLRASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SE:QM/if/30).
[0226] An exemplary To12 transposon of the disclosure, including inverted repeats, subterininal sequences and the To12 transposase, is encoded by a nucleic acid sequence comprising the following:

301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA. GTGACGTCAT GTCACATCTA

1321 AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAA.kA

2461 GATCATGAAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACCACAA.0 2641 TTTTGAGGTA CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAAA. TACGTGACAA

2941 TAACCTAGTC TCAA.GCGTTG ATGCCC.AAAA AGCTCTCTC.A AATGAA.CACT ACAA.GAAACT

3541 AAGATCCTGA GATCATAGCA GCTGCC.ATCC TTCTCCCTAA. ATTTCGGACC TCTTGGAC.AA

4081 ATGATGAAGA TTTTTTCGCT TCTTTGAAAC CGA.CAACAC.A TGAAGCCAGC AAAGAGTTGG

4381 CATTAGATTG TCTGTCTTAT AGTTTGATAA TTAAATACAA ACAGTTCT.A.A. AGCAGGATAA

4681 TG (SEQ ID NO: 31).
Homologous Recombination [0227] In certain embodiments of the methods of the disclosure, 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. In certain embodiments of the disclosure, 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.
102281 In certain embodiments of the methods of the disclosure, homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a "genomic safe harbor" site. In certain embodiments, a mammalian genomic sequence comprises the genomic safe harbor site. In certain embodiments, a primate genomic sequence comprises the genomic safe harbor site. In certain embodiments, a human genomic sequence comprises the genomic safe harbor site.
[0229] 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 (CCR.5) gene and the site of the human ortholog of the mouse Rosa26 locus.
[0230] In certain embodiments of the methods of the disclosure, 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). In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the ME-IC disrupts the endogenous gene, and optionally, replaces part of the coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MI-IC disrupts the endogenous gene, and optionally, replaces the entire coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments of the methods 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. In certain embodiments of the methods 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 or the therapeutic protein to a transcriptional or translational regulatory element. In certain embodiments of the methods 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 or the therapeutic protein to a transcriptional regulatory element. In certain embodiments, the transcriptional regulatory element comprises an endogenous T cell 5' U'TR.
102311 In certain embodiments of the introduction step comprising a homologous recombination, 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%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of the total number of primary T cells in 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 each primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a single strand break. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a double strand break. In certain embodiments of the introduction step comprising a homologous recombination, the introduction step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments, 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. In certain embodiments, 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 1400, or at least 1500 bp, at least 1600 bp, at least 1700 bp, at least 1800 bp, at least 1900 bp, at least 2000 bp in length or any length of base pairs (bp) in between, inclusive of the end points. 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 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. 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.
In certain embodiments of the genomic editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic Acid (D) at position 10 (DIM) and a substitution of Alanine (A) for Histidine (H) at position 840 (H840A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32, comprising a substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (Dl OA) and a substitution of an Alanine (A) for an Asparagine (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a type IIS
endonuclease. In certain embodiments of the genomic editing composition, the type IIS
endonuclease comprises AciI, Mn1I, AlwI, BbvI, Bed, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, MbolI, My 11, He!, SfaNI, Acid, BciVI, BfuAI, BmgBI, Bnut BpmI, BpuEl, BsaI; BseRI, BsgI, BsmI; BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Btsl, Ear!, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQI, SapI, Bad, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments, the type ITS endonuclease comprises Clo051. In certain embodiments of the genomic editing composition, 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. In certain embodiments of 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.
[0232] In certain embodiments of the methods of homologous recombination of the disclosure, 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 homodimer or a heterodimer. In certain embodiments, 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. In certain embodiments, the Cas9 is a catalytically inactive or "inactivated"
Cas9 (dCas9). In certain embodiments, the Cas9 is a catalytically inactive or "inactivated"
nuclease domain of Cas9. In certain embodiments, 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.
[0233] In certain embodiments, the inactivated, small, Cas9 (dSaCas9) operatively-linked to an active nuclease. In certain embodiments, 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).
In certain embodiments, the dSaCas9 of the disclosure comprises the mutations DlOA and (underlined and bolded) which inactivate the catalytic site. In certain embodiments, the dSaCas9 of the disclosure comprises the amino acid sequence of.

1021 ASKTQSIKKY STDILGNLYE VKSKKHPQII KKG (SEQIDNID:32).
[0234] In certain embodiments, the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphyloccocus pyogenes. In certain embodiments, 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 D 1 OA and H840A. In certain embodiments, the amino acid sequence of the dCas9 comprises the sequence of:

1321 RAAFKYFDTT IDRKRYTSTK EVIDATLIHQ SITGLYETRI DLSQLGGD (SEXPE)NO:
33).
[0235] In certain embodiments of the disclosure, 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, AciI, Mn1I, AlwI, Bbvi, Bed, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo IT, Myl.I, PleI, SfaNT, AcuI, BciVI., BfuAI, BmgBI, BmrI, BprnI., BpuET, BsaI, BseRI, Bsgl, BsmI, BspM1, BsrBI, BsrBI, BsrD1, BtgZIõ Btsl, Earl, Ecil, MmeI, NmeAIII, BbvCI, Bpul OI, BspQI, SapI, Bad, BsaXI, CspCI, BfiI, Mboll, Acc36I, FokI or Clo051.
In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and Clo051.An exemplary Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:
EGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRH
LGGSRKPDGIVY STrLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVN
PNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMITGVNGSAVNWNLLLGAE
KIRSGEM'TTEELERAMFNNSEFILKY (SEQ ID NO: 34).
[0236] 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):
MAPKICKRKVEGIKSNISLLKDELRGOISHISHEYLSLIDLAFDSKONRLFEMKVLELL
VNEYGFKGRHLOGSRKPDGIVYSTTLEDNFGIIVDTKAY SEGYSLPISOADEMERY VR
ENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEOLARLSMITGVNGSAV
NVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKY GGGGSDKKESIGLAIG.TNSVGWA
T/ITDEY KVP SKKEKVIL NTDRH SIKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRIC Y
LQEIFSNEMAKVDDSFTHRLEESFLVEEDKKHERHPIFGNIVDEVAYH LKYPTIYHLRKKL
VDSTDKADLRLIYLALAIIMIKFRGHFLIEGDLNPDATSDVDKLFIQLVQTYNQLFEENP INA
S'GFDAKAILSARLSICSRRLENIJAQLPGEKKNGLFGATHALSI,GLTPNFKSIVFDLAEDAKLQ
LSKDTYDDDLDNLLAQIGDQYADIFLAAKNLSDAILLSDILRVNTEITKAPLS'ASMIKRY DE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE'EFTKEIKPILEKVIDGYE
ELLVKLNREDLIRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFIXDNREKIEKILTFRIP
YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTA,TDKNLPNEKVLP
MELLY EYFIVYNEL7KVKY VIEGMRKPAITS'GEQKKAIVDLLFKTNRKVTVKQLKEDY F.
KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREVI
lEERIKTYAHLFDDKVMKQLKIIRRYTGWGRLSRKLINGIRDKQSGKTILDFLICS'DGFANR
NFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHE HANLAGSP AIKKGILQTVKVVDELVKVM
GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VE.ATTQLQNEKL
YLYY LQNGRDMYVDQELDINRLSDY DVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQUNA KLITQRKFDM,TKAERGGLSELDKAGFIKRQLVETRQITKHV
AQlLDSRAEVTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV
VGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN
GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV.NIVKKTEVQTGGFSKESIIPKRN
SDKLlARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK
NPIDFLEAKGY KEVICKDLIIKLPKY SLIELENGRKIMASAGELQKGNE'LALPSKYVNFLY

LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHR
DKPIREQAENHHLFTLTNLGAPAAFKYFDITIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
SQLGGDGSPICKKRKVSS (SEQ ID NO: 40).
102371 In certain embodiments, 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 Xanthornonas and Ralstonia.
[0238] In certain embodiments of the disclosure, 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. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of AciI, Mn!!, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, Hphl, HpyAV, Mboll, My1I, PleI, SfaNI, AcuI, BciVI, BfuAl, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, Sap!, Bad, BsaXI, CspCT, Bfli, MboII, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the type IIS
endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID NO: 34).
102391 In certain embodiments of the disclosure, 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. In certain embodiments of the disclosure, the type IIS
endonuclease may comprise, consist essentially of or consist of AciT, Mn!!, AlwI, BbvI, BccI, BceAT, BsmAI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, MbolI, My 1 I, PleI, SfaNI, AcuI, BciVI, BfuAl, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, Mind, NmeAIII, BbvCI, Bpul0I, BspQI, Sap!, BaeI, BsaXI, CspCI, BfiI, Mboll, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the type IIS
endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID
NO: 34).
102401 In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be covalently linked. For example, a fusion protein may comprise the DNA binding domain and the nuclease domain.
In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be operably linked through a non-covalent linkage.
Secreted Proteins from Modified T Cells 102411 In certain embodiments of the composition and methods of the disclosure, modified T-cells express therapeutic proteins. Therapeutic proteins of the disclosure include secreted proteins. Preferably, in a therapeutic context, the therapeutic protein is a human protein, including a secreted human protein. When expressed or secreted by CAR-T cells of the disclosure, the combination comprising the CAR-T cell and the therapeutic protein secreted therefrom may be considered a monotherapy. However, 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%20proteins, 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.
102421 TABLE 1. Exemplary Human Secreted Proteins Gene En sem bl ID Gene description A1BG ENSG00000121410 Alpha-1-B glycopmtein A2M ENSG00000175899 Alpha-2-macroglobulin A2ML1 ENSG00000166535 Alpha-2-macroglobulin-like 1 A4GNI ENSG00000118017 A 1pha-1.4-N-acetylglucosain iny hransferase AADACL2 ENSG00000197953 Arylacetamide deacetylase-like AANAT EN SG00000129673 Aralky famine N-acctyltransfcrasc ABCGI. EN SG00000160179 ATP-binding casselte. sub-family G (WHITE), member 1 ABHD1 ENSG00000143994 Abhydrolase domain containing ABHDIO ENSG00000144827 Abhydrolase domain containing ABHD14A ENSG00000248487 Abhydrolase domain containing ABHD15 ENSG00000168792 Abhydrolasc domain containing ABI3BP EN SG00000154175 ABI family, member 3 (NESH) binding protein AC008641.1 ENSG00000279109 AC009133.22 ENSG00000277669 AC009491.2 ENSG00000279664 A01 513.3 EN SG00000267881 ' AC136352.5 ENSG00000277666 AC145212.4 ENSG00000277400 MaFF-interacting protein AC233755.1 ENSG00000275063 ACACB ENSG00000076555 Acetyl-CoA cartiox-y lase beta ACAN ENSG00000157766 . Aggrecart ' ACE ENS600000159640 Angiotensin 1 converting enzyme ACHE ENSG00000087085 Acetylcholinesterase (Yt blood group) AC7P2 EN SG00000134575 Acid phosphatase 2. lysosomal ACP5 ENSG00000102575 Acid phosphatase 5, tartrate resistant ACP6 ENSG00000162836 . Acid phosphatase 6, iysophosphatidic ACPP ENSG00000014257 Acid phosphatase, prostate ACR EN SG00000100312 Acrosin AC7RBP EN SG00000111644 Acrosin binding protein ACRV1 ENSG00000134940 Acrosomal vesicle protein 1 ACSF2 ENSG00000167107 Acyl-CoA synthetase family member 2 ACTT., 10 ENSG00000182584 Actin-like 10 ACVR1 EN SG00000 115170 Activin A receptor, type I
AC7VR1C EN SG00000123612 Activin A receptor, type IC
ACVRI., I ENSG00000139567 Activin A receptor type II-like 1 ACYP1 ENSG00000119640 Acylphosphatase 1, elythrocyte (common) type ACYP2 ENSG00000170634 Acylphosphatase 2, muscle type ADAM10 ENSG00000137845 ADAM metallopeptidase domain 10 ADAM12 EN SG00000148848 ADAM metallopeptidase domain 12 ADAM15 ENSG00000143537 ADAM metallopeptidase domain 15 ADAM17 ENSG00000151694 ADAM metallopeptidase domain 17 ADAM 18 ENSG00000168619 . ADAM metallopeptidase domain 18 ADAM22 ENSG00000008277 ADAM metallopeptidase domain 22 ADAM28 ENSG00000042980 ADAM metallopeptidase domain 28 ADAM29 ENSG00000168594 ADAM metallopeptidase domain 29 ADAM32 ENSG00000197140 ADAM metallopeptidase domain 32 ADAM33 ENSG00000149451 . ADAM metallopeptidase domain 33 ADAM7 ENSG00000069206 ADAM metallopeptidase domain 7 ADAMS EN SG00000151651 ADAM metallopeptidase domain 8 ADAM9 EN SG00000168615 ADAM metallopeptidase domain 9 ADAMDEC I ENSG00000134028 ADAM-like, decysin 1 ADAM.rs 1 EN SG00000154734 . ADAM metallopeptidase with thrombospondin type 1 motif, 1 .
ADAMTS 10 EN SG00000142303 ADAM metallopeptidase with thrombospondin type 1 motif, 10 ADAMTS12 EN SG00000151388 ADAM metallopeptidase with thrombospondin type 1 motif, 12 A DAMTS13 EN SG00000160323 ADAM metallopeptidase with thrombospondin type 1 motif, 13 ADAMTSI4 ENSG00000138316 ADAM metallopeptidase with thmmbospondin type 1 motif, 14 ADAM.rs15 EN SG00000166106 _ ADAM metallopeptidase with thrombospondin type 1 motif, 15 A DAMTS16 ENSG00000145536 ADAM metallopeptidase with thrombospondin type 1 motif, 16 ADAMTSI7 ENSG00000140470 ADAM metallopeptidase with thmmbospondin type 1 motif, 17 ADAM.rs18 EN SG00000140873 . ADAM inetallopepi idase with ihipmbospondin type 1 motif, 18 .
ADAMTSI9 EN SG00000145808 ADAM metallopeptidase with thrombospondin type 1 motif, 19 ADAMTS2 ENSG00000087116 ADAM metallopeptidase with thrombospondin type 1 motif, 2 ADAMTS20 EN SG00000173157 ADAM metallopeptidase with thrombospondin type 1 motif, 20 ADAMTS3 ENSG00000156140 ADAM metallopeptidase with thrombospondin type 1 motif, 3 ADAM.rs5 EN SG00000154736 . ADAM inetallopepi idase with ihminbospondin type 1 motif, 5 .
ADAmTs6 ENSG00000049192 ADAM metallopeptidase with thrombospondin type 1 motif, 6 ADAMTS7 ENSG00000136378 ADAM metallopeptidase with thrombospondin type 1 motif, 7 ADAMTS8 ENSG00000134917 ADAM metallopeptidase with thrombospondin type 1 motif, 8 ADAMTS9 ENSG00000163638 ADAM metallopeptidase with thrombospondin type 1 motif, 9 ADAM.rsLI ENSG00000178031 ADAMTS-like 1 ADAMISL2 ENSG00000197859 ' ADAMTS-like 2 ADAMTSL3 EN SG00000156218 ADAMTS-like 3 ADAM-MI.4 EN SG00000143382 ADAM=rs-like 4 ADAMTSL5 EN SG00000185761 ADAMTS-like 5 ADCK1 ENSG00000063761 AarF domain containing kinase 1 ADCYA PI ENSG00000141433 Adenylate cyclase activating polypeptide 1 (pituitary) ADCYAP IR I ENSG00000078549 Adeny late cyclase activating polypeptide 1 (pituitary) receptor type I
ADGRA3 ENSG00000152990 Adhesion (.3 protein-coupled receptor A3 ADGRB2 ENSG00000121753 Adhesion G protein-coupled receptor B2 ADGRD1 ENS(300000111452 . Adhesion G protein-coupled receptor DI
=
ADGRE3 EN SG00000131355 Adhesion G protein-coupled receptor E3 ADGRE5 EN SG00000123146 Adhesion G protein-coupled receptor E5 ADORE 1 ENSG00000153292 Adhesion (.3 protein-coupled receptor Fl ADGRG1 ENSG00000205336 Adhesion G protein-coupled receptor GI
ADGRG5 ENSG00000159618 . Adhesion G protein-coupled receptor G5 =
ADGRG6 ENSG00000112414 Adhesion G protein-coupled receptor G6 ADGRV1 EN S(300000164199 Adhesion G protein-coupled receptor VI
ADII ENSG00000182551 Aciteductone dioxygenase 1 ADIG ENSG00000182035 Adipogenin ADIPOQ EN SG00000181092 Adiponectin. C1Q and collagen domain containing =
ADM ENSG00000148926 Adrenomedullin ADM2 ENSG00000128165 Adrenomedullin 2 ADM5 ENS600000224420 Adrenomedulliti 5 (pill:give) ADPGK ENSG00000159322 ADP-dependent glucokinase ADPRHE2 ENSG00000116863 ADP-ribosylhydrolase like 2 AEBP1 ENSG00000106624 AE binding protein 1 ARI ENSG00000079557 Afamin AFT ENSG00000081051 Alpha-fetoprotein AGA, ENSG00000038002 Aspartylglucosaminidase AGER ENSG00000204305 Advanced glycosylation end product-specific receptor ,GK ENS600000006530 _ Acylglycerol kinase AGPS ENSG00000018510 Alkylglycerone phosphate synthase AGR2 ENSG00000106541 Anterior gradient 2, protein disulphide isomerase family member AGR3 ENSG00000173467 Anterior gradient 3, protein disulphide isomerase family member AGRN EN SG00000188157 Agrin AGRP ENSG00000159723 Agouti related neuropeptide AGT ENSG00000135744 Angiotensinogen (serpin peptidase inhibitor, clade A. member 8) AGTPBP1 EN SG00000135049 A7113/GTP binding protein 1 AGTRAP ENSG00000177674 Angiotensin II receptor-associated protein AFICYL2 EN SG00000158467 Adetiosylhoinocysteinase-like 2 AHSG ENSG00000145192 Alpha-2-HS-glycoprotein AIG I ENSG00000146416 Androgen-induced I
AK4 ENSG00000162433 Adertylate kinase 4 AKAPIO ENSG00000108599 A kinase (PRKA) anchor protein 10 AKR1C71 EN SG00000187134 Aldo-keto reductase family 1, member CI
AL356289.1 ENSG00000279096 AL589743.1 ENSG00000279508 ALAS2 ENSG00000158578 . 5'-aminolevulinate synthase 2 ALB ENSG00000163631 Albumin ALD119.A1 EN SG00000143149 Aldehyde dehydrogeriase 9 family, member Al ALDOA ENSG00000149925 Aldolase A, 1.i7tictose-bisphosphate ALG1. ENSG00000033011 ALGI, chitobiosyldiphosphodolichol beta-mannosyltransferase ALG5 ENSG00000120697 . ALG5, dolichyl-phosphate beta-glitcosyltransferase ALG9 ENSG00000086848 ALG9, alpha-1,2-mannosyltransferase ALKBH1 ENSG00000100601 AlkB homolog 1, historic H2A dioxygenase ALKBH5 ENSG00000091542 AlkB homolog 5, RNA demethylase ALP! ENSG00000163295 Alkaline phosphatase, intestinal ALPL ENSG00000162551 . Alkaline phosphatase, liver.tortelidney ALPP ENSG00000163283 Alkaline phosphatase, placental ALPPL2 ENSG00000163286 Alkaline phosphatase, placental-like 2 AMBN EN SG00000178522 Ameloblastin (enamel matrix protein) AN1BP ENSG00000106927 Alpha-l-microglobulinibikunin precursor ANIELX ENSG00000125363 . Amelogenin, X-linked AMELY ENSG00000099721 Atnelogenin, Y-linked AM!-! ENSG00000104899 Anti-Mullerian hormone AMICA1 EN SG00000160593 Adhesion molecule, interacts with CXADR
antigen 1 ANIPD1 ENSG00000116748 Adenosine monophosphate deaminase 1 Am-rN ENSG00000187689 . Atnelotin AMY IA ENSG00000237763 Amylase, alpha IA (salivary) AMY 1B ENSG00000174876 Amylase, alpha 1S (salivaiy) AMY IC EN SG00000187733 Amylase, alpha IC (salivary) ANIY2A ENSG00000243480 Amylase, alpha 2A (pancreatic) ANIY2B ENSG00000240038 Amylase, alpha 2B (pancreatic) ANG ENSG00000214274 Angiogenin, ribonuclease, RNase A family, 5 ANGEL I EN SG00000013523 Angel hornolog 1 (Drosophila) ANGPT1 ENSG00000154188 Angiopoiet in 1 ANGPT2 ENSG0000009 1879 . Atigiopoielin 2 ANGPT4 ENSG00000101280 Angiopoietin 4 ANGPTL I ENSG00000116194 Angiopoietin-like 1 ANGPIL2 EN SG00000136859 Angiopoietin-like 2 ANGPTL3 ENSG00000132855 Angiopoietin-like 3 ANGPTL4 ENSG00000167772 . Angiopoietin-like 4 ANGPTL5 ENSG00000187151 Angiopoietin-like 5 ANGPTL6 ENSG00000130812 Angiopoietin-like 6 ANGPTL7 EN SG000001718I9 Angiopoietin-like 7 ANKI ENSG00000029534 Ankyrin I, erythmcy tic ANKDD IA ENSG00000166839 . Ankyrin repeat and death domain containing IA
ANKRD54 EN SG00000100124 Anky rin repeat domain 54 ANKRD60 ENSG00000124227 Ankyrin repeat domain 60 ANO7 EN SG00000146205 A noctamin 7 ANOS1 ENSG00000011201 Anosmin 1 ANTXR1 ENSG00000169604 Anthrax toxin receptor 1 AOAH EN SG00000136250 Acy loxyacy I hydrolase (neutrophil) AOCI ENSG00000002726 Amine oxidase, copper containing 1 A0C2 EN SG00000131480 Amine oxidase, copper containing 2 (retina-specific) A0C3 ENSG00000131471 Amine oxidase, copper containing 3 AP000721.4 ENSG00000256100 AP000866.1 EN SG00000279342 APBB1 ENSG00000166313 Amyloid beta (A4) precursor protein-binding, family B, member 1 (Fe65) APCDD I EN SG00000154856 Adenomatosis polyposis col i down-regulated 1 APCS ENSG00000132703 Amyloid P component, serum APELA ENSG00000248329 . Apelin receptor early endogenous ligand APLN ENSG00000171388 Apelin APLP2 ENSG00000084234 Amyloid beta (A4) precursor-like protein 2 ARCM! EN SG00000118 I 37 Apolipoptotein A.4 APOA 1BP ENSG00000163382 Apolipoprotein A-I binding protein AP0A2 ENSG00000158874 . Apolipoprotein A-II
AP0A4 EN SG00000110244 Apolipoprotein A-IV
AP0A5 ENSG00000110243 Apolipoprotein A-V
APOB EN SG00000084674 Apolipoptotein B
APOC I ENSG00000130208 Apolipoprotein C-I
APOC2 ENSG00000234906 Apolipoprotein C-II
APOC3 EN SG00000110245 Apolipoprotein C-III
APOC4 ENSG00000267467 Apolipoprotein C-IV
APOC74-.APOC2 EN SG00000224916 APOC4-APOC2 teadt htough (NM)) candidate) APOD EN SG00000189058 Apolipopmein D
APOE ENSG00000130203 Apolipoprotein E
APOF EN SG00000175336 _ Apolipoprotein F

APOH ENSG00000091583 Apolipopmein H (beta-2-glycoprotein I) APOL1 ENSG00000100342 Apolipopmtein L, 1 APOL3 ENS(300000128284 . Apolipoprotein L, 3 APOM ENSG00000204444 Apolipoprotein M
APOOL ENSG00000155008 Apolipoptptein 0-like ARCN I. ENS0000000951.39 Archain 1 ARFIP2 ENSG00000132254 ADP-ribosylation factor interacting protein ARHGAP36 ENS(300000147256 . Rho GTPase activating protein 36 ARHGAP6 ENS600000047648 Rho GTPase activating protein 6 ARHGEF4 ENSG00000136002 Rho guanine nucleotide exchange factor (GEF) 4 ARL16 EN SG00000214087 ADP-ribosylation factor-like 16 ARMC5 ENSG00000140691 Armadillo repeat containing 5 ARNIE, ENS(300000133794 . Aryl hydrocarbon receptor nuclear ttanslocator-like ARSA EN SG00000100299 Aiyistilfatase A
ARSB ENS000000113273 Arylsulfatase B
ARSE ENSG00000157399 A rylsuliatase E (chondrodysplasia punctata 1) ARSG ENSG00000141337 Arylsulfatase 0 ARS1 ENSG00000183876 Arylstilfatase family, member 1 ARSK EN S600000164291 Arylsulfatase family, member I( ART3 EN SG00000156219 ADP-ribosyltransferase 3 ART4 ENSG00000 I 11339 ADP-ribosyltransferase 4 (Doinbrock blood group) ARTS ENSG00000167311 ADP-ribosyltransferase 5 ARTN ENS000000117407 Artemin ASAH1 EN SG00000104763 N-acyisphingosine arnidohydrolase (acid ceramidase) 1 ASAH2 EN SG00000188611 N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) ASCI, I. ENSG00000139352 Achaete-scute family blILH transcription factor 1 ASIP ENS000000101440 Agouti signaling protein ASPN NS000000I068 I 9 . Asporin ASTI, ENSG00000188886 Astacin-like trietallo-endopeptidase (M12 family) ATAD5 ENSG00000176208 ATPase family, AAA domain containing 5 ATATI EN SG00000137343 Alpha tubtilin acetyltransferase 1 ATG2A ENSG00000110046 Autophagy related 2A
ATG5 ENS000000057663 . Autophagy related 5 AMAIN EN SG00000166454 ATM interactor ATP13A1 ENSG00000105726 ATPase type 13A1 ATP5F1 ENS000000 I 16459 ATP synthase, H+ transporting, mitochondria! Fo complex.
subunit B1.
ATP6AP I ENSG00000071553 ATPase, H+ transporting, lysosomal accessory protein I
ATP6AP2 ENS(300000182220 . ATPase, H.4- transporting, lysosomal accessory protein 2 ATPAIT1 ENSG00000123472 ATP synthase mitochondrial Fl complex assembly factor 1 AUTH ENSG00000148090 AU RNA binding proteinknoyl-CoA hydratase MT ENS000000101200 Argi nine vasopressin AXIN2 ENSG00000168646 Axin 2 AZ,GP1 ENS(300000160862 _ Alpha-2-glycoprotein 1, zinc-binding AZU1 ENSG00000172232 Azurocidin 1 B2M ENSG00000166710 Beta-2-microglobulin B3GALNT1 ENSG00000169255 Beta-1.3-N-acetylga1actosaminy It tansferase I (globoside blood group) B3GALNT2 ENSG00000162885 Beta-1,3-N-acetylgalactosaminyltransferase B3GALT1 EN SG00000172318 UDP-Gal:betaGicNAc beta 1,3-galactosy It ransferase, poly peptide 1 B3GALT4 ENSG00000235863 UDP-Gal:betaGicNAc beta 1,3-galactosyltransferase, poly peptide 4 B3GALT5 ENSG00000183778 UDP-Gal:betaGIcNAc beta 1,3-ga1actosyltransferase, poly peptide 5 B3GALT6 ENSG00000176022 1.1DP-Gal:betitGal beta 1,3-galactosy It ransferase polypeptide 6 B3GAT3 ENSG00000149541 Beta-1,3-glucuronyltransfentse 3 B3GLCT ENSG00000187676 Beta 3-glucosyltransferase B3GNT3 ENSG00000179913 UDP-GIcNAc:betaCial beta-1,3-N-acety Ighicosaminy It ra nsfe rase 3 B3GNT4 EN SG00000176383 UDP-GIcNAc:betaGal beta-1.3-N-acetylglucosaminvItransferase 4 B3GNT6 EN SG00000198488 LIDP-G1cNAc:betaGal beta-1,3-N-acetylghicosaminvItransferase 6 B3GNT7 ENSG00000156966 UDP-GIcNAc:betaCial beta-1,3-N-acety Ighicosaminy It ra nsfe rase 7 B3GNT8 ENSG00000177191 UDP-GleNAc:betaGal beta-1,3-N-acetvIglucosaminyltransferase 8 B3GNT9 EN SG00000237172 LIDP-G1cNAc:betaGal beta-1,3-N-acetylghicosaminvItransferase 9 B4GALNTI. ENSG00000135454 Beta-1.4-N-acetyl-galactosaminy I
transferase 1 B4GALNT3 ENSG00000139044 Beta-1,4-N-acetyl-galactosaminyl transferase 3 B4GALNT4 ENSG00000182272 Beta-1,4-N-acetyl-galactosaminyl transferase 4 B4GALT4 EN SG00000121578 1.1DP-Gal:betaGicNAc beta 1,4-galactosylltansferase, poly peptide 4 B4GALT5 ENSG00000158470 UDP-Gal:betaGIcNAc beta 1,4-galactosyltransferase, poly peptide 5 B4GALT6 ENSG00000118276 UDP-Gal:betaGIcNAc beta 1,4-galactosyltransferase, poly peptide 6 B4GAT1 EN SG00000174684 Beta-1,4-gluctironyltransferase I
1391)1 EN SG00000108641 89 mteiti domain 1 BACE2 ENSG00000182240 Beta-site APP-cleaving enzyme 2 SAGES ENSG00000279973 B melanoma 'antigen family, member 5 BCAM ENSG00000187244 Basal cell adhesion molecule (Lutheran blood group) BCAN ENSG00000132692 Brevican BCAP29 ENSG00000075790 B-cell receptor-associated protein 29 BCAR1 ENSG00000050820 Breast cancer anti-estrogen resistance 1 BCHE EN SG00000114200 Buty ry Icholi riesterase BCKDIAB EN SG00000083123 Branched chain keto acid dehydrogenase El, beta polypeptide BDNF ENSG00000176697 Brain-derived neurotrophic factor BGLAP ENSG00000242252 Bone gamma-carboxyglutamate (gla) protein BGN ENSG00000182492 Biglycan BLVRB ENSG00000090013 Biliverdin teductase BMP1 ENSG00000168487 Bone morphogenetic protein 1 BMPIO EN SG00000163217 Bone morphogenetic protein 10 BMP15 ENSG00000130385 Bone morphogenetic protein 15 BMP2 ENSG00000125845 . Bone morphogenetic protein 2 BIVIP3 ENSG00000152785 Bone morphogenetic protein 3 BMP4 ENSG00000125378 Bone morphogenetic protein 4 BMP6 EN SG00000153162 Bone morphogenetic protein 6 BMP7 ENSG00000101144 Bone morphogenetic protein 7 BMP8A ENSG00000183682 . Bone morphogenoic protein 8a BMP8B ENSG00000116985 Bone morphogenetic protein 8b BMPER ENSG00000164619 BMP binding endothelial regulator BNC I ENSG00000169594 Basonuclin 1 BOC ENSG00000144857 BOC cell adhesion associated, oncogene regulated 501)1 ENSG00000145919 . Biorientat ion of chromosomes in cell division 1 BOLA1 ENSG00000178096 BolA family member 1 BPI EN SG00000101425 Bactericidal/penneability-increasing protein BPWA1 EN S000000198183 BPI fold containing family A, member 1 BPITA2 EN SG00000131050 BPI fold containing family A. member 2 BPIFA3 EN SG00000131059 BPI fold containing family A. member 3 BPIFB I ENSG00000125999 BPI fold containing family B, member 1 BPIFB2 ENSG00000078898 BPI fold containing family 13, member 2 5PW53 EN SG00000186190 BPI fold containing family B, member 3 BPITB4 EN SG0000018619 I BPI fold containing family B, member 4 BPIFB6 ENSG00000167104 BPI fold containing family B, member 6 BPIFC ENSG00000184459 BPI fold containing family C
BRF1 ENSG00000185024 BRF I, RNA polymerase III transcription initiation factor 90 kDa subunit BR INT! ENS000000078725 Bone morphogenetic proteinhetinoic acid inducible neural-specific 1 BRINP2 ENSG00000198797 Bone morphogenetic proteinketinoic acid inducible neural-specific 2 .
BRINP3 ENSG00000162670 Bone moiphogenetic proteinketinoic acid inducible neural-specific 3 BSC; EN SG00000172274) Basigin (Ok blood group) BSPH I EN SG00000188334 Binder of sperm protein homolog 1 BST). ENSG00000109743 Bone marrow stromal cell antigen 1 B15D1.7 ENS600000204347 BIB (POZ) domain containing 17 BTD ENSG00000169814 Biotinidase 5IN2A2 EN SC500000124508 Butyrophilin, subfamily 2, member A2 BIN3A1 ENSG00000026950 Bay rophilin, subfamily 3, member AI
BTN3A2 EN S00000018647() Butyrophilin, subfamily 3, member A2 5IN3A3 ENSG00000111801 Butyrophilin, subbruily 3, member A3 Cl0orf10 ENSG00000165507 Chromosome 10 open reading frame 10 C10od'99 ENSG00000188373 Chromosome 10 open reading frame 99 CI lorf1 ENSG00000137720 Chromosome 11 open reading frame 1 Cliorf24 ENSG00000171067 Chromosome 11 open reading frame 24 Cllor145 EN SG00000174370 _ Chromosome 11 open reading frame 45 CI 1orf94 ENSG00000234776 Chromosome 11 open reading frame 94 C12orf10 ENSG00000139637 Chromosome 12 open reading frame 10 Cl2or149 ENSG00000111412 . Chtomosome 12 open reading frame 49 Cl2orf73 ENSG00000204954 Chromosome 12 open reading frame 73 C I 2orf76 ENSG00000174456 Chromosome 12 open wading frame 76 C I 4orf80 EN SG00000185347 Chromosome 14 open reading frame 80 C14orf93 ENSG00000100802 Chromosome 14 open reading frame 93 C16or189 EN SG00000153446 . Chromosome 16 open reading frame 89 CI6or(90 ENSG00000215131 Chromosome 16 open reading frame 90 Cl 7orf67 ENSG00000214226 Chromosome 17 open wading frame 67 C I 7 05 ENSG00000108666 Chromosome 17 open reading frame 75 C17orf99 ENSG00000187997 Chromosome 17 open reading frame 99 CI8orl.54 ENSG00000166845 . Chromosome 18 open reading frame 54 C I9orf47 ENSG00000160392 Chromosome 19 open reading frame 47 C I 9orf70 ENSG00000174917 Chromosome 19 open wading frame 70 CI 9otf80 EN SG00000 I 30173 Chromosome 19 open reading frame 80 C 1 GALT I ENSG00000106392 Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 Clorf127 EN SG00000175262 Chromosome 1 open reading frame 127 C1orf159 EN SG00000131591 Chromosome 1 open reading frame 159 C1ot1198 EN SG00000119280 Chromosome 1 open reading frame 198 C1orf234 ENSG00000227868 Chromosome I open reading frame 234 CI or154 ENSG00000 I 18292 Chromosome 1 open reading frame 54 C I orf56 ENSG00000143443 Chromosome 1 open reading frame 56 C I QA ENSG00000173372 Complement component 1. q subcomponent, A
chain C71QB ENSG00000173369 . Complement component 1, q subcomponent, B
chain CIQC EN SG00000159189 Complement component I, q subcomponent. C
chain Cl QL1 EN SG00000131094 Complement component I, q subcomponent-like 1 CIQL2 ENSG000001441.19 Complement component 1, q subcomponent-like 2 C1QL3 ENSG00000165985 Complement component 1, q subcomponent-like C1Q1,4 ENSG00000186897 . Complement component 1, q subcomponent-like 4 C I WW1 ENSG00000173918 C lq and tumor necrosis factor related protein 1 C1QTNF2 ENSG00000145861 C iq and tumor necrosis factor related protein 2 CIQINF3 ENSG00000082196 Clq and tumor necrosis factor related protein 3 C1QTNF4 ENSG00000172247 Clq and tumor necrosis factor related protein 4 C1QTNIF5 ENSG00000223953 . Clq and tumor necrosis factor related protein 5 CIQTNF7 EN SG00000163145 C lq and tumor necrosis factor related protein 7 Cl QTNF8 ENSG00000184471 C iq and tumor necrosis factor related protein 8 C I QTINIF9 ENS600000240654 Clq and tumor necrosis factor related protein 9 C1QTNF9B ENSG00000205863 Clq and tumor necrosis factor related protein 9B
C71R ENSG00000159403 Complement component 1, r subcomponent CIRI, EN SG00000139178 Complement component 1, r subcomponent-like CIS ENSG00000182326 Complement component I, s subcomponent C2 ENSG00000166278 Complement component 2 C2 lorf33 EN SG00000160221 Chromosome 21 open reading frame 33 C2 lorf62 ENSG00000205929 Chromosome 21 open reading frame 62 C2201-115 ENSG00000169314 . Chtoinosome 22 open reading frame 15 C22orf46 ENSG00000184208 Chromosome 22 open reading frame 46 C2CD2 ENSG00000157617 C2 calcium-dependent domain containing 2 C2orf40 ENSG00000119147 Chromosome 2 open reading frame 40 C2orf66 ENSG00000187944 Chromosome 2 open reading frame 66 C2or169 ENSG00000178074 . Chtoinosome 2 open reading frame 69 C2orf78 ENSG00000187833 Chromosome 2 open reading frame 78 C3 ENSG00000125730 Complement component 3 C3or133 ENSG00000174928 Chromosome 3 open reading frame 33 C3orf58 EN SG00000181744 Chromosome 3 open reading frame 58 C74A ENSG00000244731 . Complement component 4A (Rodgers blood group) C413 ENS600000224389 Complement component 413 (Chido blood group) C4BPA ENSG00000123838 Complement component 4 binding protein, alpha C4BPB EN SG00000123843 Complement component 4 binding protein, beta C4orf26 ENSG00000174792 Chromosome 4 open reading frame 26 C4orf48 ENSG00000243449 Chromosome 4 open reading frame 48 C5 ENSG00000106804 Complement component 5 C5orf46 ENSG00000178776 Chromosome 5 open reading frame 46 C6 ENSG00000039537 Complement component 6 C6orfI20 ENSG00000185127 Chromosome 6 open reading frame 120 C6orf15 ENSG00000204542 Chromosome 6 open reading frame 15 C6orf25 ENS600000204420 Chromosome 6 open reading frame 25 C6orf58 ENSG00000184530 Chromosome 6 open reading frame 58 C7 ENSG00000112936 Complement component 7 C7orf57 ENSG00000164746 Chromosome 7 open reading frame 57 C7orf73 ENSG00000243317 Chromosome 7 open reading frame 73 C78A EN SG00000157131 . Complement component 8, alpha polypeptide C8B ENSG00000021852 Complement component 8, beta polypeptide C8G EN SG00000176919 Complement component 8, gamma polypeptide C9 ENSG00000113600 Complement component 9 C9orf47 ENSG00000186354 Chromosome 9 open reading frame 47 CA10 ENSG00000154975 . Carbonic anhydrase X
CA 11 EN SG00000063180 Carbonic anhydrase XI
CA6 ENSG00000131686 Carbonic anhydrase VI
CA9 ENSG00000107159 Carbonic anhydrase IX
CABLES1 ENSG00000134508 Cdk5 and Abl enzyme substrate 1 C.ABP1 ENSG00000157782 . Calcium binding protein 1 CACNA2D1 ENSG00000153956 Calcium channel, voltage-dependent, alpha 2/delta subunit 1 CACNA2D4 EN SG00000151062 Calcium channel, voltage-dependent, alpha 2/delta subunit 4 CADM3 ENSG00000162706 Cell adhesion molecule 3 CALCA ENSG00000110680 Calcitonin-related polypeptide alpha CA LCB ENSG00000175868 _ Calcitonin-related polypeptide beta CALCR ENSG00000004948 Calcitonin receptor CALCRL ENSG00000064989 Calcitonin receptor-like C.ALR ENS(300000179218 . Calreticulin CALR3 ENSG00000269058 Calreticulin 3 CALU ENSG00000128595 Calumenin CAMK2D EN SG00000145349 Calcitimicalmodulin-dependent protein kinase II delta CAMP ENSG00000164047 Cathelicidin antimicrobial peptide C.ANX ENS(300000127022 . Calnexin CARKD ENSG00000213995 Carbohydrate kinase domain containing CARM1 ENSG00000142453 Coactivator-associated arginine methyltransferase I
CARNS1 EN SG00000172508 Cartiositte synthase 1 CARTPT ENSG00000164326 CART prepropeptide C.ASQ1 ENS(300000143318 . Calsequestrin 1 (fast-twitch. skeletal muscle) CASQ2 ENSG00000118729 Calsequestrin 2 (cardiac muscle) CATSPERG EN5G00000099338 Catsper channel auxiliary subunit gamma CBLN I ENSG00000102924 Cerebellin 1 precursor CBLN2 ENSG00000141668 Cerebellin 2 precursor CBLN3 ENSG00000139899 Cerebellin 3 precursor CBLN4 ENS600000054803 Cerebellin 4 precursor CCBE1 ENSG00000183287 Collagen and calcium binding EGF domains 1 CCDC108 EN SG00000181378 Coiled-coil domain containing 108 CCDC112 ENSG00000164221 Coiled-coil domain containing 112 CCDC129 ENSG00000180347 Coiled-coil domain containing 129 CCDC134 ENSG00000100147 Coiled-coil domain containing 1.34 CCDC149 ENSG00000181982 Coiled-coil domain containing 149 CCDC3 EN SG00000151468 Coiled-coil domain containing 3 CCDC80 ENSG00000091986 Coiled-coil domain containing 80 CCDC85A ENSG00000055813 Coiled-coil domain containing 85A
CCDC88B ENS(300000168071 . Coiled-coil domain containing 888 CCER2 ENSG00000262484 Coiled-coil glutamate-rich protein 2 CCK EN SC500000187094 Cholecystokinin CO, I. ENSG00000108702 Chemokine (C-C motif) ligand 1 CCL11 ENSG00000172156 Chemokine (C-C motif) ligand 11 CCII 3 ENS(300000181.374 . Chemokine (C-C motif) ligand 13 CCL14 ENSG00000276409 Chemokine (C-C motif) ligand 14 CCL15 ENSG00000275718 Chemokine (C-C motif) ligand 15 CCL1.6 ENSG00000275152 Chemokine (C-C motif) ligand 16 CCL17 ENSG00000102970 Chemokine (C-C motif) ligand 17 CCII 8 ENS(300000275385 Chemokine (C-C motif) ligand 18 (pulmonary and act Mit ion-regulated) CCL19 ENSG00000172724 Chemokine (C-C motif) ligand 19 CCL2 EN SG00000108691 Chemokine (C-C motif) ligand 2 CCL20 ENSG00000115009 Chemokine (C-C motif) ligand 20 CCL21 ENSG00000137077 Chemokine (C-C motif) ligand 21 CCL22 ENSG00000102962 _ Chemokine (C-C motif) ligand 22 CCL23 ENSG00000274736 Chemokine (C-C motif) ligand 23 CCL24 ENSG00000106178 Chemokine (C-C motif) ligand 24 Ca.25 EN SG00000131142 . Chemokine (C-C moth) ligand 25 CCL26 ENSG00000006606 Chemokine (C-C motif) ligand 26 CCL27 ENSG00000213927 Chemokine (C-C motif) ligand 27 CCL28 ENSG00000 I 51882 Chemokine (C-C motif) ligand 28 CCL3 ENSG00000277632 Chemokine (C-C motif) ligand 3 C7C1,31.,3 ENSG00000276085 . Chemokine (C-C moth) ligand 3-like 3 CCL4 ENS600000275302 Chemokine (C-C motif) ligand 4 CCL4L2 ENSG00000276070 Chemokine (C-C motif) ligand 4-like 2 CCL5 EN SG00000271503 Chemokine (C-C motif) ligand 5 CCL7 ENSG00000108688 Chemokine (C-C motif) ligand 7 Ca.8 ENS(300000108700 . Cheinokitie (C-C moth) ligand 8 CCNB UPI ENSG00000100814 Cyclin RI interacting protein 1, E3 ubiquitin protein ligase CCNL1 ENSG00000163660 Cyclin Li CCNL2 EN SG00000221978 Cyclin L2 CD14 ENSG00000170458 CD14 molecule CD 160 ENSG00000117281 CD160 molecule CI) 164 ENSG00000135535 CI) 164 molecule, sialomucin CD177 ENSG00000204936 CD177 molecule CI) 1E EN SG00000158488 C7Dle molecule CD2 ENSG00000116824 CD2 molecule CD200 ENSG00000091972 CD200 molecule CD200R1 ENSG00000163606 CD200 receptor 1 CD22 ENSG00000012124 CD22 molecule CD226 EN SG00000150637 C7D226 molecule CD24 ENSG00000272398 CD24 molecule CD276 ENSG00000103855 CD276 molecule CD300A ENSG00000167851 . CD300a molecule CD300LB ENSG00000178789 CD300 molecule-like family member b CD300LF EN SG00000186074 C7D300 molecule-like family member f CD300LG ENSG00000161649 CD300 molecule-like family member g CD3D ENSG00000167286 CD3d molecule, delta (CD3-TCR complex) C7D4 EN SG00000010610 . CD4 molecule CD40 ENSG00000101017 CD40 molecule, INF receptor supeifamily member 5 CD44 ENSG00000026508 CD44 molecule (Indian blood group) CD48 ENSG00000117091 CD48 molecule CD5 ENSG00000110448 CD5 molecule CD55 ENSG00000196352 CD55 molecule, decay accelerating factor for complement (Cromer blood group) CD59 ENSG00000085063 CD59 molecule, complement regulatory protein CD5L ENSG00000073754 CD5 molecule-like CD6 ENSG00000013725 CD6 molecule CD68 EN SG00000129226 C7D68 molecule CD7 ENSG00000173762 CD7 molecule CD79A ENSG00000105369 CD79a molecule, immunoglobulin-associated alpha CD80 EN SG00000121594 . CD80 molecule CDS6 ENSG00000114013 CD86 molecule CD8A ENSG00000153563 CD8a molecule CD8B ENSG00000172116 CD8b molecule CD99 ENSG00000002586 CD99 molecule CDC23 ENSG00000094880 . Cell division cycle 23 CDC40 ENSG00000168438 Cell division cycle 40 CDC45 ENSG00000093009 Cell division cycle 45 CDCP1 EN SG00000163814 CUB domain containing protein 1 CDCP2 ENSG00000157211 CUB domain containing protein 2 CDH1 ENSG00000039068 . Cadherin 1, type 1 CDH11 ENSG00000140937 Cadherin 11, type 2, OB-cadherin (osteoblast) CDH13 ENSG00000140945 Cadherin 13 CDF117 ENSG00000079112 Cadherin 17, Li cadherin (liver-intestine) CDH18 ENSG00000145526 Cadherin 18, type 2 CDH19 ENSG00000071991 Cadherin 19, type 2 CDH23 ENSG00000107736 Cadherin-related 23 CDH5 ENSG00000179776 Cadherin 5, type 2 (vascular endothelium) (MI-1RJ ENSG00000148604) Cadherin-related family member 1 CDHR4 ENSG00000187492 Cadherin-related family member 4 CDHR5 ENSG00000099834 Cadherin-related family member 5 CDKN2A ENS600000147889 Cyclin-dependent kinase inhibitor 2A
CDNF ENSG00000185267 Cerebral dopamine netuvtrophic factor CDON EN SG00000064309 Cell adhesion associated, oncogette regulated CDSN ENSG00000204539 Corneodesmosin CEACAM16 ENSG00000213892 Carcinoembryonic antigen-related cell adhesion molecule 16 CE.ACAM18 ENSG00000213822 . Carcinoembryonic antigen-related cell adhesion molecule 18 .
CEACAM19 ENSG00000186567 Carcinoembiyonic antigen-related cell adhesion molecule 19 CEAC7.AM5 EN SG00000105388 Carcinoeinbiyortic antigen-related cell adhesion molecule 5 CEACAM7 ENSG00000007306 Carcinoembryonic antigen-related cell adhesion molecule 7 CEACAM8 ENSG00000124469 Carcinoembryonic antigen-related cell adhesion molecule 8 CECR1 ENSG00000093072 . Cat eye syndrome chromosome region, candidate 1 CECR5 ENSG00000069998 Cat eye syndrome chromosome region, candidate 5 CEL ENSG00000170835 Carboxyl ester lipase CELA2A EN SG00000142615 Chymouypsin-like elastase family, member CELA2B ENSG00000215704 Chy mouypsin-like elastase family, member C7ELA3A ENSG00000142789 . Chy mot ry psin-like elastase family, member 3A
CELA3B ENSG00000219073 Chymotrypsin-like elastase family, member CEMIP ENSG00000103888 Cell migration inducing protein, hyaluronan binding CEP89 EN SG00000121289 Centtusomal protein 89kDa CER1 ENSG00000147869 Cerbents 1, DAN family BMP antagonist C7ERCAM ENSG00000167123 _ Cerebral endothelial cell adhesion molecule CERS1 ENS000000223802 Ceratnide synthase 1 CES1 ENSG00000198848 Carboni'esterase 1 CES3 ENSG00000172828 . Carboxylesterase 3 CES4A ENSG00000172824 Carbox-ylesterase 4A
CES5A ENSG00000159398 Carboxylesterase SA
cr-m? ENS600000087237 Cholesteryl ester transfer protein, plasma CFB ENS000000243649 Complement factor B
C7FC1 ENSG00000136698 . Cripto, FRL-1, cryptic family 1 CFC1B ENSG00000152093 Cripto, FRI,-1, cryptic family 1B
CFD ENSG00000197766 Complement factor D (adipsin) CFDP1 EN SG00000153774 Crattiofacial development ptotein 1 CFH EN SG00000000971 Complement factor H
CFHR1 ENSG00000244414 . Complement factor H-related 1 CHIR2 ENS000000080910 Complement factor H-related 2 CFI-W3 ENSG00000116785 Complement factor H-related 3 CFHR4 EN SG00000134365 Complement factor II-related 4 CF}-WS ENSG00000134389 Complement factor H-related 5 CFI ENS000000205403 Complement factor I
CFP ENSG00000126759 Complement factor pmperdin CGA ENSG00000135346 Glycopmtein hormones, alpha polypeptide COB EN SG00000104827 Chorionic gonadotropin, beta polypeptide COB! ENS000000267631 Chorionic gonadotropin, beta polypeptide 1 CGB2 ENSG00000104818 Chorionic gonadotropin, beta polypeptide 2 CUBS ENSG00000189052 Chorionic gonadotropin, beta polypeptide 5 CGB7 ENSG00000196337 Chorionic gonadotropin, beta polypeptide 7 COB8 ENS000000213030 C7hotionic gonadoiropin, beta polypeptide 8 CGREF I ENSG00000138028 Cell growth regulator with FT-hand domain 1 CH507-9B2.3 ENS000000280071 CHAD ENS000000136457 . Chondroadherin CHADL ENSG00000100399 Chondroadherin-like CHEK2 ENSG00000183765 Checkpoint kinase 2 CHGA ENSG00000100604 Chromogranin A
CHUB ENSG00000089199 Chromogranin B
(MIMI ENSG00000133048 . Chit nurse 3-like 1 (cartilage glycoprotein-39) CH131,2 ENSG00000064886 Chitinase 3-like 2 CHIA ENSG00000134216 Chitinase, acidic CHID1 ENSG00000177830 Chitinase domain containing 1 CHIT1 ENSG00000133063 Chitinase 1 (chitotriosidase) GIL! ENSG00000134121 . Cell adhesion molecule LI-like CHN1 ENSG00000128656 Chimerin 1 CHPF ENSG00000123989 Chondroitin polymerizing factor CHPF2 EN SG00000033100 C7hondroitin polymerizing factor 2 CHRD ENS000000090539 Chordin CHRDL1 ENSG00000101938 _ Chordin-like 1 CHRDI,2 ENS000000054938 Chordin-like 2 CHRNA2 ENSG00000120903 Cholinergic receptor, nicotinic, alpha 2 (neuronal) CHRNA5 ENSG00000169684 . Cholinergic receptor, nicotinic, alpha 5 (neuronal) CHRNB I ENSG00000170175 Cholinergic receptor, nicotinic, beta I
(muscle) CHRND ENSG00000135902 Cholinergic receptor, nicotinic, delta (muscle) GIST!. ENSG00000175264 Carbohydrate (keratin sulfate Gal-6) sulfotransferase 1 CHSTIO ENSG00000115526 Carbohydrate sulfotransfentse 10 CH sT i I ENSG00000171.310 . Carbohydrate (chondroitin 4) sulfotransferase 11 CHST13 EN S600000180767 Carbohydrate (chondroitin 4) sulfotransferase 13 CHST4 EN SG00000140835 Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 4 CHST5 EN SG00000135702 Carbohydrate (N-acetylglucosarnine 6-0) sulfotransferase 5 CHST6 EN SG00000183196 Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 6 CH sr7 ENSG00000147119 . Carbohydrate (N-acetylghicosainine 6-0) sulfottartsferase 7 .
CHsT8 ENSG00000124302 Carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8 CHSY1 ENS000000131873 Chondroitin sulfate synthase 1 CHSY3 EN SG00000198108 C7hondroitin stillate synthase 3 CHTF8 ENSG00000168802 Chromosome transmission fidelity factor 8 CILP ENSG00000138615 Cartilage intermediate layer protein, nucleotide . pyrophosphohydrolase =
CILP2 ENSG00000160161 Cartilage intermediate layer protein 2 CIRH IA ENSG00000141076 Cirrhosis, autosomal recessive IA (cirhin) CKLF ENSG00000217555 Chemokine-like factor CKMTIA ENSG00000223572 Creatine kinase, mitochondrial IA
CKNIT1B ENSG00000237289 . Creatine kinase, mitochondrial 1B
=
CLCA1 EN SG00000016490 Chloride channel accessory 1 CLCF1 ENSG00000175505 Camliotrophin-like cytokine factor 1 CLDN15 ENS000000106404 Claudin 15 CLDN7 ENSG00000181885 Claudin 7 CLDND1 ENSG00000080822 . Claudio domain containing 1 =
CL EC I 1 A ENSG00000105472 C-type lectin domain family It, member A.
CLEC16A ENSG00000038532 C-type lectin domain family 16, member A
CLEC18A ENSG00000157322 C-type lectin domain family 18, member A
CLEC18B ENSG00000140839 C-type lectin domain family 18, member B
CLEC18C ENSG00000157335 . C-type lectin domain family IS, member C
=
CL EC I 9A ENSG00000261.210 C-type lectin domain family 19, member A.
CLEC2B ENSG00000110852 C-type lectin domain family 2, member B
CLEC3A EN S000000166509 C-type lectin domain family 3, member A
CLEC3B ENSG00000163815 C-type lectin domain family 3, member B
CLGN ENSG00000153132 Calmegin CLN5 EN SG00000102805 Ceroid-lipoftiscinosis, neuronal 5 CLPS ENSG00000137392 Colipase, pancreatic CLPSL1 EN SG00000204140 Colipase-like 1 CLPSL2 ENSG00000196748 Colipase-like 2 CLPX ENSG00000166855 Caseinolytic mitochondrial matrix peptidase chaperone subunit CLSTN3 EN SG00000139182 _ Calsyntenin 3 CLU ENSG00000120885 Clusterin CLULI ENSG00000079101 Clusterin-like 1 (retinal) CMA1 ENS(300000092009 . Chymase 1, mast cell CMPK1 ENSG00000162368 Cytidine monophosphate (LIMP-CIVIP) kinase 1, cytosolic CNBDI ENSG00000176571 Cyclic nucleotide binding domain containing CNDP1 ENSG00000150656 Caruosine dipeptidase I (netallopeptidase M20 family.) CNPY2 ENSG00000257727 Canopy FGF signaling regulator 2 CNPY3 EN SG00000137161 . Canopy Rif' signaling regulator 3 CNPY4 EN SG00000166997 Canopy FGT.: signaling regulator 4 CNTFR ENSG00000122756 Ciliaiy neurotrophic factor receptor CNIN1 EN SG00000018236 Contact i n 1 CNTN2 ENSG00000184144 Contactin 2 (axonal) CNIN3 ENS(300000113805 . Contactin 3 (plastnacylotna associated) CNIN4 EN SG00000144619 Contactin 4 CNTN5 ENSG00000149972 Contactin 5 CNINAP2 EN SG00000174469 Contact in associated protein-like 2 CNTNAP3 ENSG00000106714 Contactin associated protein-like 3 CNTNAP3B ENSG00000154529 Contactin associated protein-like 3B
COASY EN SG00000068120 CoA symhase COCH ENSG00000100473 Cochlin COG3 ENSG00000136152 Component of oligomeric golgi complex 3 COLIOA1 EN SG00000 123500 Collagen, type X, alpha 1 COL1 lAl. ENSG00000060718 Collagen, type XI, alpha 1 COLI1A2 ENS600000204248 Collagen, type XI, alpha 2 COLI2A1 ENSG00000111799 Collagen, type XII, alpha 1 COL14A I EN SG00000187955 Collagen. type XIV, alpha I
COL15A1 ENSG00000204291 Collagen, type XV, alpha 1 C0L16A I. ENSG00000084636 Collagen, type XVI, alpha 1 C7OL18A 1 ENS(300000182871 . Collagen, type XVIII, alpha 1 COL 19A1 ENSG00000082293 Collagen, type XIX, alpha 1 (MIA I. EN SG00000108821 Collagen. type I, alpha 1 COL1A2 ENSG00000164692 Collagen, type 1, alpha 2 COL20A I. ENSG00000101203 Collagen, type X.X. alpha!
C70L21.A1 ENS(300000124749 . Collagen, type XXI, alpha 1 COL22A1 ENSG00000169436 Collagen, type XXII, alpha 1 COL24A1 ENSG00000171502 Collagen. type XXIV, alpha 1 COL26A1 ENSG00000160963 Collagen, type XXVI, alpha 1 COL27A1 ENSG00000196739 Collagen, type XXVII, alpha 1 C701.,28A1 ENS(300000215018 . Collagen, type XXVIII, alpha 1 COL2A1 ENSG00000139219 Collagen, type II, alpha 1 COL3A1 ENSG00000168542 Collagen. type III, alpha 1 COL4A I. EN SG00000187498 Collagen. type IV, alpha 1 COL4A2 ENSG00000134871 Collagen, type IV, alpha 2 C701.,4A3 ENS(300000169031 _ Collagen, type IV, alpha 3 (Goodpasture antigen) DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
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Claims (304)

What is claimed is:
1. 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, a therapeutic protein or a sequence encoding the same 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 marker(s) of a stem memory T cell (TSCM), thereby producing a modified stem memory T cell (TSCM).
2. A method of producing a plurality of modified stem memory T cells (TSCM), comprising introducing into a plurality of primary human T cells (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding the same 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 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell thereby producing a plurality of modified stem memory T cell (TSCM).
3. The method of claim 2, 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 (TSCM).
4. 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, a therapeutic protein or a sequence encoding the same 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 marker(s) of a central memory T cell (TCM), thereby producing a modified central memory T cell (TCM).
5. A method of producing 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, a therapeutic protein or a sequence encoding the same 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 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (TCM), thereby producing a plurality of modified central memory T cell (TCM).
6. The method of claim 5, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (TCM).
7. The method of any one of claims 1-6, wherein 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.
8. The method of any one of claims 1-7, wherein the transposon is a piggyBac transposon.
9. The method of any one of claims 1-8, wherein the transposase is a piggyBac transposase.
10. The method of claim 9, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4.
11. The method of claim 9 or 10, wherein the piggyBac transposase is a hyperactive variant and wherein the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4.
12. The method of claim 11, wherein the amino acid substitution at position 30 of SEQ
ID NO: 4 is a substitution of a valine (V) for an isoleucine (I) (I30V).
13. The method of claim 11, wherein the amino acid substitution at position 165 of SEQ
ID NO: 4 is a substitution of a serine (S) for a glycine (G) (G165S).
14. The method of claim 11, wherein the amino acid substitution at position 282 of SEQ
ID NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V).
15. The method of claim 11, wherein the amino acid substitution at position 538 of SEQ
ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K).
16. The method of any one of claims 1-15, wherein the transposase is a Super piggyBac (SPB) transposase.
17. The method of claim 16, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5.
18. The method of any one of claims 1-17, wherein the sequence encoding the transposase is an mRNA sequence.
19. The method of any one of claims 1-6, wherein the transposon is a Sleeping Beauty transposon.
20. The method of any one of claims 1-6 or 19, wherein the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
21. The method of any one of claims 1-6, wherein the transposon is a Helraiser transposon.
22. The method of any one of claims 1-6 or 21, wherein the transposase is a Helitron transposase.
23. The method of any one of claims 1-6, wherein the transposon is a Tol2 transposon.
24. The method of any one of claims 1-6 or 23, wherein the transposase is a Tol2 transposase.
25. The method of any one of claims 1-6, wherein the transposon is derived or recombined from any species.
26. The method of any one of claims 1-6 or 25, wherein the transposon is synthetic.
27. The method of any one of claims 1-26, wherein the antigen receptor is a T-cell receptor.
28. The method of claim 27, wherein the T-cell receptor is naturally-occurring.
29. The method of claim 27, wherein the T-cell receptor is not naturally-occurring
30. The method of claim 29, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
31. The method of claim 29 or 30, wherein the T-cell receptor is a recombinant T-cell receptor.
32. The method of any one of claims 1-31, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
33. The method of claim 32, wherein the CAR is a CARTyrin.
34. The method of claim 32, wherein the CAR comprises one or more VHH
sequence(s).
35. The method of claim 34, wherein the CAR is a VCAR.
33. The method of any one of claims 1-32, further comprising introducing into the primary human T cell (c) a composition comprising a second transposon comprising a sequence encoding a therapeutic protein, to produce a modified T cell capable of expressing the therapeutic protein.
34. The method of claim 33, wherein the therapeutic protein is a secreted or secretable protein.
35. The method of claim 33 or 34, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.
36. The method of claim 35, wherein the sequence encoding the therapeutic protein is a DNA sequence.
37. The method of any one of claims 33-36, wherein the transposase composition of (b) mobilizes the transposon of (a) and the second transposon of (c).
38. The method of any one of claims 1-37, further comprising introducing into the primary human T cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase, wherein the second transposase of (d) is capable of transposing the transposon of (c), and wherein the second transposase composition of (d) and the transposase composition of (b) are not identical.
39. The method of claim 38, wherein the transposase composition of (b) mobilizes the transposon of (a) and the transposase composition of (d) mobilizes the transposon of (c).
40. A method of producing a modified stem memory T cell (TSCM), comprising.
(a) introducing into a primary human T cell a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same 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 marker(s) of a stem memory T cell (TSCM), thereby producing a modified stem memory T cell (TSCM).
41. 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, a therapeutic protein or a sequence encoding the same 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 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% 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 modified stem memory T cells (TSCM).
42. The method of claim 41, wherein at least 60% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM).
43. 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, a therapeutic protein or a sequence encoding the same 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 marker(s) of a central memory T cell (TCM), thereby producing a modified central memory T cell (TCM).
44. 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, a therapeutic protein or a sequence encoding the same 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 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (TCM), thereby producing a plurality of modified central memory T cells (TCM).
45. The method of claim 44, wherein at least 60% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a central memory T
cell (TCM).
46. The method of any one of claims 40-45, wherein a viral vector comprises the antigen receptor or the therapeutic protein.
47. The method of claim 46, wherein the viral vector comprises a sequence isolated or derived from a lentivirus.
48. The method of claim 46, wherein the viral vector comprises a sequence isolated or derived from a retrovirus.
49. The method of claim 48, wherein the retrovirus is a gammaretrovirus.
50. The method of any one of claims 40-46, wherein the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
51. The method of any one of claims 40-45, wherein a nucleic acid vector comprises the antigen receptor or the therapeutic protein.
52. The method of claim 51, wherein an mRNA vector comprises the antigen receptor or the therapeutic protein.
53. The method of any one of claims 40-45, wherein a nanoparticle vector comprises the antigen receptor or the therapeutic protein.
54. The method of any one of claims 40-45, wherein the introducing step comprises a homologous recombination.
55. The method of claim 54, wherein the homologous recombination comprises contacting the composition comprising the antigen receptor or the therapeutic protein, a genomic editing construct, and a genomic sequence of at least one primary human T cell of the plurality of primary human T cells.
56. The method of claim 55, wherein a vector comprises the antigen receptor or the therapeutic protein.
57. The method of claim 56, wherein the vector is an adeno-associated vector (AAV).
58. The method of any one of claims 54-57, wherein the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.
59. The method of claim 58, wherein the genomic editing construct comprises a DNA
binding domain and a type IIS endonuclease.
60. The method of claim 59, wherein the genomic editing construct encodes a fusion protein.
61. The method of claim 59, wherein the genomic editing construct encodes the DNA
binding domain and the type ITS endonuclease and wherein the expressed DNA
binding domain and the expressed type IIS endonuclease are non-covalently linked.
62. The method of any one of claims 58-62, wherein the genomic editing construct comprises a sequence derived from a Cas9 endonuclease.
63. The method of claim 62, wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain.
64. The method of claim 62 or 63, wherein the sequence derived from a Cas9 endonuclease encodes an inactive Cas9.
65. The method of claim 64, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
66. The method of any one of claims 62-65 wherein the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
67. The method of claim 66, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
68. The method of any one of claims 62-67, wherein 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).
69. The method of any one of claims 58-61, wherein the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
70. The method of claim 69, wherein the sequence derived from a TALEN is the DNA
binding domain.
71. The method of claim 58, wherein the genomic editing construct comprises a TALEN.
72. The method of any one of claims 58-61, wherein the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
73. The method of claim 72, wherein the sequence derived from a ZFN is the DNA
binding domain.
74. The method of claim 58, wherein the genomic editing construct comprises a zinc-finger nuclease (ZFN).
75. The method of any one of claims 58-74, wherein genomic editing construct targets a safe harbor site on a mammalian chromosome.
76. The method of any one of claims 58-74, wherein genomic editing construct targets a safe harbor site on a human chromosome.
77. The method of claim 75 or 76, wherein the chromosome is in vivo, in situ, ex vivo or in vitro.
78. The method of any one of claims 58-77, wherein genomic editing construct targets a sequence encoding a component of an endogenous T-cell receptor or a sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a mammalian chromosome.
79. The method of any one of claims 58-77, wherein genomic editing construct targets a sequence encoding a component of an endogenous T-cell receptor or a sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a human chromosome.
80. The method of any one of claims 40-79, wherein the antigen receptor is a T-cell receptor.
81. The method of claim 80, wherein the T-cell receptor is naturally-occurring.
82. The method of claim 80, wherein the T-cell receptor is not naturally-occurring.
83. The method of claim 82, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
84. The method of claim 82 or 83, wherein the T-cell receptor is a recombinant T-cell receptor.
85. The method of any one of claims 40-79, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
86. The method of claim 85, wherein the CAR comprises one or more Centyrin sequence(s).
87. The method of claim 86, wherein the CAR is a CARTyrin.
88. The method of claim 85, wherein the CAR comprises one or more VHH
sequence(s).
89. The method of claim 88, wherein the CAR is a VCAR.
90. The method of any one of claims 40-89, further comprising introducing into the primary human T cell a composition comprising a sequence encoding a therapeutic protein, to produce a modified T cell capable of expressing the therapeutic protein.
91. The method of any one of claims 40-90, wherein the therapeutic protein is a secreted or secretable protein.
92. The method of claim 90 or 91, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.
93. The method of claim 92, wherein the sequence encoding the therapeutic protein is a DNA sequence.
94. The method of any one of claims 90-93, wherein the introducing comprises a homologous recombination.
95. The method of any one of claims 40-94, wherein the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
96. The method of any one of claims 40-42 or 46-95, further comprising 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 marker(s) of a stem memory T cell (TSCM).
97. The method of claim 96, 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 expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TSCM).
98. The method of claim 97, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TSCM).
99. The method of any one of claims 43-95, further comprising 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 marker(s) of a central memory T cell (TCM).
100. The method of claim 99, 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 expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (TCM).
101. The method of claim 99, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (TCM).
102. The method of any one of claims 40-42 or 46-101, wherein 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%, 900/, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (TSCM).
103. The method of any one of claims 40-42 or 46-101, wherein 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 marker(s) of a stem memory T cell (TSCM).
104. The method of any one of claims 43-101, wherein 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 marker(s) of a central memory T cell (TCM).
105. The method of any one of claims 43-101, wherein 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 marker(s) of a central memory T cell (TCM).
106. The method of claim 102 or 103, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (TSCM) from the plurality of enriched modified T-cells.
107. The method of claim 106, wherein 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.
108. The method of claim 104 or 105, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (SCM) from the plurality of enriched modified T-cells.
109. The method of claim 108, wherein 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.
110. The method of any one of claims 40-109, wherein 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.
111. The method of any one of claims 40-109, wherein the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
112. The method of claim 111, wherein 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.
113. The method of claim 111, wherein 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.
114. The method of claim 111, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 µmol/kg and 640 µmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 µmol/kg and 70 µmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 µmol/kg and 75 µmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 µmol/kg and 75 µmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 µmol/kg and 25 µmol/kg, inclusive of the endpoints.
115. The method of claim 111, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 µmol/kg, palmitic acid at a concentration of about 7 µmol/kg, linoleic acid at a concentration of about 7.5 µmol/kg, oleic acid at a concentration of about 7.5 µmol/kg and a sterol at a concentration of about 2.5 µmol/kg.
116. A method of producing a modified stem memory T cell (TSCM), comprising:
(a) introducing into a primary human T cell a composition comprising an antigen receptor or a therapeutic protein 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, wherein the activated modified -T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a modified stem memory T cell (TSCM).
117. 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 or a therapeutic protein 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%, 60%, 75%, 80%, 85%, 90%, 95% or 99% 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 modified stem memory T cell (TSCM).
118. The method of claim 117, wherein at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM).
119. 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 or a therapeutic protein 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, wherein the activated modified -T cell expresses one or more cell-surface marker(s) of a central memory T cell (TCM), thereby producing a modified central memory T cell (TCM).
120. 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 or a therapeutic protein 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%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T cell (TCM), thereby producing a modified central memory T cells (TCM).
121. The method of claim 120, wherein at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T
cells (TCM).
122. The method of any one of claims 116-121, wherein the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex.
123. The method of any one of claims 116-118 or 122, further comprising 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 marker(s) of a stem memory T cell (TSCM).
124. The method of claim 123, 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 expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TSCM).
125. The method of claim 123, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TSCM).
126. The method of any one of claims 119-122, further comprising 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 marker(s) of a stem memory T cell (TSCM).
127. The method of claim 126, 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 expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TSCM).
128. The method of claim 126, wherein at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (TSCM).
129. The method of any one of claims 116-118 or 122-128, wherein 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 marker(s) of a stem memory T cell (TSCM).
130. The method of any one of claims 116-118 or 122-128, wherein 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 marker(s) of a stem memory T cell (TSCM).
131. The method of any one of claims 119-128, wherein 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 marker(s) of a stem memory T cell (TSCM).
132. The method of any one of claims 119-128, wherein 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 marker(s) of a stem memory T cell (TSCM).
133. The method of claim 129 or 130, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (TSCM) from the plurality of enriched modified T-cells.
134. The method of claim 133, wherein 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.
135. The method of claim 131 or 132, wherein the enriching step comprising isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (TSCM) from the plurality of enriched modified T-cells.
136. The method of claim 135, wherein 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.
137. The method of any one of claims 116-136, wherein 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.
138. The method of any one of claims 116-137, wherein the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol.
139. The method of claim 138, wherein 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.
140. The method of claim 138, wherein 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.
141. The method of claim 138, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 µmol/kg and 640 µmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 µmol/kg and 70 µmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 !among and 75 µmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 µmol/kg and 75 µmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 µmol/kg and 25 µmol/kg, inclusive of the endpoints.
142. The method of claim 138, wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 µmol/kg, palmitic acid at a concentration of about 7 µmol/kg, linoleic acid at a concentration of about 7.5 µmol/kg, oleic acid at a concentration of about 7.5 µmol/kg and a sterol at a concentration of about 2.5 µmol/kg.
143. The method of any one of claims 116-142, further comprising introducing into the primary human T cell (c) a composition comprising a second transposon comprising a sequence encoding a therapeutic protein, to produce a modified T cell capable of expressing the therapeutic protein.
144. The method of claim 143, wherein the therapeutic protein is a secreted or a secretable protein.
145. The method of claim 143 or 144, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.
146. The method of claim 143 or 144, wherein the sequence encoding the therapeutic protein is a DNA sequence.
147. The method of any one of claims 143-146, wherein the transposase composition of (b) mobilizes the transposon of (a) and the second transposon of (c).
148. The method of any one of claims 143-147, further comprising introducing into the primary human T cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase, and wherein the second transposase of (d) is capable of transposing the transposon of (c), and wherein the second transposase composition of (d) and the transposase composition of (b) are not identical.
149. The method of any one of claims 1-148, wherein the introducing step further comprises a composition comprising a genomic editing construct.
150. The method of claim 149, wherein the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.
151. The method of claim 150, wherein the genomic editing construct comprises a DNA
binding domain and a type IIS endonuclease.
152. The method of claim 151, wherein the genomic editing construct encodes a fusion protein.
153. The method of claim 151, wherein 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.
154. The method of any one of claims 149-153, wherein the genomic editing construct comprises a sequence derived from a Cas9 endonuclease.
155. The method of claim 154, wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain.
156. The method of claim 154 or 155, wherein the sequence derived from a Cas9 endonuclease encodes an inactive Cas9.
157. The method of claim 156, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
158. The method of any one of claims 154-157, wherein the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
159. The method of claim 158, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
160. The method of any one of claims 154-159, wherein 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).
161. The method of any one of claims 149-153, wherein the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
162. The method of clairn 161, wherein the sequence derived from a TALEN is the DNA
binding domain.
163. The method of claim 149, wherein the genomic editing construct comprises a TALEN.
164. The method of any one of claims 149-153, wherein the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
165. The method of claim 164, wherein the sequence derived from a ZFN is the DNA
binding domain.
166. The method of claim 149, wherein the genomic editing construct comprises a zinc-finger nuclease (ZFN).
167. The method of any one of claims 149-153, further comprising introducing into a primary human T cell a composition comprising a sequence encoding a therapeutic protein.
168. The method of claim 167, wherein the therapeutic protein is a secreted or a secretable protein.
169. The method of claim 168, wherein the therapeutic protein is an intracellular protein.
170. The method of claim 168, wherein the therapeutic protein is a cytosolic protein.
171. The method of claim 168, wherein the therapeutic protein is a membrane-bound protein.
172. The method of claim 168, wherein the therapeutic protein is a transmembrane protein.
173. The method of any one of claims 1-172, wherein the cell-surface markers of the modified TSCM comprise CD62L and CD45RA.
174. The method of any one of claims 1-172, wherein the cell-surface markers of the modified TSCM comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R.beta..
175. The method of any one of claims 1-172, wherein the cell-surface markers of the modified TSCM comprise one or more of CD45RA, CD95, IL-2R.beta., CR7, and CD62L.
176. The method of any one of claims 1-172, wherein the cell-surface markers of the modified TCM comprise one or more of CD45RO, CD95, IL-2R.beta., CCR7 and CD62L.
177. The method of any one of claims 96-176, wherein the plurality of expanded modified T-cells comprises a naive T-cell (modified T N) and the cell-surface markers of the CAR-T N
comprise one or more of CD45RA, CCR7 and CD62L.
178. The method of any one of claims 96-176, wherein 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.beta., CCR7, and CD62L.
179. The method of any one of claims 96-176, wherein 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, CD9S, and IL-2R.beta..
180. The method of any one of claims 96-176, wherein the plurality of expanded modified T-cells comprises an effector T-cell (modified TEFF) and the cell-surface markers of the CAR-T EFF comprise one or more of CD45RA, CD95, and IL-2R.beta..
181. The method of any one of claims 1-39 or 116-180, wherein 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.
182. The method of claim 181, wherein the introducing further comprises a composition comprising an mRNA sequence encoding a transposase.
183. The method of claim 181 or 182, wherein the transposon is a piggyBac transposon.
184. The method of any one of claims 181-183, wherein the transposase is a piggyBac transposase.
185. The method of claim 184, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4.
186. The method of claim 184 or 185, wherein the piggyBac transposase is a hyperactive variant and wherein the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4.
187. The method of claim 186, wherein the amino acid substitution at position 30 of SEQ
ID NO: 4 is a substitution of a valine (V) for an isoleucine (I) (I30V).
188. The method of claim 186, wherein the amino acid substitution at position 165 of SEQ
ID NO: 4 is a substitution of a serine (S) for a glycine (G) (G165S).
189. The method of claim 186, wherein the amino acid substitution at position 282 of SEQ
ID NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V).
190. The method of claim 186, wherein the amino acid substitution at position 538 of SEQ
ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K).
191. The method of any one of claims 183-190, wherein the transposase is a Super piggyBac (SPB) transposase.
192. The method of claim 191, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5.
193. The method of any one of claims 1-39 or 116-180, wherein the transposon is a Sleeping Beauty transposon.
194. The method of claim 193, wherein the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
195. The method of any one of claims 1-39 or 116-180, wherein the transposon is a Helraiser transposon.
196. The method of claim 195, wherein the transposase is a Helitron transposase.
197. The method of any one of claims 1-39 or 116-180, wherein the transposon is a Tol2 transposon.
198. The method of claim 197, wherein the transposase is a Tol2 transposase.
199. The method of any one of claims 1-39 or 116-198, wherein the sequence encoding the transposase is an mRNA sequence.
200. The method of any one of claims 1-39 or 116-180, wherein the transposon is derived or recombined from any species.
201. The method of any one of claims 1-39 or 116-180, wherein the transposon is synthetic.
202. The method of any one of claims 1-39 or 116-180, wherein the transposon further comprises a selection gene.
203. The method of claim 202, wherein the T-cell expansion composition further comprises a selection agent.
204. The method of any one of claims 1-203, wherein the antigen receptor is a T-cell receptor.
205. The method of claim 204, wherein the T-cell receptor is naturally-occurring.
206. The method of claim 204, wherein the T-cell receptor is not naturally-occurring.
207. The method of claim 206, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
208. The method of claim 206 or 207, wherein the T-cell receptor is a recombinant T-cell receptor.
209. The method of any one of claims 1-203, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
210. The method of claim 209, wherein the CAR comprises one or more Centyrin sequence(s).
211. The method of claim 210, wherein the CAR is a CARTyrin.
212. The method of claim 209, wherein the CAR comprises one or more VHH
sequence(s).
213. The method of claim 212, wherein the CAR is a VCAR.
214. The method of any one of claims 1-39 and 116-213, wherein the introducing step comprises an electroporation or a nucleofection.
215. The method of any one of claims 1-39 and 116-213, wherein the introducing step comprises a nucleofection and wherein the nucleofection comprises the steps of:
(a) contacting a transposon composition, a transposase composition, and a 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.
216. The method of claim 215, wherein the transposon is a first transposon or a second transposon.
217. The method of claim 215 or 216, wherein the transposase composition is a first transposase composition or a second transposase composition.
218. The method of any one of claims 214-217, wherein the transposon composition is a 0.5 µg/µl solution comprising nuclease free water and wherein the cuvette comprises 2 µl of the transposon composition to yield 1 µg of transposon.
219. The method of claim 218, wherein the transposon composition comprises a piggyBac transposon.
220. The method of claim 219, wherein the transposon composition comprises a Sleeping Beauty transposon.
221. The method of claim 219 or 220, wherein the transposase composition comprises 5 µg of transposase.
222. The method of claim 221, wherein the transposase composition comprises a Super piggyBac (SPB) transposase.
223. The method of claim 221, wherein the transposase composition comprises a hyperactive Sleeping Beauty (SB100X) transposase.
224. The method of claim 219, wherein the transposon comprises a Helraiser transposon
225. The method of claim 224, wherein the transposase composition comprises a Helitron transposase.
226. The method of claim 219, wherein the transposon comprises a Tol2 transposon.
227. The method of claim 226, wherein the transposase composition comprises a Tol2 transposase.
228. The method of any one of claims 215-227, wherein 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.
229. The method of claim 228, wherein 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.
230. The method of claim 228, wherein the buffer maintains or enhances a level of cell viability and/or a stern-like phenotype of the primary human T cells during the nucleofection.
231. The method of claim 228, wherein 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.
232. The method of any one of claims 228-231, wherein the buffer comprises a P3 primary cell solution.
233. The method of any one of claims 228-231, wherein the buffer comprises one or more of KCl, MgCl2, CINa, Glucose and Ca(NO3)2 in any absolute or relative abundance or concentration.
234. The method of claim 228, wherein the buffer further comprises a supplement selected from the group consisting of HEPES, Tris/HCl, and a phosphate buffer.
235. The method of claim 228 or 229, wherein the buffer comprises 5 mM KCl, 15 mM
MgCl2, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2.
236. The method of claim 235, wherein the buffer further comprises a supplement comprising 20 mM HEPES and 75 mM Tris/HCl.
237. The method of claim 236, wherein the buffer further comprises a supplement comprising 40 mM Na2HPO4/NaH2PO4 at pH 7.2.
238. The method of claim 215 or 228-237, wherein the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and/or CD19.
239. The method of any one of claims 215-238, wherein the composition comprising primary human T cells comprises 100 µI of the buffer and between 5x10 6 and 25x10 6 cells.
240. The method of any one of claims 215-239, wherein the method is performed in one or more cuvette(s) simultaneously.
241. The method of any one of claims 215-240, wherein the incubating step comprises incubating the composition comprising the plurality of primary human T cells in a pre-warmed T-cell expansion composition.
242. The method of any one of claims 215-241, wherein the incubation step has a period of 2 days.
243. The method of any one of claims 40-242, wherein the activation supplement comprises one or more cytokine(s).
244. The method of claim 243, wherein the one or more cytokine(s) comprise IL-2.
245. The method of any one of claims 96-244, wherein the expansion supplement comprises one or more cytokine(s).
246. The method of claim 245, wherein the one or more cytokine(s) comprise IL-2.
247. The method of any one of claims 1-246, wherein the method further comprises introducing into a modified T SCM cell or a modified T CM cell a composition comprising a genomic editing construct.
248. The method of claim 247, wherein the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.
249. The method of claim 248, wherein the genomic editing construct comprises a DNA
binding domain and a type IIS endonuclease.
250. The method of claim 249, wherein the genomic editing construct encodes a fusion protein.
251. The method of claim 249, wherein the genomic editing construct encodes the DNA
binding domain and the type Hs endonuclease and wherein the expressed DNA
binding domain and the expressed type IIS endonuclease are non-covalently linked.
252. The method of any one of claims 247-251, wherein the genomic editing construct comprises a sequence derived from a Cas9 endonuclease.
253. The method of claim 252, wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain.
254. The method of claim 253, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A).
255. The method of any one of claims 252-254, wherein the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.
256. The method of claim 255, wherein the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
257. The method of any one of claims 252-256, wherein 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).
258. The method of any one of claims 247-251, wherein the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
259. The method of claim 258, wherein the sequence derived from a TALEN is the DNA
binding domain.
260. The method of claim 247, wherein the genomic editing construct comprises a TALEN.
261. The method of any one of claims 247-251, wherein the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN).
262. The method of claim 261, wherein the sequence derived from a ZFN is the DNA
binding domain.
263. The method of claim 247, wherein the genomic editing construct comprises a zinc-finger nuclease (ZFN).
264. The method of any one of claims 1-263, wherein the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R.beta..
265. The method of any one of claims 1-263, wherein the primary human T cell is a naive T-cell (T N) and wherein the T N expresses one or more of CD45RA, CCR7 and CD62L.
266. The method of any one of claims 1-263, wherein the primary human T cell is a T
memory stem cell (T SCM) and wherein the T SCM expresses one or more of CD45RA, CD95, IL-2R.beta., CR7, and CD62L.
267. The method of any one of claims 1-263, wherein the primary human T cell is a central memory T-cell ( TCM) and wherein the T CM expresses one or more of CD45RO, CD95, IL-2R.beta., CCR7, and CD62L.
268. The method of any one of claims 1-263, wherein the primary human T cell is an effector memory T-cell (T EM) and wherein the T EM expresses one or more of CD45RO, CD95, and IL-2R.beta..
269. The method of any one of claims 1-263, wherein the primary human T cell is an effector T-cell (T EFF) and wherein the T EFF expresses one or more of CD45RA, CD95, and IL-2R.beta..
270. The method of any one of claims 1-269, wherein the primary human T cell expresses CD4 and/or CD8.
271. A composition comprising a modified-T SCM produced by the method of any one of claims 1-270.
272. A composition comprising a modified-T CM produced by the method of any one of claims 1-270.
273. A use of the composition of claim 271 or 272 for the manufacture of a medicament to treat a subject in need thereof.
274. The use of claim 273, wherein the modified T SCM or modified T CM is autologous.
275. The use of claim 274. wherein the modified T SCM or modified T CM is allogeneic.
276. The use of any one of claims 273-275, wherein the antigen receptor is a T-cell receptor.
277. The use of claim 276, wherein the T-cell receptor is naturally-occurring.
278. The use of claim 276, wherein the T-cell receptor is not naturally-occurring.
279. The use of claim 278, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
280. The use of claim 278 or 279, wherein the T-cell receptor is a recombinant T-cell receptor.
281. The use of any one of claims 273-275, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
282. The use of claim 281, wherein the CAR comprises one or more Centyrin sequence(s).
283. The use of claim 282, wherein the CAR is a CARTyrin.
284. The method of claim 283, wherein the CAR comprises one or more VHH
sequence(s).
285. The method of claim 284, wherein the CAR is a VCAR.
286. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of claim 271 or 272.
287. The method of claim 286, wherein the modified T SCM or modified T CM is autologous.
288. The method of claim 286, wherein the modified T SCM or modified T CM is allogeneic.
289. The method of any one of claims 286-288, wherein the antigen receptor is a T-cell receptor.
290. The method of claim 289, wherein the T-cell receptor is naturally-occurring.
291. The method of claim 289, wherein the T-cell receptor is not naturally-occurring.
292. The method of claim 291, wherein the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor.
293. The method of claim 291 or 292, wherein the T-cell receptor is a recombinant T-cell receptor.
294. The method of any one of claims 286-288, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
295. The method of claim 294, wherein the CAR comprises one or more Centyrin sequence(s).
296. The method of claim 295, wherein the CAR is a CARTyrin.
297. The method of claim 294, wherein the CAR comprises one or more VHH
sequence(s).
298. The method of claim 297, wherein the CAR is a VCAR.
299. The method of claim any one of claims 286-298, wherein the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen.
300. The method of claim any one of claims 286-298, wherein the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antigen.
301. The method of claim any one of claims 286-298, wherein the disease or disorder is a disease or disorder characterized by a lack of an activity or low abundance of a secretory protein or wherein the disease or disorder is a disease or disorder is treated by increasing an activity or an abundance of a secretory protein.
302. The method of claim 301, wherein the secretory protein comprises a coagulation factor VIII or coagulation factor IX protein.
303. The method of claim 301 or 302, wherein the abundance of the secretory protein is determined at a local site.
304. The method of claim 303, wherein the local site is accessible by a modified T SCM cell or a modified T CM cell.
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Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190119636A1 (en) 2017-10-23 2019-04-25 Poseida Therapeutics, Inc. Modified stem cell memory t cells, methods of making and methods of using same
JP2019528760A (en) * 2016-09-30 2019-10-17 ポセイダ セラピューティクス， インコーポレイテッド Modified stem cell memory T cell, method for producing the same and method for using the same
CA3047313A1 (en) 2016-12-16 2018-06-21 B-Mogen Biotechnologies, Inc. Enhanced hat family transposon-mediated gene transfer and associated compositions, systems, and methods
US11278570B2 (en) 2016-12-16 2022-03-22 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
EP3351263A1 (en) 2017-01-20 2018-07-25 Universitätsklinikum Hamburg-Eppendorf Pharmaceutical preparation for treating or preventing tissue adhesion
SG11202001445XA (en) 2017-08-18 2020-03-30 Neutrolis Inc Engineered dnase enzymes and use in therapy
WO2019246486A2 (en) 2018-06-21 2019-12-26 B-Mogen Biotechnologies, Inc. ENHANCED hAT FAMILY TRANSPOSON-MEDIATED GENE TRANSFER AND ASSOCIATED COMPOSITIONS, SYSTEMS, AND METHODS
EP3864147A4 (en) 2018-10-08 2022-07-06 Neutrolis, Inc. Engineering of dnase enzymes for manufacturing and therapy
US11058724B2 (en) * 2018-10-08 2021-07-13 Neutrolis, Inc. Methods of using DNASE1-like 3 in therapy
US10988746B2 (en) 2018-10-08 2021-04-27 Neutrolis, Inc. Manufacturing and engineering of DNASE enzymes for therapy
US11352613B2 (en) 2019-02-04 2022-06-07 Neutrolis, Inc. Engineered human extracellular DNASE enzymes
EP3959305A1 (en) * 2019-04-26 2022-03-02 Allogene Therapeutics, Inc. Methods of manufacturing allogeneic car t cells
CN110358734B (en) 2019-06-13 2020-08-25 首都医科大学宣武医院 CAR-T preparation method taking Tcm as main effect component and application thereof
WO2021173925A1 (en) * 2020-02-28 2021-09-02 Wisconsin Alumni Research Foundation Nonviral generation of genome edited chimeric antigen receptor t cells
EP4298205A1 (en) * 2021-02-23 2024-01-03 Poseida Therapeutics, Inc. Genetically modified induced pluripotent stem cells and methods of use thereof
CN114990061B (en) * 2022-06-30 2023-08-29 河南省人民医院 Culture method for inducing expansion of central memory T cells

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
GB8601597D0 (en) 1986-01-23 1986-02-26 Wilson R H Nucleotide sequences
US5266491A (en) 1989-03-14 1993-11-30 Mochida Pharmaceutical Co., Ltd. DNA fragment and expression plasmid containing the DNA fragment
US5580734A (en) 1990-07-13 1996-12-03 Transkaryotic Therapies, Inc. Method of producing a physical map contigous DNA sequences
US5733761A (en) 1991-11-05 1998-03-31 Transkaryotic Therapies, Inc. Protein production and protein delivery
US5641670A (en) 1991-11-05 1997-06-24 Transkaryotic Therapies, Inc. Protein production and protein delivery
WO1995030413A1 (en) * 1994-05-10 1995-11-16 The Kitasato Institute Hematopoietic stem cell proliferation accelerator
US6218185B1 (en) 1996-04-19 2001-04-17 The United States Of America As Represented By The Secretary Of Agriculture Piggybac transposon-based genetic transformation system for insects
US6835394B1 (en) 1999-12-14 2004-12-28 The Trustees Of The University Of Pennsylvania Polymersomes and related encapsulating membranes
US7105343B1 (en) 2000-10-31 2006-09-12 University Of Notre Dame Du Lac Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector Piggybac
US6962810B2 (en) 2000-10-31 2005-11-08 University Of Notre Dame Du Lac Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggyBac
IL152904A0 (en) * 2002-01-24 2003-06-24 Gamida Cell Ltd Utilization of retinoid and vitamin d receptor antagonists for expansion of renewable stem cell populations
AU2003250519A1 (en) * 2002-08-19 2004-03-03 Gamida-Cell Ltd. Ex-vivo expansion of hematopoietic stem cell populations in mononuclear cell cultures
US8399643B2 (en) * 2009-02-26 2013-03-19 Transposagen Biopharmaceuticals, Inc. Nucleic acids encoding hyperactive PiggyBac transposases
US8808748B2 (en) 2010-04-20 2014-08-19 Vindico NanoBio Technology Inc. Biodegradable nanoparticles as novel hemoglobin-based oxygen carriers and methods of using the same
ES2718308T3 (en) 2011-01-07 2019-07-01 Poseida Therapeutics Inc Compositions and methods for administration to tumors of high oxygen affinity binding agents
EP3594245A1 (en) * 2012-02-13 2020-01-15 Seattle Children's Hospital d/b/a Seattle Children's Research Institute Bispecific chimeric antigen receptors and therapeutic uses thereof
CA2906970C (en) * 2013-03-21 2021-05-18 Ospedale San Raffaele Srl Targeted disruption of t cell receptor genes using engineered zinc finger protein nucleases
KR102452767B1 (en) * 2013-05-14 2022-10-12 더 보드 오브 리젠츠 오브 더 유니버시티 오브 텍사스 시스템 Human application of engineered chimeric antigen receptor (car) t-cells
ES2939760T3 (en) * 2014-03-15 2023-04-26 Novartis Ag Cancer treatment using a chimeric receptor for antigens
DK3129487T3 (en) * 2014-04-09 2020-11-30 Dna Twopointo Inc IMPROVED NUCLEIC ACID CONSTRUCTIONS FOR EUKARYOT GENEPRESSION
CN107075483A (en) * 2014-07-15 2017-08-18 朱诺治疗学股份有限公司 The engineered cell treated for adoptive cellular
ES2822557T3 (en) 2014-08-08 2021-05-04 Poseida Therapeutics Inc Compositions and their use to induce nanoparticle-mediated microvascular embolization of tumors
DK3189132T3 (en) * 2014-09-04 2020-08-10 Stemcell Tech Inc Soluble antibody complexes for T cell or NK cell activation and expansion
EP3018200A1 (en) * 2014-11-07 2016-05-11 Fondazione Matilde Tettamanti e Menotti de Machi Onlus Improved method for the generation of genetically modified cells
AU2015358413A1 (en) 2014-12-05 2017-06-29 Poseida Therapeutics, Inc. Compositions and methods for inducing nanoparticle-mediated microvascular embolization of tumors
US10273300B2 (en) * 2014-12-29 2019-04-30 The Trustees Of The University Of Pennsylvania Methods of making chimeric antigen receptor-expressing cells
US20170000743A1 (en) 2015-07-02 2017-01-05 Vindico NanoBio Technology Inc. Compositions and Methods for Delivery of Gene Editing Tools Using Polymeric Vesicles
US10456452B2 (en) 2015-07-02 2019-10-29 Poseida Therapeutics, Inc. Compositions and methods for improved encapsulation of functional proteins in polymeric vesicles
JP2019528760A (en) * 2016-09-30 2019-10-17 ポセイダ セラピューティクス， インコーポレイテッド Modified stem cell memory T cell, method for producing the same and method for using the same

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