CA3036926C - 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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CA3036926C
CA3036926C CA3036926A CA3036926A CA3036926C CA 3036926 C CA3036926 C CA 3036926C CA 3036926 A CA3036926 A CA 3036926A CA 3036926 A CA3036926 A CA 3036926A CA 3036926 C CA3036926 C CA 3036926C
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cell
cells
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CA3036926A1 (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

DEMA_NDE 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.
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 Date Recue/Date Received 2021-01-12 and multipotent capacity to derive central memory (Tcm), effector memory (TEA) and effector T cells (TO, 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 VHI-1, 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.
107] 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 (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
- 2 -more cell-surface marker(s) of a stem memory T cell (Tscivi), 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 I 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 (Tsai). 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 (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 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 I 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 (Tsai). 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 1L-2143. In certain embodiments, the cell-surface markets of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2R.0, 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
- 3 -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 piggyBac Tm (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBacTm (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.
[091 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 piggyBac Tm (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 Ill 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 /0 or any percentage in between identical to:
- 5 -541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
5).
10131 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 piggy-BacTm 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 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 tryptophan (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
- 6 -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 praline (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 mednonine (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 lysinc (K) for a prolinc (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) for a 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 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 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 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 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 qsteine (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). hi 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 phenylalaninc (F) for a lcucinc (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 Ill 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).
10141 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 argininc (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 asparaginc (N) for an aspartic acid (D). In certain cmbodimcnts, 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 Ill 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) 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.
[015] 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 (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%, 950/0, 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 I 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 stern memory T
cells (Tsai). 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 (TsciA). 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 (Tsc1,4), 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 stein 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-21213. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, 1L-2143, 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).
10161 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 HQLCQEEKAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7).
(018] The disclosure provides a method of producing a modified stem memory T
cell (Tsai), 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 (Tsam). The disclosure provides a method of producing a plurality of modified stein memory T cells (Tsai), 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 /0, 60%, 65%, 70%, 75%, 80%, 85 /0, 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 I 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 stein 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 (Tsar), 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 (Tsc-m). 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 I 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). hi 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-2R13. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2RO, 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.
10191 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 gcnomc 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 ACATGCAAAT TGACCATCCC TCCGCTACGC TCAA.GCCACG
- 13 -61 CCCACCAGCC AATCAGAAGT GACTA.TGCAA ATTAAC:CCAA CAAAGATGGC AGTTAAATTT
121 GCATACGCAG GTGTCAAGCG CCCCA.GGAGG CAACGGCGGC CGCGGGCTCC CAGGACCTTC

421 GTCCCACCCC CACA.GAATCA GCCAGPATCA GCCGTTGGTC A.GAC.AGCTCT CAGCGGCCTG

841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG AAC.ATAGTTG TGGTGGAATG
901 ACTGTTCGAT GTGAA.TTTTG CCTATCACTA AATTTCTCTG ATGAAAAACC ATCCGATGGG

1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGAC.AGTAA AAATTTCATG

1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAA.TACACG GACAAGTTTA TCACCGTACT
1201 GGAACTTTA.0 ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT
1261 ACAGCCGAA.G CTACAAGTAA AAGATTAGCA ATGCCAGAAA ACCAGGGCTG CTCAGAAAGA.
1321 CTC,ATGATCA ACATCAACAA CCTC.A.TGCAT GAAATAAATG AATTAACAAA ATCGTACAAG

1681 GGTGAAAAAG GCTGGGGAAC AGATATTGCA TTAAGACTCA GAGAC.AACAG TGTAATCGAC

1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGAT.AA ATTTCATCAA AGCAAACCAA

2041 AGAAA.TATGC AGCAGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC
2101 GATTTA.TTCA TAACCATGAC ATGCAACCCC AAATGGGCAG ATATTACAAA CAATTTACAA
- 14 -2461 TGTGGAATAC AAAATCCAAA TAGTCCATGT ATGGAAAATG G.AAAATGTTC AAAGGGATAT
2521 CCAAAAGAAT TTCAAAATGC GACC.ATTGGA AATATTGATG GATATCCCAA ATACAAACGA

2641 TATAA.' CCCGT ATTTGTGCCT TAAATATAAC TGTCA.TATAA ATGTTGAAGT CTGTGCATCA

2761 CAAATTTCTG AAAAAAATAT TATCAA.TCAT GACGAAGTAC AGGACTTCAT TGACTCCAGG

2381 CA.TGCAATCA CAAGATTAGC TATTCATTTG CCAAATGATC AGAATTTGTA TTTTCATACC

3121 GGTAGACTGT TCACTGTGAG CTTTA.GAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT

3241 GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC TTGA.TGACAC TATCTGGAAA

3361 TATATATGTG TGTTTGGATG TCCTTCTGCT GCAGACAAAT TATGGGATGA GAATA_AATCT

3781 CGTGGTGGTA CTGTTTTACC CAC:AGCATCT ACAGGAATTG CTGCAAATTT ACTTCTTGGT

4081 CAATGTCTCA GTATTGTACC ACATGCTATG CGA.TCGGCCA TAGTACAAAC GAGTTTAAAG
4141 TACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA AAACAAATAT GAGATCAGA.G

4261 CATTTAGGAA TGGATATTAT TGAAA.TCCCC CATGAAATGA TTTGTAACGG ATCTATTATT
4321 GAAGCTACCT TTGGAAATAG TATATCTATA GATAATATTA AAAA.TATATC TAAACGTGCA
- 15 -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 LPEPVFAHGQ LYVAFSRVRR ACDVKVKWN TSSQGKLVKH SESVFTLNVV YREILE (SEQ ID
NO: 28).
10221 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 covalendy 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 ED NO: 29).
10231 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 transposasc composition comprising a transposase or a sequence encoding the transposasc; 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 (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 transposasc composition comprising a transposasc or a sequence encoding the transposasc; to produce a plurality of modified T cells, wherein at least 2%, 5%, 100/0, 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 (Tsavi), 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 I 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-2Rf3. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2R, 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.
10241 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 -541 PT,DHKKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV SDTREST.LTF P.AICSLSIKT
631 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEQ ID NO: 30).
[025] An exemplary To12 transposon of the disclosure, including inverted repeats, subtemunal sequences and the To12 transposase, is encoded by a nucleic acid sequence comprising the following:

121 TTTTTTTAGA. . 'AAAAAAAGTA. CTTTTTACTC CTTACAATTT TATTTACAGT C:AAAAAGTAC
181 TTATTTTTTG GAGA.TCACTT CA.TICTATTT TCCCTTGCTA TTACC.AAACC .AATTGAATTG
241 CGCTGATGCC CAGTTTA.ATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT

361 TTACCACAAT GCACAGCACC TTGAC'CTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG

721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAA.T GCATGCGATT
781 GAC,'AATATAA. GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT
941 CCACGGGAAA. TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT
- 19 -1681 TCGAATACAT TTTGGTCCAA AAA.TAACAAA ACCTA.C:GACT TTA.TTCGGCA TTGTATTCTC
1741 TTCCGGGTCT GTTGTCAA.TC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA
1801 AGTCGTAGGT TTTGTTA.TTT TTGGACCAAA .ATGTATTTTC GATGCTTCAA ATAATTCTAC

2101 G.AGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA AATATTTATT TGTTGTTTTT

2221 GATCGGGACC TCCACCCATG CTTCCA'GCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC

2341 ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCAT'TTAAA GAGCTGATTA GTACACTGCA

2461 GATCATGAAA. CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACCA.CAAC

2701 GGTTGTTTGC ACAA.CCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG
2761 TGTGGAAAAC P.ATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGTGATG ACACTGATTC

2941 TAACCTAGTC TCAA.GCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAAACT
-20 -4081 ATGATGAAGA TTTTTTCGCT TCTTTGAAAC CGACPACACA TGPAGCCAGC AA.AGAGTTGG
4141 ATGGATATCT GGCCTGTGTT TCAGA.CACCA GGGAGTCTCT GCTCACGTTT CCTGCTATTT

4261 TCAGCA.CTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT AACAATTTTG

4381 CATTAGATTG TCTGTCTTAT AGTTTGAT.AA TTAAATACAA ACAGTTCTAA AGCAGGATAA

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 1-cell (Tem), thereby producing a modified central memory T-eel! (Tem). The disclosure provides a method of producing a plurality of modified central memory 1-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 (Tem). hi 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 (Tem), 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 1-cell (Tem), 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 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 memory T-cells (Tem). 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 (Tem), 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 (Tem), thereby producing a plurality of modified central memory 1-cells (Tem).
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 (Tem), 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 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory 1-cell (Tcm), thereby producing a plurality of modified central memory 1-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 (Tem). In certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, CCR7, and CD62L. hi 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. 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 Tem, wherein the plurality of modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R13 and the plurality of modified Tem expresses one or more CD45RO, CD95, IL-2Rf3, 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 (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 1-cells (Tscm) 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 (Tscm) comprise at least 90% of the total number of cells of the composition and the modified central memoq 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 1-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 1-cells (Tscm) comprise at least 80% of the total number of cells of the composition and the modified central memory 1-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 1-cells (Tsaq) comprise at least 30% of the total number of cells of the composition and the modified central memory 1-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 17-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 1-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 stern memory 1-cells (Tsai) comprise at least 60% of the total number of cells of the composition and the modified central memory 1-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 1-cells (Tscm) comprise at least 50% of the total number of cells of the composition and the modified central memory 1-cells (Tcm) comprise at least 50% of the total number of cells of the composition. In certain embodiments of this method, the transposon is a plastnid 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. hi 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 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.
10291 hi certain embodiments of the methods of the disclosure, the antigen receptor is a 1-cell receptor. In certain embodiments, the 1-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 1-cell receptor is not naturally-occurring, the T-eell receptor comprises one or more mutation(s) compared to a wild-type 1-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 1-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.
[0301 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). hi 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). hi 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, hi 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 piggyBacill 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 1-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 1-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 the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified 1-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 I 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 I
cells (Tsc:m). 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 (Tsar), 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 (Tsc-m), 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 (Tscm), 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-2R13. In certain embodiments, the cell-surface markers of the activated modified Tscm comprise one or more of CD45RA, CD95, IL-2R[3, 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 'F-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 1-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 market(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%õ 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 /0 of modified T-cells that express cell-surface marker(s) of a stem memory T cell (Tscm). 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, linolcic 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;
linolcic 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 timol/kg and 640 timol/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.7 tunol/kg and 70 mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 mol/kg and 75 timol/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 pmol/kg and 25 mol/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 mol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 timol/kg, oleic acid at a concentration of about 7.5 tunol/kg and a sterol at a concentration of about 2.5 tunol/kg.
[033] The disclosure provides a method of producing a modified central memory 1-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 1-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 1-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 (Tau), 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 1-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 (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 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 (Tcm). 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 (Term), 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 (Tem). 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 (Tai), 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% 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 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% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory I cell (Tem), thereby producing a plurality of activated
-30 -modified central memory T cell (TcNi). In certain embodiments, the cell-surface markers of the activated modified Tcm comprise one or more of CD45RO, CD95, 1L-2n, CCR7, and CD62L.
[034] hi 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 1-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 1-cells, wherein at least 2% of the plurality of expanded modified 1-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 (TeNi). 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 I 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 (Tem) from the plurality of enriched modified 1-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), di isopropyl adipate (DIPA), n-butyl-benzenesulfonamide, I ,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 p.mol/kg and 640 Amol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 timol/kg and 70 tunol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 punol/kg and 75 timol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 tunol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 timol/kg and 25 ginol/kg, inclusive of the endpoints.
In certain embodiments of this method, the T-ccll 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 tunol/kg, linoleic acid at a concentration of about 7.5 umol/kg, oleic acid at a concentration of about 7.5 timol/lcg 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 (rscm) 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 1-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 1-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-2R13 and the plurality of activated modified Tcm expresses one or more CD45RO, CD95, 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 (Tscm) comprise at least 10% of the total number of cells of the composition and the modified central memory 1-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 I-cells (Tc:m) 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 (Tso4) 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 (1'cim) 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.
10361 In certain embodiments of methods of the disclosure of producing a composition comprising a plurality of modified stem memory T-cells (Tscm) and a plurality of modified central memory T-cells (Tcm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T
cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (Tscm) and a plurality of activated modified central memory T-cells (Tcm), 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 (Tcm) with a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified 'f-cells, wherein the plurality of expanded modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD
127, CD45RO, CD95, CD95 and IL-2R13 and the plurality of expanded modified Tcm expresses one or more CD45RO, CD95, IL-2R13, 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 1-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 TCNI 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-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. hi 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 ing/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
-35-and 640 pmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 timol/kg and 70 pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 p.mol/kg and 75 p.mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol/kg and 75 iAmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 prnol/kg and 25 imol/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 641=01/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 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 (Tservi) 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 (Tscis,4) 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 (Tsc-m) 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 1-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 (Tam) comprise at least 70% of the total number of cells of the composition and the
-36 -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.
[037] 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 1-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%, 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 genornic 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 (D 10A) 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 Asparaginc (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
cndonucicase comprises AciI, Mn11, Alwl, Bbvl, Bccl, BccAl, BsmAl, Bsm.F1, BspCN1,
- 38 -BsrI, 13tsCI, Hgal, Hphl, HpyAV, MbolI, My!!, Plc!, SfaNI, AcuI, BciVI, BfuAI, BingBI.
BmrI, BpmI, BpuEl, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Btsl, EarI, EciI, Mme!, NmeAIII, BbvCI, Bpul 0I, BspQI, Sap!, Bad, BsaXI, CspCI, Bfil, Mboll, 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.
10381 in certain embodiments of the methods of producing an activated modified TSCIA or Tag 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.
10391 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 primal), 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
-39 -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 lentivinis.
[0401 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, AAV I 0 or AAV11. 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 AAV
I, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV I 0 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-I,K03, rAAV-NP59 and rAAV-NP84.
10421 In certain embodiments of the methods of producing an activated modified Tscm or Tcm 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.
10431 In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, a nanopaiticle 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.
10441 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 1-cell receptor comprises one or more mutation(s) compared to a wild-type 1-cell receptor. In certain embodiments, and, in particular, those embodiments wherein the 1-cell receptor is not naturally-occurring, the T-eell receptor is a recombinant 1-cell receptor. hi 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 VI-IH
sequence(s). In certain embodiments, the CAR is a VCAR.
-41 -10451 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, 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.
10461 The disclosure provides a method of producing a modified stem memory T
cell (Tsem), comprising: (a) introducing into a primal), 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 stem memory T cell (Tsem), thereby producing a modified stem memory T cell (Tsem). 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 inonospecific 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 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 (Tsc-m), 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 1-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 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 senim albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM,
-43 -and an expansion supplement to produce a plurality of expanded modified 1-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 1-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-methylpmpyl) 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 1-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, palmitie 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 1-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 iumol/kg and 640 ttmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 mol/kg and 70 mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 1=01/kg and 75 tunol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 1.1mol/kg and 75 tunol/kg, inclusive of the endpoints; and a sterol at a concentration of
- 44 -between 0.25 tunol/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 pmol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 tunol/kg, oleic acid at a concentration of about 7.5 pinol/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 timol/kg, palmitic acid at a concentration of about 7.27 mon& linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 timol/kg. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 timol/kg and a sterol at a concentration of 2.61 timol/kg. In certain embodiments, at least 2%, 5%, 100%, 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 1-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%, 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). 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 1-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 transfcrrin, 2-Mercaptocthanol, lscov-c's MOM, 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, nicotinamidc, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipatc
-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 1-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 steml 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 1-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 1-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 timol/kg and 70 !Among, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 mol/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 p.mol/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 tunol/kg, palmitic acid at a concentration of about 7 timol/kg, linoleic acid at a concentration of about 7.5 1..tmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 tunol/kg. In certain embodiments, the 1'-cell expansion
- 46 -composition comprises one or more of octanoic acid at a concentration of about 63.75 umol/kg, palmitic acid at a concentration of about 7.27 iimol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of about 7.56 innol/kg and a sterol at a concentration of about 2.61 !Among. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 innol/kg, palmitic acid at a concentration of about 7.27 p.mol/kg, linoleic acid at a concentration of about 7.57 1=01/kg, oleic acid at a concentration of 7.56 pniol/kg and a sterol at a concentration of 2.61 timol/kg.
10471 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 1-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 (Tcm), comprising:
(a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 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% 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 monospccific tetrameric antibody complex. In certain embodiments, the methods farther comprises the step of. (c) contacting the activated modified T cell and a 1-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human
- 47 -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 (1'civi). 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), di isopropyl adipate (DIPA), n-butyl-benzenesulfonamide, I ,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 ing/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 turnol/kg and 640 limol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 limol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 imnol/kg and 75 tunol/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
-48 -sterol at a concentration of between 0.25 pmol/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 gmol/kg, palmitic acid at a concentration of about 7 Ltmol/kg, linoleic acid at a concentration of about 7.51.imolVkg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 prnol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 pnol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of about 7.56 p.mol/kg and a sterol at a concentration of about 2.61 pmol/kg.
in certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 lAmol/kg, palmitic acid at a concentration of about 7.27 mol/kg, linoleic acid at a concentration of about 7.57 1=01/1cg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61p.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 central memory T cell (Tcm). 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 (Tcm). 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 (Tcm). 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 (Tcm). 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 (Tcm) from the plurality of enriched modified T-cells.
In certain embodiments, 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 transfcrrin, 2-Mercaptocthanol, Iscov-e's MOM, and an expansion supplement to produce a plurality of expanded enriched modified Tcm.
In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamidc, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipatc
- 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 1-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 steml 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 1-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 1-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 timol/kg and 70 !Among, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 mol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 p.mol/kg and 25 timol/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 7 gmol/kg, linoleic acid at a concentration of about 7.5 mol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 tunol/kg. In certain embodiments, the 1'-cell expansion
-50 -composition comprises one or more of octanoic acid at a concentration of about 63.75 lima& palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 pimol/kg, oleic acid at a concentration of about 7.56 innol/kg and a sterol at a concentration of about 2.61 !Among. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 punol/kg, palmitic acid at a concentration of about 7.27 p.mol/kg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of 7.56 ttmol/kg and a sterol at a concentration of 2.61 timol/kg.
10481 The disclosure provides a method of producing a composition comprising a plurality of modified stem memory T-cells (Tscm) and a plurality of modified central memory T-cells (Tc.m), 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 stein 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-2R13 and the plurality of activated modified Tat expresses one or more CD45RO, CD95, 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 transfcrrin, 2-Mercaptoethanol, Iscovc's MDM, and an expansion supplement to produce a plurality of expanded modified 1-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 (Tuu). 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 1-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 1-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 iumol/kg and 640 timol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 tunol/kg and 75 tunol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 1.1mol/kg and 75 tunol/kg, inclusive of the endpoints; and a sterol at a concentration of
-52 -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 pniol/kg, palmitic acid at a concentration of about 7 pmol/kg, linoleic acid at a concentration of about 7.5 tunol/kg, oleic acid at a concentration of about 7.5 iimol/kg and a sterol at a concentration of about 2.5 iimol/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 mon& linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 p.mol/kg. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 iimol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 p.mol/kg and a sterol at a concentration of 2.61 mol/kg. In certain embodiments, at least 2%, 5%, 100%, 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 Tsem and a plurality of expanded modified TCM expresses cell-surface marker(s) of a stem memory T cell (Tsai). 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 Tsem and a plurality of expanded modified Tem 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 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 (Tsai). 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 1-cells that express cell-surface marker(s) of a central memory T cell (Tem). hi 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 (Tsem) from the composition or isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tem) from the composition.
In certain embodiments, the enriching step further comprises isolating modified 1-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 (Tem) from the composition. In certain embodiments, the enriching step further comprises contacting the isolated modified Tscm and/or 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 composition comprising a plurality of expanded enriched modified Tscm and/or Tem. hi certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetrarnethy1-5-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 stem! 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 1-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 steno! 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, linolcic 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 -1/m01/kg and 640 mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 i/mol/kg and 70 mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 mol/kg and 75 p.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 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 mot/kg, palmitic acid at a concentration of about 7 mot/kg, linoleic acid at a concentration of about 7.5 mol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 p.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 limol/kg, oleic acid at a concentration of about 7.56 mol/kg and a sterol at a concentration of about 2.61 mol/kg.
In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 molikg, 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 mot/kg and a sterol at a concentration of 2.61 mol/kg. In certain embodiments of this method, the modified stem memory 1-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 1-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 1-cells (Fe!) 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 (Tscm) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (Tcm) comprise at least 30% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (Tcm) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (Tcm) comprise at least 40% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (Tcm) comprise at least 50% of the total number of cells of the composition.
[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 die method produces a modified I 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 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 is a To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a ToI2 transposon, the transposase is a To12 transposase.
[0501 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 -10511 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 paIindromic repeats (CRISPR) associated protein 9 (Cas9) DNA
endonuclease. hi 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 (D
10A). 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 gcnomic 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 piggyBacTM (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) (G165S). In certain embodiments, the amino acid substitution at position 282 of SEQ ID 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.
10531 In certain embodiments of the methods of the disclosure, the transposon further comprises a selection gene. ha 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 1-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 1-cell receptor is not naturally-occurring, the T-eell receptor comprises one or more mutation(s) compared to a wild-type 1-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 1-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.
[055] 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-2143. In certain embodiments, the cell-surface markers of the modified Tscm comprise one or more of CD45RA, CD95, IL-2RO, CR7, and CD62L.
[056] In certain embodiments of the methods of the disclosure, the plurality of expanded modified 1-cells comprises a naïve 1-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 1-cells comprises a central memory T-cell (modified TcNi) and the cell-surface markers of the CAR-Tcm comprise one or more of CD45RO, CD95, IL-2R. CCR7, and CD62L. In certain embodiments, the plurality of expanded modified T-cells comprises an effector memory 1-cell (modified TEm) and the cell-surface markers of the CAR-TEm comprise one or more of CD45RO, CD95, and IL-2141. 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-211.13.
[057] In certain embodiments of the methods of the disclosure, the plurality of expanded modified T-cells comprises a central memory T-cell (modified Tem) and the cell-surface markers of the CAR-Tem comprise one or more of CD45RO, CD95, IL-2R13, 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 Tem) and the cell-surface markers of the CAR-Tem comprise one or more of CD45RO, CD95, IL-2R13, 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 Tem), the plurality of expanded modified 1-cells comprises a Taw 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-21113.
[0581 The disclosure provides a method of producing a modified stem memory I
cell (Tsc-m), 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 stein 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 (Tsai), thereby producing a plurality of activated CAR stern memory T cells (Tsai). 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 stern 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-I 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 (Tsai), 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 (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 CD621, and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tsar comprise one or more of CD62L, CD45RAõ
CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-24. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-2R. CR7, and CD62L. The disclosure provides a method of producing a modified stem memory T cell (Tsai), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-I
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 I cell (Tscm), thereby producing a CAR-expressing stem memory T cell (Tscm) (CAR-Tscm).
10591 The disclosure provides a method of producing a plurality of modified stem memory T cells (Tscm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T
cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%.
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsovi), 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 I cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-I 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 I 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 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 (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, CDI 27, 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-2R3, CR7, and CD62L.
1060] 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 serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded CAR-T
cells, wherein at least 2% of the plurality of expanded CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsai) (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, pal mitic 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, linolcic 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;
linolcic 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 ing/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 1-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 tunol/kg and 70 1=01/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 1=01/kg and 75 tunol/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 timoUkg, 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 timol/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 timol/kg, oleic acid at a concentration of about 7.56 gmol/kg and a sterol at a concentration of about 2.61 grriol/kg. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 timol/kg, palmitic acid at a concentration of about 7.27iumoUkg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of 7.56 tunol/kg and a sterol at a concentration of 2.611=01/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-2Rf3. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2R13, 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, IL-2R13, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-TEm) and the cell-surface markers of the CAR-TEm comprise one or more of CD45RO, CD95, and IL-2Rf3. 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-2Rf3. Additional cell-surface markers are described in Gattinoni et al. (Nat Med. 2011 Sep 18; 17(10): 1290-7.
[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 -Date Recue/Date Received 2021-01-12 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 1-cell activator composition comprising one or more of an anti-human CD3 monospecific tetratneric 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 market(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 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). hi 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
-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-2Rp. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, EL-2R(. CR7, and CD62L.
[0621 In certain embodiments of the methods of the disclosure, the plurality of expanded CAR-T cells comprises a naïve 1-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, IL-2RP, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-Ti) and the cell-surface markers of the CAR-TEm comprise one or more of CD45RO, CD95, and IL-2R3. 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-2Rp.
[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-regulatory 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.
19651 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 -10661 In certain embodiments of the methods of producing a modified TSCht 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 I-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 ginol/kg and 640 p.mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 mol/ke and 70 latnol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 prnol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 p.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 gmol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 p.mol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 jamoUkg. 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 Knol/kg, oleic acid at a concentration of about 7.56 mol/kg and a sterol at a concentration of about 2.61 mollg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 prriol/kg, palmitic acid at a concentration of about 7.27 lamol/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 i.unol/kg. In certain embodiments of the nucleofection, the transposon composition is a 0.5 pg/ 1 solution comprising nuclease free water and the cuvette comprises 2 I of the transposon composition to yield 1 pg 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 pg 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 Helraiscr 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.
10671 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 electroporation, 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 compriscs 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 Tsem 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 KCI, 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 KCI, 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 mM HEPES and 75 mM
Tris/HC1. In certain embodiments, the buffer comprises 5 mM KCI, 15 mM MgCl2, 90 mM
CINa, 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 1.d of the buffer and between 5x106 and 25x106 cells.
[068] In certain embodiments of the methods of producing a modified Tscivi of the disclosure, the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and/or CD! 9. In certain embodiments, the composition comprising primary human T cells comprises 100 1.11 of the buffer and between 5x106 and 25x106 cells.
10691 As used herein, the terms "supplemented T-cell expansion composition" or "1-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 ((IMDM); available at ThermoFisher Scientific as Catalog number 12440053).
[070] As used herein, the terms "supplemented 1-cell expansion composition" or "1-cell expansion composition" may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transfenin, 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 1'-cell expansion composition" or "T-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, 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 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 "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), alkanes (e.g., nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). ln certain embodiments, the terms "supplemented 1-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-tetramehy1-5-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), stcaric 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 1-cell expansion composition" or "1-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 MUM, 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 1-cell expansion composition" or "1-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 IvIDIvI, 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 1-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 /0), 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 1%), 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%), scrine (about 1.6%), glutamine (about 67.01%), argininc (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 1-cell expansion composition" or "1-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 1-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 "1-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 1-cell expansion composition" or "1-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 1-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 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 1-cell expansion composition" or "1-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 6.4 Amol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 timol/kg and 70 Lunol/kg, inclusive of the endpoints; linolcic 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 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 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 timol/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 tunol/kg. In certain embodiments, the terms "supplemented 1-cell expansion composition" or "1-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 gmol/kg, pahnitic acid at a concentration of about 7.27 gmolikg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of about 7.56 tunol/kg and a sterol at a concentration of about 2.61 pmol/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 gmolikg, palmitic acid at a concentration of about 7.27 Limon& linoleic acid at a concentration of about 7.571.1mo1/kg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 timol/kg.
10751 As used herein, the term "P3 buffer" may be used interchangeably with a buffer comprising one or more of KC1, MgCl2, CINa, 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 KC1, 15 mM
MgCl2, 90 mM CINa, 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 CINa, 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 KC1, 15 mM MgCl2, 90 mM
CINa, 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 "1-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 2HCI, 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 FICI, vitamin B12, D-Glucose, Glutathione (reduced), L-Glutamine and Mercaptoethanol in any absolute or relative abundance or concentration. The teims "supplemented RPM-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 HC1, 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.
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 2HCI, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isolcucine, L-Lcucine, L-Lysinc HCl, L-Methionine, L-Phenylalanine, L-Prolinc, L-Scrinc, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 21-120, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Arninobenzoic acid, D-Panthothenic acid (hemicalcium), pyridoxine HC1, riboflavin, thiamine HC1, 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 HCI, L-Methionine, L-Phenylalanine, L-Pmline, 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.
10781 As used herein, the term "hnmunoCultTM 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 "lmmunoCultTM
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-Glutaminc, L-Histidinc 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 pantothcnate, 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 GSK3D 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 (BLUEBIRDBIOn1).
10801 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 permeable to the nucleic acid, transposon, vector or composition of the disclosure.
[0811 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 1, 2, 3, 4, 5, 6 or 7 days or any number/portion of days in between. The incubation step may have a period of at least 1 week. 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) EI-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, E0-151, E0-148, E0-156, E0-210, E0-213, and FI-156. In certain embodiments, the applying step may comprise applying one or more of the following
- 80 -
81 program(s) El-115, El-151, El-156, EI-I58, EG-I15, EG-I42, EG-I51, ES-115, ES-151, E0-151, E0-148, EO-156, E0-210, EO-213, and FI-156, or a program that provides the same number of electrical pulses, each pulse having the same duration and intensity, and a substantially similar interpulse duration of time. In certain embodiments, the applying step may be performed using a known electroporation/nucleofection device, including, but not limited to, Lonza Amaxa, MaxCyte technology, BTX 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.
10831 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. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (1L-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.
10841 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 lyrnpokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (1L-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (1L-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.
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 naïve T cells. In certain embodiments, the efficiency of production of modified Tscm and/or Ten,r 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.
[0861 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.
10871 In certain embodiments of the methods of producing a modified Tsem 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 Tsem and/or To,' of the disclosure, the primary human T cell is a naïve 1-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 Tem of the disclosure, the primary human T
cell is a modified Tsem a T memory stem cell (modified Tscm) and the modified Tsem expresses one or more of CD45RA, CD95, IL-2R, 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 1-cell (modified Tem) and the modified Tem expresses one or more of CD45RO, CD95, IL-2RO, CCR7, and CD62L. In certain embodiments of the methods of producing a modified Tsem and/or Tem of the disclosure, the primary human I cell is an effector memory 1-cell (modified TEm) and the modified TEM
expresses one or more of CD45RO, CD95, and IL-2R(. In certain embodiments of the methods of producing a modified Tsem and/or TCM of the disclosure, the primary human T
cell is an effector 1-cell (modified TEFF) and the modified Tuff, expresses one or more of CD45RA. CD95, and IL-2RO.
[0891 In certain embodiments of the methods of producing a modified Tsem 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.
[090] In certain embodiments of the methods of producing a modified Tsem and/or Tcm of the disclosure, the primary human I 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.
10911 Primary human T cells of the disclosure may comprise pan T cells. As used herein, pan 1-cells include all T lymphocytes isolated from a biological sample, without sorting by subtype, activation status, maturation state. or cell-surface marker expression.
10921 In certain embodiments of the methods of the disclosure, the method further comprises introducing into a modified Tsai or Tag cell a composition comprising a genomic editing construct or composition. In certain embodiments, the genomic editing constnict 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 1IS endonuclease and wherein the expressed DNA binding domain and the expressed type US
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 endonucicasc 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 I 0 (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 Flistidine (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 11S 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 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).
10931 The methods of making modified Tscm and/or Tcm cells of the disclosure may be optimized to produce a greater number or greater proportion of modified Tscm and/or Tcm cells. 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 naive T
cells. As the number and/or proportion of nave T cells increases in the population of T
cells subjected to the methods of the disclosure, the number and/or proportion of modified Tscm and/or Tcm cells of the disclosure produced also increases. 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 Tcm cells of the disclosure produced by the method increases. The length of time or duration required for a method of disclosure to precede, or
- 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 T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R13. In certain embodiments, the primary human T
cell is a naive 1-cell (TN) and the IN 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-2113, CR7, and CD62L. In certain embodiments, the primary human T cell is a central memory T-cell (Tcm) and wherein the Tcm expresses one or more of CD45RO, CD95, IL-2R3, CCR7, and CD62L. In certain embodiments, the primary human T cell is an effector memory 1-cell (TEm) and the EM
expresses one or more of CD45RO, CD95, and In certain embodiments, the primary human T cell is an effector 1-cell (TEFF) and the TEFF expresses one or more of CD45RA, CD95, and IL-2R. In certain embodiments, the primary human T cell expresses CD4 and/or CD8.
[095] The disclosure provides a composition comprising a modified Tscm produced a method of the disclosure. The disclosure provides a composition comprising a modified Tcm produced a method of the disclosure. The disclosure provides a composition comprising a modified TSCM and a modified TcNi produced a method of the disclosure. In certain embodiments of the composition comprising a modified Tscm and a modified Tcm produced a method of the disclosure, a plurality of TSCM may comprise at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% or the composition. . In certain embodiments of the composition comprising a modified Tscm and a modified Tcm produced a method of the disclosure, a plurality of Tcm may comprise at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% or the composition.
10961 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 usc, the modified Tscm and/or Tcm is autologous. In certain embodiments of this use, the modified Tscm and/or Tcm is allogeneic.
In certain embodiments, the antigen receptor is a 1-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-eeli 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 VHFI sequence(s). In certain embodiments, the CAR is a VCAR.
10971 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 Teis,i 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, hi certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, in those embodiments wherein the T-eell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. hi certain embodiments, and, in particular, in those embodiments wherein the 1-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. hi 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.
10991 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.
101.001 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.
101011 Figure 4 is a series of plots depicting an enrichment of the CAR-Tscm phenotype at Day 19 of the method described in Example 1. Cells were nucleofected with a surrogate CARTyrin plasmid. CAR-Tscm cells express CD62L and 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 -101041 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.
10101 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.
101061 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.
101071 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+), Tern (CD62L+CD45RA-), Tern (CD62L-CD45RA-) and Teff (CD62L-CD45RA+). All CAR+ T cells were CD95+.
101081 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 11 flow cytometer) into naïve (CD62L+CD45RA+), Tern (CD62L+CD45RA-), Tern (CD62L-CD45RA-), and Tcff (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 -101091 Figure 9 is a pair of graphs showing that the piggyBacTm manufacturing process yields high levels of Tscm in samples from multiple myeloma (MM) patients even when naive T cells are rare. T cells from MM patients (triangles) and healthy donors (circles) were characterized for memory marker expression by flow cytometry before (left) and after (right) the Poseida manufacturing process. Expression of CD45RA and CD62L was assessed by FACS and plots are shown for the MM patients and a healthy donor. it is known that T cells from MM patients generally have lower frequencies of naive and TSCM cells, but higher frequencies of Teff, unlike those from healthy normal donors which are the opposite.
Regardless of the input frequency of naive and Ts= 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).
101.101 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 114 of either a Sleeping Beauly 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 (jig) of the Sleeping Beauty transposon SB100x, (second from top) 5 lag of SB100x, (3rd from top) 10 jig of SB100x, (second from bottom) 5 iv 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 103 incrementing by powers of 10.
The final right hand column shows CD62L expression, with markings reading from 0 to 103 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.
[0111] 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 fluorescein 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 cells.
[0114] 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) TTAA-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 depict SEQ ID NOs 41 and 42 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 -Date Recue/Date Received 2021-06-10 transgenes were selected by melliotrexate 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 I cells receiving transposition reagents, RNP
plus HR or MMEJ donor plasmids, but not in T cells receiving donor plasmids alone.
[0118] Figure 17A-C is a series of graphs showing the phenotype of primary human pan T
cells modified by HR and MMEJ at the AAVS I 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 (Tem)), CD45RA-CD62L- (effector memory T cells (Tern)), and CD45RA+CD62L- (T effectors (Teff)). B) Percentage of total (3FP+ 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 AAVS1 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 5'-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 AAVS1 region beyond the 500 bp homologue arm at the 5'-end AAVS-3r CTGCACCACGTGATGTCCTC (SEQ ID NO: 37), yielding a 0.73 kb DNA fragment for both HR or MMEJ) and 3'-end junction (with one primer priming the polyA
signaling region SV40pA-lr GTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38) and the other priming the AAVS I 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 AAVS I target site. PCR products were displayed on Agarosc gel. Non-specific bands in HR samples arc the result of only a single round of PCR
and would likely have been resolved given additional rounds.
DETAILED DESCRIPTION
-92 -101201 The disclosure provides a method for producing human chimeric antigen receptor (CAR) expressing-T cells using the piggyBac TM Transposon System under conditions that preserve or induce stem-cell memory T cells (Tsem) with potent CAR activity (referred to herein as a CAR-Isem. Compositions comprising CAR-Tscm produced using the methods of the disclosure comprise? 60% CAR-Tsem 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 memory CAR-T cells (CAR-TEm), effector CAR-T cells (CAR-TB), and terminally-differentiated effector CAR-T cells (CAR-TIE). A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN) > Tsai > Tem > Ti >
TE > TTE, whereby TN is the parent precursor cell that directly gives rise to Tsem, which then, in turn, directly gives rise to Tem, etc. Compositions comprising CAR-Tsc:m, CARTyrin-Tsem and/or VCAR,Isem of the disclosure may comprise one or more of each parental CAR-T
cell subset with CAR-Tsai being the most abundant (e.g. TSCM > Tem > TEm > TE > TrE).
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 Tsem, compositions of the disclosure comprising non-naturally occurring CAR-Tsc-m are more potent and efficacious in treating patients against diseases and cancers.
101211 Immunotherapy 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 CD l9 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
stem cell-like CAR-T would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (Tem), effector memory (TEm) and effector T cells (TE), thereby producing better tumor eradication and long-term CAR-T engraftment.
-93 -101221 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.
101231 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 VI-IH
expression by blocking improper gene activation or silencing.
101241 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.
1012.51 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 transgcnc 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 memory 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 101261 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).
(01271 In certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD36, CD3s, 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).
101281 In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD35, CD3F., 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 IYINVAPLAGTCGVLLLSLVITLYC (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 atctacatttgggcaccactggccgggacctgtsgagtgctgctgctgagcctggtcatcacactgtactgc (SEQ
ID NO:
11).
101291 In certain embodiments of the CARS of the disclosure, the endodomain may comprise a human CD3t; endodomain.
101301 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 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ

PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRIIKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising
-96 -cgcgtgaaetttagtcgatcagcagatgccccagcttacaaacaeegacagaaccagctgtataacgagctgaatctgg gccgccga gaggaatatgaegtgctggataageggagaggacgcgaccccgamtgggaggcaagcccaggcgcaannnccetcagga agg cctgtataacgagctgcagaaggacaaantggcagaagcctattctgagatcggcatgaagggggagegacggagaggc aaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctc,c aagg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKILYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID
NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYTFKQPFMRPVQ1TQEE1DGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgetgtatattttcaaacagccatcatgcgceccgtgcagactacccaggaggaagaegg gtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (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 actaccacaccagcacctagaccaccaactccagetccaaccatcgcgagtcagcccctgagtctgagacctgaggcct gcaggcc agetgcaggaggagctgtgcacaccaggggectggacttcgcctgcgac (SEQ ID NO: 17).
[0132] The disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
101331 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. 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), DFIFR (encoding Dihydmfolate 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 I family, member Al), FRANCF, RAD5 IC
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NIC2 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 VKQTLNF'DLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQ'TLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 700/0, 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 A.TNFSLLKQAGDVEENPGP (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 /o, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-12A
peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagectgctgacctstggagacgtggaggasancccaggacca (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 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 VKQ'TINFDLLKLAGDVESNPGP (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).
[01371 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.
101381 Transposons of the disclosure may comprise piggyBac transposons.
Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
[01391 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.
[01401 Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovims, 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 -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-D.1.8). 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). MGNfT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDHI (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RADS IC
(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. hi 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 (]SG-E2A peptide, an F2A peptide, a (]SO-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 ggatctggagagggaaggggaaecctgctgacctgteeagacgtggaggaaaacccaggacca (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).
101431 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 1-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.
[01441 The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is a nanoparticic. Exemplary nanoparticle vectors of the disclosure include, but are not limited to, nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g. polymersomes), micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any
- 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 Thymidylate Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RADS IC
(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-12A 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 ggatctggagagggaaggggaagectgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QC'TNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 900/0, 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).
101471 The disclosure provides a composition comprising a vector of the disclosure.
CARTyrins 101.481 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-IBB 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 atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (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, CD36, 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 transmembranc 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 atctacatttgggcaccactggccgggacctgtggaggctgctgctgagcctggtcatcacactgtactgc (SEQ
ID NO:
11).
[01521 In certain embodiments of the CARS of the disclosure, the endodomain may comprise a human CD3t endodomain.
[01531 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 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST.ATKDTYDALHMQALP
PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagettacaaacagggacagaaccagctgtataacgagctgaatctgg gccgccga gaggaatatgacgtgctggatangeggagaggacgcgaccccgaaatgggaggcaageccaggcgcvannecctcagga agg cctgtataacgagetgcagaaggacaaaatggcagaagcctattctgagateggcatgaagggggagcgacggagaggc aaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgactgcatatgcaggcactgcctccaa gg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYTFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID
NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-IBB costimulatoty domain may be encoded by the nucleic acid sequence comprising aagagaggcaggangaaactgctgtatattttcaaacagccettcatgcgccccgtgcagactacccaggaggaagacg ggtocc tgtcgattccctgaggaagaggaaggegggtgtgagctg (SEQ ID NO: 15). The 4-1BB
costimulatory domain may be located between the transmembrane domain and the CD28 costimulatoiy domain.
- 106 -101541 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 111 PA PR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID
NO: 16). The human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagetecaaccatcgegagtcagcccctgagtctgagacctgaggcct gcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).
101551 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 MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEATNLTVPGSERSYD
LTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). The consensus sequence may encoded by a nucleic acid sequence comprising atgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacg cagccttcg atagttttatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtectgacagtgccagggtecgaacgcte ttatgacctg acagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggeaatatcagettecetetgtecg caatettcaz 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)-(1). Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type 111 (FN3) domains or at least 15 fibronectin type 111 (FN3) domains. The scaffold may bind an antigen with at least one affinity selected from a KD of less than or equal to 10-9M, less than or equal to 10-' 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 10-14M, and less than or equal to 10-15M. The KD may be determined by surface plasmon resonance.
101561 The disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
101571 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)-methylguaninc-DNA methyltransferasc), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide s.ynthase), and NKX2.2 (encoding NK2 Homcobox 2).
101591 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 T'2A 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 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 700/0, 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 /0, 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 EGRGSIITCGDVEENPGP (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 II NO: 19). A
GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagg,gaaggagaagectgctgacctgtggagacgtggaggnamcccaggacca (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).
- 110 -101611 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.
101631 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 serotypes (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.
101651 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 Thymidylatc Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR I), ALDHI (encoding Aldehyde dehydrogenase 1 family, member A l ), FRANCF, RADS IC
- Ill -(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 ggatctggagaggsaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (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 VKQUNFDLLICLAGDVESNPGP (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 Ill 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).
[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 1-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. polymersomes). micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any combination thereof. A nanoparticle vector may be passively or actively transported across a cell membrane.
[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 Thymidylatc Synthetase), MGMT
(encoding 0(6)-mediylguanine-DNA methyltransferase), multidrug resistance gene (MDRI), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RADS 1C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramidc 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. hi 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 11 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 ggatctegagagggaaegggaagcctgctgacctgtggagacgtggagganaacccaggacca (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 GSGVKQMNFDLLKLAGDVESNPGP (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).
101711 The disclosure provides a composition comprising a vector of the disclosure.
Scaffold Proteins 101721 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.
101.731 Protein scaffolds of the disclosure may be derived from a fibronectin type 111 (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.
101.741 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.
101751 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.
101761 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.

101771 The disclosure provides a method of generating libraries of a protein scaffold based on a fibronectin type 111 (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.
101.781 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.
101791 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.
101801 The protein scaffolds of the disclosure offer advantages over conventional therapeutics, such as ability to administer locally, orally, or cross the blood-brain barrier, ability to express in E. Coli allowing for increased expression of protein as a function of resources versus mammalian cell expression ability to be engineered into bispecific or tandem molecules that bind to multiple targets or multiple epitopes of the same target, ability to be conjugated to drugs, polymers, and probes, ability to be formulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.

101811 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., affmity matured, to improve certain binding or related properties.
101821 Three of the six loops of the protein scaffold of the disclosure correspond topologically to the complementarily 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 affinity. One or more of these loop regions are randomized with other loop regions and/or other strands maintaining their sequence as backbone portions to populate a library and potent binders can be selected from the library having high affinity for a particular protein target. One or more of the loop regions can interact with a target protein similar to an antibody CDR interaction with the protein.
101831 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.
101841 Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a VHE). 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.
101871 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.
[0188] 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 ale 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 affmity 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 foiming 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 thennostability 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 Aylmer 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.
101941 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.
101951 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.
101961 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. Ktmitz 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 at.. 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).
101991 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.
102001 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 102011 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 piggyBac" (SPB) transposase of the disclosure simultaneously prior to introduction of the transposon and transposase into the cell. In certain preferred embodiments, the Super piggyBac" (SPB) transposase is an mRNA sequence encoding the Super piggyBac"
(SPB) transposasc.
102021 Additional disclosure regarding piggyBac transposons and Super piggyBacTm (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.
[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 piggyBacIm or a Super piggyBacIm (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBacIm (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:

361 EPYKLTIVGT VRSNKREIFE VLKNSRSRPV GTSMFCFDGE, LTLVSYKPKE, AKMVYLLSSC

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 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:

Date Recue/Date Received 2021-01-12 541 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 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 (Cl). 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 piggyBac TM (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). hi certain embodiments, the Super pig,gyBacTm (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).
102091 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 threonine (T) for an alanine (A). hi certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (I). 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 tyrosine (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 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 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 (1-1) 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 (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 methioninc (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).
102101 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 valinc (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 alaninc (A) for a lysinc (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 (5) 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 piggy BacTm 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 ED NO: 4.
[0211] 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.
[02121 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.
102131 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.
102141 By "transient transformation" is intended that a poly-nucleotide construct introduced into the host does not integrate into the genome of the host.

102151 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 Tcm.
Additional Transposon 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 1-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 I cell, wherein the modified T
cell expresses one or more cell-surface marker(s) of a modified stem memory 1-cell (Tscm) or a modified central memory T-cell (Tcm), thereby producing a modified stem memory 1-cell (Tscm) or a modified central memory T-cell (Tcm). The disclosure provides a method of producing a plurality of modified stem memory T-cells (Tscm) or a plurality of modified central memory T-cells (Tcm), comprising introducing into a plurality of primary human T
cells (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%

or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T-cell (Tscm) or a central memory 1-cell (Tcm), thereby producing a plurality of modified stem memory T-cells (Tscm) or a plurality of modified central memory T-cells (Tcm).
102181 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 transposasc (SB100X).
102191 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).
(02201 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).
102211 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:

301 GAGCAGGAGG CCGCTGGACA TAGAGC.AGAG CGA.GAGAGAG GGTGGCTTGG AGGGCGTGGC

421 GTCCCACCCC CACAGAATCA GCCAGAATCA GCCGTTGGTC AGACAGCTCT C.AGCGGCCTG

541 TCTAAAGAAC AACTGTTGAT ACAAC'GTAGC TCTGCAGCCG AAAGATGCCG GCGTTATCGA

661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA. ACAGCGGCGT
721 CATCGACAGA AAATGTCTAA AGA.CCAACGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA
781 CGACAGAATA TGTCTAGAGA ACAGTCATCA. ACAAGTACTA CCAATACCGG TAGGAACTGC

1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGTAA AAATTTC.ATG

1201 GG.AACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT

1381 ATGCTACATG AGGTAGAAAA GGAAGCCC.AA TCTGAAGCAG CAGCAAAAGG TATTGCTCCC
1441 ACAG.AAGTAA CAATGGCGAT TAAAT.ACGAT CGTAACAGTG A.CCC.AGGTAG .ATATAATTCT

1561 AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CAAA.TGCCAC TAAAATGAAA

1741 AATAATACTA GACAAAATGT AAGGA.CACGA GTCACACAAA TGCAGTATTA TGGATTTCAT
1801 CTCTCTGTGC GGGACACGTT CAATCCTATT TTAAATGCAG GAAAATTAAC TC.AACAGTTT

1921 TCTAAGTTG'A GAGTTGAAAA ATATAGTGGT TTGATGGATT ATCTCAAATC TAGATCTGAA.

2041 AGAAATATGC AGC.AGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC
2101 GATTTATTCA. TAACCATGAC ATGCAACCCC AAATGGGCAG ATATTACAAA CAATTTACAA
2161 CGCTGGCAAA. P.AGTTGAAAA. CAGACCTGAC TTGGTAGCCA, GAGTTTTTAA TATTAAGCTG
2221 AATGCTCTTT TAAATGATAT ATGTAAATTC CA.TTTATTTG GCAAAGTAAT AGCTAAAATT

2701 ATTA.AAAGTG TCAAATATTT ATTTAAATAC ATCTATAAAG GGCACGATTG TGCAAATATT
2761 CAAATTTCTG AAAAAAAT.AT TATCAATCAT GACGAAGTAC AGGACTTCAT TGACTCCAGG
2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA. TGCGAATGCA TGACCAATCT

3061 CATTATGTGT TTAATAATTC TTTGTGGACA AAACGCCGAA AGGGTGGGAA. TAAAGTATTA
3121 GGTAGA.CTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT

3481 GAAATGCATG C C CT TAAC GA AATTCAGGAG GTATTC:ACAT TGCATGGAAT GAAATGTTCA
3541 CATTTCAAAC T T CC GGACTA TC CTT TAT TA ATGAATGCAA ATACATGTGA TC.AATTGTAC
3601 GAGCAACAAC AG GCAGA.GGT TT T GATAAA.T TCTCTGAATG ATGAACAGTT GGCAGCCTTT

3341 GGAAGAACCT T T CA.T TC C CA ATATIAAT TA CCAATTCCAT TADATGAAAC TTCAATTTCT
3901 AGACTCGATA TAAAGAGT GA .AGT TGCTAAA ACCATTAAAA AGGCCCAACT TCTCATTATT

CGA

4201 GATT CTGCTT ATAGTGAATG GT TAGTAAAA CTTGGAGA.TG GCAAACTTGA TAGC.AGTTTT
4261 CATTTAGGAA T G GATAT T.AT TGtAAATCC CC CAT GAAAT GA T TT GTAACGG
ATCTATTATT

GATGCCAGCA
4801 TTTGCGATGA CTATTAATAA AT C:ACAAGGA CAAACTCTAG A(:A.GAGTAGG AATATTCCTA

TCGAAGAGCA

TTTTATATCA

GTATTA

AGAGAAAGGG

5281 TGCACCGGGC CACTAG ( SEQ ID NO: 27 ) .
10222] 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.
[0223] An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:

1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVINVN TSSQGKLVKH SESVFTLNVV YREILE (SEQU) 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 covalent;
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 tenninate 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).
[02251 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 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEX:p".)/s10:30).
[0226] An exemplary To12 transposon of the disclosure, including inverted repeats, subterminal sequences and the To12 transposase, is encoded by a nucleic acid sequence comprising the following:

121 TTTTTTTAGA. AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC
181 TTATTTTTTG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACC.AAACC AATTGAATTG

301 TATATGAAA' .T TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA
361 TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAA.TCAGTG

541 AAATAAAGAT TCATTCAAGA TGtAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC

781 GACAATATAA. GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT
841 CCACGGGAAA TTTGGCGCCT ATTGC.AGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT
901 TGTGTTTGAA TTCATGCAAA .ACACAAGAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT

1081 AATAGAAATA CAGATGTTCA. TTGTTTGTTC ATGTTA.GTTC ACAGTGCATT AACTAATGTT
1141 AAC.AAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGCA GTTCATTATT
1201 AGTTCA.TGTT AACTAATGTA GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCA.AA
1261 ACTAATGTAA TGAAA.TCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC

1321 AATATAATCA GAAATAAAAT TAATGTTTGA. TTGTCACTAA ATGCTACTGT ATTTC:TAAAA
1381 TCAACAAGTA TTTAACATTA TAAAGTGTGC AATTGGCTGC AAA.TGTCAGT TTTATTAAAG

1501 GCCCGTAAG'A CCTCCGTTCA TCTTCAGAA.0 AC.AGTTTAAG ATATTTTAGA TTTAGTCCGA

1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTA.CAA TGCTGAATAA

1981 ATCTTAAACT GTGTTCCGAA GATGAACGGA GGTGT'TACGG GCTTGGAACG ACATGAGGGT
2041 GAGTCATT.AA TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA
2101 GAGTGTATGT GTAATTGTTA CATTTATTGC A.TACAATATA AATA.TTTATT TGTTGTTTTT
2161 ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA TTGACAGCAC AGAAGAGAAA.

2461 GATCATGAAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACCACAA.0 2701 GGTTGTTTGC A.CAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG

2881 CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT GTC,ACTTACT
2941 TAACCTAGTC TCAA.GCGTTG ATGCCC.AAAA AGCTCTCTCA AATGAACACT ACAAGAAA.CT

3121 GTGGAATTCA ACTTTTATGG CTGTTGACAG AATTCTTC.AA ATTTGCAAAG AAGCAGGAGA
3181 AGGCGCACTT CGGAATATAT GCACC'TCTCT TGAGGTTCCA ATGTAAGTGT TTTTCCCCTC
3241 TATCGATGTA AACA.AATGTG GGTTGTTTTT GTTTAATACT CTTTGATTAT GCTGATTTCT

3421 TGCCT.AGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGGTACT

3541 AAGATCCTGA GATCATAGCA GCTGCC.ATCC TTCTCCCTAA ATTTCGGACC TCTTGGACAA

3721 TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATC:TCAA TATGAATATT GATGTAAAGT
3781 ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAA.CCAAACT CATATGTATC
3841 ATATGTGGTT TGGAAATGCA GTTAGATTTT .ATGCTAAAAT AAGGGATTTG CATGATTTTA
3901 GATGTA.GATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAATT TGTTCCCAGT

4381 ATGATGAA.GA TTTTTTCGCT TCTTTGAAAC CGACAACACA TGAAGCCAGC AAAGAGTTGG

4381 CATTAGATTG TCTGTCTTAT AGTTTGATAA TTAAANACAA AC:A.GTTCTAA AGCAGGATAA
4441 AACCTTGTAT GCATTTCATT TAATGTTTTT TGAGATTAAA AGCTTAAACA AG.AATCTCTA
4501 GTTTTCTTTC TTGCTTTT.AC TTTTACTTCC TTAATACTCA AGT.ACAATTT TAATGGAGTA

4681 TG (SEQ ID NO: 31).
Homologous Recombination 102271 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.
102291 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 ((CR5) gene and the site of the human oitholog of the mouse Rosa26 locus.
102301 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 1-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 MHC disrupts the endogenous gene, and optionally, replaces part of the coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MHC disrupts the endogenous gene, and optionally, replaces the entire coding sequence of the endogenous gene with a donor sequence composition of the disclosure. 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' UTR.
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, hi 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 gcnomic 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 gcnomic 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 (Dl OA) 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, Alvvl, BbvI. Bed, BceAI, BsmAI, BsmFI, BspCNI, BsrI, 13tsCI, Hgal, HphI, HpyAV, Mbo II, My II, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, Bind, BpmI, BpuEl, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrD1, BtgZI, Btsl, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQI, Sap', 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 gcnomic editing composition.
102321 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 gcnomic 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 D1OA 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 (SWIDNID: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 10A and H840A. In certain embodiments, the amino acid sequence of the dCas9 comprises the sequence of:

1021 MIAKSEQE1G KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG ElVWDKGROF

1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD (SEQII)Dal 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, Mn II. AlwI, BbvI, Bed, BceAl, BsmAI, BsmFI, BspCNI, Bsrl, BtsCI, Hgal, HphI, HpyAV, Won, My II, PleI, SfaNT, AcuI, BciVI, BfuAI, BmgBI, Bind, BpmT, BpuEI, Bsal, BseRI, Bsgl, Bsml, BspM1, BsrBI, BsrB1, BsrD1, BtgZI, Btsl, Earl, Ecil, Mmcl, NmcA111, BbvCI, Bpul0I, BspQI, SapI, Bad, BsaXI, CspCI, Bfil, Mboll, Acc36I, Fokl or Clo051.
In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and C1o051.An exemplary Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:
EGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSICQNRLFEMKVLELLVNEYGFKGRH
LGGSRKPDGIVY STILEDNF'GIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVN
PNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE
KIRSGEMTIEELERAMFNNSEFILKY (SEQ ID NO: 34).
102361 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):
MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKONRLFEMKVLELL
VNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAY SEGYSLPISQADEMERYVR
ENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEOLRRLSMTTGVNGSAV
NVVNLLLGAEK I RSGEMTIEELERAMFNNSEFILKYGGGGSDKKYS/GLA /GTNSVG WA
VITDEYKVPSICKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY
LQEIPSNEMAKVDDSKFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL
VDSIDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTTIVQLFEENPINA
..S'GPDA KAILSARLSKSRRLENLIAQUGEKKNGIFGAVALSI.GLT.PNFKSNEDIAEDA KLQ
LSKDTYDDDLDNILAQIGDQYADIFLAAKNISDAILLSDILRVNTEITKAPIS'ASMIKRYDE

ELLVKLVREDLIRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFIXDNREKIEK1L.TFRIP
YYVGPLARGNSRFAWMIRKSEETITPWNFEEVVDKGASAQSFIERATINTDKVITNEKVLP
KHSLLYEYFIVYNELTKVKYV7EGMRKPAfiLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF
KKIECFDSVELS'GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRDI
IEERLICITAHLFDDKLAIKQIXRRRYTGWGRIAS'RKLINGIRDKQSGKTILDFLKS'DGFANR

GRHKPENIVIEMARENQTTQKGQKNSRERIVKRIEEG1KE'LGSQ1LKEHPPEATQLQNEKL
YLYYLQNGRAVYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKFLIRSDKNRGKSDNVP
SEEVVKKAIKNYWRQLLNAKLITQRKFDIVLIKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRA/LVIKYDENDKLIREVKVI7'LKSKLVSDFRKDIWYKVREINNYHHAHDAYLNAV
VGDILIKKYPKLESEFVYG'DYKVYDVRKMIAKSAQEIGKA7AKYPFTSNIMNFPKTETIL4N
GEIRKRPLIETNGETGEIVWDKGRDFATVRKVISMPQVVVKKIEVQTGGESKESIIPKRN
SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE'KGKS'KKLKSVKELLGITIMERSSFEK
NPIDFLEAKGYKEVKKDLIIKLPKYS'LFELENGRKRAILASAGELQKGNELALPSKYVNFLY

LAS'HYEKLKGSPEDNAQKQLFVEQHKHYLDEMEQ1SEFSKRVILADANLDKVLSAY1VKHR
DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
SQLGGDOSPKKKRKVSS (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 hornodimer or a heterodimer. Nuclease domains of the genomic editing compositions or constructs of the disclosure may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a transcription-activator-like effector nuclease (TALEN). TALENs are transcription factors with programmable DNA
binding domains that provide a means to create designer proteins that bind to pre-determined DNA
sequences or individual nucleic acids. Modular DNA binding domains have been identified in transcriptional activator-like (TAL) proteins, or, more specifically, transcriptional activator-like effector nucleases (TALENs), thereby allowing for the de novo creation of synthetic transcription factors that bind to DNA sequences of interest and, if desirable, also allowing a second domain present on the protein or polypeptide to perform an activity related to DNA. TAL proteins have been derived from the organisms Xanthomonas and Ralstonia.
102381 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 II, AIM, BbvI, Bed, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, Hgal, Hphl, HpyAV, Mboll, My!!, PleI, SfaINI, Acul, BciVI, BfuAl, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQI, Sap!, Bad, BsaXI, CspCI, BfiI, 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 firmer nuclease (ZFN) and a type US
endonuclease. In certain embodiments of the disclosure, the type 11S
endonuclease may comprise, consist essentially of or consist of AciT, Mull, AlwI, BbvI, Bed, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, Hgal, HphI, HpyAV, IvIbolI, My1I, Pie!, SfaNI, ActiI, BciVI, BfuAl, BmgB1, Bmrl, Bpml, BpuEl, Bsal, BscRI, Bsgl, Bsml, BspMI, BsrBI, BsrBI, BsrD1, BtgZI, BtsI, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the type ITS
endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID
NO: 34).
[0240] 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 [0241] 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.
Exemplary human secreted proteins are provided, but are not limited to the human secreted proteins, in Table 1.
[0242] TABLE 1. Exemplary Human Secreted Proteins Gene Ensembl ID Gene description AlBG ENSG00000121410 Alpha-1-B glycoprotein A2M ENSG00000175899 Alpha-2-macroglobulin A2ML1 ENSG00000166535 Alpha-2-macroglobulin-like 1 A4GNT ENSG00000118017 Alpha-1,4-N-acetylglucosaminyltransferase AADACL2 ENSG00000197953 Arylacetamide deacetylase-like AANAT ENSG00000129673 Aralkylamine N-acetyltransferase ABCG1 ENSG00000160179 ATP-binding cassette, sub-family G (WHITE), member 1 ABHD1 ENSG00000143994 Abhydrolase domain containing ABHD10 ENSG00000144827 Abhydrolase domain containing ABHD14A ENSG00000248487 Abhydrolase domain containing ABHD15 ENSG00000168792 Abhydrolase domain containing ABI3BP ENSG00000154175 ABI family, member 3 (NESH) binding protein AC008641.1 ENSG00000279109 Date Recue/Date Received 2021-01-12 AC009133.22 ENSG00000277669 AC009491.2 ENSG00000279664 AC011513.3 ENSG00000267881 AC136352.5 ENSG00000277666 AC145212.4 ENSG00000277400 MaFF-interacting protein AC233755.I ENSG00000275063 ACACB ENSG00000076355 Acetyl-CoA carboxy lase beta ACAN EN SG00000157766 . Aggrecan ACE ENSG00000159640 Angiotensin I converting enzyme ACHE ENSG00000087085 Acerylcholinesterase (Yt blood group) ACP2 ENSGONK81114575 Acid phosphatase 2. lysosoinal ACP3 ENSG00000102375 Acid phosphatase 5, tanrate resistant ACP6 ENSG00000162836 . Acid phosphatase 6, lysophosphatidic ACPP ENSG00000014257 Acid phosphatase, prostate ACR EN SG00000100312 Acrosin ACRBP ENSG00000111644 Acrosin binding protein ACRV1 ENSG00000134940 Acrosomal vesicle protein I
ACSF2 ENSG00000167107 Acyl-CoA synthetase family member 2 A cm t 0 EN SG00000182584 Actin-like 10 ACVR1 ENSG00000115170 Act ivin A receptor, type!
ACVR I C EN SG00000123612 Activin A receptor, type IC
ACVRL I ENSG00000139367 Activin A receptor type II-like 1 ACYP I ENSG00000119640 Acylphosphatase 1, elythrocyte (common) type ACYP2 EN SG00000170634 Acylphosphatase 2, muscle type ADAM 10 ENSG00000 137845 ADAM metallopeptidase domain 10 ADAM12 EN SG00000148848 ADAM metallopeptidase domain 12 ADAM15 ENSG00000143537 ADAM inetallopeptidase domain 15 ADAM 17 ENSG00000151694 ADAM metallopeptidase domain 17 ADAM 18 ENSG00000168619 . ADAM metallopeptidase domain 18 ADAM22 ENSG00000008277 ADAM metallopeptidase domain 22 ADAM28 ENSG00000042980 ADAM metaliopeptidase domain 28 ADAM29 EN SG00000168594 ADAM metallopeptidase domain 29 ADAM32 ENSG00000197140 ADAM metallopeptidase domain 32 ADAM33 ENSG00000149451 . ADAM metallopeptidase domain 33 ADAM7 ENSG00000069206 ADAM metallopeptidase domain 7 ADA2vI8 ENSG00000151651 ADAM metallopeptidase domain 8 ADAM9 ENSG00000168615 ADAM metallopeptidase domain 9 ADAMDEC1 ENSG00000134028 ADAM-like, decysin 1 ADANITS1 ENSG00000154734 . ADAM metallopeptidase with ihrombospondin type 1 motif, 1 ADAMTSIO ENSG00000142303 ADAM metallopeptidase with thrombospondin type 1 motif, 10 ADAMTS 12 EN SG00000151388 ADAM metallopeptidase with thromhospondin type 1 motif, 12 ADAMIS13 ENSG00000160323 ADAM metallopeptidase with thromhospondin type 1 motif 13 ADAMTS14 ENSG00000138316 ADAM metallopeptidase with thrombospondin type 1 motif, 14 A DAMTS15 ENSG00000166106 ADAM metallopeptidase with Iluombosixindin type 1 motif, 15 A DAMTS16 ENSG00000145536 ADAM metallopeptidase with thrombospondin type I motif, 16 ADAMTS17 ENSG00000140470 ADAM metallopeptidase with thrombospondin type 1 motif, 17 ADANITSI8 ENSG00000140873 . ADAM metallopeptidase with thrombospondin type 1 motif, 18 ADAMTS19 ENSG00000145808 ADAM metallopeptidase with thrombospondin type 1 motif. 19 ADAMTS2 ENSG00000087116 ADAM metallopeptidase with thrombospondin type 1 motif, 2 ADAMTS20 ENSG00000173157 ADAM metallopeptidase with thrombospondin type I motif, 20 ADAMTS3 ENSG00000156140 ADAM metallopeptidase with thrombospondin type 1 motif, 3 ADAM=rs5 EN S(300000154736 . ADAM metallopeptidase with iltiombospondin type 1 motif, 5 .
ADAmTs6 EN SG00000049192 ADAM metallopeptidase with thrombospondin type 1 motif, 6 ADAMTS7 ENSG00000136378 ADAM metallopeptidase with thrombospondin type 1 motif, 7 A DAMTSR ENSGONK)0134917 ADAM metallopeptidase with thrombospondin type I motif, 8 ADAMTS9 ENSG00000163638 ADAM metallopeptidase with thrombospondin type 1 motif, 9 ADANITS1..1 EN S600000178031 ADAMTS-like 1 ADAmTsL2 EN SG00000197859 ' ADAMTS-like 2 ADAMTSI.3 EN SC100000156218 ADAMTS-like 3 ADAMTSIA ENSG00000143382 ADANIFS-like 4 ADAMTSL5 EN SG00000185761 ADAMTS-like 5 ADCK I ENSG00000063761 AarF domain containing kinase I
ADCYAP I EN SG00000141433 Adenyiate cyclase activating polypeptide 1 (pituitary) ADCYA.P IR I ENSG00000078549 Adeny late cyclase activating polypeptide 1 (pituitary) receptor type 1 ADGR A3 ENSG00000 152990 Adhesion G protein-coupled receptor A3 ADGRB2 ENSG00000121753 Adhesion G protein-coupled receptor 132 ADGRD1 ENS(300000111452 . Adhesion G protein-coupled receptor DI
ADGRE3 EN SG00000131355 Adhesion G protein-coupled receptor E3 ADGRE5 ENSG00000123146 Adhesion G protein-coupled receptor 5 ADGRFI ENSG00000153292 Adhesion G protein-coupled receptor F!
ADGRG1 EN5G00000205336 Adhesion G protein-coupled receptor G1 ADGR(35 ENS(3000001596 I 8 . Adhesion G protein-coupled receptor (35 ADGRG6 ENSG00000112414 Adhesion G protein-coupled receptor (36 ADGRV I ENSG00000164199 Adhesion G protein-coupled receptor VI
ADII EN SG00000182551 Aciredtictone dioxygenase 1 AD1G ENSG00000182035 Adipogenin AD1POQ ENSG000)0181092 Adiponectin. C IQ and collagen domain containing ADM ENSG00000148926 Adrenomedid lin ADM2 ENSG00000128165 Adrenomedul lin 2 ADIOS ENSG00000224420 Adrenomeduliin 5 (pulative) ADPGK ENSG00000159322 ADP-dependent glucokinase ADPRI1L2 ENSG00000116863 ADP-ribosylhydrolase like 2 AEBP1 ENSG00000106624 AB binding protein I
AFM ENSG00000079557 Afamin AR ENSG00000081051 Alpha-fetoprotein AGA ENSG00000038002 Aspartylghicosaininidase AGER ENSG00000204305 Advanced glycosylation end product-specific receptor AGK ENSG00000006530 Acylglycerol kinase AGPS EN 86000000185 It) Alkylglycercme phosphate sy,,nthase AGR2 ENSG00000106541 Anterior gradient 2, protein disulphide isomerase family member AGR3 EN SG00000173467 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) AGIMBP1 EN SG00000135049 ATP/GIP binding protein 1 AGTRAP ENSG00000177674 Angiotensin Il receptor-associated protein AFICYL2 ENSG00000158467 Adenosylhomocysteinase-like 2 AHSG ENSG00000145192 Alpha-2-HS-glycoprotein AIG I ENSG00000146416 Androgen-induced I
AK4 EN SG00000162433 Adenyiate kinase 4 AKAP10 ENSG00000108599 A kinase (PRKA) anchor protein 10 AKRICI ENSG00000187134 A ldo-keto reductase family I, member CI
AL356289.1 ENSG00000279096 AL589743.1 ENS600000279508 ALAS2 ENSG00000158578 . 5'-aminolevulinate synthase 2 ALB ENSG00000163631 Albumin ALD1-19.A1 EN SO00000143149 Aldehyde debydrogenase 9 family, member Al ALDOA ENSG00000149925 Aldolase A, fructose-bisphosphate ALGI. ENS600000033011 .4161, chitobiosyldiphosphodolichol beta-mannosyltransferase AL.G5 ENSG00000120697 . ALG5, dolichyl-phosphate beta-glueosvitraneerase ALG9 ENS600000086848 ALGO, alpha-1,2-mannosyltransferase ALKBH I ENSG00000100601 ALkB homolog 1, histone H2A dioxygenase ALKBH5 EN SG00000091542 AlkB homolog 5, RNA demethy lase ALP1 ENSG00000163295 Alkaline phosphatase, intestinal ALPL EN SG00000162551 . Alkaline phosphatase, liver/bone/kidney ALPP ENSG000001632R3 Alkaline phosphatase, placental ALPPL2 ENSG0000016:3286 Alkaline phosphatase, placental-like 2 AMBN EN S600000178522 Arneloblastin (enamel matrix protein) AMBP ENSG00000106927 Alpha-l-mic roglobulialbikunin precursor AMELX ENSG00000125363 . Amelogenin. X-linked AMELY EN 860000009972 I Amelogenin, Y-linked AMH ENSG00000104899 Anti-Mullerian hormone AM1CA I ENSG00000160593 Adhesion molecule, interacts with CXADR
antigen I
ANIPD I ENSG00000116748 Adenosine monophosphate deaminase I
ANITN EN SG00000187689 . Amelmin AMY IA ENSG00000237763 Amylase, alpha IA (salivary) AMY 1B ENSG00000174876 Amylase, alpha 1.B (salivary) AMY IC ENSG00000187733 Amylase, alpha IC (saliva))) ANIY2A EN8600000243480 Amylase, alpha 2A (pancreatic) ANIY2B ENS600000240038 . Amylase, alpha 2B (pancreatic) .ANG ENSG00000214274 Angiogenin, ribonuclease, RNase A family, 5 ANGELI ENSG00000013523 Angel homolog 1 (Drosophila) ANGPTI ENSG00000154188 Angiopoietin 1 ANGPT2 EN SG00000091879 . Angiopoiet in 2 ANGPT4 ENSG00000101280 Angiopoietin 4 ANGPTL1 EN SG00000116194 Angtopoietin-like 1 ANGPTL2 ENSG00000136859 Angiopoietirt-like 2 ANGPTL3 ENSG00000132855 Angiopoietin-like 3 ANGPTL4 ENSG00000167772 . Angiopoietin-like 4 ANGPTL5 EN SG00000187151 Angiopoietin-like 3 ANGPTL6 ENSG00000130812 Angiopoietin-like 6 A NGP*11.7 ENSGONKI0171819 Angiopoieti n-I ike 7 ANK I ENSG00000029534 Ankyrin 1, elythwcytic ANKDD IA ENSG00000166839 . Anky ri n repeat and death domain comaining IA
ANKRD54 EN SG00000100124 Ankyrin repeat domain 54 ANKRD60 ENSG00000124227 A nkyri n repeal domain 60 ANcr ENSG00000146205 Anoctamin 7 ANOS1 ENSG00000011201 Anosmin 1 ANTXR1 ENSG00000169604 Anthrax toxin receptor 1 A0A11 EN SG00000136250 Acyloxyacyl hydroiase (nettimphil) AOC1 ENSG00000002726 Amine oxidase, copper containing I
A0C2 ENSG001)00131480 Amine oxidase, copper containing 2 (retina-specific) A0C3 ENSG00000131471 Amine oxidase, copper containing 3 AP000721.4 ENSG00000256100 AP000866.1 EN SG00000279342 APBB I ENSG00000166313 Antyloid beta (A4) precursor protein-binding. faintly. B. member 1 (Fe65) APCDD1 ENSG00000154856 Adenornatosis polyposis colt down-regulated APCS ENSG00000132703 Amy fold P component. serum APELA ENSG00000248329 . Apelin receptor early endogenous ligand APLN EN SG00000 I 71.3g8 Apelin APLP2 ENSG00000084234 Amyloid beta (A4) precursor-like protein 2 APOA 1 ENSG00000 118137 Apotipoptoteirt A-I
APOA1BP ENSG00000163382 Apolipoprotein A-I binding protein AP0A2 EN SG00000158874 . Apolipoprotein A-II
AP0A4 ENSG00000110244 Apolipoprotein A-IV
AP0A5 ENSG00000110243 Apolipoptotein A-V
APOB ENSG00000084674 Apolipoptotein B
APOC1 ENSG00000130208 Apolipoprotein C-I
APOC2 ENSG00000234906 Apolipoprotein C-II
APOC3 ENSG00000110245 Apolipoprotein C-III
APOC4 ENSG00000267467 Apolipoprotein C-IV
APOC.'4-.A POC2 ENSG00000224916 APOC4-APOC2 readthrough (NMD candidate) APO!) ENSG00000189058 Apolipoprotcin D
APOE EN SG00000130203 Apolipoprotein E
APOF ENSG00000175336 Apolipoprotein F

APOH EN SG00000091583 Apolipoprotein H kbeta-2-glycoproIeirt 1) APOL I ENSG00000100342 Apolipoprotein L, 1 APOL3 ENSG00000128284 . Apolipoprotein L. 3 APOM ENSG00000204444 Apolipoprotein M
APOOL EN SG00000155008 Apolipoprotein 0-like ARCN I ENSG00000095139 Archain 1 ARFIP2 ENSG00000132254 ADP-ribosylation factor interacting protein ARTIGAP36 ENSG00000147256 . Rho GTPase activating protein 36 ARHGAP6 EN SG00000047648 Rho GTPase activating protein 6 ARHGEF4 EN SG00000136002 Rho guanine nucleotide exchange factor (GEF) 4 ARLIE> EN SG00000214087 ADP-ribosylation factor-like 16 ARMC5 ENSG00000140691 Armadillo repeat containing 5 ARNTL ENSG00000133794 . Aryl hydrocarbon receptor nuclear translocator-like ARSA EN SG00000100299 Arylsulfatase A
ARSE ENS000000113273 Atylsulfatase B
ARSE ENSGO0tX)0157399 A ry Isullatase E (cliondrodysplasia plinctrita 1) ARSG ENSG00000141337 Arylsulfatase G
ARSI ENSG00000183876 Arylsulfatase family, member 1 ARSK EN SG00000164291 Arylsulfatase family, member K
ART3 ENSG00000156219 ADP-ribosyltransferase 3 ART4 EN SG00000111339 ADP-nbosyltransferase 4 (Dombrock blood group) ARTS ENSG00000167311 ADP-ribosyltransferase 5 ARTN ENSG000001 I 7407 Artemin ASAH1 ENSG00000104763 N-acylsphingosinc amidohydrolase (acid ceramidase) 1 ASAH2 EN SG00000188611 N-acylsphingosine amidohydrolase (non-lysosonial cerainidase) ASO, I. ENSG00000139352 Achtiete-scute faintly bIlLEI transcription factor I.
ASIP ENSG00000101440 Agouti signaling protein ASPN EN SG00000106819 . Asporin =
ASTI, ENSG00000188886 Astacin-like inetallo-endopeptidase (M12 family) ATAD5 ENSG00000176208 ATPase family, AAA domain containing 5 ATAT1 EN SG00000137:343 Alpha tubrdin acetyltransferase 1 ATG2A ENSG00000110046 Autophag related 2A
ATG5 ENS000000057663 . Autophagy related 5 ATMIN ENSG00000166454 ATM interactor ATP13A1 EN 5000000105726 AlT'ase type 13AI
ATP5F1 EN SG00000116459 ATP synthase, Ff.. transporting, mitochondria! Fo complex.
subunit El A1P6AP1 EN5000000071553 ATPase,H+ transporting. lysosomal accessory protein 1________ ¨;;171-3-67j;1--------EVS-i3i00000-182220i¨ITT17,;:t7-Fr+-7;;-;;;;;iWtItTlysosoinai accessory protein;-i .
ATPAF1 ENSG00000123472 ATP synthase mitochondria! Ft complex assembly factor 1 ALT! ENSG00000148090 AU RNA binding proteinienoyl-CoA hydratase MT ENS000000101200 Argi nine vasopressin AXIN2 ENSG00000168646 Axin 2 .....
--A-zeip-i---------Ewsziiii(roliZiZT-7¨A-517412-glycoprotein 1, zinc-binding - ISO-AZU I EN SG00000172232 Azurocidin 1 132M ENSG00000166710 Beta-2-microglobulin B3GALNT1 ENSG00000169255 Beta-L3-N-acetylgalactosarninyltransferase 1 (globoside blood group) B3GALNT2 ENSG00000162885 Beta-1,3-N-acetylgalactosaminyltransferase 2 B3GALT1 ENSG00000172318 1.1DP-Gal:hetirGIcNAc beta 1,3-galactosyltransferase, poly peptide 1 B3GALT4 ENSG00000235863 LIDP-Gal:betaGIcNAc beta 1,3-galactosyltninsferase, polypeptide 4 B3GALT5 ENSG00000183778 UDP-Gtd:betaGIcNAc beta 1,3-galactosyltransferase, polypeptide 5 B3GALT6 ENSGOOt)00176022 1.1DP-Gal:betaGal beta 1,3-ga1ac105ylinin5fer45e polypemide 6 B3GAT3 EN SO00000149541 Beta-1,3-g1ucuronyltransferase 3 B3GLCT ENSG00000187676 Beta 3-glucosy Itransferase aceiy Ightcosami try ItranSferase 3 B3GNT4 ENSG00000176383 UDP-GIcNAc:betaGal beta-1,3-N-acety Ighicosaminyltransferase 4 133GNT6 ENSG00000198488 UDP-G1cNAc:betaGal beta-1,3-N-acety IgiucosaminvItransferase 6 1330NT7 EN S600000156966 ITDP-OlcNAc:betaGal beta-1,3-N-aceiy IghtCOSaini try ItranSle tase 7 B3GNT8 ENSG00000177191 LIDP-GIcNAc:betaGal beta-1,3-N-acety Iglucosaminyltransferase 8 133(3NT9 EN SG00000237172 (.113P-GIcNAc:betitGal beitt-1,3-N-acetylglucosaminvitransferase 9 B4GALNT1 ENSG00000135454 Beta-L4-N-acetyl-galactosaminyl transfcrase B4GALNT3 EN SG00000139044 Bent-14-N-acetyl-galactosaminyltninsfcrase B4GALNT4 ENSG00000182272 Bent-1,4-N-acetyl-galactosaminyltransferase B4GALT4 EN SG00000121578 1.1DP-GaLnetaGicNAc beta 1,4-galactosyltransferase, poly peptide 4 B4GALT5 ENSG00000158470 UDP-Gal:betaGicNAc beta 1,4- galactosyln-ansfetase, poll/peptide 5 B4GALT6 EN SG00000118276 111)P-Ga1:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 B4GAT1 ENSG00000174684 Beta-1,4-g1uctironyltransferase 1 1391)1 ENSG00000108641 139 piotein domain I.
BACE2 ENSG00000182240 Beta-site APP-cleaving enzyme 2 BAGE5 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 BCAR 1 ENSG00000050820 Breast cancer anti-estrogen resistance 1 BCHE EN SG00000114200 Buty ry !cholinesterase BCK1)11B ENSG00000081121 Branched chain keto acid dehydrogenase El.
beta polypeptide BDNE: ENSG00000176697 Brain-derived neurotrophic factor BGLAP ENSG00000242252 Bone gamma-carboxyglutainate (gia) protein BGN ENSG00000182492 Biglycan BLVRB ENSG00000090013 Bilivendin teductase B
BMP1 ENSG00000168487 Bone toorptiogenetic protein I

BMPIO ENSG00000163217 Bone morphogenetic protein 10 13NEP15 ENSG00000130385 Bone morphogenetic protein 15 BMP2 ENSG00000125845 . Bone morphogenetic protein 2 BMP3 ENSG00000152785 Bone morphogenetic protein 3 BMP4 ENSG00000125378 Bone morphogenetic protein 4 BMP6 EN SG00000153162 Bone morphogenetic protein 6 BMP7 ENSG00000101144 Bone morphogenetic protein 7 BMPSA ENSG00000 183682 . Bone morphogenetic protein 8a BMP8B EN SG00000 116985 Bone morphogenetic protein 8b BMPER EN SG00000164619 BMP binding endothelial regulator BNC1 ENSGONK)0169594 Basonuclin 1 BOC ENSG00000144857 BOC cell adhesion associated, oncogene regulated BOD1 ENSG00000145919 . Biorientation of chromosomes in cell division 1 BOLA 1 ENSG00000 178096 BolA. family member I
BPI ENSG00000101425 Bactericidal/penneability-ineteasing protein 8PD:A 1 EN SG00000198183 BPI fold containing family A, member I
BP1EFA2 EN SG00000131050 BPI fold containing family A. member 2 BPIFA3 EN SG00000131059 BPI fold containing family A. member 3 BPIFB1 ENSG00000 125999 BPI fold containing family B, member 1 BPIF:132 ENSG00000078898 BPI fold containing family B, member 2 BPIFB3 ENSG00000 186190 BPI fold containing family B, member 3 BPITB4 ENSG00000186191 BPI fold containing family B, member 4 BPIFB6 ENSG00000167104 BPI fold containing family B, member 6 BPIFC EN SG00000184459 BPI fold containing family C
BRE, ENSG00000185024 BR.F1, RNA polyinerase III transcription initiation factor 90 kDa subunit BRINP1 ENSG00000078725 Bone morphogenetic proteinfretinoic acid inducible neural-specific 1 BRINP2 ENSG00000198797 Bone morphogenetic protchthetinoie acid inducible neural-specific 2 =
BRINP3 ENSG0000016.2670 Bone moiphogenetic proteinIretinoic acid inducible neural-specific 3 BSC/ ENSG00000 172274) Basigin (Ok blood group) BSPH I ENSG00000188334 Binder of sperm protein homolog I
BST1. ENSG00000109743 Bone marrow stromal cell antigen 1 BTBD17 ENSG00000204347 BIB (POZ) domain containing 17 BTD ENSG00000169814 Biotinidase BTN2A2 ENSG00000 124508 Butyrophilin, sublatraly 2, member A2 BTN3A.1 ENSG00000026950 .Butyrophilin, subfamily 3, member Al --1E-STICI3E------------0 -6116(5.67)-18-674-7()------ -filay ro-piTiiiii5aC-6WiiiTT,TIZTi713-;-A-2-----BTN3A3 EN SG00000111801 Butyrophilin, sublhinily 3, member A3 ClOoif 10 ENSG00000165507 Chromosome 10 open leading frame 10 CI 0ot199 ENSGOOt)00 188373 Chromosome 10 open reading frame 99 CI lorfl ENSG00000137720 Chromosome 11 open reading frame I
Cl loif24 ENSG00000171067 Chromosome 11 open reading frame 24 CI lor145 ENSG00000174370 Chromosome 11 open reading frame 45 CI lort94 ENSG00IX/0234776 Chromosome 11 open reading frame 94 Cl2orf10 ENSG00000139637 Chromosome 12 open reading frame 10 CI 2 449 EN SG00000111412 . Chromosome 12 open reading frame 49 Cl2orf73 ENSG00000204954 Chromosome 12 open leading frame 73 Cl 2or176 ENSG00000174456 Chromosome 12 open leading frame 76 CI 4orf80 ENSG00000185347 Chromosome 14 open reading frame 80 CI4olf93 ENSG00000100802 Chromosome 14 open reading frame 93 Cleorf89 ENSG00000153446 . Chromosome 16 open reading frame 89 CI6orf90 ENSG00000215131 Chromosome 16 open reading frame 90 CI 7o rf67 ENSG00000214226 Chromosome 17 open leading frame 67 CI 7orf75 ENSGOOmo 108666 Chromosome 17 open reading frame 75 CI 7o1f99 ENSG00000187997 Chromosome 17 open reading frame 99 CI8or154 ENSG00000166845 . Chromosome 18 open reading frame 54 C19orf47 ENSG00000160392 Chromosome 19 open reading frame 47 CI 9or170 ENSG00000174917 Chromosome 19 open leading frame 70 CI 9orl80 ENSGO0tX)0130173 Chromosome 19 open reading frame 80 C I GALTI ENSG00000106392 Core 1 synthase, glyeoprotein-N-acetylgalactosamine 3-beta-galactosyltransfemse 1 Clorf127 EN SG00000175262 Chromosome 1 open reading frame 127 Clotf159 EN SG00000131591 . Chromosome (open reading frame 159 C1otf198 ENSG00000 119280 Chromosome 1 open reading frame 198 C1orf234 ENSG00000227868 Chromosome 1 open reading frame 234 CI or154 ENSG00000 118292 Chromosome 1 open reading frame 54 CI orf56 ENSG00000143443 Chromosome 1 open reading frame 56 CIQA ENSG00000 173372 Complement component 1. q subcomponent, A
chain CIQB ENSG00000173369 . Complement component I. q subcomponent, B
chain C1QC ENSG00000159189 Complement component I, q subcomponent. C
chain CI QL1 ENSG00000131094 Complement component 1, q subcomponent-like CI QL2 EN SG00000144119 Complement component 1, q subcomponent-like C1QL3 ENSG00000165985 Complement component 1, q subcomponent-like CIQL4 ENSG00000186897 Complement component I. q subcomponent-like CIQINF I ENSG00000173918 C lq and tumor necrosis factor related protein 1 CI QTNF2 ENSG00000145861 C lq and tumor necrosis factor related protein 2 C I QINF3 ENSG00000082196 Clq and tumor necrosis factor related protein 3 CIQTNF4 ENSG00000172247 Clq and tumor necrosis factor related protein 4 CIQTNF5 ENS600000223953 . C I q and tumor necrosis factor related protein 5 Cl QTNF7 ENSG00000163145 C It] and tumor necrosis factor related protein 7 CI Q1'NF8 ENSG00000184471 C lq and tumor necrosis factor related protein 8 CI QINF9 ENSG00000240654 C 1 q and tumor necrosis factor related protein 9 CIQTNF9B ENSG00000205863 Clq and tumor necrosis factor related protein 9B
CIR ENSG00000159403 Complement component I. r subcomponent CIRL EN SC100000 139178 Complement component 1, r subcomponent-like CIS ENSG00000182326 Complement component 1, 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 C22orf15 ENSG00000169314 . Chromosome 22 open reading frame 15 C22orf46 ENSG00000184208 Chromosome 22 open leading frame 46 C2CD2 ENSG00000157617 C2 calcium-dependent domain containing 2 C2or140 ENSG00000 1191.47 Chromosome 2 open reading frame 40 C2orf66 ENSG00000187944 Chromosome 2 open reading frame 66 C2orf69 ENSG00000178074 . Chromosome 2 open reading frame 69 C2orf.78 ENSG00000187833 Chromosome 2 open reading frame 78 C3 ENSG00000125730 Complement component 1 C3or133 ENSG00000174928 Chromosome 3 open reading flame 33 C3orf58 EN 5G00000181744 Chromosome 3 open reading frame 58 C4A ENSG00000244731 . Complement component 4A (Rodgers blood group) C413 ENSG00000224389 Complement component 413 (Undo 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 C4orl48 ENSG00000243449 Chromosome 4 open reading frame 48 C5 ENSG00000106804 Complement component 5 C5orl46 ENSG00000178776 Chromosome 5 open reading frame 46 C6 ENSG00000039537 Complement component 6 C6orf120 EN5G00000185127 Chromosome 6 open reading frame 120 C6orf15 ENS600000204542 Chromosome 6 open reading frame 15 C6orf25 ENSG00(X)0204420 Chromosome 6 open reading frame 25 C6orf58 ENSG00000184530 Chromosome 6 open reading frame 58 C7 EN SG00000112936 Complement component 7 C7orf57 ENSG00000164746 Chromosome 7 open reading frame 57 C7otf73 EN SG00000243317 Chromosome 7 open reading frame 73 C8A EN SG00000157131 . Complement component 8, alpha polypeptide C8B ENSG00000021852 Complement component 8, beta polypeptide C8G ENSG00000176919 Complement component 8, gamma polypeptide C9 ENSG00000113600 Complement component 9 C9orf47 ENSG00000186354 Chromosome 9 open reading frame 47 CA10 EN SG00000154975 . Carbonic anhydrase X
CA 11 EN SG00000063180 Carbonic anhydrase XI
CA6 EN SG00000131686 Carbonic anhydrase VI
CA9 ENSG00000107159 Carbonic anhydrase IX
CABLES I ENSG00000134508 Cdk5 and Abl enzyme substrate 1 CABP1 EN SG00000157782 . 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 CADIV13 ENSG00000162706 Cell adhesion molecule 3 CALCA ENSG00000110680 Calcitonin-related polypeptide alpha CALCB ENSG00000175868 CaIcitomn-related poly peptide beta CALCR ENSG00000004948 Calcitonin receptor CALCRL ENSG00000064989 Calcitonin receptor-like CALR ENSG00000179218 . Calteticulin CALR3 ENSG00000269058 Calrcticulin 3 CALU ENSG00000128595 Calumenin CAMK2D ENSG00000145349 Caicitimicaltriodulin-dependent protein kinase 11 delta CAMP ENSG00000164047 Cathelicidin antimicrobial peptide CANX EN S600000127022 . Calnexin CARKD EN SG00000213995 Carbohydrate kinase domain containing CARM1 ENSG00000142453 Coactivator-associated argi nine met hy ltransferase 1 CA RN S I EN SCM000172508 C7arnosi tie synthase i CARTPT EN SG00000164326 CART prepropeptide CA SQ1 ENSG00000143318 . Calsequestrin 1 (fast-twitch. skeleial muscle) CA SQ2 EN SG00000118729 Calsequestrin 2 (cardiac muscle) CATSPERG ENSG00000099318 Caisper channel auxiliary subunit gamma CBLN1 ENSGO0tX)0102924 Cerebellin 1 precursor CBLN2 ENSG00000141668 Cerebellin 2 precursor CBLN3 ENSG00000139899 Cerebellin 3 precursor CBLN4 ENSG00(8)0054803 Cerebellin 4 precursor CCBE I ENSG00000183287 Collagen and calcium binding EGF domains 1 CCDC108 EN SG00000181378 Coiled-coil domain containing 108 CCDC 112 EN SG00000164221 Coiled-coil domain containing 112 CCDC 129 EN SG00000180347 Coiled-coil domain containing 129 CCDC1.34 .ENSG00000100147 Coiled-coil domain containing 1.34 CCDC149 EN SG00000181982 Coiled-coil domain containing 149 CCDC3 EN SG00000151468 Coiled-coil domain containing 3 CCDC80 ENSG00000091986 Coiled-coil domain containing 80 CCDC85A EN SG00000055813 Coiled-coil domain containing 85A
CCDC8813 EN SG00000168071 . Coiled-coil domain containing 88B
CCER2 ENSG00000262484 Coiled-coil glutamate-rich protein 2 CCK ENSG00000187094 Cholecystokinin CCL 1. ENSG00000108702 Chentokine (C-C motif) ligand 1 CCL11. EN SG00000172156 Chemokine (C-C motif) ligand 11 CCI, 13 ENSG00000181.374 . Chemokine (C-C motif) ligand 13 CCL14 ENSG00000276409 Chentokinc-: (C-C motif) ligand 14 CCL1.5 ENSG00000275718 Chemokine (C-C motif) ligand 15 CCL16 ENSG00000275152 Chemokine (C-C motif) ligand 16 CCL17 ENSG00000102970 Chemokine (C-C motif) ligand 17 Ca.18 ENSG00000275385 Chemokine (C-C motif) ligand 18 (pulmonary and act ivalion-regulated) CCL19 ENSG00000172724 Chemokine (C-C motif) ligand 19 CCL2 ENSG00000108691 Chernokine (C-C motif) ligand 2 CCL20 ENSG00000115009 Chentokine (C-C motif) ligand 20 CCL21. EN SG00000137077 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 CCL25 EN SG0000013 1142 . Cheinokine (C-C motif) ligand 25 CCL26 ENSG00000006606 Chemokine (C-C motif) ligand 26 CCL27 ENSG00000213927 Chernokine (C-C motif) ligand 27 CCL28 EN SG00000151882 Chentokine (C-C motif) ligand 28 CCL3 ENSG00000277632 Chemokine (C-C motif) ligand 3 CCI31.3 ENSGOO(X)0276085 . Cheinokine (C-C motif) ligand 3-like 3 CCL4 EN S(3(}0000275302 Chemokine (C-C motif) ligand 4 CCL4L2 ENSG00000276070 Chernokine (C-C motif) ligand 4-like 2 CCL5 EN SG00000271503 C7hentokine (C-C motif) ligand 5 CCL7 ENSG00000108688 Chemokine (C-C motif) ligand 7 CC,L8 ENSG00000108700 . Cheinokine (C-C motif) ligand 8 CCNB I IPI EN SG00000100814 C'yclin B1 interacting protein 1, E3 ubiquitin protein ligasc CCNL 1 ENSG00000163660 Cycl in LI
CCNL2 ENSG00000221978 Cyclin L2 CD14 ENSG00000170458 CD14 molecule CD160 ENSG00000117281 CD160 molecule CD 164 EN SG00000135535 CI) 164 molecule, sialormicin CD177 ENSG00000204936 CD177 molecule CD 1E ENSG00000158488 C7D le molecule CD2 ENSG00000116824 CD2 molecule CD200 ENSG00000091972 CD200 molecule CD200R I EN SG00000163606 CD200 receptor 1 CD22 ENSG00000012124 CD22 molecule CD226 ENSG00000150637 C1)226 molecule CD24 ENSG00000272398 CD24 molecule CD276 ENSG00000103855 CD276 molecule CD300A EN SG00000167851 . CD300a molecule CalOOLB ENSG00000178789 CD300 molecule-like family member b CD3OOLF ENSG00000186074 CD300 molecule-like family member f CD3OOLG ENSG00000161649 CD300 molecule-like family member g CD3D EN SG00000167286 CD3d molecule, delta (CD3-TCR complex) CD4 ENSG00000010610 . CD4 molecule CD40 ENSG00000101017 CD40 molecule, TNF receptor superfamily member 5 CD44 ENSG00000026508 CD44 molecule (Indian blood group) CD48 ENSG000001 17091 CD48 molecule CD5 ENSG00000110448 CD5 molecule CD55 EN SG00000196352 CD55 molecule, decay accelerating factor for complement (Cromer blood group) CD59 ENSG00000085063 CD59 molecule, complement regulatoy protein CD51.. EN SG00000071754 COS molecule-like CD6 ENSG0000001:3725 CD6 molecule CD68 EN SG00000129226 (068 molecule CD7 ENSG00000173762 CD7 molecule CD79A ENSG00000105369 CD79a molecule, immunoglobulin-associated alpha CD80 EN SG00000121594 . CD80 molecule CD86 ENSG00000114013 CD86 molecule CD8A ENSG00000153563 CD8a molecule CD8B ENSG000001721.16 CD8b molecule CD99 ENSG00000002586 CD99 molecule CDC23 ENSG00000094880 . Cell division cycle 23 CDC40 EN SG00000168438 Cell division cycle 40 CDC45 ENSG00000093009 Cell division cycle 43 CDCP1 ENSG00000163814 CUB domain containing protein I
CDCP2 ENSG00000157211 CUB domain containing protein 2 CDH1 ENSG00000039068 . Cadherin 1, type I
CDH11 .ENSG00000140937 Cadherin II, type 2. OB-cadherin (osteoblast) CDH13 ENSG00000140945 Cadherin 13 CD1117 ENS000000079112 Cadherin 17, LI eadherin (In er-intestine) CDH18 ENSG00000145526 Cadherin 18, type 2 CDH19 ENSG00000071991 Cadherin 19, type 2 CD1123 EN SG00000107736 Cadherin-related 23 CDH5 ENSG00000179776 Cadherin 5, type 2 (vascular endothelium) CDHR1 ENSG00000148600 Cadherin-related family member 1 CDHR4 ENSG00000187492 Cadherin-related family member 4 CDHR5 ENSG00000099834 Cadherin-related family member 5 CDKN2A .ENSG00000147889 Cyclin-clependent kinase inhibitor 2A
CDNF ENSG00000185267 Cerebral dopamine nemm rophic factor CDON ENSG00000064309 Cell adhesion associated, oncogene regulated CDSN ENSG00000204539 Corneodesmosin CEACAM16 ENSG00000213892 Carcinoembryonic antigen-related cell adhesion molecule 16 CEACAM18 ENSG00000213822 . Carcinoembryonic antigen-related cell adhesion molecule 18 CEACAM19 ENSG00000186567 Carcinoembiyonic antigen-related cell adhesion molecule 19 CEAC.AM5 ENSG00000105388 Carcinoembiyortic antigen-re bled cell adhesion molecule 5 CEACAM7 EN SG00000007306 Carcinoembryonic antigen-related cell adhesion molecule 7 CEACAM8 EN SG00000124469 Carcinoembryonic antigen-related cell adhesion molecule 8 CECR I ENS600000093072 . Cat eye syndrome chromosome region, candidate 1.
CECR5 ENSG00000069998 Cat eye syndrome chromosome region.
candidate 5 CEL ENSG00000170835 Carboxyl ester lipase CELA2A ENSG00000142615 Chytnotrypsin-like elastase family, member CELA2B ENSG00000215704 Chymottypsin-like elastase family, member 2B
CELA3A ENSG00000142789 . Chy mottypsin-like elastase family, member CELA3B ENSG00000219073 Chy mot rypsin-like elastase family, member CEMIP ENSG0000010:3888 Cell migration inducing piotein, hyaluronan binding CEP89 ENSG00000121289 Centiosomal protein 89kDa CER1 ENSG00000147869 Cerbents 1, DAN family BMP antagonist CERCAM EN SG00000167123 Cerebral endothelial cell adhesion molecule CER.S1 ENSG00000223802 Ceratnide synthase 1 CES I ENS000000198848 Carboxylesterase 1 CES3 ENSG00000172828 . Catboxylesterase 3 CES4A ENSG00000172824 Carboxy (esterase 4A
CES5A EN SG00000159398 Carboxylestemse 5A
CETP ENS600000087237 Cholesteryl ester transfer protein, plasma CFB ENS000000243649 Complement factor B
CFC1 ENSG00000136698 Cripto, FRL-I, clyptic family 1 CFCIB EN S000000152093 ' Cripto, FRL-1, cryptic family 113 CFI) ENSG00000197766 Complement factor D (adipsir) CFDP I ENSGONK)0153773 Crattiorkial development protein I
CFH ENS000000000971 Complement factor H
CFHR I ENSG00000244414 . Complement factor H-related I
CFHR2 .EN S0000000809 I 0 Complement factor H-related 2 CFTIR3 ENSG00000116785 Complement factor H-related 3 CFI1R4 ENSG00000134365 Complement factor H-related 4 CFHR5 ENS000000134389 Complement factor H-related 5 CFI ENS000000205403 Complement factor I
CEP EN S000000126759 Complement factor properdin CGA ENSG00000135346 Glycoprotein honnones, alpha poly pept ide COB ENSG00000104827 Chonoitic gonadotropin, beta polypeptide CGB1 ENS00000026763I Chotionic gonadotropin. beta polypeptide 1 CGB2 ENS6000001048 18 Chorionic gonadotropin. beta polypeptide 2 C0135 .EN S000000189052 Chorionic gonadotropin, beta polypeptide 5 CGB7 ENSG00000 196337 Chorionic gonadotropin, beta polypeptide '7 C0/38 ENSG00000213030 Chotionic gonadoiropin, beta polypeptide 8 COREFI ENS000000138028 Cell growth regulator with EF-hand domain 1 CH507-9B2.3 ENS000000280071 CHAD EN S000000136457 Chondroadherin CHADL ENSG00000100399 ' Chondroadherin-like CHEK2 EN SG00000183765 Checkpoint kinase 2 CHGA ENS000000100604 Chromogranin A
CHGB ENSG00000089199 Chromogranin B
CHM! ENSG00000133048 . Chilinase 3-like 1 (cartilage glycoprotein-39) CHI3L2 ENS0000000648136 Chitinase 3-like 2 CHIA ENSG00000134216 Chitinase, acidic CHID! ENSG00000177830 Chitinase domain containing 1 CHIT! ENSG00000133063 Chitinase I (chitotriosidase) CHI, I EN S000000134121 . Cell adhesion molecule L.1-like CHN I ENSG00000128656 Chimerin 1 CHPF ENSG0000012:3989 Chondroitin polymerizing factor CHPF2 EN S000000033100 Chondroitin polymerizing factor 2 CHRD ENSG00000090539 Chordin CHRDL 1 ENSG00000101.938 Chordin-like I

CHRDL2 ENSG00000054938 Chordin-like 2 CHRNA2 ENSG00000120903 Cholinergic receptor, nicotinic, alpha 2 (neuronal) CHRNA5 ENSG00000169684 . Cholinergic receptor, nicotinic, alpha 5 (neuronal) CHRNB I ENSG00000170175 Cholincrgic receptor, nicotinic, beta I
(muscle) CHRND ENSG00000135902 Choliner& receptor, nicotinic, delta (muscle) CHST1. ENSG00000175264 Carbohydrate (keratin sulfate (.ial-6) sulfotrausferase 1 CHSTIO ENSG00000115526 Carbohydrate sulfotransferase 10 CHST11 ENSG000001713 Jo . Carbohydrate (chondtoitin 4) sulfotransferase 11 CHST13 ENSG00000180767 Carbohydrate (chondroitirt 4) sulfotninsferase 13 CHST4 ENSG00000140835 Carbohydrate (N-acetylglucosamine 6-0) sulfotransfemse 4 CI-TST5 ENSG00000135702 Carbohydrate (N-acety Ighicosamine 6-0) sal fotransfe rase 5 CHST6 ENSG00000183196 Carbohydrate (N-acety iglucosamine 6-0) sulfotransferase 6 CH sT7 ENSG00000147119 . Carbohydrate (N-acetylgIncosainine 6-0) suifotransferase 7 CHST8 .ENSG00000124302 Carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8 CHSY1 ENS000000131873 Chondroitin sulfate synthase I
CHSY3 ENS000000198108 Chondrottin stable synthase 3 CHTF8 ENSG00000168802 Chromosome transmission fidelity factor 8 CILP ENSG00000138615 Cartilage intermediate layer protein, nucleotide . pyrophosphohydrolase .
CILP2 ENSG0000016016I Cartilage intermediate layer protein 2 CIRH I A ENSG00000141076 Cirrhosis, autosomal recessive IA (cirhin) CKLF ENS6000002 17555 Chemokine4 ike factor CKMTI A ENS000000223372 Creatine kinase, mitochondria! IA
CKMT1B EN S(300000237289 . Creatine kinase, mitochondria! 1B
CLCA 1 ENSG00000016490 Chloride channel accessory 1 CLCF I ENSG00000175505 Candiotiophin-like eytokine factor I
CLDN15 ENSG00000106404 Claudin 15 CLDN7 ENSG00000181885 Claudin 7 C71..DNDI EN S(300000080822 . Claudio domain containing 1 CLEC I IA ENSG00000105472 C-type lectin domain family 11, member A.
CLEC I 6A ENSG00000038532 C-type 'coin domain family 16, member A
CLEC18A EN SG00000157 :322 C-type lectin domain family 18, member A
CLEC I8B ENS000000140839 C-type lectin domain family 18, member l3 CLEC I8C EN SG00000157335 . C-type lectin domain family 18, member C
CLECI9A ENSG00000261.210 C-type lectin domain family 19, member A.
CLEC2B ENS000000 110852 C-type lectin domain family 2, member B
CLEC3A ENSG00000166509 C-type lectin domain family 3, member A
CLEC3B ENSG00000163815 C-type lectin domain family 3, member B
CLGN ENSG00000153132 Calmegin CLN5 ENSG00000102805 Ceroid-lipofttscinosis, neuronal 5 CLPS ENSG00000137392 Colipase, pancreatic CLPSL1 EN SG00000204140 Col ipase-like 1 CLPSL2 ENSG00000196748 Colipase-like 2 CLPX EN SG00000166855 Caseinolytic mitochondria, matrix peptidase chaperone subunit CLSTN3 ENSG00000139182 Calsvntenin 3 CLU EN SG00000120885 Clusterin CLUL1 ENSG00000079101 Clusterin-like 1 (retinal) CMA I. ENSG00000092009 . Chymase 1, mast .v1 CMPK1 ENSG00000162368 Cytidine monophosphate (LIMP-CMP) kinase 1, cytosolic CNBEt1 ENSG00000176571 Cyclic nucleotide binding domain containing CNDP1 EN SG00000150656 Camosine dipeptidase 1 (meiallopeptidase M20 family) CNPY2 ENSG00000257727 Canopy FGF signaling regulator 2 CNPY3 ENSG00000137161 . Canopy RIF signaling regulator 3 CNPY4 EN SG00000166997 Canopy MT: signaling regulator 4 CNTFIt ENSG00000122756 Ciliary neurotrophic factor receptor eNTNI EN S0)0000018236 Contactin 1 CNTN2 ENSG00000184144 Contactin 2 (axonal) c:NTN 3 EN sci00000 113805 . Coniactin 3 (plastnacylotna associated) CNTN4 EN SG00000144619 Contactin 4 CNTN5 ENSG00000149972 Contact in 5 CNTNAP2 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 synthase COCH ENSG00000100473 Cochlin C063 EN SC1001)00136152 Component of oligorrteric golgi complex 3 COLIOA1 ENSG00000123500 Collagen, type X, alpha 1 COLI1A1. ENSG00000060718 Collagen, type XI, alpha 1 COLI1A2 EN SG00000204248 Collagen, type XI, alpha 2 COL12A.1 E'NSG00000111799 Collagen, type XII, alpha 1 COL14A1 EN SG00000187955 Collagen. type XIV, alpha 1 COL15A1 EN 5000000204291 Collagen, type XV, alpha 1 COL16A1. ENSG00000084636 Collagen, type XVI, alpha 1 COL18A1 ENSG00000182871 . Collagen, type XVIII, alpha 1 COL19A 1 ENSG00000082293 Collagen, type XIX. alpha 1 COLIA1 EN SG00000108821 Collagen. type I, alpha 1 COL1A.2 EN SG00000164692 Collagen, type I, alpha 2 COL20A1. ENSG00000101203 Collagen, type VC, alpha 1 COL21.A1 ENSG00000124749 . Collagen, type XXI, alpha 1 COL22A1 ENSG00000169436 Collagen, type XXII, alpha 1 COL24A1 ENSG00000171502 Collagen. type XXIV, alpha 1 COL26A1 EN SG00000160963 Collagen, type XXVI. alpha 1 C0L27A1 ENSG00000196739 Collagen, type XXVII, alpha 1 COL28A1 ENSG00000215018 . Collagen, type XXVIII, alpha 1 COL2 A I ENSG00000139219 Collagen, type II, alpha 1 COL3A I ENSG00000168542 Collagen. type III, alpha 1 COL4A1 EN SG00000187498 Collagen, type IV. alpha I.
COL4A2 ENSG00000134871 Collagen, type IV, alpha 2 COL4A3 EN SG00000169031 Collagen, type IV, alpha 3 (Goodpasture antigen) DEMA_NDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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Claims (23)

What is claimed is:
1. A method of producing a plurality of modified T cells, comprising (a) introducing into a plurality of primary human T cells (i) a transposon composition comprising a transposon comprising an antigen receptor or a nucleic acid sequence encoding the antigen receptor and (ii) a transposase composition comprising a transposase or a sequence encoding the transposase;
(b) contacting the modified T cells with a T cell activator composition; and (c) culturing the modified T cells, under conditions that produce a plurality of modified T cells such that at least 25% of the plurality of modified T cells express the antigen receptor and express cell-surface markers of a stem memory T cell (Tscm) or a Tscm-like cell; and wherein the cell-surface markers comprise CD45RA and CD62L.
2. The method of claim 1, wherein a nucleic acid vector comprises the nucleic acid sequence encoding the antigen receptor.
3. The method of any one of claims 1-2, wherein the introducing step comprises a homologous recombination.
4. The method of claim 3, wherein the homologous recombination comprises contacting the transposon composition comprising the nucleic acid sequence encoding the antigen receptor and a genomic editing construct with a genomic sequence of at least one primary human T cell of the plurality of primary human T cells.
5. The method of claim 4, wherein the genomic editing construct comprises a guide RNA
and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease.

Date Recue/Date Received 2021-09-24
6. The method of claim 5, wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease.
7. The method of claim 4, wherein the genomic editing construct comprises an amino acid sequence derived from a Cas9 endonuclease, an amino acid sequence derived from a transcription activator-like effector nuclease (TALEN) or an amino acid sequence derived from a zinc-finger nuclease (ZFN).
8. The method of claim 7, wherein the amino acid sequence derived from a Cas9 endonuclease is the DNA binding domain or encodes an inactive Cas9.
9. The method of claim 4, wherein the genomic editing construct targets a safe harbor site on a human chromosome, a nucleic acid sequence encoding a component of an endogenous T
cell receptor or a nucleic acid sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a human chromosome.
10. The method of any one of claims 1-9, wherein the transposon is a piggyBac transposon, a Sleeping Beauty transposon, a Helraiser transposon or a To12 transposon.
11. The method of any one of claims 1-10, wherein the transposase is a piggyBac transposase, a super piggyBac transposase, a Sleeping Beauty transposase, a hyperactive Sleeping Beauty transposase (SB100X), a Helitron transposase or a To12 transposase.
12. The method of any one of claim 1-11, wherein the sequence encoding the transposase is an mRNA sequence.
13. The method of any one of claims 1-12, wherein the antigen receptor is a T cell receptor (TCR).
14. The method of claim 13, wherein the TCR is not naturally-occurring.

Date Recue/Date Received 2021-09-24
15. The method of any one of claims 1-12, wherein the antigen receptor is a Chimeric Antigen Receptor (CAR).
16. The method of claim 15, wherein the CAR comprises at least one of a single chain variable fragment (scFv), a single domain antibody, a VHH, an antibody mimetic, a protein scaffold or a Centyrin.
17. The method of any one of claims 1-12, wherein the antigen receptor is a therapeutic protein which is a secreted or secretable protein.
18. The method of any one of claims 1-17, wherein the T cell activator composition comprises one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement.
19. The method of claim 18, wherein the T cell activator composition further comprises an anti-human CD2 monospecific tetrameric antibody complex.
20. The method of any one of claims 1-19, wherein the conditions comprise culturing the modified T cells in a medium comprising a sterol; an alkane; phosphorus and one or more of an octanoic acid, a palmitic acid, a linoleic acid, and an oleic acid.
21. The method of claim 20, wherein the medium comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints or between 6.4 gmol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints or between 0.7 mol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints or between 0.75 mol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints or between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration Date Recue/Date Received 2021-09-24 of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints or between 0.25 mol/kg and 25 mol/kg, inclusive of the endpoints.
22. The method of any one of claims 1-21, wherein at least 30%, at least 40%, at least 50%
or at least 60% of the plurality of modified T cells expresses the cell-surface markers of a stem memory T cell (Tscm) or a Tscm-like cell.
23. The method of any one of claims 1-21, wherein at least 25% or at least 30% of the modified T cells of the population further express one or more of CD28, CCR7, CD127, CD45RO, CD95 and IL-2R13.

Date Recue/Date Received 2021-09-24
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