CA3154236A1

CA3154236A1 - Modified stem cell memory t cells, methods of making and methods of using same

Info

Publication number: CA3154236A1
Application number: CA3154236A
Authority: CA
Inventors: Eric Ostertag; Devon SHEDLOCK
Original assignee: Poseida Therapeutics Inc
Current assignee: Poseida Therapeutics Inc
Priority date: 2016-09-30
Filing date: 2017-10-02
Publication date: 2018-04-05
Anticipated expiration: 2037-10-02
Also published as: CA3154236C; CN110199015A; IL287286B; JP2019528760A; SG10202007963SA; AU2020227020A1; AU2022235633A1; CA3105873A1; KR20200108927A; IL276907A; KR20190063468A; JP2024096417A; IL265157B; IL287286A; CA3036926C; CA3036926A1; AU2017337147A1; IL265157A; KR102588469B1; IL276907B

Abstract

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

Description

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

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

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

MODIFIED STEM CELL MEMORY T CELLS, METHODS OF MAKING AND
METHODS OF USING SAME
RELATED APPLICATIONS
[01] This application claims the benefit of provisional applications USSN
62/402,707 filed September 30, 2016, USSN 62/502,508 filed May 5, 2017, USSN 62/553,058 filed August 31, 2017 and USSN 62/556,309 filed September 8, 2017.
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 2022-04-05 and multipotent capacity to derive central memory (Tcm), effector memory (Tim) and effector I cells (TB), thereby producing better tumor eradication and long-term modified-T cell engraftment. Modified-T cells of the disclosure include, but are not limited to, those cells that express an antigen receptor comprising a protein scaffold of the disclosure.
Modified-T cells of the disclosure include, but are not limited to, those cells that express a chimeric antigen receptor (CAR) (i.e. CAR-T cells of the disclosure). Chimeric antigen receptors (CARs) of the disclosure may comprise one or more sequences that each specifically bind an antigen, including, but not limited to, a single chain antibody (e.g. a scFv), a sequence comprising one or more fragments of an antibody (e.g. a VF1H, 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.
1071 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 product 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 I 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 -Date Recue/Date Received 2022-04-05 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 (Tsc.m), 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 I 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 (TRIO. 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). hi 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 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 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments; the cell-surface markers of the CAR-Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2R3, 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 -Date Recue/Date Received 2022-04-05 cis-regulatory insulator elements. In certainembodiments,thetransposon isapiggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac or a Super piggyBacTM
(SPB)transposase.
gmq In certain embodiments of the methodsofthe disclosure,thetransposon is aplasmid DNAtransposonwithasequence encodingtheantigen receptororthetherapeuticprotein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. Incertainembodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBa& or a Super piggyBacTm (SPB) transposase. In certain embodiments, and, in particular, those embodiments whereinthetransposase is a SuperpiggyBacTm(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 PVMHKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:

4).
[010] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBacTm (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:

Date Recue/Date Received 2022-04-05 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
4).
[011] In certain embodiments, the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO:
4. In certain embodiments, the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO:
4. In certain embodiments, the transposase enzyme is a piggyBacTM (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ
ID NO: 4. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N).
[0121 In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBacTm (SPB) transposase enzyme. In certain embodiments, the Super pig,gyBacTm (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 senine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBacTm (SPB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

- 5 -Date Recue/Date Received 2022-04-05 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
5).
[013] in certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacim 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, 42.1,436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ
ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTM or Super piggyBacTm transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO:
4 or SEQ ID
NO: 5 is a substitution of a serine (5) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (I) 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 (5). 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 al an i tic (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of

- 6 -Date Recue/Date Received 2022-04-05 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 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 prolinc (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

- 7 -Date Recue/Date Received 2022-04-05 NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ TD NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ 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 TD NO: 4 or SEQ ID NO: 5 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for an arginine (R)In certain embodiments, the amino acid substitution at position 319 of SEQ 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 ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C).
In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a lysine (K) for a serine (5). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ

- 8 -Date Recue/Date Received 2022-04-05 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 TD 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 pig,gyBacTm 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 piggyBacrm 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 prolinc (P) for a scrinc (5). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S). hi certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for an

- 9 -Date Recue/Date Received 2022-04-05 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 piggyBac TM 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.
[0151 The disclosure provides a method of producing a modified stem memory T
cell (Tscm), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a modified stem memory T cell (Tscm). The disclosure provides a method of producing a plurality of modified stein memory T cells (Tscm), comprising introducing into a plurality of primary human T
cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%,

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the Date Recue/Date Received 2022-04-05 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 (Tscm). in certain embodiments, the method produces a plurality of modified T
cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscn,i). 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 stein memory T cell (Tsai), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). 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-2Rii. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, IL-2RP, CR7, and CD62L. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
[016] In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

- 11 -Date Recue/Date Received 2022-04-05 301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 6).
[017] In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7).
[018] The disclosure provides a method of producing a modified stem memory T
cell (Tsc.m), 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 stein memory T cell (Tscm). The disclosure provides a method of producing a plurality of modified stein memory T cells (Tscm), comprising introducing into a plurality of primary human T
cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 700/e, 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 I 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

- 12 -Date Recue/Date Received 2022-04-05 stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm).
In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of modified T
cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62.L
and CD45RA. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD621,, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-21t13. 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 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 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:

- 13 -Date Recue/Date Received 2022-04-05 61 CCCACCAGCC AATCAGAAGT GACTATGCAA ATTAACCCAA CAAAGATGGC AGTT:AAATTT

181 GCTGGCCCCG GGAGGCGAGG CCGGCCGCGC CTAGCCA.CAC CCGCGGGCTC CCGGGACCTT

481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC AGACCGTGAC AGTAGAGAGC; TGGGACTATG

601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAAA GAAGGCGGCG CCTGCAACA.G

841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG AACA.TAGTTG TGGTGGAATG

1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT CCATGGGTGC AAATATTGCA.

1441 ACAGAAGTAA CAATGGCGA.T TAAATACGAT CGTAACAGTG ACCCAGGTAG ATATAATTCT

1681 GGTGAAAAA.G GCTGGGGAAC AGATATTGCA TTAA.GACTCA GAGAC.AACAG TGTAATCGAC
1741 AATAATACTA GACAAAATGT AAGGACACGA GTCACACAAA TGCAGTA.TTA TGGATTTCAT

1861 ATTGTGGATT CATATTCAAA. AATGGAGGCC AATCGGATAA ATTTCATCAA AGCAAACCAA

2041 AGAAATATGC AGCAGCGATA TCAGGATGCT A.TGGCAATTG TAACGAAGTA TGGC.AAGCCC
2101 GATTTATTCA TAACCATGAC ATGCAACCCC AAATGGGCAG ATATTACAAA. CAATTTACAA
2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC TTGGTAGCCA GAGTTTTTAA. TATTAAGCTG

2281 CATGTCATTG AATTTCAGAA ACGCGGA.CTG CCTCACGCTC ACATATTATT GATATTAGAT

2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA ATTGTA.AAAT CAAATATGGT ACATGGACCA

- 14 -Date Recue/Date Received 2022-04-05 2521 CCAAAAGAAT TTCAAAATGC GAC:CATTGGA AATATTGATG GATATCCCAA. ATACAAACGA

2701 ATTAA_AAGTG TCAAATATTT ATTTAAATAC ATCTATAAAG GGCACGATTG TGCAAATATT

3001 TTCTTATTGA ATAGAGAAGA TTCTGATGCA CGTAATTATT ATTATTGGGA GATTCCACA.G

3241 GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC TTGA.TGACAC TATCTGGAAA

3421 CATTTTATTG AAGATTTCTG TTGGAAA.TTA CACCGAAGAG AAGGTGCCTG TGTGAACTGT
3481 GAAATGCATG CCCTTAACGA AATTCAGGAG GTATTCACAT TGCATGGAAT GAAATGTTCA.

3721 GGTCCAGGTG GTAGTGGAAA AA.CATATCTG TATAAAGTTT TAACACATTA TATTAGAGGT

3841 GGAAGAACCT TTCATTCCCA ATATAAATTA CCAATTCCAT TAAA.TGAAAC TTCAATTTCT
3901 AGACTCGATA TAAAGAGTGA AGTTGCTAAA ACC.ATTAAAA AGGCCCAACT TCTCATTATT

4141 T.ACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA AAACAAATAT GAGATCAGAG

4441 GATGCAGATT TTCACACATA TTTGAGTGAT GATTCCATTG ATTCAACAGA TGATGCTG.AA

4561 AAATTAAAAT TGAAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA. TAGTAAATGG

4681 GAAGTATTAA CAGGATCTGC AGAGGGA.GAG GTTGTTCTGA TTCCAAGAAT TGATTTGTCC

- 15 -Date Recue/Date Received 2022-04-05 4921 TGTGACGTTA, AAGTTAAAGT TGTAAATACT TCATCACAAG GGAAATTAGT CAAGCACTCT
4981 GAAAGTGITT TTACTCTTAA. TGTGGTATAC AGGGAGATAT TAGAATAAGT TTAATCACTT

5281 TGCACCGGGC CACTAG (SEQ ID NO: 27).
[020] 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.
[021) An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:

241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY KMLHEVEKEA QSEAAAKGIA.

- 16 -Date Recue/Date Received 2022-04-05 1441 LPEPVFAHGQ LYVAFSRVRR AC DVKVKVVN T SSQGKLVKH SESVFTLNVV YREI LE (SEQ ID
NO: 28) .
10221 In Helitron transpositions, a hairpin close to the 3' end of the transposon functions as a tenninator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5'-TC/CTAG-3' motif. A 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence GT GCAC GAATTT CGTGCACC GGGCCACTAG (SEQ ID 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 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 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 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 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 -Date Recue/Date Received 2022-04-05 more cell-surface marker(s) of a stern 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 stern 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 I 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 stern 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, C.D95, CD95 and IL-2R[3. 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 of this method, the transposon is a To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
[024] In certain embodiments of the methods of the disclosure, the transposase is a To12 transposase. To12 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family. Exemplary To12 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the To12 transposase, which contains four exons. An exemplary To12 transposase of the disclosure comprises an amino acid sequence comprising the following:
MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSFKMKCVIC LPLNKEISAF

- 18 -Date Recue/Date Received 2022-04-05 TFEVLA.SAMN

SEGCGEGSDG
301 VE FQDAS RVL DQDDGFEFQL PKHQKCA.CHL LiNLVS SVDAQ KALSNEHYKK LYRSVEGICCQ
361 ALWNKSSRSA LAAEAVES ES RLQLLRPNQT RWNSTFMAVD RI LQI CKEAG EGALFtNICTS

541 PLDHKKELAN SS SDDEDFFA SLKPTTHE.AS KELDGYLACV SDTRESLLTF PAI CS L S I KT
601 NT PL PASAAC ERLFSTAGLL FS PKRAP.LDT NNFENQLLLK LNLREYN FE (SEQ ID NO:
30).
[0251 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 CTTACA_ATTT TATTTACAGT CAAAAAGTAC
181 TTATTTTTTG GAGA.TCACTT CATTCTATTT TCCCTTGCTA TTACCAAACC AATTGAATTG

60]. GGCCTTCAAA AGTTCGCCAT CAAA.CCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT

721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCC TAT TTTCA,CTAAT GCATGCGATT

1141 AACAAGATAT AAA.GTATTAG T.AAA.TGTTGA AATTAACATG TATACGTGCA. GTTCATTATT

1381 TCAACAAGTA TTTAACATTA TA_AAGTGTGC AATTGGCTGC AAATGTCAGT TTTATTAAAG
1441 GGTTAGTTCA CCCAAAAATG AAAATAA.TGT CATTAAT GA.0 TCGCCCTCAT GTCGTTCCAA.
1501 GCCCGT.AAGA CCTCCGTTCA TCTTCAGAA.0 ACAGTTTAAG ATATTTTAGA TTTA.GTCCGA

- 19 -Date Recue/Date Received 2022-04-05 1681 TCGAATACAT TTTGGTCC.A.A. AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC

1921 AGTATACCGT ACATACATTT TCAGTGGAGG GACAG_A_AAGC TCTCGGACTA AATCTAAAAT

2881 CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT GTCACTTA.CT

-20 -Date Recue/Date Received 2022-04-05 4441 AACCTTGTAT GCATTTCATT TAATGTTTTT TGAGATTAAA AGCTTAAACA AGAA.TCTCTA.
4501 GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA AGTACAATTT TAATGGAGTA.

4681 TG (SEQ ID NO: 31).
[026] The disclosure provides a method of producing a modified central memory T-cell (Tcm), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified Tee!! expresses one or more cell-surface marker(s) of a central memory T-cell (Tcm), thereby producing a modified central memory T-cell (Tcb,f). The disclosure provides a method of producing a plurality of modified central memory T-cells (Tcm), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T-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 25% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tcm), thereby producing a plurality of modified central memory T-cells (Tcm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tcm), thereby producing a plurality of modified central memory T-cells (Tcm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tcn.i), thereby producing a plurality of

- 21 -Date Recue/Date Received 2022-04-05 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 T-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 1-cells (Teri). In certain embodiments, the method produces a plurality of modified T
cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Teri), 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 1-cell (Tem), thereby producing a plurality of modified central memory T-cells (Tem). hi certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, CCR7, and CD62L. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super 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 -Date Recue/Date Received 2022-04-05 [0271 The disclosure provides a method of producing a composition comprising a plurality of modified stem memory T-cells (Tscm) and a plurality of modified central memory T-cells (Tcm), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a composition comprising a plurality of modified Tscm and a plurality of modified Tcm, wherein the plurality of modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R3 and the plurality of modified Tcm expresses one or more CD45RO, CD95, 1L-2R13, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified Tscm and a plurality of modified Tem. In certain embodiments of this method, the modified stem memory 1-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 1-cells (Tscm) comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (Tcm) comprise at least 90 /0 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 90% of the total number of cells of the composition and the modified central memory 1-cells (fcm) comprise at least 10% 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 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 (Tcm) 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 (Tscm) comprise at least 30% of the total number of cells of the composition and the modified central memory 1-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 1-cells

- 23 -Date Recue/Date Received 2022-04-05 (Tscm) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (lei) 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 ntunber of cells of the composition and the modified central memory 1-cells (Tem) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 60% of the total number of cells of the composition and the modified central 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 1-cells (Tscm) comprise at least 50% of the total number of cells of the composition and the modified central memory 1-cells (Tem) comprise at least 50% of the total number of cells of the composition. In certain embodiments of this method, the transposon is a plasmid DNA
transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super 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.
[028] In certain embodiments of the methods of the disclosure, the transposon may be derived or recombined from any species. Alternatively, or in addition, the transposon may be synthetic.
[029] in certain embodiments of the methods of the disclosure, the antigen receptor is a 1-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the 1-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein the 1-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

- 24 -Date Recue/Date Received 2022-04-05 certain embodiments, and, in particular, those embodiments wherein the 1-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR
comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
[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). In certain embodiments, this methods fiuther comprises introducing into the primary human T
cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase. In certain embodiments, the second transposase composition transposes the transposon of (c). In certain embodiments, the transposase composition of (b) transposes the transposon of (a) and the transposase composition of (d) transposes the transposon of (c). In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments. the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super piggyBacTM
(SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon

- 25 -Date Recue/Date Received 2022-04-05 is a To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
[031] The disclosure provides a method of producing a modified stem memory T
cell (Tscm), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a stem memory T
cell (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: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory I cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 50% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsc.m), thereby producing a plurality of activated modified stem memory T
cells (Tscm). In

-26 -Date Recue/Date Received 2022-04-05 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 800/i of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T
cells (Tscm). in certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 85% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T cells (Tscm). in certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 95% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (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-2R.11. In certain embodiments, the cell-surface markers of the activated modified Tscm comprise one or more of CD45RA, CD95, IL-2Rfl, CR7, and CD62L.
[0321 In certain embodiments of the methods of the disclosure of producing a modified stem memory T cell (Tscm), comprising: (a) introducing into a primary human T
cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, this method further comprises the step of (c) contacting the activated modified T-cell and a T-cell expansion

-27 -Date Recue/Date Received 2022-04-05 composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stein memory T
cell (Tscm). In certain embodiments of this method, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments of this method, at least 60% of the plurality of expanded modified 1-cells expresses cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, this method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surf-ace marker(s) of a stem memory T cell (Tscm). In certain embodiments of this method, the enriching step comprises isolating modified 1-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 1-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, pahnitic 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 1-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
linoleic acid at

- 28 -Date Recue/Date Received 2022-04-05 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 1-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 1-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 tunol/kg and 640 ttmol/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.7 mol/kg and 70 innol/kg, 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 timol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 timol/kg and 25 mol/kg, inclusive of the endpoints. In certain embodiments of this method, the 1-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 timol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 turiol/kg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 timol/ka.
[033] The disclosure provides a method of producing a modified central memory T-cell (Tcm), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified 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, wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a central memory 1-cell (Tcm), thereby producing a central memory 1-cell (Tcm). The disclosure provides a method of producing a plurality of modified central memory 1-cell (Tcm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a 1-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific

-29 -Date Recue/Date Received 2022-04-05 tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T-cell (Tcm), thereby producing a plurality of activated modified central memory T-cell (Tcm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 25 /0 of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T cell (Tcm). hi 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 (Tcm), thereby producing a plurality of activated modified central memory T cell (Tcm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T
cell (Tcm). 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 (Tcm), thereby producing a plurality of activated modified central memory T cell (Tcm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T
cell (Tcm). In certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 85% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T cell (Tcm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 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 (Tcm). 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 T cell (Tcm), thereby producing a plurality of activated

- 30 -Date Recue/Date Received 2022-04-05 modified central memory T cell (1'cm). In certain embodiments, the cell-surface markers of the activated modified Tem comprise one or more of CD45RO, CD95, CCR7, and CD62L.
[0341 In certain embodiments of the methods of the disclosure of producing a modified central memory T cell (Teri), 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 1-cell, the 1-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 1-cell and a 1-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (Tem). 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 1-cells expresses cell-surface marker(s) of a central memory T cell (Tai). In certain embodiments of this method, at least 60% of the plurality of expanded modified 1-cells expresses cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, this method further comprises the step of (d) enriching the plurality of expanded modified 1-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 400/0, 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 (Tem). 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 1-cells that express cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments of this method, the enriching step comprises isolating modified 1-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 -Date Recue/Date Received 2022-04-05 isolated modified TcNi and a 1-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified Tcm. In certain embodiments of this method, the 1-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further compriscs 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 1-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 gmol/kg and 640 tunol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 tunol/kg and 25 gmol/kg, inclusive of the endpoints.
In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 gmol/kg, linoleic acid at a concentration of about 7.5 'Among, oleic acid at a concentration of about 7.5 lAmol/kg and a sterol at a concentration of about 2.5 gmol/kg.
10351 The disclosure provides a method of producing a composition comprising a plurality of modified stem memory 1-cells (Tscm) and a plurality of modified central memory T-cells

- 32 -Date Recue/Date Received 2022-04-05 (rcm), 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 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 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 (Tam), wherein the plurality of activated modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rl3 and the plurality of activated modified Tcm expresses one or more CD45RO, CD95, IL-2R13, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified Tscm and a plurality of modified Tcm. In certain embodiments of this method, the modified stem memory 1-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 /0 of the total number of cells of the composition and the modified central memory T-cells (Tcm) comprise at least 90% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory 1-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 1-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 1-cells (Tscm) comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells

- 33 -Date Recue/Date Received 2022-04-05 (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.
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 I 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 tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (Tscsi) and a plurality of activated modified central memory 1-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 1-cells (Tscm) and the plurality of activated modified central memory 1-cells (Tcsi) with a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein the plurality of expanded modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2120 and the plurality of expanded modified Tcm expresses one or more CD45RO, CD95, CCR7, and CD62L, thereby producing a composition

- 34 -Date Recue/Date Received 2022-04-05 comprising a plurality of expanded modified ISOM and a plurality of expanded modified Icm.
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 or isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step 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 and isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step further comprises contacting the composition comprising the isolated modified TSCM
and the isolated modified Tayi with a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a composition comprising a plurality of expanded enriched modified Tscm and a plurality of expanded enriched modified Taw 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, pahnitic acid, linoleic acid, oleic acid, stearic acid hydrazide, olcamidc, a sterol and an alkanc. 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 1-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 mgikg, 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 1-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 gmol/kg

- 35 -Date Recue/Date Received 2022-04-05 and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 ginol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 limolikg 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 of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 gmol/kg, linoleic acid at a concentration of about 7.5 gmol/lcg, oleic acid at a concentration of about 7.5 tunol/kg and a sterol at a concentration of about 2.5 gmol/kg. In certain embodiments of this method, the modified stem memory 1-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 (Tsc.m) comprise at least 10% of the total ntunber of cells of the composition and the modified central memory 1-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 1-cells (Tscm) comprise at least 90% of the total number of cells of the composition and the modified central memory 1-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 (Tscm) 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 1-cells (Tam) comprise at least 70% of the total ntunber of cells of the composition and the

- 36 -Date Recue/Date Received 2022-04-05 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 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 1-cells (Tscm) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (Tam) 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 T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, the introducing step comprises a homologous recombination.
In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of a portion of primary I cells of the plurality ofT 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 Date Recue/Date Received 2022-04-05 composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor, a 5 genomic sequence and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 5' to the break point induced by the genomic editing composition and the 3' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3' to the break point induced by the genomic editing composition. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence sequentially, and the genomic editing composition is provided first. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA
binding domain and a nuclease domain. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a TALEN. In certain embodiments of the genomic editing composition, the DNA
binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genoinic 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 CIO (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 (D10A) and a substitution of an Alanine (A) for an Asparagine (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a type IIS
endonuclease. In certain embodiments of the genomic editing composition, the type IIS
endonuclease comprises Acii, Mn11, AlwI, Bbvi, Bed, BceAl, BsmAI, BsmF1, BspCNI,

- 38 -Date Recue/Date Received 2022-04-05 Bsrl, BtsCI, Hgal, Hphl, HpyAV, Mboll, My II, Piet, SfaN1, AcuI, BciVI, BfuAl, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, Earl, EciI, MrneI, NmeAIII, BbvCI, Bpul0I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc361, Fokl or Clo051. In certain embodiments, the type IIS endonuclease comprises Clo051. In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a TALEN or a nuclease domain thereof. In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a ZFN or a nuclease domain thereof. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition induces a break in a genomic sequence and the donor sequence composition is inserted using the endogenous DNA repair mechanisms of the primary T cell. In certain embodiments of the introduction step comprising a homologous recombination, the insertion of the donor sequence composition eliminates a DNA binding site of the genomic editing composition, thereby preventing further activity of the genomic editing composition.
[038] in certain embodiments of the methods of producing an activated modified Tscm or 'fcm 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 tetramcric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises die antigen receptor. In certain embodiments, the viral vector comprises one or more sequences isolated, derived, or recombined from an RNA virus. In certain embodiments, the RNA virus is a single-stranded or a double-stranded virus. In certain embodiments, the viral vector comprises one or more sequences isolated, derived, or recombined from a DNA virus. In certain embodiments, the DNA virus is a single-stranded or a double-stranded virus. In certain embodiments, the virus is replication-defective.

[039] in certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator Date Recue/Date Received 2022-04-05 composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus.
In certain embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus.
[0401 In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus.
In certain embodiments, the viral vector comprises a sequence isolated or derived from a gamma retrovirus.
[0411 In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In certain embodiments, the AAV is a serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV1I. In certain embodiments, the AAV comprises a sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11. In certain embodiments, the AAV
comprises a sequence isolated, derived, or recombined from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11. In certain embodiments, the AAV comprises a sequence isolated, derived, or recombined from AAV2.
In certain embodiments, including those in which the vector crosses the blood brain barrier

-40 -Date Recue/Date Received 2022-04-05 (BBB), the AAV comprises a sequence isolated, derived, or recombined from AAV9.
Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV
hybrids containing the genome of one serotype and the capsid of another serotype (e.g.
AAV2/5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03, rAAV-NP59 and rAAV-NP84.
[042] In certain embodiments of the methods of producing an activated modified Tscm or 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.
[043] In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, a nanoparticle vector comprises the antigen receptor.
Nanoparticles may be comprised of polymers disclosed in, for example, international Patent Publication No. WO 2012/094679, International Patent Publication No. WO 2016/022805, International Patent Publication No. WO/2011/133635, International Patent Publication No.
WO/2016/090111, international Patent Publication No. WO/2017/004498, WO/2017/004509, International Patent Application No. PCT/US2017/030271, US Patent No.
6,835,394, US
Patent No. 7,217,427, and US Patent No. 7,867,512.
[044] in certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type 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 T-cell receptor. In certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHH
sequence(s). In certain embodiments, the CAR is a VCAR.

- 41 -Date Recue/Date Received 2022-04-05 10451 In certain embodiments of the methods of producing an activated modified Tscn..1 or Tod of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, the method further comprises introducing into the primary human T
cell, a composition comprising a therapeutic protein to produce a modified T
cell capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the introducing step comprises a homologous recombination and a donor sequence comprises a sequence encoding the therapeutic protein.
In certain embodiments, the donor sequence that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first donor sequence comprises the antigen receptor and a second donor sequence comprises the therapeutic protein. in certain embodiments, a vector comprises a sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle. In certain embodiments, the vector that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first vector comprises the antigen receptor and a second vector template comprises the therapeutic protein.
[046] The disclosure provides a method of producing a modified stem memory T
cell (Tscp.1), 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 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 and an activation supplement to produce an activated modified T-cell, wherein the activated modified-T cell expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a modified stem memory T cell (Tscm). 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 -Date Recue/Date Received 2022-04-05 and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T
cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscn,i), 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-Tsc0. The disclosure provides a method of producing a plurality of modified stem memory T cells (Tscm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T
cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T
cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stein memory T cells (Tscm). In certain embodiments, the methods further comprises the step of: (c) contacting the activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM,

-43 -Date Recue/Date Received 2022-04-05 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 T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the 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 1-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 1-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 I
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 1-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 mol/kg and 640 jAmol/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 gmol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 tunol/kg and 75 mot/kg, inclusive of the endpoints; and a sterol at a concentration of

- 44 -Date Recue/Date Received 2022-04-05 between 0.25 timol/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 timol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 timol/kg, oleic acid at a concentration of about 7.5 ttmol/kg and a sterol at a concentration of about 2.5 nmol/kg. hi certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 u.mol/kg, palmitic acid at a concentration of about 7.27 tunol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of about 7.56 prnol/kg and a sterol at a concentration of about 2.61 ttmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 trmol/kg, palmitic acid at a concentration of about 7.27 ttmol/kg, linoleic acid at a concentration of about 7.57 trmol/kg, oleic acid at a concentration of 7.56 umol/kg and a sterol at a concentration of 2.61 poiol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 400/o, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 900/o, 95%, 99%
or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, at least 60%
of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T
cell (Tscm). In certain embodiments, the method further comprises the step of:
(d) enriching the plurality of expanded modified 1-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 1-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 1-cells that express cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, the enriching step further comprises isolating modified 1-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tscm) from the plurality of enriched modified T-cells. In certain embodiments, the enriching step further comprises contacting the isolated modified Tscm and a 1-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified Tscm. In certain embodiments, the 1-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-cliol (TMDD), diisopropyl adipate

-45 -Date Recue/Date Received 2022-04-05 (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g.
cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints;
pahnitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg =
parts per million). In certain embodiments, the 1-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 1-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, thc T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and 70 pmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 gmol/kg and 75 pmol/kg, inclusive of the endpoints; and a stem! at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the 1-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 mol/kg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 gmol/ka. In certain embodiments, the 1-cell expansion

-46 -Date Recue/Date Received 2022-04-05 composition comprises one or more of octanoic acid at a concentration of about 63.75 wnol/kg, palmitic acid at a concentration of about 7.27 gmolikg, linoleic acid at a concentration of about 7.57 pawl/kg, oleic acid at a concentration of about 7.56 gmol/kg and a sterol at a concentration of about 2.61 gmol/kg. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 gmol/kg, palmi tic acid at a concentration of about 7.27 gmol/kg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of 7.56 gmol/kg and a sterol at a concentration of 2.61 gmol/kg.

[047] The disclosure provides a method of producing a modified central memory T cell (Tem), 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 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 and an activation supplement to produce an activated modified T-cell, wherein the activated modified-T cell expresses one or more cell-surface marker(s) of a central memory T cell (Tem), thereby producing a modified central memory T cell (Tavi). The disclosure provides a method of producing a plurality of modified central memory T cells (Tem), comprising:
(a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a 1-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospccific 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 (Tem), thereby producing a modified central memory T cell (Tem). 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 (Tem). 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 activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, hwnan Date Recue/Date Received 2022-04-05 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 T-cells expresses one or more cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, the T-cell expansion composition comprises or thrther 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 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 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 [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 1-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 innol/kg and 6401..tmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 pimol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 timol/kg and 75 molikg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pinol/kg and 75 tunol/kg, inclusive of the endpoints: and a

-48 -Date Recue/Date Received 2022-04-05 sterol at a concentration of between 0.25 i.tmol/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 641.1mol/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 i.tmol/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 gmol/kg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 mol/kg. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 Lunolikg, palmitic acid at a concentration of about 7.27 mol/kg, linoleic acid at a concentration of about 7.571Amol/kg, oleic acid at a concentration of 7.56 mol/kg and a sterol at a concentration of 2.61 pimol/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 (Teri). In certain embodiments, at least 60% of the plurality of expanded modified 1-cells expresses cell-surface marker(s) of a central memory T cell (Teis,r). 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%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified 1-cells that express cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, the enriching step further comprises isolating modified 1-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, the enriching step further comprises contacting the 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, the 1-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-cliol (TMDD), diisopropyl adipate

-49 -Date Recue/Date Received 2022-04-05 (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g.
cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints;
pahnitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg =
parts per million). In certain embodiments, the 1-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 1-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, thc T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pmol/kg and 640 1=01/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 p.mol/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 gmol/kg and 75 pmol/kg, inclusive of the endpoints; and a stem! at a concentration of between 0.25 pmol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 timol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 p.mol/ka. In certain embodiments, the 1-cell expansion

- 50 -Date Recue/Date Received 2022-04-05 composition comprises one or more of octanoic acid at a concentration of about 63.75 ptinol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 p.molfkg, oleic acid at a concentration of about 7.56 gmol/kg and a sterol at a concentration of about 2.61 gmol/kg. In certain embodiments, the 1-cell expansion composition comprises octanoic acid at a concentration of about 63.75 ttmol/kg, palmi tic acid at a concentration of about 7.27 mol/kg, linoleic acid at a concentration of about 7.57 ttmol/kg, oleic acid at a concentration of 7.56 timol/kg and a sterol at a concentration of 2.61 mol/kg.
[048] The disclosure provides a method of producing a composition comprising a plurality of modified stem memory 1-cells (Tscm) and a plurality of modified central memory T-cells (Tcm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a composition comprising a plurality of modified stem memory T-cells (Tscm) and a plurality of modified central memory 1-cells (Tcm), wherein a transposon comprises the antigen receptor, and (b) contacting the composition 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 and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (Tscm) and a plurality of activated modified central memory T-cells (Tcm), wherein the plurality of activated modified TSChl expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RD 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 1-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the methods further comprises the step of:
(c) contacting the composition and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified 1-cells, wherein at least 2% of the composition comprising a plurality of expanded modified 1-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 -Date Recue/Date Received 2022-04-05 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 (Tem). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the 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 1-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 1-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 I
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 1-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 mol/kg and 640 jAmol/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 gmol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 Lunol/kg and 75 mot/kg, inclusive of the endpoints; and a sterol at a concentration of

- 52 -Date Recue/Date Received 2022-04-05 between 0.25 ptmol/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 timol/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 mmol/kg, oleic acid at a concentration of about 7.5 ttmol/kg and a sterol at a concentration of about 2.5 tunol/kg. hi 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 ttmol/kg, linoleic acid at a concentration of about 7.57 ttmol/kg, oleic acid at a concentration of about 7.56 umol/kg and a sterol at a concentration of about 2.61 ttmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 p.mol/kgõ
palmitic acid at a concentration of about 7.27 ttmol/kg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of 7.561Amol/kg and a sterol at a concentration of 2.61 p.mol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 400/o, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 900/o, 95%, 99%
or any percentage in between of cells the composition comprising a plurality of expanded modified Tscm and a plurality of expanded modified Tcm expresses cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of cells the composition comprising a plurality of expanded modified Tscm and a plurality of expanded modified Tcm expresses cell-surface marker(s) of a central memory T
cell (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%õ 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 (Tscb,i). In certain embodiments, the method further comprises the step of: (d) enriching the composition to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%

or any percentage in between of modified T-cells that express cell-surface marker(s) of a central memory T cell (Tcm). In certain embodiments, the enriching step further comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tscm) from the composition or isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the composition.
In certain embodiments, the enriching step further comprises isolating modified 1-cells that express one

- 53 -Date Recue/Date Received 2022-04-05 or more cell-surface marker(s) of a stem memory T cell (Tscm) from the composition and isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the composition. In certain embodiments, the enriching step further comprises contacting the isolated modified Tscm and/or Tcm and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a composition comprising a plurality of expanded enriched modified Tscm and/or Tcm. In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyri-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 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 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 1-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

- 54 -Date Recue/Date Received 2022-04-05 gmol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 gmol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/kg and 75 gmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 gmol/kg and 75 ginol/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 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 gmol/kg, linoleic acid at a concentration of about 7.5 timol/kg, oleic acid at a concentration of about 7.5 gmol/kg and a sterol at a concentration of about 2.5 i.umol/kg. In certain embodiments, the 1-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 gmol/kg, oleic acid at a concentration of about 7.56 iimol/kg and a sterol at a concentration of about 2.61 pinol/kg.
In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 gmol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of 7.56 gmol/kg and a sterol at a concentration of 2.61 gmol/kg. In certain embodiments of this method, the modified stem memory 1-cells (Ism) 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 (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 1-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

- 55 -Date Recue/Date Received 2022-04-05 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 stein memory T-cells (Tscm) comprise at least 30% of the total munber of cells of the composition and the modified central memory 1-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 1-cells (Tcp,) 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 1-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 1-cells (Tay) 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 1-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 tetratneric antibody complex and an activation supplement to produce an activated modified 1-cell, the method further comprises introducing into the primary human T cell (c) a second transposon composition comprising a transposon comprising a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the method further comprises introducing a transposase composition. In certain embodiments, the transposase composition transposes the transposon of (a) and the second transposon. In certain embodiments, the method comprises introducing a first transposase composition and a

- 56 -Date Recue/Date Received 2022-04-05 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 pig,gyBac 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.
[050] in certain embodiments of the methods of the disclosure, including those wherein the method comprises introducing into a primary human T cell (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetratneric 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.
hi 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 -Date Recue/Date Received 2022-04-05 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 palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA
endonuclease. In certain embodiments, the genomic editing construct comprises a DNA binding domain and a type I1S 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 [IS 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 (Dl OA). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alaninc (A) for a Histidinc (H) at position 840 (H840A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dCas9 (SEQ ID
NO: 33). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dSaCas9 (SEQ ID NO: 32). In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS
endonuclease, the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN). in certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS
endonuclease, the sequence derived from a TALEN is the DNA binding domain. In certain embodiments,

- 58 -Date Recue/Date Received 2022-04-05 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).
[052] 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 piggyBac' (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBac Tm (SPB) transposase, the sequence encoding the transposase is an mRNA
sequence. In certain embodiments, the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4. In certain embodiments, the piggyBac transposase is a hyperactive variant and the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4. In certain embodiments, the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I) (I30V). In certain einbodiments, the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of a senile (S) for a glycine (G) (G165S). In certain embodiments, the amino acid substitution at position 282 of SEQ TD NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V). In certain embodiments, the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N) (N538K). In certain embodiments, the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5. In certain embodiments, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments, the transposon is a Helraiser transposon. In certain embodiments, in particular those embodiments wherein the transposon is a Helraiser transposon, the

- 59 -Date Recue/Date Received 2022-04-05 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. In certain embodiments, the 1-cell expansion composition further comprises a selection agent.
[054] In certain embodiments of the methods of the disclosure, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein the 1-cell receptor is not naturally-occurring, the T-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 1-cell receptor is a recombinant T-cell receptor. in certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR
comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
10551 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-21213. In certain embodiments, the cell-surface markers of the modified Tscm comprise one or more of CD45RA, CD95, IL-21213, 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 1-cell (modified Tem) and the cell-surface markers of the CAR-Tcm comprise one or more of CD45RO, CD95, IL-2Rf1 CCR7, and CD62L. In certain embodiments, the plurality of expanded modified 1-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-2R13. In certain embodiments,

-60 -Date Recue/Date Received 2022-04-05 plurality of expanded modified T-cells comprises an effector T-cell (modified IEEE) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2R[3.
[057] In certain embodiments of the methods of the disclosure, the plurality of expanded modified T-cells comprises a central memory 1-cell (modified 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 most abundant cell in the plurality of expanded modified 1-cells is a central memory T-cell (modified Tcm) and the cell-surface markers of the CAR-Tcm comprise one or more of CD45RO, CD95, IL-2R13, CCR7, and CD62L. In certain embodiments, wherein the most abundant cell in the plurality of expanded modified 1-cells is a central memory 1-cell (modified Tcm), the plurality of expanded modified 1-cells comprises a Tscm cell and the cell-surface markers of the Tscm cell comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R[3.
[058] 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 tetramcric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce an activated CAR-T cell, wherein the activated CAR-T cell expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a CAR-expressing stem memory T cell (Tscm) (CAR-Tscm). The disclosure provides a method of producing a plurality of modified stem memory T cells (Tscm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T
cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T
cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at

- 61 -Date Recue/Date Received 2022-04-05 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 (Tsam), thereby producing a plurality of activated CAR stem memory T cells (Tsar). In certain embodiments, the method produces a plurality of activated CA R-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 inemory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tsai). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RO. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-2R11 CR7, and CD62L. The disclosure provides a method of producing a modified stem memory T cell (Tscm), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-T
cell and (b)

-62 -Date Recue/Date Received 2022-04-05 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).
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 I 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-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-I cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 80%
of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells

-63 -Date Recue/Date Received 2022-04-05 (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 (Tsc.m). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R13. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-2R13, CR7, and CD62L.
[060] In certain embodiments, this method may farther 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 (Tscm) (CAR-Tscm). In certain embodiments, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g.
cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints;
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 nig/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 -Date Recue/Date Received 2022-04-05 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 punol/kg and 640 gmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 timol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/lcg and 75 gmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 gmol/kg and 75 mot/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 gmol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 iamol/kg, palmitic acid at a concentration of about 7 innol/kg, linoleic acid at a concentration of about 7.5 mol/kg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 pmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of about 7.56 mol/kg and a sterol at a concentration of about 2.61 gmol/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 7.27 gmol/kg, linoleic acid at a concentration of about 7.57 gmol/kg, oleic acid at a concentration of 7.56 mol/kg and a sterol at a concentration of 2.61iamol/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 -Date Recue/Date Received 2022-04-05 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 2022-04-05 comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T
cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR
stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TKO, 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 embodirnents, the method produces a plurality of activated CAR-T cells, wherein at least 80%
of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95%
of the

-67 -Date Recue/Date Received 2022-04-05 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 (Tsem). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tsem comprise one or more of CD62L, CD45RA, CD28, CCR7, CDI27, CD45RO, CD95, CD95 and IL-2Rf3. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-2R13, CR7, and CD62L.
[062] In certain embodiments of the methods of the disclosure, the plurality of expanded CAR-T cells comprises a naive T-cell (CAR-TN) and the cell-surface markers of the CAR-TN
comprise one or more of CD45RA, CCR7 and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises a central memory T-cell (CAR-Tem) and the cell-surface markers of the CAR-Tem comprise one or more of CD45RO, CD95, IL-21213, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-TO and the cell-surface markers of the CAR-TEm comprise one or more of CD45RO, CD95, and IL-2RI3. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector T-cell (CAR-TEI..0 and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2R13.
[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 chitneric 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 1-cell expansion composition of the methods of the disclosure may further comprise a selection agent to simultaneously select and expand an activated or modified T cell of the disclosure.
[065] In certain embodiments a CAR of the disclosure may be a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.

-68 -Date Recue/Date Received 2022-04-05 [066] In certain embodiments of the methods of producing a modified Tscm of the disclosure, the introducing step may comprise an electroporation or a nucleofection. When the introducing step comprises a nucleofection, the nucleofection may comprise the steps of:
(a) contacting a transposon composition, a transposase composition, and a composition comprising a plurality of primary human T cells in a cuvette; (b) applying one or more electrical pulses to the cuvette, and (c) incubating the composition comprising the plurality of primary human T cells in a composition comprising a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37 C. In certain embodiments, the 1-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-43-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 1-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 1-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 -Date Recue/Date Received 2022-04-05 (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 timol/kg and 640 timol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 mmol/kg and 70 ttmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 timol/kg and 75 tunol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 lAmol/kg and 75 timol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 ttmol/kg and 25 mol/kg, inclusive of the endpoints. In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 timol/kg, palmitic acid at a concentration of about 7 timol/kg, linoleic acid at a concentration of about 7.5 timol/kg, oleic acid at a concentration of about 7.5 timol/kg and a sterol at a concentration of about 2.5 tunol/kg. in certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 mot/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 tunol/kg, oleic acid at a concentration of about 7.56 timol/kg and a sterol at a concentration of about 2.61 ttmol/kg.
In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 mollkg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 famol/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 of the nucleofection, the transposon composition is a 0.5 tig/ 1 solution comprising nuclease five water and the cuvettc comprises 2 p1 of the transposon composition to yield 1 tig 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 ttg of transposase. The transposase composition may comprise a hyperactive piggyBacTM or Super piggyBacTm (SPB) transposase. The transposase composition may comprise a hyperactive Sleeping Beauty (SB100X) transposase.
In certain embodiments, the transposon may comprise a Helraiser transposon and the transposase composition may comprise a Helitron transposase. In certain embodiments, the transposon may comprise a To12 transposon and the transposase composition comprises a To12 transposase.
[067] In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition and a first transposase

-70 -Date Recue/Date Received 2022-04-05 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 primaiy 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 primaty human T cells in a cuvette.
In certain embodiments, the transposon composition comprises a sequence encoding the

- 71 -Date Recue/Date Received 2022-04-05 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 transposasc arc contacted with the composition comprising a plurality of primary human T cells second. In certain embodiments of the methods of producing a modified Tscm of the disclosure, the composition comprising primary human T
cells comprises a buffer that maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells prior to the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells during the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and/or a stem-like phenotype of the primary human T cells following the nucleofection. In certain embodiments, the buffer comprises a P3 primary cell solution

-72 -Date Recue/Date Received 2022-04-05 (Lonza). In certain embodiments, the buffer comprises one or more of KCI, MgC12, CINa, 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 KC!, 15 mM MgCl2, 90 mM CINa, 10 inM Glucose and 0.4 mM Ca(NO3)2. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgC12, 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 KC1, 15 mM MgCl2, 90 mM
CINa, 10 mM Glucose and 0.4 rnIvi Ca(NO3)2 and a supplement comprising 40 mM
Na21-IP04/Nati2PO4 at pH 7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 p1 of the buffer and between 5x106 and 25x106 cells.
10681 In certain embodiments of the methods of producing a modified Tscm of the disclosure, the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and/or CD19. In certain embodiments, the composition comprising primary human T cells comprises 100 p1 of the buffer and between 5x106 and 25x106 cells.
[0691 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 I-cell expansion composition" or "T-cell expansion composition"
may be used interchangeably with a media comprising one or more of phosphorus, an octanoic fatty acid, a palmitic fatty acid, a linoleic fatty acid and an oleic acid. In certain embodiments, the media comprises an amount of phosphorus that is 10-fold higher than may be found in, for example, Iscove's Modified Dulbecco's Medium ((TMDM); available at ThermoFisher Scientific as Catalog number 12440053).
[0701 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 "1-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 -Date Recue/Date Received 2022-04-05 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), olearnide (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 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-tetramethy1-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No.

94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS
No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), alkanes (e.g., nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No.
98-92-0), 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS
No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid

- 74 -Date Recue/Date Received 2022-04-05 hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS
No. 143-74-8) and lanolin alcohol.
[072] As used herein, the terms "supplemented T-cell expansion composition" or "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 1-cell expansion composition" or "1-cell expansion composition"
may be used interchangeably with a media comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate and phosphate.
[073] As used herein, the terms "supplemented T-cell expansion composition" or '7-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 1-cell expansion composition" or "1-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, threoninc, alanine, proline, cysteine, lysine, tyrosine, methionine, valinc, isolcucine, leucine, phenylalanine and tryptophan. hi 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 the following free amino acids in the corresponding average mole percentages: histidine (about 1%), asparagine (about 0.5%), serine (about 1.5%), glutamine (about 67%), arginine (about 1.5%), glycine (about 1.5%), aspartic acid (about 1%), glutamic acid (about 2%), threonine (about 2%), alanine (about 1%), proline (about 1.5%), cysteine (about 1.5%), lysine (about 3%), tyrosine (about 1.5%), methionine (about 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 1-cell expansion composition" or '7-cell expansion composition" may be used interchangeably with a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about .78%), asparagine (about 0.4%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine

-75 -Date Recue/Date Received 2022-04-05 (about 3.14%), leucine (about 3.3%), phenylalanine (about 1.64%) and tryptophan (about 0.37%).
[074] As used herein, the terms "supplemented T-cell expansion composition" or "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 T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, the terms "supplemented 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 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1 mg/kg (wherein mg/kg = parts per million). ). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg =
parts per million). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the terms "supplemented T-cell expansion composition" or "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 tunol/kg and 640 mol/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 mol/kg

-76 -Date Recue/Date Received 2022-04-05 and 75 grnol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 gmol/kg and 75 mol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 !Among and 25 iimol/kg, inclusive of the endpoints. In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition"
may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 64 mol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 gmol/kg, oleic acid at a concentration of about 7.5 tanol/kg and a sterol at a concentration of about 2.5 mol/kg. In certain embodiments, the terms "supplemented T-cell expansion composition" or "F-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27 ttmol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of about 7.56 tnnol/kg and a sterol at a concentration of about 2.61 mol/kg.
In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27 gmol/lcg, linoleic acid at a concentration of about 7.57 ttmol/kg, oleic acid at a concentration of 7.56itmolikg, and a sterol at a concentration of 2.61 ttmol/kg.
[075] 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 KCl, 15 mM
MgC12, 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 ClNa, 10 mM Glucose and 0.4 mM
Ca(NO3)2 and a supplement comprising 20 mM HEPES and 75 mM Tris/HC1. The term "P3 buffer"
may be used interchangeably with a buffer comprising 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.
[076] As used herein, the terms "supplemented RPMI-1640 media" or "T-cell conditioned media (TCCM)" may be used interchangeably with a media comprising one or more of

-77 -Date Recue/Date Received 2022-04-05 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-Glutarnine, L-Arginine, L-Aspar-agine (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HCI, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2H20, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid. D-Panthothenic acid (hemicalcium), pyridoxine HCI, riboflavin, thiamine HCl, vitamin B12, D-Glucose, Glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration. The terms "supplemented RPM1-1640 media" or "T-cell conditioned media (TCCM)" may be used interchangeably with a media comprising water, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HCI, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 21-120, L-Valine, D-Biotin, choline chloride, folic acid. Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid (hemicalcium), pyridoxine HCI, riboflavin, thiamine HC1, vitamin B12, 13-Glucose, Glutadiione (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 AIMV"
media may be used interchangeably with a media comprising one or more of water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine serum. HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutainine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydmxy-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 HCI, riboflavin, thiamine HCl, vitamin B12, D-Glucose,

-78 -Date Recue/Date Received 2022-04-05 elutathione (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 HC1, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2H20, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid (hemicalcium), pyridoxine HC1, riboflavin, thiamine HCl, vitamin B12, D-Glucose, glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration.
[0781 As used herein, the term "ImmunoCultrm medium" may be used interchangeably with a medium comprising one or more of water, human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycinc, L-Alaninc, 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-Pheny-lalanine, L-Prolinc, L-Scrine, L-Thre.onine, 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-lnositol, calcium chloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in any absolute or relative abundance or concentration. The term "ImmunoCultTm medium" may be used interchangeably with a medium comprising water, human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, L-Glutamine, L-Histidine hydrochloride H20, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Ttyptophan, L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-Calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine

-79 -Date Recue/Date Received 2022-04-05 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 1-cells of the disclosure, including modified Tsem and/or Teri of the disclosure, may be incubated, cultured, grown, stored, or otherwise, combined at any step in the methods of the procedure with a growth medium comprising one or more inhibitors a component of a PI3K pathway. Exemplary inhibitors a component of a PI3K
pathway include, but are not limited to, an inhibitor of GSK3f3 such as TWS119 (also known as GSK
3B inhibitor XII; CAS Number 601514-19-6 having a chemical formula CisH14N402).
Exemplary inhibitors a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIOTm).

[080] As used herein, the terms "electroporation" and "nucleofection" are meant to describe alternative means to deliver a nucleic acid, transposon, vector or composition of the disclosure to a cell by providing an electric pulse that induces a cell membrane (the cell membrane, nuclear membrane, or both) to become permeable or to become more penneable to the nucleic acid, transposon, vector or composition of the disclosure.

[081] 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. hi 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. hi 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) El-151, El-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, EO-151, EO-148, EO-156, EO-210, EO-213, and FI-156. In certain embodiments, the applying step may comprise applying one or more of the following Date Recue/Date Received 2022-04-05 program(s) E1-115, E1-151, El-156, El-158, EG-115, EG-142, EG-151, ES-115, ES-151, E0-151, EO-148, EO-156, EO-210, E0-213, and Fl-156, or a program that provides the same number of electrical pulses, each pulse having the same duration and intensity, and a substantially similar interpulse duration of time. In certain embodiments, the applying step may be performed using a known electroporation/nucleofection device, including, but not limited to, Lonza Amaxa, MaxCyte technology, B'FX 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.

[082] 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.
[0831 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 (1L-2), interleukin-3 (IL-3), interleukin-4 (1L-4), interleukin-5 (1L-5), interleukin-6 (IL-6), interleukin-7 (EL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFT). The one or more cytokine(s) may comprise IL-2.
[084] In certain embodiments of the methods of producing a modified T cell (e.g. a Them and/or Tcm) of the disclosure, the expansion supplement comprises one or more cytokine(s).
The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (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 (INFO. The one or more cytokine(s) may comprise IL-2.
[0851 In certain embodiments of the methods of producing a modified T cell (e.g. a Tscm and/or To,i) 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%, Date Recue/Date Received 2022-04-05 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between of naive T cells. In certain embodiments, the efficiency of production of modified Tscm and/or Tem of the disclosure may be increased by increasing a proportion or percentage of naïve T
cells in a cell population to which the methods of the disclosure are applied.
[086] In certain embodiments of the methods of producing a modified Tscm and/or Tem of the disclosure, the primary human T cell is a memory T cell.
[087] In certain embodiments of the methods of producing a modified TSCN1 and/or Tai of the disclosure, the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp.
[088] In certain embodiments of the methods of producing a modified Tscm and/or Teri of the disclosure, the primary human T cell is a naive T-cell (modified TN) and the modified TN
expresses one or more of CD45RA, CCR7 and CD62L. In certain embodiments of the methods of producing a modified Tscm and/or Tcm of the disclosure, the primary human T
cell is a modified Tscm a T memory stem cell (modified Tsem) and the modified Tsem expresses one or more of CD45RA, CD95, IL-2R3, CR7, and CD62L. In certain embodiments of the methods of producing a modified TSCM and/or Tem of the disclosure, the primary human T cell is a central memory T-cell (modified 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 T cell is an effector memory T-cell (modified TEm) and the modified Ti 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 I-cell (modified TEFF) and the modified TER, expresses one or more of CD45RA. CD95, and1L-2RP.
[089] In certain embodiments of the methods of producing a modified Tscm and/or Teri 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 Tscm and/or Tem of the disclosure, the primary human T cell may be isolated, prepared or derived from for Date Recue/Date Received 2022-04-05 example, whole blood, peripheral blood, umbilical cord blood, lymph fluid, lymph node tissue, bone marrow, and cerebral spinal fluid (CSF). The term "peripheral blood" as used herein, refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow.
Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver or bone marrow. The terms "umbilical cord blood", "umbilical blood" or "cord blood", which can be used interchangeably, refers to blood that remains in the placenta and in the attached umbilical cord after child birth. Cord blood often contains stem cells including hematopoietic cells.
[091] Primary human T cells of the disclosure may comprise pan T cells. As used herein, pan T-cells include all T lymphocytes isolated from a biological sample, without sorting by subtype, activation status, maturation state, or cell-surface marker expression.
[0921 In certain embodiments of the methods of the disclosure, the method further comprises introducing into a modified Tscm or Tovi cell a composition comprising a genomic editing construct or composition. In certain embodiments, the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease. In certain embodiments, the genomic editing construct comprises a DNA binding domain and a type IIS
endonuclease. In certain embodiments, the genomic editing construct encodes a fusion protein.
In certain embodiments, the genomic editing construct encodes the DNA binding domain and the type IIS endonuclease and wherein the expressed DNA binding domain and the expressed type 11S
endonuclease are non-covalcntly linked. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a Cas9 endonuclease. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS
endonuclease, the sequence derived from a Cas9 endonuclease is the DNA binding domain. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes an inactive Cas9. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes a truncated Cas9. In certain embodiments, the

- 83 -Date Recue/Date Received 2022-04-05 sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (DI 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 IIS endonuclease, the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
in certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a TALEN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a TALEN. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type 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-fmger nuclease (ZFN).
10931 The methods of making modified Tsai and/or Tem 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 naive T cells increases in the population of T
cells subjected to the methods of the disclosure, the number and/or proportion of modified Tscm and/or Tcm cells of the disclosure produced also increases. 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 -Date Recue/Date Received 2022-04-05 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 T-cell (TN) and the TN expresses one or more of CD45RA, CCR7 and CD62L.
In certain embodiments, the primary human T cell is a T memory stem cell (Tscm) and the Tsrm 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, CCR7, and CD62L. In certain embodiments, the primary human T cell is an effector memory T-cell (Tim) and the EM
expresses one or more of CD45RO. CD95, and IL-24. In certain embodiments, the primary human T cell is an effector T-cell (TEFF) and the TIFF expresses one or more of CD45RA, CD95, and IL-2R13. 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 Tcm 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.
[096] The disclosure provides a use of a composition comprising a modified Tscm and/or Tcm produced a method of the disclosure for the manufacture of a medicament to treat a subject in need thereof. In certain embodiments of this use, the modified Tscm and/or Tcm is autologous. In certain embodiments of this use, the modified TSCNland/or Tcm is allogeneic.
In certain embodiments, the antigen receptor is a T-cell receptor. In certain embodiments, the 1-cell receptor is naturally-occurring. In certain embodiments, the 1-cell receptor is not

- 85 -Date Recue/Date Received 2022-04-05 naturally-occurring. In certain embodiments, and, in particular, in 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 T-cell receptor. In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VFIFI sequence(s). In certain embodiments, the CAR is a VCAR.
[097] The disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a modified Tscm and/or Tcm 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-cell 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 T-cell receptor is not naturally-occurring, the 1-cell receptor is a recombinant 1-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 VIAH sequence(s). In certain embodiments, the CAR is a VCAR. In certain embodiments of this method, the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen. In certain embodiments of this method, the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antigen. In certain embodiments, the disease or disorder is a disease or disorder caused by a lack of an activity or an insufficient amount of a secretory protein. In certain embodiments, the disease or disorder is a disease or disorder treated by a replacement of an activity of a therapeutic protein or by an increase in an amount of the therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein. In certain embodiments, the secretory protein is lacking an activity or a sufficient amount within a local area of a body. In certain embodiments, the local area of a

- 86 -Date Recue/Date Received 2022-04-05 body is accessible by a native T-cell or a modified 1-cell. In certain embodiments, the modified T-cell is produced in vivo, ex vivo, in vitro or in situ.
BRIEF DESCRIPTION OF THE DRAWINGS
[098] Figure 1 is a series of plots depicting the emergence of the CAR-Tscm phenotype at Day 11 of the method of Example 1. Cells were nucleofected with a surrogate CARTyrin plasmid. CAR-Tscm cells express CD62L and CD45RA as shown in the bottom two plots.
[099] Figure 2 is a series of plots depicting the purity of the CAR-Tscm produced by the method of Example 1 at day 19. The population of CAR-Tscm cells produced by the method described in Example I at day 19 contained no B cells or lymphocytes. The majority of the cells are CD3+ 1-cells. Only 1.1% are Natural Killer cells and 1.7% are Natural Killer T-cells.
[0100] Figure 3 is a plot showing that at Day 11 of the method described in Example 1, the majority of the T-cells produced express the CARTyrin.
[01011 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-Tsai 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 PD I, 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-BCMA), 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 -Date Recue/Date Received 2022-04-05 [0104] Figure 6B is a series of plots depicting T cells transposed with a plasmid containing a sequence encoding a green fluorescent protein (GFP). Left-hand plots depict live T cells exposed to transposase in the absence of the plasmid. Right-hand plots depict live T cells exposed to transposase in the presence of the plasmid. Cells were exposed to either a hyperactive transposase (the "Super piggyBac") or a wild type piggyBac transposase.
[01.05] Figure 6C is a table depicting the percent of transformed T cells resulting from transposition with WT versus hyperactive piggyBac transposase. T cells contacted with the hyperactive piggyBac transposase (the Super piggyBac transposase) were transformed at a rate 4-fold greater than WT transposase.
[0106] Figure 6D is a table depicting the percent of transformed T cells resulting from transposition with WT versus hyperactive piggyBac transposase 5 days after nucleofection. T
cells contacted with the hyperactive piggyBac transposase (the Super piggyBac transposase) were transformed at a rate far greater than WT transposase.
[0107] Figure 7 is a graph showing a phenotypic difference between piggyBacTM
and lentivirus-produced CAR+ T cells. CAR+ T cells were produced using either piggyBac transposition or lentivirus transduction. Human pan T cells were transposed with piggyBac encoding CAR, stimulated with anti-CD3/CD28 beads at day 2 post-transposition, expanded, and examined on day 19 post-transposition. For production using lentivirus, pan T cells were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding CAR (MOT
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 naïve (CD62L+CD45RA+), Tern (CD62L+CD45RA-), Tern (CD62L-CD45RA-) and Teff (CD62L-CD45RA+). All CAR-e- T cells were CD95+.
[0108] Figure 8A-B is a pair of graphs showing that piggy BacTm preferentially transposes naive T cells. Human pan T cells were sorted (using a BD FACSAria 11 flow cytometer) into naive (CD62L+CD45RA+), Tern (CD62L+CD45RA-), Tern (CD62L-CD45RA-), and Teff (CD62L-CD45RA+) subsets. The sorted subsets were each either transposed with piggyBac-GFP or transduced with lentivirus-GFP. For the former, each sorted subset was transposed with PiggyBac-GFP, stimulated with anti-CD3/CD28 beads at day 2 post-transposition, expanded, and examined on day 19 post-transposition. For the latter, the sorted subsets were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding GFP (MO!
5), expanded, and examined on day 19 post-stimulation. n=3 donors.

- 88 -Date Recue/Date Received 2022-04-05 101091 Figure 9 is a pair of graphs showing that the piggyBacim manufacturing process yields high levels of Tscm in samples from multiple myeloma (MM) patients even when naïve T cells are rare. T cells from MM patients (triangles) and healthy donors (circles) were characterized for memory marker expression by flow cytometiy before (left) and after (right) the Poseida manufacturing process. Expression of CD45RA and CD62L was assessed by FACS and plots are shown for the MM patients and a healthy donor. It is known that T cells from MM patients generally have lower frequencies of 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 Tscm from different MM
patients, production of P-BCMA-101 using the Poseida manufacturing process resulted in a product that exhibited a high level of CD8+ Tscm (E). This was also true for a MM patient who was actively receiving treatment (red triangle).
[0110] Figure 10 is a series of Fluorescence Activated Cell Sorting (FACs) plots characterizing T and Tscm cell markers in human pan T cells transformed with the Sleeping Beauty (SB I00x) transposition system and the methods of the disclosure.
Sleeping Beauty (SB I 00x) Transposition yields predominately Tscm phenotype using Poseida manufacture process. Human pan T cells were transposed using 1 Lig of either a Sleeping Beauty or piggyBac transposon plasmid and SB100x or SPB mRNA, respectively as shown.
Following transposition, cells were expanded ex vivo and all non-transposed cells were depleted using the Posoida 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 (Lig) of the Sleeping Beauty transposon SB100x, (second from top) 5 jig of SB100x, (3 from top) 10 jig of SB100x, (second from bottom) 5 jig of the piggvBac transposon P-BCMA-101 and at bottom, an unstained control. The x-axis, in order from left to right, in the first and second columns shows Forward Scatter (FSC), units from 0 to 250 thousand (abbreviated "k"), in increments of 50k. The x axis of the third column from the left shows CD8 expression, with markings reading from 0 to 105 incrementing by powers of 10.
The final right hand column shows CD62L expression, with markings reading from 0 to 105 incrementing by powers of 10. The y-axis, in the first column, shows Side Scatter (SSC), in units from 0 to 250k in increments of 50k. The y-axis in the second column from the left

- 89 -Date Recue/Date Received 2022-04-05 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 CD4512A, from 0 to 105 incrementing by powers of 10.
[01.111 Figure 11 is a schematic diagram showing the human coagulation pathway leading to blood clotting. Contact activation, for example by damaging an endothelium, activates an intrinsic clotting pathway. Tissue factors activate an extrinsic clotting pathway, for example following trauma. Both pathways converge onto the conversion of Prodnombin 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.
101.121 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 flumscein isothiceyanate (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 -Date Recue/Date Received 2022-04-05 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 piggyBacIm 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 piggyBacIm donor plasmid, GFP-2A-DHFR gene expression cassette is flanked by piggyBacIm 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 piggyBacIm 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 2022-04-05 transgenes were selected by methotrexate addition using the DHFR selection gene encoded in the bi-cistronic GFP-2A-DHFR integration cassettes. GFP+ cell percentage was assessed at Day 11 post-nucleofection by flow cytomeny and data are summarized in bar graph. GFP+
cells were highly enriched via selection in pan T cells receiving transposition reagents, RNP
plus HR or MMEJ donor plasmids, but not in T cells receiving donor plasmids alone.
(01181 Figure 17A-C is a series of graphs showing the phenotype of primary human pan T
cells modified by FIR and MMEJ at the AAVS1 site. The phenotype of GFP+ CD8+
pan T
cells was analyzed at Day 11 post-nucleofection by flow cytometry. A) Cells were stained with 7AAD (cell viability), CD4, CD8, CD45RA and CD62L, and FACS plots show gating strategy. CD8+ T cell subsets were defined by expression of CD45RA+CD62L+
(stem cell memory T cells (Tscm)), CD45RA-CD62L+ (central memory T cells (Tem)), CD45RA-CD62L- (effector memory T cells (Tern)), and CD45RA+CD62L- (T effectors (Teti)). B) Percentage of total GFP+ CD8+ T cells in each T cell subset is summarized in bar graph. An enriched population of GFP+ Tscm was achieved in all cases using either the piggyBacTm transposon system, or HR and MMEJ in combination with Cas9 RNP. C) The total number of pan T cells was analyzed at day 13 post-nucleofection and data are summarized in bar graph.
[01191 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 AAVS I 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 AAVS I region beyond the 500 bp homologue ann at the 5'-end AAVS-3r CTGCACCACGTGATGTCCTC (SEQ ID NO: 37), yielding a 0.73 kb DNA fragment for both FIR or MMEJ) and 3'-end junction (with one primer priming the polyA
signaling region SV40pA-Ir 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 AAVS1 target site. PCR products were displayed on Agarose gel. Non-specific bands in HR samples are the result of only a single round of PCR
and would likely have been resolved given additional rounds.
DETAILED DESCRIPTION

-92 -Date Recue/Date Received 2022-04-05 101201 The disclosure provides a method for producing human chimeric antigen receptor (CAR) expressing-T cells using the piggvBacTM Transposon System under conditions that preserve or induce stem-cell memory T cells (Tsem) with potent CAR activity (referred to herein as a CAR-Tscm. Compositions comprising CAR-Tscm produced using the methods of the disclosure comprise > 60% CAR-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-TE), and terminally-differentiated effector CAR-T cells (CAR-TTE). A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN) > Tsem > Tem > Tam > TE > TIE, whereby IN is the parent precursor cell that directly gives rise to Tsem, which then, in turn, directly gives rise to Tem, etc. Compositions comprising CAR-Tsem, CARTyrin-Tsem and/or VCAR-Tsem of the disclosure may comprise one or more of each parental CAR-T
cell subset with CAR-Tsem being the most abundant (e.g. Tscm > Tem > Tam > In > TTE).
While, the absolute quantities/abundances and relative proportions of each parental T
cell subset may vary among samples of patient blood and naturally-occurring cell populations, and naturally-occurring cell populations may have a high abundance and/or proportion of Tsem, compositions of the disclosure comprising non-naturally occurring CAR-Tsem are more potent and efficacious in treating patients against diseases and cancers.
101.211 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 CD19+ hematological malignancies. Unlike traditional biologics and chemotherapeutics, CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments. To achieve this, CAR-T cells must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses.
Thus, intensive efforts have been focused on the development of CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as of a CAR-T
product containing early memory cells, especially stern cell memory (Tscm). A
stem cell-like CAR-I would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (Tem), effector memory (Tiuk4) and effector T cells (In), thereby producing better tumor eradication and long-term CAR-T engraftment.

-93 -Date Recue/Date Received 2022-04-05 [0122] CAR-Tscm of the disclosure may comprise a Centyrin-based CAR, referred to as a CARTyrin (and hence, the cell may be referred to as a CARTyrin-Tscm).
Centyrins are alternative scaffold molecules based on human consensus tenascin FN3 domain, are smaller than scFv molecules, and can be selected for monomeric properties that favor stability and decrease the likelihood of tonic signaling in CAR molecules. CARTyrins of the disclosure may be introduced to T cells using a plasmid DNA transposon encoding the CARTyrin that is flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking improper gene activation or silencing.
[0123] CAR-Tscm of the disclosure may comprise a VHH-based CAR, referred to as a VCAR (and hence, the cell may be referred to as a VCAR-Tscm). VCARs of the disclosure may be introduced to T cells using a plasmid DNA transposon encoding the VHH
that is flanked by two cis-regulatory insulator elements to help stabilize VHH
expression by blocking improper gene activation or silencing.
[0124] In certain embodiments of the methods of the disclosure, the piggyBacTM
(PB) Transposon System may be used for stable integration of antigen-specific (including cancer antigen-specific) CARTyrin or VCAR into resting pan T cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme (although the transposon and transposase would be comprised in separate compositions until they were introduced into a cell), called Super piggyBacTm (SPB), in a single electroporation reaction.
Delivery of piggyBacrm transposon into untouched, resting primary human pan T cells resulted in 20-300/0 of cells with stable integration and expression of PB-delivered genes.
Unexpectedly, a majority of these modified CARTyrin-expressing T cells were positive for expression of CD62L and CD45RA, markers commonly associated with stem memory T-cells (Tscm cells).
To confirm that this phenotype was retained upon CAR-T cell stimulation and expansion, the modified CARTyrin-expressing T cells positive for expression of CD62L and CD45RA were activated via stimulation of CD3 and CD28. As a result of stimulation of CD3 and CD28, >
60% of CARTyrin+ T cells exhibited a stem-cell memory phenotype. Furthermore, these cells, which expressed a CARTyrin specific for a cancer antigen, were fully capable of expressing potent anti-tumor effector function.
[0125] To determine whether or not the PB system directly contributed to enhancing the expression of stem-like markers, the phenotype of CAR-T cells generated either by PB
transposition or lentiviral (LV) transduction was compared. To do this, a new vector was constructed by subcloning the CARTyrin transgene into a common LV construct for

- 94 -Date Recue/Date Received 2022-04-05 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, cytokine production, and lytic capability in response to target tumor cells. Unlike the PB-transposed CARTyrin+ I 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 [0126] 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 referred to in the context of a CAR as a VCAR), an antibody mimic, and a Centyrin (referred to in the context of a CAR as a CARTyrin).
[0127] In certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD38, CD3c, 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 -Date Recue/Date Received 2022-04-05 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 atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagetagacca (SEQ ID NO:
9).
[0128] In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD38, CD38, CD3y, CD3; CD4, CD8a, CDI9, 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 IYTWAPLAGTCGVILLSLVTTLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8a transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ
ID NO:
11).
[0129] In certain embodiments of the CARs of the disclosure, the endodomain may comprise a human CD31; cndodomain.
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
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising

-96 -Date Recue/Date Received 2022-04-05 cgcgtgaagtttagtcgatcagcagatgccccagettacaaacagggacagaaccagctgtaraw,gagetgaatctgg gccgccga gaggaatatgacgtgctggataageggapggacgcgaccccgannigggaggcaagcccaggcgcaaaaaccctcagga agg cctgtataacgagagcagaaggacaaaatggcagaagcetattctgagateggcatgaagggggagcgacggagaggca aagg gcacgatgggetgtac,cagggactgagcaccgccacaaaggacacctatgatgctctgeatntgcaggcactgcctcc aagg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID
NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYTFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-I BB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattttcaaacagccetteatgcgccccgtgcagactacccaggaggaagaca gtgctcc tgtcgattecctgaegaagageaaegcgggtgtgagetg (SEQ ID NO: 15). The 4-1BB
costimulatory domain may be located between the transmembrane domain and the C.D28 costimulatory domain.
[01311 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 TTTPA PRPPTPA PTIASQPLSLRPEACRPA AGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising TrrPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID
NO: 16). The human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcct gcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).
[01321 The disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
[01331 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 -Date Recue/Date Received 2022-04-05 encode any gene product (e.g. transcript, protein, enzyme) essential for cell viability and survival. Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for conferring resistance to a drug challenge against which the cell is sensitive (or which could be lethal to the cell) in the absence of the gene product encoded by the selection gene. Exemplary selection genes encode any gene product (e.g.
transcript, protein, enzyme) essential for viability and/or survival in a cell media lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene.
Exemplary selection genes include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding 'Thymidylate Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RADS IC
(encoding RADS! Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).
[01351 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 GSGEGRGSLETCGDVEENPGP
(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 ggataggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggaeca (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

-98 -Date Recue/Date Received 2022-04-05 comprising GSGQC'FNYALLKLAGDVESNPGP (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).
[01361 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 sccond self-cleaving peptide located, for example, downstream of the one or more of a sequence that specifically binds an antigen of the disclosure. The first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO:
18)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ
ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A
peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagectgctgacctgtggagacgtggaggaaaaeccaggacca (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 -Date Recue/Date Received 2022-04-05 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'TLNFDLLKLAGDVESNPGP (SEQ ID NO: 023). A G50-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 ATNFST.,LKQAGDVEENPGP (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.
[01381 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.
[0140] Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovinis, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof.
The viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (ITR) sequences located in cis next to one or more of a sequence that specifically binds an antigen. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to all serotypes (e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).

- 100 -Date Recue/Date Received 2022-04-05 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.
[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), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof [0142] Viral vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a self-cleaving peptide is located between a CAR and a selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR. The self-cleaving peptide may comprise, for example, a 12A 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

- 101 -Date Recue/Date Received 2022-04-05 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 GSGATNFSLLKQAGDVEENPOP (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 T-cell and the mRNA vector comprising the CAR of the disclosure. The T cell comprising the mRNA
vector, the modified T cell, may then be administered to a subject.
[0144] 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

- 102 -Date Recue/Date Received 2022-04-05 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, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidnig resistance gene (MDR1), ALDHI (encoding Aldehyde dehydrogenase I family, member Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.
[01461 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 nanoparticic vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and a selection gene. The self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a

- 103 -Date Recue/Date Received 2022-04-05 sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90 /0, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID
NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[0147] The disclosure provides a composition comprising a vector of the disclosure.
CARTyrins [0148] The disclosure provides a chimeric antigen receptor (CAR) comprising:
(a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one Centyrin; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. As used throughout the disclosure, a CAR
comprising a Centyrin is referred to as a CARTyrin. In certain embodiments, the antigen recognition region may comprise two Centyrins to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three Centyrins to

- 104 -Date Recue/Date Received 2022-04-05 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 mimeties 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.
[01501 in certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD38, CD3e, CD37, CD3, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD8a signal peptide. The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID
NO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggetctgctgctgcacgcagctagacca (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, CD38, CD3e, CD37, CD3t,", CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. The CD8a transmembrane domain may comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8a

- 105 -Date Recue/Date Received 2022-04-05 transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatagggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ
ID NO:
11).
[01521 In certain embodiments of the CARS of the disclosure, the endodomain may comprise a human CD3 endodomain.
[01.531 In certain embodiments of the CARS of the disclosure, the at least one costimulatory domain may comprise a human 4-11313, 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
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGIYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL.P
PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagettacaaacagggacagaaccagctgtataacgagctgaatctgg gccgccga gaggaatatgacgtgctggataageggagaggacgcgaccecgaaatgggaggcaagcccaggcgcaaaaaccetcagg aagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagateggcatgaagggggagcgacggagaggc aaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctcca agg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPF.MRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID
NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggaapantgctgtatattacaaacagcccttcatmcmcgtgcagactacccaggaggaagacgggtgct ec tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BB
costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

- 106 -Date Recue/Date Received 2022-04-05 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 TITPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID
NO: 16). The human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagetccaaccatcgcgagtcagcocctgagtctgagacctgaggcct 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 111 (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 MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYD
LTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). The consensus sequence may encoded by a nucleic acid sequence comprising atgctgectgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacg cagcatcg atagattatcategtgtaccgggagaacatcgaaaccggcgaggccattgtectgacagtgccagggtecgaacgetct tatgacctg acagatctgaagcccsgaactgagtactatgtgcagatcgccggcgicaaaggaggcaatatcagettccctctgtccg caatcttcac 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; (t) a F-G loop

- 107 -Date Recue/Date Received 2022-04-05 comprising or consisting of the amino acid residues KGGHRSN at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type lii (FN3) domains. The scaffold may bind an antigen with at least one affinity selected from a Ku of less than or equal to 10-9M, less than or equal to 10-1 M, less than or equal to 10-11M, 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.
[0156] The disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
[0157] The disclosure provides a transposon comprising the CAR of the disclosure.
Transposons of the disclosure be episomally maintained or integrated into the genome of the recombinant/modified cell. The transposon may be part of a two component piggyBac system that utilizes a transposon and transposase for enhanced non-viral gene transfer.
[01581 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 drag 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, enzyine) essential for viability and/or survival in a cell media lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene.
Exemplary selection genes include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), Tyms (encoding Thymidylate Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MORI), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).
[0159] Transposons of the disclosure may comprise at least one self-cleaving peptide(s) located, for example, between on or more of a protein scaffold, Centyrin or CARTyrin of the disclosure and a selection gene of the disclosure. The at least one self-cleaving peptide may

- 108 -Date Recue/Date Received 2022-04-05 comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A
peptide. A 12A
peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP
(SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ 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 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 GSGAINFSLLKQAGDVEENPGP (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 -Date Recue/Date Received 2022-04-05 protein scaffold, Centyrin or CARTyrin of the disclosure the second self-cleaving peptide located, for example, downstream of the one or more of a protein scaffold, Centyrin or CARTyrin of the disclosure. The first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A
peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A
peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID
NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A
peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP
(SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A
GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ 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 GSGAINFSLLKQAGDVEENPGP (SEQ ID NO: 26).

- 110 -Date Recue/Date Received 2022-04-05 [0161] The disclosure provides a composition comprising the transposon the disclosure. In certain embodiments, a method introducing the composition may further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme.
The sequence encoding a transposase enzyme may be an mRNA sequence.
[0162] Transposons of the disclosure may comprise piggyBac transposons.
Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
[0163] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.
[0164] Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovims, 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 genomc of one serotype and the capsid of another scrotype (e.g.
AAV2/5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.
[0165] Viral vectors of the disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.
Exemplary selection genes of the disclosure may include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDHI (encoding Aldehyde dehydrogenase I family, member Al), FRANC F, RAD5 IC

Date Recue/Date Received 2022-04-05 (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.
[01.661 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 12A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLITCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-'T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ 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 Date Recue/Date Received 2022-04-05 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 GSGA'TNFSLLKQAGDVEENPGP (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).
[01.671 The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is an mRNA vector. The vector may be a recombinant mRNA
vector. T cells of the disclosure may be expanded prior to contacting the T-cell and the mRNA vector comprising the CAR of the disclosure. The T cell comprising the mRNA
vector, the modified T cell, may then be administered to a subject.
[0168] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is a nanoparticle. Exemplary nanoparticle vectors of the disclosure include, but are not limited to, nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g. 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.
101.691 Nanoparticle vectors of the disclosure may comprise a selection gene.
The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound. Exemplary selection genes of the disclosure may include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGNIT
(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.
[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-Date Recue/Date Received 2022-04-05 cleaving peptide and wherein a self-cleaving peptide is located between a CAR
and the nanoparticle. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located between a CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and a selection gene. The self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgeteacctgtggagacgtggagganaacccaggacca (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 Date Recue/Date Received 2022-04-05 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[0171] The disclosure provides a composition comprising a vector of the disclosure.
Scaffold Proteins [0172] A Centyrin is one example of a protein scaffold of the disclosure. An antigen recognition region of a CAR of the disclosure may comprise at least one protein scaffold.
[0173] Protein scaffolds of the disclosure may be derived from a fibronectin type III (FN3) repeat protein, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods of making and using them. In a preferred embodiment, the protein scaffold is comprised of a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter "Tenascin"). In a further preferred embodiment, the protein scaffold of the present invention is a consensus sequence of 15 FN3 domains. The protein scaffolds of the disclosure can be designed to bind various molecules, for example, a cellular target protein. In a preferred embodiment, the protein scaffolds of the disclosure can be designed to bind an epitope of a wild type and/or variant form of an antigen.
[0174] Protein scaffolds of the disclosure may include additional molecules or moieties, for example, the Fe region of an antibody, albumin binding domain, or other moiety influencing half-life. In further embodiments, the protein scaffolds of the disclosure may be bound to a nucleic acid molecule that may encode the protein scaffold.
[0175] The disclosure provides at least one method for expressing at least one protein scaffold based on a consensus sequence of multiple FN3 domains, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one protein scaffold is expressed in detectable and/or recoverable amounts.
[0176] The disclosure provides at least one composition comprising (a) a protein scaffold based on a consensus sequence of multiple FN3 domains and/or encoding nucleic acid as described herein; and (b) a suitable and/or pharmaceutically acceptable carrier or diluent.

Date Recue/Date Received 2022-04-05 [0177] The disclosure provides a method of generating libraries of a protein scaffold based on a fibronectin type HT (FN3) repeat protein, preferably, a consensus sequence of multiple FN3 domains and, more preferably, a consensus sequence of multiple FN3 domains from human Tenascin. The library is formed by making successive generations of scaffolds by altering (by mutation) the amino acids or the number of amino acids in the molecules in particular positions in portions of the scaffold, e.g., loop regions.
Libraries can be generated by altering the amino acid composition of a single loop or the simultaneous alteration of multiple loops or additional positions of the scaffold molecule. The loops that are altered can be lengthened or shortened accordingly. Such libraries can be generated to include all possible amino acids at each position, or a designed subset of amino acids.
The library members can be used for screening by display, such as in vitro or CIS display (DNA, RNA, ribosome display, etc.), yeast, bacterial, and phage display.
[0178] Protein scaffolds of the disclosure provide enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations;
they may be expressed and folded in prokaryotic systems, such as E. coil, in eukaiyotic systems, such as yeast, and in in vitro transcription/translation systems, such as the rabbit reticulocyte lysate system.
[0179] The disclosure provides an isolated, recombinant and/or synthetic protein scaffold based on a consensus sequence of fibronectin type HI (FN3) repeat protein, including, without limitation, mammalian-derived scaffold, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding protein scaffold based on the consensus FN3 sequence. The disclosure further includes, but is not limited to, methods of making and using such nucleic acids and protein scaffolds, including diagnostic and therapeutic compositions, methods and devices.
[0180] The protein scaffolds of the disclosure offer advantages over conventional therapeutics, such as ability to administer locally, orally, or cross the blood-brain barrier, ability to express in E. Coli allowing for increased expression of protein as a function of resources versus mammalian cell expression ability to be engineered into bispecific or tandem molecules that bind to multiple targets or multiple epitopes of the same target, ability to be conjugated to drugs, polymers, and probes, ability to be formulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.

Date Recue/Date Received 2022-04-05 [0181] Moreover, the protein scaffolds possess many of the properties of antibodies in relation to their fold that mimics the variable region of an antibody. This orientation enables the FN3 loops to be exposed similar to antibody complementarity determining regions (CDRs). They should be able to bind to cellular targets and the loops can be altered, e.g., affinity matured, to improve certain binding or related properties.
[0182] Three of the six loops of the protein scaffold of the disclosure correspond topologically to the complementarity determining regions (CDRs 1-3), i.e., antigen-binding regions, of an antibody, while the remaining three loops are surface exposed in a manner similar to antibody CDRs. These loops span at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO: I. Preferably, the loop regions at or about residues 22-28, 51-54, and 75-81 are altered for binding specificity and affmity. One or more of these loop regions are randomized with other loop regions and/or other strands maintaining their sequence as backbone portions to populate a library and potent binders can be selected from the library having high affinity for a particular protein target. One or more of the loop regions can interact with a target protein similar to an antibody CDR interaction with the protein.
[0183] Scaffolds of the disclosure may comprise a single chain antibody (e.g.
a scFv). Single chain antibodies of the disclosure may comprise three light chain and three heavy chain CDRs of an antibody. In certain embodiments, the single chain antibodies of the disclosure comprise three light chain and three heavy chain CDRs of an antibody, wherein the complementarity-determining regions (CDRs) of the single chain antibody are human sequences. The disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one single chain antibody (e.g. a scFv); (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region may comprise two single chain antibodies (e.g.
two scFvs) to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three single chain antibodies (e.g. three scFvs) to produce a tri-specific CAR.
In certain embodiments, the ectodomain may further comprise a signal peptide.
Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
[0184] Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a Sequence comprising one or more fragments of an antibody of the disclosure may comprise two heavy chain variable regions of an antibody. In Date Recue/Date Received 2022-04-05 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-1); (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 Willis) 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 affiilin, an affimer, an affitin, an alphabody, an anticalin, and avimer (also known as avidity multimer), a DARPin (Designed Ankyrin Repeat Protein), a Fy-nomer, a Kunitz domain peptide, and a monobody.
[0187] Affibody molecules of the disclosure comprise a protein scaffold comprising or consisting of one or more alpha helix without any disulfide bridges.
Preferably, affibody molecules of the disclosure comprise or consist of three alpha helices. For example, an affibody molecule of the disclosure may comprise an immunoglobulin binding domain. An affibody molecule of the disclosure may comprise the Z domain of protein A.
[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.

Date Recue/Date Received 2022-04-05 Affilin molecules functionally mimic an antibody's affinity to antigen, but do not structurally mimic an antibody. In any protein scaffold used to make an affilin, those amino acids that are accessible to solvent or possible binding partners in a properly-folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.
[0189] Affimer molecules of the disclosure comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide a high affinity binding site for a specific target sequence. Exemplary affimer molecules of the disclosure comprise a protein scaffold based upon a cystatin protein or tertiary structure thereof.
Exemplary affimer molecules of the disclosure may share a common tertiary structure of comprising an alpha-helix lying on top of an anti-parallel beta-sheet.
[0190] Affitin molecules of the disclosure comprise an artificial protein scaffold, the structure of which may be derived, for example, from a DNA binding protein (e.g. the DNA
binding protein Sac7d). Affitins of the disclosure selectively bind a target sequence, which may be the entirety or part of an antigen. Exemplary a.ffitins 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.
[0191] Alphabody molecules of the disclosure may also be referred to as Cell-Penetrating Alphabodies (CPAB). Alphabody molecules of the disclosure comprise small proteins (typically of less than 10 kDa) that bind to a variety of target sequences (including antigens).
Alphabody molecules are capable of reaching and binding to intracellular target sequences.
Structurally, alphabody molecules of the disclosure comprise an artificial sequence forming single chain alpha helix (similar to naturally occurring coiled-coil structures). Alphabody molecules of the disclosure may comprise a protein scaffold comprising one or more amino acids that are modified to specifically bind target proteins. Regardless of the binding specificity of the molecule, alphabody molecules of the disclosure maintain correct folding and thermostability.
[0192] Anticalin molecules of the disclosure comprise artificial proteins that bind to target sequences or sites in either proteins or small molecules. Anticalin molecules of the disclosure Date Recue/Date Received 2022-04-05 may comprise an artificial protein derived from a human lipocalin. Anticalin molecules of the disclosure may be used in place of, for example, monoclonal antibodies or fragments thereof. Anticalin molecules may demonstrate superior tissue penetration and thermostability than monoclonal antibodies or fragments thereof Exemplary anticalin molecules of the disclosure may comprise about 180 amino acids, having a mass of approximately 20 kDa.
Structurally, anticalin molecules of the disclosure comprise a barrel structure comprising antiparallel beta-strands pairvvise 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 painvise connected by loops and an attached alpha helix.
[0193] Avimer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). Avimers of the disclosure may recognize multiple binding sites within the same target or within distinct targets. When an avimer of the disclosure recognize more than one target, the avimer mimics function of a bi-specific antibody. The artificial protein avimer may comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. Amino acid sequences of one or more of the peptides of the avimer may be derived from an A domain of a membrane receptor. Avimers have a rigid structure that may optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure may demonstrate greater heat stability compared to an antibody.
[01941 DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high alYmity for a target sequence. In certain embodiments, DARPins of the disclosure arc derived from ankyrin proteins and, optionally, comprise at least three repeat motifs (also referred to as repetitive structural units) of the ankyrin protein. Ankyrin proteins mediate high-affinity protein-protein interactions. DARPins of the disclosure comprise a large target interaction surface.
[0195] Fynomers of the disclosure comprise small binding proteins (about 7 kDa) derived from the human Fyn SW domain and engineered to bind to target sequences and molecules with equal affinity and equal specificity as an antibody.
[0196] Kunitz domain peptides of the disclosure comprise a protein scaffold comprising a Kunitz domain. Kunitz domains comprise an active site for inhibiting protease activity.
Structurally. Kunitz domains of the disclosure comprise a disulfide-rich alpha+beta fold. This Date Recue/Date Received 2022-04-05 structure is exemplified by the bovine pancreatic trypsin inhibitor. Kunitz domain peptides recognize specific protein structures and serve as competitive protease inhibitors. Kunitz domains of the disclosure may comprise Ecallantide (derived from a human lipoprotein-associated coagulation inhibitor (LAC)).
[0197] Monobodies of the disclosure are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable in size to a single chain antibody. These genetically engineered proteins specifically bind target sequences including antigens.
Monobodies of the disclosure may specifically target one or more distinct proteins or target sequences. In preferred embodiments, monobodies of the disclosure comprise a protein scaffold mimicking the structure of human fibronectin, and more preferably, mimicking the structure of the tenth extracellular type III domain of fibronectin. The tenth extracellular type III domain of fibronectin, as well as a monobody mimetic thereof, contains seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three complementarily 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 FG. Monobodies of the disclosure may comprise an adnectin.
Production and Generation of Scaffold Proteins [0198] At least one scaffold protein of the disclosure can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);
Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).
[0199] Amino acids from a scaffold protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.
[0200] Optionally, scaffold proteins can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, the scaffold proteins can be optionally prepared by a process of analysis of the parental sequences and various Date Recue/Date Received 2022-04-05 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.. Inununofilter program of Xencor, Inc. of Monrovia, Calif.). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability of the candidate scaffold protein to bind its antigen.
In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved.
Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used.
piggyBac Transposon System [0201] The methods of the disclosure produce a modified Tscm of the disclosure regardless of the method used for introducing an antigen receptor into a primary human T
cell of the disclosure. The methods of the disclosure produce a modified Tscm of the disclosure with greater efficacy and/or a greater abundance, proportion, yield of modified -Tscm of the disclosure when the antigen receptor or the therapeutic protein of the disclosure is introduced to the primary human T cell using the piggyBac transposon system. A piggyBac transposon system of the disclosure may comprise a piggyBac transposon comprising an antigen receptor of the disclosure. Preferably, the primary human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a transposase of the disclosure simultaneously (or in very close temporal proximity, e.g. the primary human T
cell, the transposon and the transposasc arc contained in the same container (such as a cuvettc) prior to introduction of the transposon and transposase into the cell ¨ however they would not be permitted to interact in the absence of the cell. Preferably, the primary human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a Super piggyBacTm (SPB) transposase of the disclosure simultaneously prior to introduction of the transposon and transposase into the cell. In certain preferred embodiments, the Super piggyBacTm (SPB) transposase is an mRNA sequence encoding the Super piggN,-Baerm (SPB) transposase.
[0202] Additional disclosure regarding piggyBac transposons and Super piggyBac Tm (SPB) transposases may be found in International Patent Publication WO 2010/099296, US Patent No. 8,399,643, US Patent No. 9,546,382, US Patent No. 6,218,185, US Patent No.
6,551,825, Date Recue/Date Received 2022-04-05 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 piggyBacIm (PB) transposase enzyme. The piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
4).
[0206] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBacIm (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 2022-04-05 541 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 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 (Cl), 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 Date Recue/Date Received 2022-04-05 an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP A1d4VYLLSSC

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
5).
[02091 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 piggyBac rm or Super piggyBacim 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 pig,gyBacTm 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). 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 TD NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid Date Recue/Date Received 2022-04-05 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 phcnylalaninc (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 mcthioninc (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 ttyptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at Date Recue/Date Received 2022-04-05 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) fora 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) fora 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 glycinc (G) fora cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C).
In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a lysine (K) for a serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). in certain embodiments, the amino acid substitution at Date Recue/Date Received 2022-04-05 position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V).
In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a glutamine (Q).
[0210] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacrm 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 pigrp.,BacTm transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super piggyBacTm transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID
NO: 5. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ TD 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 Date Recue/Date Received 2022-04-05 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 TD NO: 5 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBacTm transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID
NO: 4. In certain embodiments, including those embodiments wherein the piggvBacTM 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.
[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 arc known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
[0212] As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.
[0213] By "stable transformation" is intended that the polynucleotide construct introduced into a plant integrates into the genome of the host and is capable of being inherited by progeny thereof.
[0214] By "transient transformation" is intended that a poly-nucleotide construct introduced into the host does not integrate into the genome of the host.

Date Recue/Date Received 2022-04-05 [0215] In preferred embodiments, the piggyBac transposon system is used to introduce exogenous sequences into a primary human T cell by stable transformation to generate a modified Tscm or Tem.
Additional 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 1-cell (Teri) , comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T
cell expresses one or more cell-surface marker(s) of a modified stem memory T-cell (Tscm) or a modified central memory 1-cell (Tem), thereby producing a modified stem memory (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 (Tem), comprising introducing into a plurality of primary human T
cells (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 150/0, 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 1-cell (Tscm) or a central memory T-cell (Tcm), thereby producing a plurality of modified stem memory 1-cells (Tscm) or a plurality of modified central memory T-cells (Tem).
[0218] In certain embodiments of the methods of the disclosure, the transposon is a Sleeping Beauty transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon. the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).
[0219] In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

Date Recue/Date Received 2022-04-05 121 PLLQNRHKKA RLRFATAHGD KDRTEWPNVI., WSDETKIELF GHNDHRYVWR KKGEACKPKN

301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 6) .
[0220] In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

61 RRYLSPRDER TLVP.KVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSARKK

241 FQHDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVP. ARRPTNLTQL
301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7) .
[02211 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 Hel raiser transposon of the disclosure includes Helibatl, which comprises a nucleic acid sequence comprising:

61 CCCACCAGCC AATCAGtAAGT GACT.ATGCAA A.TTAACCCAA CAAAGATGGC AGTTAAATTT

361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA. CTGACAGCAG GGAGGAGGAA

661 AATGTATC.TG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA ACAGCGGCGT
721 CATCGACAGA AAATGTCTAA AGACCAACGT GCCTTTGAAG TTGAA.AGAAG GCGGTGGCGA

Date Recue/Date Received 2022-04-05 1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG GACAAGTTTA TCACCGTA.CT
1201 GGAACTTTAC ATCCTTCGGA. TGGTGTTTCT CGGAAGTTTG CTCAA.CTCTA TATTTTGGAT

1501 CCCCGTGTAA. CCGAGGTTGC TGTCATATTC AGAAACGAAG ATGGAGAACC TCCTTTTGAA

1621 CAAATCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT ATCCTATTCT TTTTCCACA.T

1741 AATAATACTA GACAAAA.TGT AAGGACACGA GTCACACAAA TGCAGTATTA TGGATTTCAT

2101 GATTTATTCA TAACCATGAC ATGCAACCCC AAATGGGCAG ATATTACAAA CAATTTACAA.
2161 CGCTGGCAAA AAGTTGAAAA. CAGACCTGAC TTGGTAGCCA GAGTTTTTAA TATTAAGCTG

2341 AGTGAGTCCA AATTACGTTC AGAAGA.TGAC ATTGACCGTA TAGTTAAGGC AGAAATTCCA
2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA. ATTGTAAAAT CAAATATGGT ACATGGACCA

252]. CCAAAAGAAT TTCAAAATGC GACCATTGGA AATATTGATG GATATCCCAA. ATACAAACGA

2641 TATAACCCGT ATTTGTGCCT T.AAATATAAC TGTCATATAA ATGTTGAAGT CTGTGCATCA

2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA. TGCGAA.TGCA TGACCAATCT

3061 CATTATGTGT TTAATAATTC TTTGTGGAC.A AAACGCCGAA AGGGTGGGAA TAAAGTA.TTA
312]. GGTAGACTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT

Date Recue/Date Received 2022-04-05 3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA ATACATGTGA. TCAATTGTAC
3601 GAGCAA.CAAC AGGC.AGAGGT TTTGATAAAT TCTCTGAATG ATGAA.CAGTT GGCAGCCTTT
3661 CAGACTATAA CTTCAGCCAT CGAA.GATCAA ACTGTACACC CCAAATGCTT TTTCTTGGAT
3721 GGTCCAGGTG GTAGTGGAAA AA.CATATCTG TATAAAGTTT TAACACATTA TATTAGAGGT

4261 CATTTAGGAA TGGATATTA.T TGAAATCCCC CATGAAATGA TTTGTAACGG ATCTATTATT

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

Date Recue/Date Received 2022-04-05 1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVKVVN TSSQGKLVEH SESVFTLNVV YREILE (SEQID
NO: 28).
[02241 In Helitron transpositions, a hairpin close to the 3' end of the transposon fiinctions as a terminator. However, this hairpin can be bypassed by the transpogase, resulting in the transduction of flanking sequences. hi addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5'-TC/CTAG-3' motif. A 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 29).
102251 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 Date Recue/Date Received 2022-04-05 encoding the To12 transposase, which contains four exons. An exemplary Tol2transposase of the disclosure comprises an amino acid sequence comprising the following:

601 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SWIDNO:34 [0226] An exemplary To12 transposon of the disclosure, including inverted repeats, subtemiinal sequences and the To12 transposase, is encoded by a nucleic acid sequence comprising the following:

241 CGCTGN.PGCC CAGTTTAATI".PAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT

961 TGTATTGTCA AAACGGTAA.0 ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA

1141 AACAAGATAT AAAGTATTA.G TAAATGTTGtA AATTAACA.TG TATA.CGTGCA GTTCATTATT
1201 AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTA.TT GTAAAAGTGT TACCATCAAA
126]. ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC

Date Recue/Date Received 2022-04-05 1381 TCAACAAGTA TTTAACATTA T.AAA.GTGTGC AATTGGCTGC AAATGTC.AGT TTTATTAAAG

1501 GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATA.TTTTAGA TTTAGTCCGA

1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA.

1861 CTAACCCACT GATGTCACAT GGACTACTTT GATGTTTTTA TTACCTTTCT GGACATGGA.0 2101 GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA AATA.TTTATT TGTTGTTTTT

2281 AGTCAAA.CAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA TCATTCAAGG
2341 ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATTA GTACACTGCA.

2461 GATCATGAAA. CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACCACAAC

2641 TTTTGAGGTA CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAAA. TACGTGACAA
2701 GGTTGTTTGC ACAACCACA.G ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG

2941 TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAG.AAA.CT
3001 CTACAGATCT GTCTTTGGCA AATGCCAAGC TTTATGGAAT AAAAGCA.GCC GATCGGCTCT

3541 AAGATCCTGA GATCATAGCA GCTGCCA.TCC TTCTCCCTAA ATTTCGGACC TCTTGGACAA
3601 ATGATGAAAC CATCATAAAA CGAGGTAAA.T GAATGCAAGC AACATACACT TGACGAATTC

Date Recue/Date Received 2022-04-05 3721 TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATCTCAA TATGAATATT GATGI'AAAGT
3781 ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACC.AAACT CATATGTATC
3841 ATATGTGGTT TGGAAATGCA. GTTAGATTTT ATGCTAAAAT AAGGGATTTG CATGATTTTA
3901 GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCA.TAAAATT TGTTCCCAGT

4021 ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAG_AAGGA. ATTGGCCAAC AGTTCATCTG

4501 GTTTTCTTTC TTGCTTTTA.0 TTTTACTTCC TTAATACTCA AGTA.CAATTT TAATGGAGTA

4681 TG (SEQ ID NO: 31).
Homologous Recombination [02271 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.
[02281 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.
[02291 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 Date Recue/Date Received 2022-04-05 (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 (CCR5) gene and the site of the human ortholog 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 (ME-IC). 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 1-cell receptor or the MI-IC disrupts the endogenous gene, and optionally, replaces the entire coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor to an endogenous T cell promoter. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional or translational regulatory clement. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional regulatory element. In certain embodiments, the transcriptional regulatory element comprises an endogenous T cell 5' U'TR.
102311 In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of a portion of primary T cells of the plurality of T cells. In certain embodiments, Date Recue/Date Received 2022-04-05 the portion of primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of the total number of primary T cells in the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of each primary T cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a single strand break. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a double strand break. In certain embodiments of the introduction step comprising a homologous recombination, the introduction step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor, a 5' genomic sequence and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 5' to the break point induced by the genomic editing composition and the 3' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3' to the break point induced by the genomic editing composition. In certain embodiments, the 5' genomic sequence and/or the 3' genomic sequence comprises at least 50 bp, 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 1100 bp, at least 1200 bp, at least 1300 bp, at least 1400, or at least 1500 bp, at least 1600 bp, at least 1700 bp, at least 1800 bp, at least 1900 bp, at least 2000 bp in length or any length of base pairs (bp) in between, inclusive of the end points. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence sequentially, and the genomic editing composition is provided first. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA
binding Date Recue/Date Received 2022-04-05 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 (FT840A)). 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 (DIOA) 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 Acil, Mull, AlwI, BbvT, Bed, BceAl, BsmAI, BsmFI, BspCNI, Bsrl, BtsCI, Hgal, Hphl, HpyAV, Mboll, Myll, Plol, SfaNI, Acul, BciVI, BfuAl, BmgBI, Bind, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, 13tgZI, Btsl, Earl., Ecu. MmeT, NmeAIII, BbvCI, Flpul0I, BspQI, SapI, Rae!, BsaXT, CspCI, BfiI, MboTI, Acc36I, Fokl or Clo051. In certain embodiments, the type IIS endonucicase comprises Clo05 1. 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 ZEN or a nuclease domain thereof. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition induces a break in a genomic sequence and the donor sequence composition is inserted using the endogenous DNA repair mechanisms of the primary T cell. In certain embodiments of the introduction step comprising a homologous recombination, the insertion of the donor sequence composition eliminates a DNA binding site of the genomic editing composition, thereby preventing further activity of the genomic editing composition.
[0232] In certain embodiments of the methods of homologous recombination of the disclosure, the nuclease domain of a genomic editing composition or construct is capable of introducing a break at a defined location in a genomic sequence of the primary human T cell, Date Recue/Date Received 2022-04-05 and, furthermore, may comprise, consist essentially oforconsistof, ahomodimerora 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 oforconsistof, a Cas9, aCas9nuclease domainora fragment thereof. In certain embodiments, the Cas9 isacatalytically inactive or "inactivated" Cas9 (dCas9). In certain embodiments, the Cas9 isacatalytically inactive or"inactivated"nuclease domain of Cas9. In certain embodiments, the dCas9 is encoded by ashortersequence that is derived fromafull length, catalytically inactivated, Cas9, referredtoherein as a"small"dCas9or dSaCas9.
[0233] In certain embodiments, the inactivated, small, Cas9 (dSaCas9) operatively-linked to an active nuclease. In certainembodiments,the disclosure provides afusionprotein comprising, consisting essentially oforconsistingofaDNA binding domain and molecule nuclease, wherein the nuclease comprises asmall, inactivated Cas9 (dSaCas9).
In certain embodiments, the dSaCas9 ofthedisclosure comprises the mutations DlOAandN580A
(underlined and bolded) which inactivate the catalytic site. In certain embodiments, the dSaCas9 of the disclosure comprises the amino acid sequence of 121 VNEVEEDTGN ELSTKEQISR NSKATJEINC(VAELQLERLKK DGEVRGSINR FKTSDYVKEA

541 IPLEDLLNNP FNYEVDHIIP RSVSFDNSFN NKVLVKQEEh. SKKGNRTPFQ YLSSSDSKIS

1021 ASKTQSIKKY STDILGNLYE VKSKKHPQII KKG (SEQ ID NO: 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 Date Recue/Date Received 2022-04-05 dCas9 with substitutions at positions 10 and 840ofthe amino acid sequence of the dCas9 which inactivate the catalytic site. In certain embodiments, these substitutions are Dl OA and I-1840A. Incertainembodiments, the amino acid sequence of the dCas9 comprises the sequence of:

61 ATRLKRTARR RYTRRKNRIC YLOEIFSNEM AEVDDSFFHR LEESFLVEED KEliERHPIFG

1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD (SEQ ID NO:
33).
[02351 In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dCas9 or a dSaCas9 and a type IIS
endonuclease. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and a type IIS endonuclease, including, but not limited to, AciI, Mn1I, AlwI, BbvI, Bed, BceAI, BsmAI, BsmFI, BspCNI, Bsrl, BtsCI, HgaI, HphI, HpyAV, Mbo 1I, Myl I, Nei, SfaM, AcuT, BciVI, BfuAT, BmgBI, BmrT, BpmT, BpuEI, BsaT, BseRI, Bsgl, BsmI, BspMI, BsrBI, BsrBI, BsrD1, BtgZI, Btsl, Ear!, Ecil, Mind, NmeAIII, BbvCI, Bpul OI, BspQI, SapI, Bad, BsaXI, CspCI, Bffl, MboII, Acc36I, FokI or Clo051.
In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or Date Recue/Date Received 2022-04-05 consist of a dSaCas9 and Clo051.An exemplary Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:
EGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVIELLITNEYGFKGRH
LOGSRKPDGIVY STrLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVN
PNKWWENFSEEVICKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE
KIRSGEM'TTEELERAMFNNSEFILKY (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):
MAPKKKRKVEGIKSNISLLKDELRGOISHISIIEYLSLIDLAFDSKONRLFEMKVLELL
VNEYGFKGRHLGGSRKPDGI'VYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVR
ENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEOLRRLSMTTGVNGSAV
NVVNLI,I,G A EK IR SG EMTI EELERA 114 INNS EFT LKY GGGGSDKKES7GLAIGTNSVGWA
VITDEYKVPSKKFKVLGNTDRHSIKICNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY
LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERIIPIFGNIVDEVAYHEICYPTIYHLRKKL
VDSTDKADLRL1YLAIAHMIKFRGHMEGDIRPDNS'DVDKLFIQLVQTYNQLFEENPTNA
..SGIDAKAILS'ARLSKS'RRLENLIAQI,PGEKKNGLEGMIALSWEIPNEKSATDLAEDAKLQ
LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKVGYAGYIDGGASQE'EF'YKF1KPILEICVDG1E
ELLVKLIVREDLIRKQRIFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILIFRIP
YYVGPLARGNSRFAWMTRKSEE7'ITPWNFEEWDKGASAQSHERATTNFDKNLPNEKVLP
KHSLLYEYFIVY.NEL7KVKYV7EGMRKPAITSGEQKKAIVDLLFKTNRKVIVKOLKEDYF
KKIE'CIDSVEISGVEDRFNASEGITHDLLKIIKDKDIIDNEENADILEDIVEIL1LFEDREM
IEERLICTYAHLFDDKVAIKQLKIRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS'DGFANR
NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVICYM
GRHKPENIVIEMARENQTIQKGQKNSRE.RAIKRIEEGIKELGSQILKEHPVENIQLQ.NEKL
YLYYLQNGRLIVIYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKATRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AOLDSRALATIKYDENDKLIREVICVITLKSKLVSDFRKDIWYKVREINNYHHAHDAYLNAV
VGTALIICKYPKLESEFVYGDYKVYDVRKNIZAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN

SDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEK
ATIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKIMILASAGELQKGNELALPSKYVNFLY

Date Recue/Date Received 2022-04-05 LASHYEKLKGSPEDNEQKQLITEQHKHYLDEBEQISEFSKIWILADANLDKPL&IYNKHR
DKPIREQAENIIHLF __________________________________________________ LTIVLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
SQLGGDGSPKIUCRKVSS (SEQ ID NO: 40).
[02371 In certain embodiments, the nuclease capable of introducing a break at a defined location in the genomic DNA of the primary human T cell may comprise, consist essentially of or consist of, a homodimer or a heterodimer. Nuclease domains of the genomic editing compositions or constructs of the disclosure may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a transcription-activator-like effector nuclease (TALEN). TALENs are transcription factors with programmable DNA
binding domains that provide a means to create designer proteins that bind to pre-determined DNA
sequences or individual nucleic acids. Modular DNA binding domains have been identified in transcriptional activator-like (TAL) proteins, or, more specifically, transcriptional activator-like effector nucleases (TALENs), thereby allowing for the de nova 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. TAI, proteins have been derived from the organisms Xanthomonas and Ralstonia.
[02381 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 HS
endonuclease. in certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of AciI, Mn!!, AlwI, BbvI, Bed, BceAI, BsmAI, BsinFI, BspCNI, Bsrl, BtsCI, Hgal, Hphl, HpyAV, Mbol I, My 11, Plel, SfaNI, AcuI, BciVI, BfuAl, BmgBI, Bmrl, Bpml, BpuEl, Bsal, BscR1, BsgI, Bsml, BspM1, BsrB1, BsrBI, BsrDI, BtgZI, Btsl, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQT, Sap!, Bad, BsaXI, CspCI, Bfil, 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 fmger nuclease (ZFN) and a type IIS
endonuclease. In certain embodiments of the disclosure, the type IIS
endonuclease may comprise, consist essentially of or consist of AciT, Mn!!, AlwI, BbvI, BccI, BceAT, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, Hgal, IIphI, HpyAV, Mboll, My 11, PleI, SfaNI, AcuI, BciVI, BfitAl, BmgBI, BmrI, BpmI, BpuEl, Bsal, BseR1, Bsgl, Bsml, BspMI. BsrB1, BsrBI, BsrDI, Date Recue/Date Received 2022-04-05 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 2022-04-05 AC009133.22 ENSG00000277669 AC009491.2 ENSG00000279664 AC136352.5 ENSG00000277666 AC145212.4 ENSG00000277400 MaFF-interacting protein AC233755.1 ENSG00000275063 ACACB ENSG00000076555 Acetyl-CoA calboxy lase beta At:AN ENts600000157766 Aggrecan ACE ENSG00000159640 Angiotensin I convening enzyme ACHE ENSG00000087085 Acetylcholinesterase (Yt blood group) ACP2 EN S000000134575 Acid phosphatase 2. Ivsosomal ACP5 ENSG00000102575 Acid phosphatase 5, tartrate resistant ACP6 ENSG00000162836 Acid phosphatase 6, lysophosphatidic At:PP ENSG00000014257 Acid phosphatase, prostate ACR ENSG00000100312 Ac rosin ACRBP ENS000000111644 Acrosin binding protein ACRV1 ENSG00000134940 Acrosomal vesicle protein 1 ACSF2 ENSG00000167107 Acyl-CoA synthetase family member 2 ACTLIO ENSG00000182584 Actin-like 10 ACVR I ENSG00000115170 Activin A receptor, type I
ACVR1C ENS(100000123612 Activin A mcemor, type IC
ACVRL I ENSG00000139567 Activin A receptor type II-like 1 ACYP I E.NSG00000119640 Acylphosphatasc 1, clytluocylc (common) type ACYP2 ENSG(' )0000170634 Acylphosphatase 2, muscle type ADAM 10 ENSG00000137845 ADAM metallopeptidase domain 10 ADAM12 EN S000000148848 ADAM metallopeptidase domain 12 ADAM13 ENSG00000143537 ADAM metallopeptidase domain 15 ADAM17 ENSG00000151694 ADAM metallopeptidase domain 17 ADAM 18 ENS(300000168619 ADAM metallopeptidase domain 18 ADAM22 ENSG00000008277 ADAM metallopeptidase domain 22 A DAM28 ENS000000042980 ADAM metallopeptidase domain 28 ADAM29 ENSG00000168594 ADAM metallopeptidase domain 29 ADAM32 ENSG00000197140 ADAM metallopeptidase domain 32 --;i15-A-ITI-33-------EST a00000144I51-----Aii-AVin-e--till10¨pe--piZie---&-imai n 33 ADAM? ENSG00000069206 ADAM metallopeptidase domain 7 ADAM8 EWA-00)00151651 ADAM .metailopeptidasc domain 8 ADAM9 EN SG00000168615 ADAM metallopeptidase domain 9 ADAMDEC I ENS0)0000134028 ADAM-like, decysin 1 AD.AMTS1 ENSG000001547 :34 ADAM metallopeptidase with ihtombospondin type 1 motif, 1 ADAMTSIO ENSG00000142303 ADAM metallopeptidase with thrombospondin type 1 motif. 10 ADAMTS12 EN8G00000151388 ADAM metallopeptidase with thrombospondin type 1 motif, 12 ADA.MTS13 EN S000000160323 ADAM metallopeptidase with thrornbospondin type I motif, 13 ADAMTS14 EN SG00000138316 ADAM metallopeptidase with thrombospondin type 1 motif, 14 ADAMTS15 ENSG00000166106 ADAM metallopeptidase with thtombospondin type 1 motif, 15 Date Reeue/Date Received 2022-04-05 ADAMTS16 EN SG00000145536 ADAM metallopeptidase with thrombospondin type I motif, 16 A DAMTS17 EN S600000140470 ADAM metallopeptidase with thrombospondin type 1 motif, 17 ADAMTS18 ENSG00000140873 ADAM metallopeptidase with thumbospondin type 1 motif, 18 ADAMTS19 ENSG00000145808 ADAM metallopeptidase with thiombospondin type 1 motif, 19 ADAIvITS2 ENSG00000087116 ADAM metallopeptidase with thrombospondin type I motif, 2 ADAMTS20 ENSG00000173157 ADAM metallopeptidase with thrombospondin type I motif, 20 ADAMTS3 ENS000000156140 ADAM metallopeptidase with thrombospondin type 1 motif, 3 ADAMTS5 ENts600000154736 ADAM metallopeptidase with thtombospondin type 1 motif, 5 ADAmTs6 ENSG00000049192 ADAM metallopeptidase with thtombospondin type 1 motif, 6 AD.A.MTS7 ENSG00000136378 ADAM metallopeptidase with thrombospondin type I motif, 7 ADAMIS8 EN S(300000134917 ADAM metallopeptidase with thrombospondin type 1 motif 8 ADAMTS9 ENSG00000163638 ADAM metallopeptidase with thrombospondin type 1 motif, 9 ADAMISL 1 EN SG0000017803 I ADAMTS-like 1 ADAMTSI..2 ENSG00000197859 ADM/ITS-I ike 2 ADAMTSL3 ENSG00000156218 ADAMTS-like 3 ADAMISL4 ENS000000143382 ADANTFS-like 4 ADAMTSL5 ENSG00000185761 ADAMTS-like 5 ADCK1 ENSG00000063761 AarF domain containing kinase ADCYAP1 EN SC1000001414:33 Adeny late cyclase activating polypeptide 1 (pituitary) ADCYAP1R I ENSG00000078549 Adeny late cyclase activating polypeptide I (pituitaty) receptor type I
ADGRA3 ENSG00000152990 Adhesion 0 protein-coupled receptor A3 ADGRB2 ENS(300000121753 Adhesion G protein-coupled receptor B2 ADGRD I ENSG00000111452 Adhesion G protein-coupled receptor 1)1 ADGRE3 ENSG1)0000131355 Adhesion G protein-coupled receptor El ADORES EN S000000123146 Adhesion 0 protein-coupled receptor E5 ADGRE1 ENS000000153292 Adhesion (3 protein-coupled receptor Fl ADGRG1 ENS000000205336 Adhesion G protein-coupled receptor G1 ADGR05 ENSG00000159618 Adhesion (3 protein-coupled receptor (35___________________ --A-15-671-i-0-6----------ESIS-a{)000i0i 12414 Adhesion G protein-coupled receptor 06 ADGRV I ENSG00000164199 Adhesion (3 protein-coupled receptor VI
ADIl ENS(10000018255 I Acireductone dioxygenase 1 ADIG ENSG00000182035 Adipogenin ADIPOQ ENSG00000181092 Adiponectin. C IQ and collagen domain containing ADM ENS010000148926 Adrenomedullin ADM2 ENSG00000128165 Adrenomedullin 2 Ai A15 ENS000000224420 Adrenoinedullin i (pulative) ADP0K ENS(300000159322 ADP-dependent glucokinase ADPRHL2 ENS000000116863 ADP-ribosylhydrolase like 2 AEBP1 ENSG00000106624 AE binding protein 1 AIN ENS000000079557 Afarnin AEP EN S(300000081051 Alpha-letoprotein AGA. ENS000000038002 Aspartylglecosaminidase AGER ENSG00000204305 Advanced glycosylation end product-specific receptor AGK ENSG00000006530 Acylglyceroi kinase Date Recue/Date Received 2022-04-05 AGPS ENSG00000018510 Alkylg1ycerone phosphate synthase AGR2 ENS600000106541 Anterior gradient 2, protein disulphide isornerase family member AGRI.; ENSG00000173467 Anterior gradient 3, protein disulphide isonienise family member AGRN EN SG00000188157 Agrin AGRP ENSG00000159723 Agouti related neuropeptide AGT ENSG00000135744 Angiotensinogen (serpin peptidase inhibitor, clade A. member 8) AGIPBP I ENSG00000135049 ATPIGTP binding omen' I
AGTRAP ENS(300000177674 Angiotensin II receptor-associated protein AFICYL2 ENSG00000158467 Adetiosylhomocysieinase-like 2 AHSG ENSG00000145192 Alpha-2-HS-glycoprotein AIG I ENSG00000146416 Androgen-induced I
AK 4 EN S(300000162431 Adeny late kinase 4 AKAPIO ENSGu0000108599 A kinase (PRKA) anchor protein 10 AKR ICI EN SG00000187134 Aldo-keto reductase family 1, member CI
AL356289.1 ENSG00000279096 AL589743.1 ENS600000279508 AI,AS2 ENSG(' 10000158578 5'-aminoievtilinate synthase 2 ALB ENSG00000163631 Albumin ALD119A I ENSG00000143149 Aldehyde deity drogenase 9 family, member Al ALDOA ENSG00000149925 Aldolase A, fructose-bisphosphate ALG I ENSG000000330 11 ALG I, chitobiosyldiphosphodolichol beta-maimosyltransfera.se ALG5 ENS(300000120697 ALG5, dolichyl-phosphate beta-glitcosyltransferase ALG9 ENSG00000086848 ALG9, alpha-1,2-mannosyltransferase ALKBH1 ENSG00000100601 AlkB homolog 1, histone H2A dioxygenase ALKBH5 ENSG00000091542 AlkB hornolog 5, RNA demethylase ALP! ENSG00000163295 Alkaline phosphatase. intestinal ALPL ENS(300000162551 Alkaline phosphatase, liver/bone/kidney ALPP ENSG00000163283 Alkaline phosphatase, placental ALPPL2 ENSG00000163286 Alkaline phosphaiase, placental-like 2 AMBN ENSG00000178522 Ameloblastin (enamel matrix protein) AMBP ENSG00000106927 Alpha-1-microglobulinibikunin precursor AMELX ENS600000 125361 Amelogenin, X-linked AMEX ENSG0000009972 I Amelogenin. Y-linked AMH ENSG00000104899 Anti-Mullerian hormone AMICA1 ENSG00000160593 Adhesion molecule, interacts with CXADR
antigen 1 AMPD1 ENSG00000116748 Adenosine monophosphate deaminase 1 ANIFN ENS(300000 187689 A tnelot in AMY IA ENSG00000237763 Amylase, alpha IA (salivary) AMY 1B ENSG00000174876 Amylase, alpha 1B (salivary) AMY IC ENS600000 I 87733 Amylase, alpha IC (salivary) AMY2A ENSG00000243480 Amylase, alpha 2A (pancreatic) AMY2B ENSG00000240038 Amylase. alpha 2B (pancreatic) ANG ENSG00000214274 Angiogenin, ribonuclease, RNase A family, Date Recue/Date Received 2022-04-05 ANGEL I ENSG00000013523 Angel hontolog 1 (Drosophila) ANGPT I ENSG00000 154188 A ngiopoiet in 1 ANGPT2 ENSG00000091879 A tigiopo tett ii 2 ANGPT4 ENSG00000101280 Angiopoietin 4 ANGPTL1 ENSG00000116194 Angiopoietin-like 1 ANGPTL2 ENSG00000 136859 Angiopoictin-like 2 ANGP1'I.3 ENSG00000132855 Angiopoietin-like 3 ANGPTL4 ENS600000167772 A ngiopo ieti n-like 4 ANGP11,5 ENSG00000187151 Atigiopoietin-like 5 ANGPTL6 ENSG00000130812 Angiopoietin-like 6 ANGPTL7 ENSG00000 171819 Angiopoictin-like 7 ANK I ENSG00000029534 Anky rin 1, erythrocytic ANKDD IA ENSG00000166839 Auk y rin repeat and death domain containing IA
ANK R D54 EN SG00000100124 A tiky ri n repeat domain 54 ANKRD60 ENSG00000124227 Ankyrin repeat domain 60 ANO7 ENS000000146205 A nociamin 7 ANOS1 ENSG00000011201 Anosmin I
ANTXR 1 ENSG00000169604 Anthrax toxin receptor 1 AOAH ENSG00000136250 Acy loxyacyl hydrolase (neutrophil) AOC1 ENSG00000002726 Amine oxidase, copper containing 1 A0C2 ENS000000131480 Amine oxidase, copper containing 2 (retina-specific) A0C3 ENSG00000131471 Amine oxidase, copper containing 3 AP000721.4 ENSG00000256100 AP000866.1 ENSG00000279342 APBB1 ENSG00000166313 Amyloid beta (A4) precursor protein-binding, family B, member 1 (Fe65) APCDD I ENSG00000154856 Adenoinatosis polyposis coli down-regulated 1 APCS ENSG00000132703 Amyloid P component, serum APELA ENSG00000248329 Aydin receptor early endogenous ligand -7,017S1------------iNgiiiiiiiiiiiiiiiiii-----A¨pe¨ii-17---------------------------------------------APLP2 ENSG00000084234 Amyloid beta (A4) precursor-like protein APOA 1 ENSG0000011.8137 Apolipoptotein A-I
APOA1BP ENSG00000163382 Apolipoprotein A-I binding protein AP0A2 ENSG00000158874 Apolipoprotein A-II
AP0A4 fiN SG00000110244 Apolipoprotein A-IV
APOA 5 ENSG00000110243 Apolipoptotein A-V
A MB ENSG00000084674 Apolipoprotein B
APOC1 ENSG00000130208 Apolipoprotein C-I
APOC2 ENSG00000234906 Apolipoprotein C-II
APOC3 EN SG00000110245 Apolipoprotein C-I1 1 APOC4 ENSG00000267467 Apolipoprotein C-IV
APOC4-APOC2 ENSG00000224916 APOC4-APOC2 readthrough (NMD candidate) APOD ENSG00000 189058 Apolipoptotein D
APOE ENSG00000130203 Apolipoprotein E
APOF ENSG00000175336 Apo lipoprotein F

Date Recue/Date Received 2022-04-05 APOH ENSG00000091583 Apolipoprotein H (beta-2-glycopr)tcin I) APOL I ENSG00000 I 00342 Apolipoprotein L, I
APOL3 ENS(300000128284 Apolipoprotein L, 3 APOM ENS000000204444 Apolipoprotein M
APOOL ENSG00000 155008 Apolipoprotein 0-like ARCN1 ENSG00000095139 Archain I
ARFIP2 ENSG00000 I 32254 ADP-ribosylation factor interacting protein 2 ARHGAP36 ENS600000147256 Rho GTPase activating protein 36 ARHGAP6 ENSG00000047648 Rho OTPase activating protein 6 ARHGEF4 ENSG00000 136002 Rho guanine nucleotide exchange factor (GEF) 4 ARL16 ENS000000214087 ADP-nbosy !anon factor-like 16 ARMC5 ENSG00000140691 Armadillo repeat containing 5 ARNIL ENS(300000133794 Aryl hydrocarbon receptor nuclear translocator-like ARSA EN SC100000100299 AtyIs& fatase A
ARSB ENS000000 I 13273 Atylsulfatase B
ARSE ENS000000157399 A rylsulfatase E (chondrodysplasia punctala 1) ARSG ENSG00000141337 Arylsulfatase 0 ARSI ENSG00000183876 Arylstilfatase family, member I
AR SK EN S(30000016429 I Atylsulfittase family., member K
ART3 ENSGu0000156219 ADP-ribosy Itransferase 3 ART4 ENS000000111339 ADP-nbosy liransferase 4 (Dombrock blood group) ARTS ENS0000001673 11 ADP-ribosyltransferase 5 ARTN ENS600000117407 Artemin ASAH1 ENS000000104763 N-acylsphingosine amidohydrolase (acid ceramidase) 1 ASAH2 ENSG00000188611 N-acylsphingosine amidohydrolase (non-lysosomal cerarnidase) ASCL1 ENS000000139352 Achaete-scute family bIlLii transcription factor I.
AS!? ENS000000101440 Agouti signaling protein ASPN ENS(300000106819 _ Aspori n_______________________________________________ --Vs'ff.-.-------------T-N ¨ciiiiiii-aiiiiiiiiiii;-- Astacin-like tnetallo-endopeptidase (M12 family) ATAD5 ENS000000 176208 ATPase family, AAA domain containing 5 ATAT I ENS000000 137343 Alpha tubitlin acetyltrattsfetase I
ATG2A ENSG00000110046 Autophagy related 2A
ATG5 ENS000000057663 Autophagy related 5 ATMIN IiNSCR10000166454 ATM intentctor ATPI3A1 ENSG00000105726 ATPase type 13A1 ATP5F I ENSG00000116459 ATP synthase, F1+ transporting, mitochondrial Fo complex.
subunit Hi ATP6AP I ENSG00000071553 ATPase, H+ transporting, lysosoraal accessory protein I
ATP6AP2 ENS(300000182220 ATPase, Hi- transporting, lysosoinal accessory protein 2 ATPAF I ENSG00000123472 ATP synthase mitochondrial Fl complex assembly factor 1 ALM ENSG00000148090 AU RNA binding proteinknoyi-CoA
Itydratase AVP ENS000000 I 01200 Arginine vasopressin AXIN2 ENS000000168646 Axin 2 AZGPI ENS(300000160862 Alpha-2-glycoprote in 1, zinc-butding - ISO-Date Recue/Date Received 2022-04-05 A.Z1.11 ENSG00000172232 Azurocidin I
B2M EN SG00000166'710 Beta-2-inicroglobul in B3GALNT1 ENSG00000169255 Beta-1..3-N-acety Igaiactosaminy It tansferase 1 (globoside blood group) B3GALNT2 ENSG00000162885 Beta-1,3-N-acetylgalactosaminy It ransferase 2 B3GALT1 ENSG00000172318 UDP-Gal:betaGicNAc beta 1,3-ga1ac105y It ninsferase, poly peptide 1 B3GALT4 ENSG00000235863 UDP-GakbetaGIcNAc beta 1,3-galactosyltninsferase, poly peptide 4 B3GALT5 ENSG00000183778 UDP-Ottl:betaGicNAc beta 1,3-g,alactosyltrattsferase, poly peptide 5 B.3GALT6 ENSG00000176022 LIDP-Gal:beta(Ial beta 1,3-galactosylininsferase polypeptide 6 B3GAT3 ENSG0000014954 I Bcta-1.3-glucuromit ransfcntse 3 B3GLCT ENSG00000187676 Beta 3-glucosyltransferase B3G NT3 ENts600000179913 UDP-OlcNAc:beiaCial beta-1,3.N-aceiy Iglitcosaminy It ransfe tine 3 B3GNT4 ENSG00000176383 LTDP-GIcNAc:betaGal beta-1.3-N-acetylglucosaininv Itransfcrase 4 B3GNT6 ENSG00000198488 LIDP-GIcNAc:bettiGal beta-1,3-N-acety Igl UCOSaIll i ITV Itransferase 6 B3ONT7 ENSG00000156966 UDP-01cNAc:beiaGal beta- I ,3-N-acei) Ighteosaminy It ransfetase 7 B3ONT8 ENSG00000177191 UDP-OlcNAc:beiaGal beta- I.3-N-acetv Iglucosaminv It ransferase 8 B3GNT9 ENSG00000237172 UDP-GIcNAc:bettiGal beta-1,3 -N-acetylglucosant inv It ninsfentse 9 B4GALNT1. ENSG00000135454 Beta-1.4-N-acetyl-galaciosaininy I
transfonse 1 B4GALNT3 ENsG00000139044 Beta- I,4-N-acetyl -galactosaminyl t ntnsferase 3 B4GALNT4 ENSG00000182272 Beta-1,4-N-acetyl-galactosaminyltransferase 4 B4GALT4 ENSG00000121578 1.1DP-Gal:betaGicNAc beta 1,4-galactosyliransfentse, poly peptide 4 B4GALT5 ENSG00000158470 UDP-Gal:betaGIcNAc beta 1,4-galactosyltransferase, poly pent ide 5 B4GALT6 ENSG00000 118276 LIDP-GakbetaGicNAc beta 1,4-galactosyltransferase, poly peptide 6 B4GAT1 ENSG00000174684 Beia-1,4-glucuronyltransferase 1 B9D I ENSG0000010864 I 139 protein domain 1 B.ACE2 ENSG00000182240 Beta-site APP-cleaving enzyme 2 BAGE5 ENS600000279973 B melanoma antigen family, member 5 BCAM ENSG00000187244 Basal cell adhesion molecule (Lutheran blood group) BCAN ENSG00000132692 Brevican BCAP29 ENSG00000075790 B-cell receptor-associated protein 29 BCAR1 ENSG00000050820 Breast cancer anti-estrogen resistance 1 BCHE ENSG00000114200 Butyry !cholinesterase BCKDHB ENSG00000083123 Branched chain keto acid dehydrogenase El. beta polypeptide BDNF ENSG00000176697 Brain-derived neurotrophic factor BGLAP ENSG00000242252 Bone gamma-carboxy glutamate (gla) protein BON EN8600000182492 Big,lycart BLVRB ENS(300000090013 Bilivendin teductrise B
BMP I ENSG00000168487 Bone morphogenetic protein I

Date Recue/Date Received 2022-04-05 BMP 10 ENSG00000163217 Bone morphogenctic protein 10 BMP15 ENS600000130385 Bone morphogenetic protein 15 BMP2 ENS(300000125845 Bone morphogenetic protein 2 BMP3 ENS000000152785 Bone morphogenetic protein 3 BMP4 ENSG00000125378 Bone morphogenetic protein 4 BMP6 ENSG00000153162 Bone morphogenetic protein 6 BMP7 ENSG00000101144 Bone morphogenetic protein 7 BMP8A ENs600000183682 Bone inorphogenetic protein Ka BMP8B ENSG00000116985 Bone morphogenetic protein 8b BMPER ENSG00000164619 BMP binding endothelial regulator BNC1 EN S000000169594 Basonuclin 1 BOC ENSG00000144857 BOC cell adhesion associated, oncogene regulated BOD I ENSG00000145919 Biorientat ion of chromosomes in cell division 1 BOLA! ENS(300000178096 BolA family member I
BPI ENS(300000101425 Bactericidal/permeability-increasing protein BPIFA1 EN S000000198183 BPI fold containing family A, member I
BPIFA2 ENSG00000131050 BPI fold containing family A. member 2 BPIFA3 ENSG00000131059 BPI fold containing family A. member 3 BP1FB I ENSG00000125999 BPI fold containing family B, member I
BPIFB2 ENS0u0000078898 BPI fold containing family B, member 2 BPIFIE33 EN SG00000186190 BPI fold containing family B, member 3 BPIFB4 EN SCi0000018619 I BPI fold containing family B, member 4 BPIFB6 E.NS000000167104 BPI fold containing family B. member 6 BP1FC ENSCR)0000184459 BPI fold containing family C
BRF1 ENSG00000185024 BRFI, RNA poly merase III transcription initiation factor 90 kDa subunit BR. INT! ENS000000078725 Bone morphogenetic proteitilietinoic acid inducible neural-specific I
BRINP2 ENSG00000198797 Bone morphogenetic protein/retinoic acid inducible neural-specific 2 BRINP3 ENSG00000162670 Bone moiphogcnetic proteiniretinoic acid inducible neural-specific 3 BSG ENSG00000172270 Basigin (Ok blood group) BSPH1 ENSG00000188334 Binder of sperm protein homolog 1 BST1. ENSG00000109743 Bone marrow stromal cell antigen 1 BTBDI.7 ENSCR10000204347 BTB (P02) domain containing 17 BTD ENSG00000169814 Biotinidase BIN2A2 ENS600000124508 Butyrophilin, subfamily 2, member A2 BIN3A1 ENSG00000026950 Butyrophilin, subfamily 3, member Al BTN3A2 ENS00000018647() Butyrophilin, subfamily 3, member A2 BTN3 A3 ENS000000111801 Butyrophilin, subfamily 3, member A3 Cl0orf10 ENSG00000165507 Chromosome 10 open reading frame 10 CIO r1=99 ENSG00000188373 Chromosome 10 open reading frame 99 Cl 10111 ENSG00000137720 Chromosome 11 open reading frame 1 Cl lorf24 ENSG00000 I 71067 Chromosome II Open reading frame 24 Cl 1ot-145 ENS(300000174370 Chromosome II open reading frame 45 Date Recue/Date Received 2022-04-05 CI lorD4 ENSG00000234776 Chromosome 11 open reading frame 94 C I 2orf10 EN SG00000139637 Chromosome 12 open reading frame 10 Cl2or149 ENSG00000 111412 Chromosome 12 open reading frame 49 Cl2orf73 ENSG00000204954 Chromosome 12 open reading frame 73 Cl 2orl76 ENSG00000 174456 Chromosome 12 open reading frame 76 C140'1780 EN SG00000185347 Chromosome 14 open reading frame 80 C I 4orf93 ENSG00000100802 Chromosome 14 open reading frame 93 CI6or189 ENS600000153446 Chromosome 16 open reading frame 89 C16ort90 ENSG00000215131 Chromosome 16 open reading frame 90 C I 7orf67 ENSG00000214226 Chromosome 17 open reading frame 67 Cl 7orf75 ENS000000108666 Chromosome 17 open reading frame 75 CI7orf99 ENSG00000187997 Chromosome 17 open reading frame 99 CI8or154 ENSG00000166845 Chromosome 18 open reading frame 54 C I 9or147 ENSG00000160392 Chromosome 19 open reading frame 47 CI9or170 ENSG00000174917 Chromosome 19 open reading frame 70 CI 90 00 EN S000000130173 Chromosome 19 open reading frame 80 CIGALT1 ENSG00000106392 Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 Clorf127 ENSG00000175262 Chromosome 1 open reading frame 127 Clorf159 E.NSG00000131591 Chromosome 1 open reading frame 159 C1011198 ENSG00000119280 Chromosome I open reading frame 198 C1ort234 ENSG00000227868 Chromosome 1 open reading frame 234 C 1 or154 ENSG00000118292 Chromosome 1 open reading frame 54 Cl ort36 ENSG00000143443 Chromosome 1 open reading frame 56 C IQA ENSG00000173372 Complement component 1. q subcomponent, A chain C1QB ENS000000173369 Complement component I. q subcomponent, B chain CIQC ENSG00000159189 Complement component 1, q subcomponent. C chain CIQL1 ENSG00000131094 Complement component I, q subcomponent-like I
C I QL2 ENSG00000144119 Complement component 1, q subcomponent-like 2 C1%3 ENSG00000165985 Complement component 1, q subcomponent-like 3 C1QL4 ENSG00000186897 Complement component I. q subcomponent-like 4 CIO:INF 1 ENSG00000173918 Clq and tumor necrosis factor related protein I
C I QTNF2 ENSG00000 145861 Ciq and tumor necrosis factor related protein 2 C I Q11µ1F3 ENSG00000082196 C lq and tumor necrosis factor related protein 3 C I QTNF4 ENS600000172247 Clq and tumor necrosis factor related protein 4 C1QTNF5 ' ENSG00000223953 Clq and tumor necrosis factor related protein 5 CIQINF7 ENSG00000163145 Clq and tumor necrosis factor related protein 7 CI QTNF8 ENSG00000 184471 Ciq and tumor necrosis factor related protein 8 CI QTN F9 ENS000000240654 Clq and rumor necrosis factor related protein 9 C I QINF9B ENSG00000205863 Clq and rumor necrosis factor related protein 9B
C1R EN scit r0000159403 Complement component 1, r subcomponent CIRL ENSG00000139178 Complement component I. r subcomponent-like C IS ENSG00000182326 Complement component 1, s subcomponent C2 ENS000000166278 Complement component 2 Date Recue/Date Received 2022-04-05 C2 lorf33 EN SG0000016022 I Chromosome 21 open reading frame 33 C2 lorf62 ENS600000205929 Chromosome 21 open reading .frame 62 C22or115 ENSG00000169314 Chromosome 22 open reading frame 15 C22orf46 ENSG00000184208 Chromosome 22 open reading frame 46 C2CD2 ENSG00000157617 C2 calcium-dependent domain containing 2 C2orf40 ENSG00000119147 Chromosome 2 open reading frame 40 C2orf66 ENSG00000187944 Chromosome 2 open reading frame 66 C2orf69 ENS600000178074 Chromosome 2 open reading frame 69 C2otf78 ENSG00000187833 Chromosome 2 open reading frame 78 C3 ENSG00000125730 Complement component 3 C3or133 ENSG00000174928 Chromosome 3 open reading frame 33 C3orf58 EN SG00000181744 Chromosome 3 open reading frame 38 C74A ENSG0000024473 I Complement component 4A
(Rodgers blood group) CM ENSG00000224389 Complement component 4B (Chido blood group) C4BPA ENSG00000123838 Complement component 4 binding protein, alpha C4BPB ENSG00000123843 Complement component 4 binding protein, beta C4orf26 ENSG00000174792 Chromosome 4 open reading frame 26 C4orf48 ENSG00000243449 Chromosome 4 open reading frame 48 C5 EN SCI00000 106804 Complement component 5 C5orf46 ENSG00000178776 Chromosome 5 open reading frame 46 C6 ENSG00000039537 Complement component 6 C6orf120 ENSG00000185127 Chromosome 6 open reading frame 120 C6orf15 ENS600000204542 Chromosome 6 open reading frame 15 C6orf25 ENso)0000204420 Chromosome 6 open reading frame 25 C6orf58 ENSG00000184530 Chromosome 6 open reading frame 58 C7 ENSG00000112936 Complement component 7 C7ort37 ENSG00000164746 Chromosome 7 open reading frame 57 C7orf73 ENSG00000243317 Chromosome 7 open reading frame 73 C8A, ENSG0000015713 I Complement component 8, alpha polypeptide C8B ENSG00000021852 Complement component 8, beta polypeptide C86 ENSG00000176919 Complement component 8, gamma polypeptide C9 EN SG00000113600 Complement component 9 C9orf47 ENSG00000186354 Chromosome 9 open reading frame 47 154975 anhydrase x CA!! ENSG00000063180 Carbonic anhydrase XI
CA6 ENSG00000131686 Carbonic anhydrase VI
CA9 ENSG00000107159 Carbonic anhydrase IX
CABLES! ENS0)0000134508 Cdk5 and Abl enzyme substrate CAB? I ENSG00000157782 Calcium binding protein 1 CACNA2D1 ENSG00000153956 Calcium channel. voltage-dependent, alpha 2/delta subunit 1 CACNA2D4 ENSG00000151062 Calcium channel, voltage-dependent, alpha 2/delta subunit 4 CADM3 ENSG00000162706 Cell adhesion molecule 3 CALCA ENSG00000 I 10680 Calcitonin-relmed polypeptide alpha CALCB ENSG00000175868 Calettonin-related poly peptide beta Date Recue/Date Received 2022-04-05 CALCR ENSG00000004948 Calcitonin receptor CALCRL ENSG00000064989 Calcitonin receptor-like CALR ENS(300000179218 Calreticulirt CALR3 ENSG00000269058 Calreticulin 3 CALU ENSG00000128595 Calumenin CAMK2D ENSG00000145349 Calciumicaltnodulin-dcpendent protein kinase II delta CAMP ENSG00000164047 Cathelicidin antimicrobial peptide CANX ENts600000127022 Calnexin CARKD ENSG00000213995 Carbohydrate kinase domain containing CARM1 ENSG00000142453 Coactivator-associated arginine methyltransferase 1 CARNS1 ENS000000172508 Camosine sy nthase 1 CARTPT ENSG00000164326 CART prepropeptide CASQ1 ENS(300000143318 Calsequestnn 1 (fast-twilch. skeletal muscle) CA SQ2 ENS(300000118729 Calsertuestrin 2 (cardiac muscle) CATSPERG ENS(300000099338 Catsper channel auxiliary subunit gamma CBLN I ENS000000102924 Cerebellin 1 precursor CBLN2 ENSG00000141668 Cerebellin 2 precursor CBLN3 ENSG00000139899 Cercbellin 3 precursor CBLN4 ENS(300000054803 Cerebelli I) 4 precursor CCBE1 ENSG00000183287 Collagen and calcium binding EGF domains CCDC108 EN S000000181378 Coiled-coil domain containing 108 CCDC112 ENSG00000164221 Coiled-coil domain containing 112 CCDC129 ENSG00000180347 Coiled-coil domain containing 129 CCDC134 ENSG(' )0000100147 Coiled-coil domain containing 134 CCDC149 ENSG00000181982 Coiled-coil domain containing 149 CCDC3 ENS000000151468 Coiled-coil domain containing 3 CCDC80 ENSG00000091986 Coiled-coil domain containing 80 CCDC85A ENSG00000055813 Coiled-coil domain containing 85A
CCDC88B EN1S(300000168071 Coiled-coil domain containing 88B
CCER2 ENSG00000262484 Coiled-coil glutamate-rich protein 2 CCK ENS000000187094 Cliolecystokinin CCL1. ENSG00000108702 Chemokine (C-C motif) ligand 1 CCU 1 ENSG00000172156 Chemokine (C-C motif) ligand 11 --aYi-..13----------EC- a0000018-1374------CceITTokine (c-c. motif) ligand 13 CCL14 ENSG00000276409 Chemokine (C-C motif) ligand 14 CCL15 ENSG00000273718 Chemokine (C-C motif) ligand 15 CCL16 ENSG00000275152 Chernokine (C-C motif) ligand 16 CCL17 ENS0)0000102970 Chemokine (C-C motif) ligand 17 CC1.18 ENS(300000275385 Chemokine (C-C motif) ligand 18 (pulmonary and act ivia ion-tegulated) CCL19 ENSG00000172724 Chemokine (C-C motif) ligand 19 CCL2 EN SG00000108691 Chemokine (C-C motif) ligand 2 CCL20 ENSG00000115009 Chemokine (C-C motif) ligand 20 CCL21. ENSG00000137077 Chemokine (C-C motif) ligand 21 CCL22 ENsG00000102962 Chemokine (C-C motif) ligand 22 Date Recue/Date Received 2022-04-05 CCL23 ENSG00000274736 Chernokine (C-C motif) ligand 23 CCL24 EN SG00000106178 Chemokine (C-C motif) ligand 24 CC125 ENSG000001:31.142 Chemokine (C-C motif) ligand 25 CCL26 ENSG00000006606 Chemokine (C-C motif) ligand 26 CCL27 ENSG00000213927 Chemokine (C-C motif) ligand 27 CCL28 ENSG00000151882 Chernokine (C-C motif) ligand 28 CCL3 ENSG00000277632 Chemokine (C-C motif) ligand 3 CCL3L3 ENS600000276085 Chemokine (C-C moth) ligand 3-like 3 CCL4 ENSG00000275302 Chemokine (C-C motif) ligand 4 CCL4L2 ENSG00000276070 Chemokine (C-C motif) ligand 4-like 2 CCL5 ENSG00000271503 Chemokine (C-C motif) ligand 5 CCL7 ENSG00000108688 Chemokine (C-C motif) ligand 7 CCL8 ENS(300000108700 Chemokine (C-C motif) ligand 8 CCNB I TP1 ENSG00000100814 C'yclin B1 interacting protein I. E3 tibiquit in protein ligase CCNL1 ENSG00000163660 Cyclin Li CCNL2 ENS000000221978 Cyclin L2 CD14 ENSG00000170458 CD14 molecule CD160 ENSG00000117281 CD160 molecule CD164 EN SC100000135535 CD164 molecule, sialornt ici it CD177 ENSGu0000204936 CD177 molecule CD 1E ENSG00000158488 CD 1 c molecule CD2 ENSG00000116824 CD2 molecule CD200 E.NSG00000091972 CD200 molecule CD200R1 ENSG(' )0000163606 CD200 receptor 1 CD22 ENSG00000012124 CD22 molecule CD226 ENSG00000150637 CD226 molecule CD24 ENSG00000272398 CD24 molecule CD276 ENSG00000103855 CD276 molecule CD300A ENSG00000167851 CD300a molecule CD300LB ENSG00000178789 CD 300 molecule-like family member b CD300LF ENSG00000186074 CD300 molecule-like family member f CD300L6 ENSG00000161649 CD300 molecule-like family member g CD3D ENSG00000167286 CD3d molecule, delta (CD3-TCR complex) 7154-EST a00000iii110---a-i74-717,-;iicule ----------------------------CD40 ENSG00000101017 CD40 molecule. TNT receptor superfamily member 3 CD44 ENSG00000026508 CD44 molecule (Indian blood group) CD48 ENSG00000117091 CD48 molecule CD5 ENSG(' )0000110448 CD3 molecule CD55 ENSG00000196352 CD55 molecule, decay accelerating factor for complement (Cromer blood group) CD59 ENS600000085063 CD59 molecule, complement regulatory protein CD5L ENSG00000073754 CD5 molecule-like CD6 ENSG00000013725 CD6 molecule CD68 ENSG00000129226 CD68 molecule Date Recue/Date Received 2022-04-05 CD7 ENSG00000173762 or molecule CD79A ENSG00000105369 CD79a molecule, immunoglobulin-associated alpha CD80 ENS(300000121594 CD80 molecule CD86 ENSG00000114013 CD86 molecule CD8A ENSG00000153563 CD8a molecule CD8B ENSG00000172116 CD8b molecule CD99 ENSG00000002586 0D99 molecule CDC23 ENS600000094880 Cell division cycle 23 CDC40 ENSG00000168438 Cell division cycle 40 CDC45 ENSG00000093009 Cell division cycle 45 CDCP1 ENSG00000163814 CUB domain containing protein 1 CDCP2 ENSG00000157211 CUB domain containing protein 2 CDH I ENSG00000039068 Cadherin 1, type I
CDH II ENSG00000140937 Cadherin II. type 2, OB-cadherin (osteoblast) CDH13 ENSG00000140945 Cadherin 13 CD1117 EN S000000079112 Cadherin 17, Li cadherin (liver-intestine) CDH18 ENSG00000145526 Cadherin 18, type 2 CDH19 ENSG00000071991 Cadherin 19, type 2 CD11123 ENSG00000107736 Cadherin-related 23 CDH5 ENSG00000179776 Cadherin 5, type 2 (vascular endothelium) CDHR1 ENS000000148600 Cadherin-related family member 1 CDHR4 ENSG00000187492 Cadherin-related family member 4 CDHR5 ENS600000099834 Cadherin-related family member 5 CDKN2A ENSG(' )0000147889 Cychu-dependent kinase inhibitor 2A
CDNF ENSG00000185267 Cerebral dopamine neumtrophic factor CDON ENS000000064309 Cell adhesion associated, oncogene regulated CDSN ENSG00000204539 Corneodesmosin CEACAM16 ENSG00000213892 Carcinoembryonic antigen-related cell adhesion molecule 16 CEACAM18 ENSG00000213822 Carcirtoembryonic antigen-related cell adhesion molecule 18 CEAC.AM19 ENSG00000186567 Carcinoembryonic antigen-related cell adhesion molecule 19 CEACAM5 ENS000000103388 Careinoembryonic antigen-related cell adhesion molecule 5 CEACAM7 ENSG00000007306 C:areinoembryonic antigen-related cell adhesion molecule 7 CEACAM8 ENSG00000124469 _Carcinoembryonic antigen-related cell adhesion molecule 8....._ --a-7:752--1--------ESaiiiiiiii(579-5i772--- Cat eye syndrome chromosome region, candidate 1 CECR5 ENSG00000069998 Cat eye syndrome chromosome region, candidate 5 CEL ENSG00000170835 Carboxyl ester lipase CELA2A ENSG00000142615 City inottypsin-like elastase family, member 2A
CELA2B ENS0)0000215704 Chymotrypsin-like elastase family, member CELA3A ENSG00000142789 Clay motry psin-like elastase family.
member 3A
CELA3B ENSG00000219073 Chymottypsin-like elastase family. member CEIVIIP ENSG00000103888 Cell migration inducing plotein.
hyaluronan binding CEP89 ENS000000121289 Centiosoinal protein 89kDa CER.1 ENSG00000147869 Cerbenis I. DAN family BMP antagonist CERCAM ENS(300000167123 Cerebral endothelial cell adhesion molecule Date Recue/Date Received 2022-04-05 CER.S1 ENS000000223802 CeratMde synthase I
CES I ENSG00000198848 Carboxylesterase 1 CES3 ENS(300000172828 Carboxylesterase 3 CES4A ENSG00000172824 Carbox-ylesterase 4A
CES5A ENSG00000159398 Carboxylesterase SA
CET? EN5600000087237 Cholestetyl ester transfer protein, plasma CFB ENS000000243649 Complement factor B
CFC1 EN S600000136698 Cripto, FRL- I , cryptic family 1 CFC1B ENS(300000152093 Cripto, FRL-1, cryptic family CFD ENSG00000197766 Complement factor D (adipsin) CFDP1 ENS000000153774 Crattiolacial development piotein 1 CFH EN SG00000000971 Complement factor H
CFEIR I ENS(300000244414 Complement factor H-related 1 CEHR2 ENSG00000080910 Complement factor H-related 2 CEHR3 ENSG00000116785 Complement factor H.-mimed 3 CIFI1R4 ENS000000134365 Complement factor H-related 4 CEHR5 ENSG00000134389 Complement factor H-related 5 CFI ENS000000205403 Complement factor I
CFP ENSG00000126759 Complement factor properdin CGA ENSG00000135346 Glycoprotein hormones, alpha polypeptide COB ENS000000104827 Chorionic gonadot ropin, beta poly peptide COB! EN SG00000267631 Chorionic gonadotropin, beta polypeptide 1 CGB2 E.NS600000104818 Chorionic gonadotropin, beta poly peptide 2 COBS ENs(30000018905 2 Chorionic gonadotropitt, beta polypeptide 5 CGB7 ENSG00000196337 Chorionic gonadotropin, beta polypeptide 7 COB8 ENSG00000213030 Chorionic gonadotropin, beta poly pept ide 8 CGREF1 ENSG00000138028 Cell growth regulator with EF-hand domain 1 01507-9B2.3 ENSG00000280071 CHAD ENS(300000136457 Chondroadhe tin CHADL ENSG00000100399 Chondroadherin-like CH EK 2 ENS000000183765 Checkpoint kittase 2 CHGA ENSG00000100604 Chromogranin A
CHGB ENSG00000089199 Cht=omogranin B
.....
-6:113Li----------ESTR0i0000i33048 Chit inase 3-like I (cartilage glycoprotein-39) CHI3L2 ENS000000064886 Chitinase 3-like 2 CHIA ENSG00000134216 Chitinase, acidic CHID! ENSG00000177830 Chitinase domain containing 1 CHIT! ENSO )0000133063 Chitinase 1 (chitotriosidase) CHL I ENS(30000013412 I Cell adhesion molecule L 1-like CI1N1 ENSG00000128636 Chimerin 1 CHPF ENSG00000123989 Chondroitin polymerizing factor CHPF2 ENS000000033100 Chondroitin polymerizing factor 2 CHRD ENS000000090539 Chordin CHRDL1 ENS(300000101938 anordin-like 1 Date Recue/Date Received 2022-04-05 CHRDL2 ENSG00000054938 Chordin-likc 2 CHRN.A2 ENS600000 I 20903 Cholinergic receptor, nicotinic, alpha 2 (neuronal) CHRNA5 ENSG00000169684 Cholinergic receptor, nicotinic, alpha 5 (neuronal) CHRNB1 ENSG00000170175 Cholinergic receptor, nicotinic, beta 1 (muscle) CHRND ENS000000135902 Cholinergic receptor, nicotinic, delta (muscle) GIST! ENSG00000175264 Carbohydrate (keratin sulfate (ial-6) sulfotransferase I
CHSTI 0 ENS000000115326 Carbohydrate sulfotransferase 10 CH ST1 I ENS600000171.310 Carbohydrate (chondroitin 4) sulibtransie rase II
CHST13 ENSG00000180767 Carbohydrate (chondroitin 4) sulfotransferase 1.3 CHST4 ENSG00000140835 Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 4 CHST5 ENSG00000135702 Carbohydrate (N-acetylglucostintine 6-0) sulfotransferase 5 CHST6 ENSG000001831% Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 6 CHST7 ENSG00000147119 Carbohydrate (N-acetylgiticosamine 6-0) sulfotransferase 7 CHST8 ENSG00000124302 Carbohydrate (N-acety Igalactosamine 4-0) sit! fotransierase 8 CHSY I ENSG00000131873 Chondroitin sulfate synthase 1 CHSY3 ENS000000198108 Cliondroitin sulfate synthase 3 CHTF8 ENSG00000168802 Chromosome transmission fidelity factor 8 CILP ENSG00000138615 Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase CILP2 ENSG0000016016 I Cartilage intermediate layer protein 2 CIRH IA ENSG00000141076 Cirrhosis, amosomai recessive IA (cirbin) CKLF ENSG00000217555 Chernokine-like factor CKMT1A ENSG00000223572 Creatine kinase, mitochondria( IA
CKMT1B ENSG00000237289 Creatine kinase, mitochondria' 1B
CLCA 1 E'NSG110000016490 Chloride channel accessory I
CLCF1 ENSGU0000175505 Cardionophin-like cytokine factor 1 CLDN15 ENSG00000106404 Claudia 15 CLDN7 ENSG00000181885 Claudia 7 C7LDND1 ENSG00000080822 _ Claudia domain containing .1 --(97617-------iNgiiiiiiiiiiiiTi-5-4727-- C-type lectin domain fliriiily 11, member A
CLEC16A EN5000000038532 C-type lectin domain family 16, member A
CLEC18A ENSG00000157322 C-type lectin domain family 18, member A
CLEC I8B ENSG00000140839 C-type lectin domain family 18, member B
CLEC18C ENSG00000157335 C-type lectin domain family 18, member C
CLEC19A IiNS(300000261.210 C-type lectin domain family 19, member A
CLEC2B ENSG00000110852 C-type lectin domain family 2, member B
CI.EC1 A ENS600000166509 ('-type lectin domain family 3, member A
CLEC3B EN SG00000163815 C-type lectin domain family 3, member B
CLGN ENSG00000153132 Calmegin CLN5 ENSG00000102805 Ceroid-lipofuscinosis, neuronal 5 CLPS ENSG00000137392 Colipase, pancreatic CLPSL I ENSG00000204140 Colipase-like 1 CLPSL2 ENSG00000196748 Colipase-like 2 CLPX ENSG00000166855 Caseinolytic mitochondria' matrix peptidase chaperone subunit CLSTN3 ENS(300000139182 Calsy Merlin 3 Date Recue/Date Received 2022-04-05 CLU ENSG00000120885 Clustcrin CLUL1 ENSG0000007910 I Clusierin-like I (retinal) (MA I. ENSG00000092009 Clay mase I. mast cell CMPK I ENSG00000162368 Cytidine monophosphate (UMP-CMP) kinase 1, cytosolic CNBDI ENSG00000176571 Cyclic nucleotide binding domain containing 1 CNDP1 ENSG00000150656 Carnosine dipeptidasc 1 trnetallopeptidase M20 family) CNPY2 ENSG00000257727 Canopy FGF signaling regulator 2 CNPY3 ENS(Li0000013716 I Canopy FGF signaling regulator:
CNPY4 ENSG00000166997 Canopy FGT.' signaling regulator 4 CNTFR ENSG00000122756 Ciliary neurotrophic factor receptor CNTN1 ENSG00000018236 Contad in I
CNTN2 ENSG00000184144 Contactin 2 (axonal) CNTN3 ENSG00000113805 Contactin 3 (plastnacy Ionia associated) CNTN4 ENSG00000144619 Contact in 4 CNTN5 ENSG00000149972 Contactin 5 CNTNAP2 ENSG00000174469 Contact in associated protein-like 2 CNINAP3 ENSG00000106714 Contactin associated protein-like 3 CNTNAP3B ENSG00000154529 Cornactin associated protein-like 3B
COASY ENSG00000068120 CoA synthase COCH ENSGu0000100473 Cochlin COG3 EN SG00000136152 Component of oligorneric golgi complex 3 COLIOA I ENSG00000123500 Collagen, type X, alpha 1 COLI lAl. E.NSG00000060718 Collagen, type XI, alpha 1 COLI1A2 '4:m0)0000204248 Collagen, type XI, alpha 2 COL 12A1 ENSG00000111799 Collagen, type XII, alpha I
C01..14A 1 ENSG00000187955 Collagen. type XIV, alpha 1 COL 15A1 ENS00000020429 I Collagen, type XV, alpha 1 COL 16A1 ENSG00000084636 Collagen, type XVI, alpha 1 COL18A 1 ENSG0000018287 I Collagen, type XVIII. alpha 1 COL 19A1 ENSG00000082293 Collagen, type XIX, alpha 1 COLIA1 ENSG00000108821 Collagen, type 1, alpha 1 COL IA2 ENSG00000164692 Collagen, type I, alpha 2 C0L20A I. ENSG00000101203 _Collagen, type X.X.alpha 1 --651:2T-A-1-----ESIST300-000i17477-4-9-- Collagen, type XXI, alpha 1 COL22A1 ENSG00000169436 Collagen. type XXII, alpha 1 COL24A1 ENSG00000 171502 Collagen, type XXIV, alpha 1 COL26A1 ENSG00000160963 Collagen, type XXVI, alpha I
COL27A1 ENS0)0000196739 Collagen, type )0CVII, alpha 1 C01.28A1 ENSG00000215018 Collagen, type XXVEll, alpha 1 COL2AI ENSG00000139219 Collagen, type II. alpha 1 COL3A I ENSG00000168542 Collagen. type III, alpha 1 COL4A1 ENSG00000187498 Collagen. type IV, alpha I
COL4A 2 ENS60000013487 I Collagen, type IV. alpha 2 COL4A3 ENSG0000016903 I Collagen, type IV, alpha 3 (Goodpasture antigen) Date Recue/Date Received 2022-04-05 COL4A4 ENSG00000081052 Collagen, type IV. alpha 4 COL4A 5 EN SG00000188133 Collagen, type IV. alpha 5 COI3A6 ENSG00000197565 Collagen, type IV, alpha 6 COL5A1 ENSG00000130635 Collagen, type V. alpha 1 COL5A2 ENSG00000204262 Collagen, type V. alpha 2 COL5A3 ENS600000080573 Collagen, type V. alpha 3 COL6A1 ENSG00000142156 Collagen, type VI. alpha 1 C01,6A2 ENts600000142 173 Collagen, type VI, alpha 2 COL6A3 ENSG00000163359 Collagen, type VI, alpha 3 COL6A5 ENSG00000172752 Collagen. type VI, alpha 5 a)1..6A6 ENSG00000206384 Collagen. type VI. alpha 6 COL7A1 ENSG00000114270 Collagen, type VII, alpha 1 C0L8A1 ENSG00000144810 Collagen, type VIII, alpha i COL8A2 ENS300000171812 Collagen, type VIII, alpha 2 COL9A1 ENSG00000112280 Collagen, type IX, alpha 1 a)1-9A2 ENS000000049089 Collagen. type IX, alpha 2 COL9A3 ENSG00000092758 Collagen, type IX. alpha 3 COLEC 10 ENSG00000184374 Collectin sub-family member 10 (C-iy pc lectin) (Dux: 1 I ENscl0000n 118004 Collectin sub-family member COLGALT1 ENSG00000130309 Collagen beta(1-0)galactosyltransferase 1 COLGALT2 ENSG00000198756 Collagen beta(1-0)galactosy liransfe lase 2 COLO ENSG00000206561 Collagen-like tail subunit (single strand of homotrimer) of asynunetric acetylcholinestentse COMP ENSG00000105664 Cartilage oligomeric matrix protein COP% ENSG00000168090 C0P9 signalosome subunit 6.________________________ --Ei.5-0-6--------N-SC-1165660I1-477-5 Coenly me Q6 monooxygenase CORT ENSG00000241563 Cortistatin CP ENSG00000047457 Centloplasinin (ferioxidase) CPA!. ENSG00000091704 Carboxypeptidase Al (pancreatic) CPA2 ENSG00000158516 Carboxypeptidase A2 (pancreatic) CPA3 ENSG00000163751 Carboxypeptidase A3 (masi cell) CPA4 ENSG00000128510 Calbox-ypeptidase A4 CPA6 ENSG00000165078 Carboxypeptidase A6 CPAM.D8 EN SG000001601 II C3 and PZP-Iike. alpha-2-macrog1obulin domain containing 8 CPB1 ENSG00000153002 Qtrboxy peptidase B1 (tissue) CPB2 ' ENSG00000080618 Carboxypeptidase B2 (plasma) CPI-3, ENSG00000109472 Carboxypeptidase E
CPM ENSG00000135678 Calboxypeptidase M
CPN I ENSG00000120054 Carboxs.peptidaw N.
polypeptide 1 CPN2 ENSG00000178772 Carbon. pept idase N.
polypeptide 2 CPO EN S600000144410 Carboxypeptidase 0 CPQ ENSG00000104324 Carboxypeptidase Q
CPVL ENSG00000106066 Carboxypeptidase, vitellogenic-like CPX41 ENS600000088882 Carboxypeptidase X (M14 family), member 1 Date Recue/Date Received 2022-04-05 cpxm2 ENSG00000121898 Carbox-ypeptidase X (M14 family), member 2 CPZ ENSG00000109625 Carboxy peptidase Z
CR1L ENS(300000197721 Complement component (3b,14b) receptor 1-like CRB2 ENSG00000148204 Climbs family member 2 CREG1 ENSG00000143162 Cellular repressor of EIA-stimulated genes 1 CREG2 ENSG00000175874 Cellular repressor of EIA-stimulated genes 2 CRELD1 ENSG00000163703 Cysteine-rich with EGF-like domains 1 CRELD2 ENS600000184164 Cysteine-rich with EGF-like domains 2 CRH ENSG00000147571 Cotticotropin releasing hormone CRHI3P ENSG00000145708 Corticotropin releasing hormone binding protein CREME ENS000000120088 Corticotropin releasing hormone receptor 1 CRHR2 ENSG00000106113 Corticotropin releasing hormone receptor 2 CRISP1 ENSG00000124812 Cysteine-rich secretory protein 1 CR ISP2 ENS(300000124490 C'ysteine-rich secretory protein 2 CRISP3 ENS(300000096006 Cysteine-rich secretmy protein 3 CR ISPLD2 ENS000000103196 Cysteine-rich secretory protein LCCL domain containing 2 CRLF1 ENSG00000006016 Cytokine receptor-like factor CRP ENSG00000132693 C-reactive protein, pentraxin-related CRTAC I ENS(300000095713 Cartilage acidic protein I
CRTAP ENSGu0000170275 Cartilage associated protein CRY2 ENS000000121671 Cryptochrome circadian clock 2 CSAD ENSG00000139631 Cysteine sulfinic acid decarboxy lase CSF1 E.NS600000184371 Colony stimulating factor I
(macrophagc) CSF1R ENS0)0000182578 Colony stimulating factor 1 receptor CSF2 ENSG00000164400 Colony stimulating factor 2 (granulocyte-macrophage) CSF2R A ENS000000198223 Colony stimulating factor 2 receptor, alpha, low-affinity (granulocyte-macrophage) CSF3 ENSG00000108342 Colons stimulating factor 3 (granulocyte) CSGALNACT I ENSG00000147408 Chondroitin sulfate N-acetylgalactostiminyltransferase 1 CSH I ENSG00000136488 Chorionic somatomammotropin hormone 1 (placental lactogen) CSH2 ENS(300000213218 Chorionic somatomammotropin hormone 2 CSHL1 ENSG00000204414 Chorionic somatomammotropin hormonc-like 1 CSN IS I ENSG00000126545 Casein alpha sl CSN2 ENSG00000 I 35222 Casein beta CSN3 ENSG00000171209 Casein kappa CST1 ' ENS(300000170373 Cystatill SN
CST I.1 ENSG00000125831 Cystatin Ii CST2 ENSG00000170369 Cystatin SA
CST3 ENS000000101439 Cystatin C
CST4 ENSG00000 I 0144 I Cystalin S
CST5 EN S600000 170367 Cystat in D
CST6 ENSG00000175315 Cystatin UM
CST7 ENSG00000077984 Cystatin F (leukocystatin) CST8 ENSG00000125815 Cystatin 8 (cystatin-related epididvmal specific) Date Recue/Date Received 2022-04-05 CST9 ENSG00000173335 Cystatin 9 (testatin) CST9L ENSG00000101435 Cystatin 9-like CSTL1 Esis600000125823 Cystatin-like 1 C155 ENSG00000169551 Cancer/testis antigen 55 CTB-60818.6 ENSG00000267335 crBs ENSG0000011715I Chitobiase, di-N-acctyl-CTD-2313N18.7 ENSG00000225805 CID-2370N5.3 EN s(300000265118 CTGE ENSG00000118523 Connective tissue growth factor CTHRC I ENSG00000 164932 Collagen triple helix repeat containing I
CTLA4 ENS000000163599 Cytotoxic T-Iymphocyte-associated protein CTNS ENSG00000040531 Cystinosin, lysosomal gstine transporter CTRB1 ENS(300000168925 Clay mot ty psinogen B I
CTRB2 ENSG00000168928 Chy inot r), psi noge it 82 CTRC ENSG00000162438 Chymotrypsin C (ealdecrin) CTRL ENS000000141086 City !nutty psi ri-like CTSA ENSG00000064601 Cathepsin A
CTSB ENSG00000164?33 Cathepsin B
CTSC ENSG00000109861 Cathepsin C
CTSD ENSG00000117984 Cathepsin D
CTSE ENS000000196188 Cat hepsi n E
CTSF ENSG00000174080 Cathepsin F
CTSG ENSG00000100448 Cathepsin G
CTSH ENSG(' )0000103811 Cathepsin H
CTSK ENSG00000143387 Cathepsin K
CTSL ENSG00000135047 Cathepsin L
CISO EN5600000256043 Cathepsin 0 CTSS ENSG00000163131 Cathepsin S
CTSV ENS(300000136943 Cathepsin V
CTSW ENSG00000172543 Cathepsin W
crsz ENS600000101160 Cat hepsi n 7 CUBN EN SG00000107611 Cubilin (intrinsic tnctor-cobalamin receptor) CUTA ENS600000112514 CutA divalent cation tolerance.homolog (E. coli)._________ ¨ff3a..-1-------ESIST:ii0(000062I0;-----E-1171-n-aTt-ie (C-X3-C motif) ligand CXADR ENSG00000154639 Coxsackie virus and adenovirus receptor CXCL1 ENSG00000163739 Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) CXCL10 ENSG00000169245 Chemokine (C-X-C motif) ligand 10 CXCL II ENS000000169248 Chemokine (C-X-C motif) ligand 11 CXCL12 ENSG00000107562 Chemokine (C-X-C motif) ligand 12 CXCL13 ENS600000156234 Chemokine (C-X-C motif) ligand 13 CXCL1.4 ENSG00000145824 Chemokine (C-X-C motif) ligand 14 CXCL 17 ENSG00000189377 Chemokine (C-X-C motif) ligand 17 CXCL2 ENSG00000081041 Chemokine (C-X-C motif) ligand 2 Date Recue/Date Received 2022-04-05 CXCL3 ENSG00000163734 Chemokine (C-X-C motif) ligand 3 CXCL5 ENSG00000163735 Chemokine (C-X-C motif) ligand 5 CXCL6 ENS(300000124875 Chemokine (C-X-C motif) ligand 6 CXCL8 ENSG00000169429 Chemokine (C-X-C motif) ligand 8 CXCL9 ENSG00000138755 Chemokine (C-X-C motif) ligand 9 CXorf.36 EN5G000001471.13 Chromosome X open reading frame 36 CYB5D2 ENSG00000167740 Cytochrome b5 domain containing 2 CY 1-1R1 EN 8600000187954 Cysteineihistidine-rich 1 CYP17A I ENSG00000148795 Cytochrome P450, family 17, subfamily A, polypeptide 1 CYP20A1 ENSG00000119004 Cytochrome P450, family 20, subfamily A, polypeptide I
CYP21A2 EN S000000231852 Cytochrome P450, family 21, subfamily A, poly peptide 2 CYP26B1 ENSG00000003137 Cytochrome P450, family 26, subfamily B.
polypeptide 1 CYP2A6 ENS(300000255974 Cytoclimine P450, family 2, subfamily A, polvpeptide 6 CYP2A7 ENS(300000198077 Cytoclimine P450, family 2, subfamily A, polypeptide 7 CYP2B6 ENS(300000197408 Cytochrome P450, family 2, subfamily B, polypeptide 6 CYP2C18 ENS000000108242 Cytochrome P450, family 2, subfamily C, potypeptide 18 CYP2C19 ENSG00000165841 Cytochrome P450, family 2. subfamily C.
polypeptide 19 CYP2C8 ENSG00000138115 Cytochromc P450. family 2, subfamily C.
polypeptidc 8 CYP2C9 ENS(300000138109 Cytochrome P450, family 2, subfamily C, polypeptide 9 CYP2EI ENSG00000130649 Cytochrome P450, family 2, subfamily E, polypeptide I
CYP2F1 ENS000000197446 Cytochrome P450, family 2, subfamily F.
polypeptide 1 CYP2J2 ENSG00000134716 Cytoehrome P450, family 2, subfamily J, polypeptide 2 CYP2R1 ENSG00000186104 Cytochrome P450. family 2. subfamily R.
polypeptide 1 CY P2S1 ENSG(' )0000167600 Cytochrome P450, family 2, subfamily S. polypeptide 1 CYP2W1 ENSG00000073067 Cytochrome P450, family 2, subfamily W.
polypeptide 1 CYP46A 1 ENS000000036530 Cytochrome P450, family 46, subfamily A, polypeptide I
CYP4F11 ENSG00000 I 71903 Cytochrome P450, family 4. subfamily F.
polypeptide 11 CYP4F2 ENSG00000186115 Cytochrome P450. family 4. subfamily F, polypeptide 2 CYR61 ENS(300000142871 Cysteine-rich. angiogertic inducer, 61 CYTL I ENSG00000 170891 Cytokine-like 1 D2I143DI4 ENSG00000180902 D-2-Itydroxyglutar4te debydrogentise DAG1 ENSG00000173402 Dystroglycan. 1 (dystrophin-associated glycoprotein 1) DAND5 ENSG00000179284 DAN domain family member 5:_BMP
antagonist -15:476--------ESIST-iiiiii65-11-oiiiiT---15:icmino-acid oxidase DAZAP2 ENSG00000183283 DAZ associated protein 2 DB1-I ENSG00000 123454 Dopamine beta-hydrox-y lase (dopamine beta-monooxygenase) DBNL ENSG00000136279 Drebrin-like DCD ENSO(' J0000161634 Denneidin DCN ENS(300000011465 Decorin DDIAS ENSG00000165490 DNA damage-induced apoptosis suppressor DDOST EN8G00000244038 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit (non-catalytic) DDR1 ENS(300000204580 Discoidin domain teceptor tyrosine kinase 1 DDR2 ENS000000162733 Discoidin domain teceptor tyrosine kinase Date Recue/Date Received 2022-04-05 DDT ENSG00000099977 D-doptichrome tantomerase DDX17 ENSG0000010020 I DEAD (Asp-Gin-Ala-Asp) box helic...use DDX20 ENS600000064703 DEAD I Asp-GM-Ala-Asp) box poly peptide DDX23 ENSG00000109832 DEAD (Asp-Glu-Ala-Asp) box helicase 23 DDX28 ENSG00000182810 DEAD (Asp-Gin-Ala-Asp) box 1).0/peptide DEMI ENSG00000177030 DEAF I transcription factor DEER ENSG00000140995 Differentially expressed in FDCP 8 homolog (mouse) DEE:\ 1 ENS000000206047 Delensin, alpha 1 DEFA113 ENSG00000240247 Defensin, alpha 113 DEFA3 ENSG00000239839 Defensin. alpha 3, nentrophil-specific DEFA4 ENS000000164821 Defensi it, alpha 4, corticostatin DEFA5 ENSG00000164816 Defensin. alpha 5, Paneth cell-specific DEFA6 ENS600000164822 Defensin, alpha 6. Paneth cell-specific DEFB 1 EN SG00000164825 Damn], beta 1 DEFB 103A ENSG00000176797 Defensin, beta 103A
DEE:13103B ENS000000177243 Defensin. beta 103B
DEFB104A EINSG00000176782 Defensin, beta 104A
DEFB104B ENSG00000177023 Dcfcnsin, beta 104B
DEFB 105A ENSG00000186562 Defensin, beta 105A
DUB 10513 ENSG00000186599 Defensin, beta 105B
DEFB106A ENS000000186579 Defensiii, beta 106A
DEFB106B ENSG00000187082 Defensin, beta 106B
DEFB107A E.NSG00000186572 Dcfcnsin, beta 107A
DEFB10713 EN SG00000198129 Defensin, beta 1078 DEFB 108B ENSG00000184276 Defensin, beta 108B
DUB 110 ENSG00000203970 Defensin. beta 110 DEFB113 ENSG00000214642 Defensin, beta 113 DEFB114 ENSG00000177684 Defensin, beta 114 DEFB 115 ENSG00000215547 Defensin, beta 115 DEFB116 ENSG00000215545 Defensin, beta 116 DEFB 118 ENSG00000131068 Defensin, beta 118 DEFI3119 ENSG00000180483 Defensin, beta 119 DEFB121 ENSG00000204548 Defensin, beta 121 15E713- iii---- EST aiii oliiii-8-6T2-4- ¨ ¨ ¨ - ¨1.5-eTe-ri-;;IiiT, beta 123 - ¨
DEFB 124 ENSG00000180383 Defensin. beta 124 DEFB125 ENSG00000 178591 Defensin. beta 125 DEFB126 ENSG00000125788 Defensin, beta 126 DEFB127 ENS000000088782 Defensin, beta 127 DEFB 128 ENSG00000185982 Defensin, beta 128 DEFB129 ENSG00000125903 Defensin. beta 129 DEFB 130 ENSG00000232948 Defensin. beta 130 DEFB13 I ENS000000186146 Defensin, beta 131 DEFB132 ENSG00000186458 Defensin, beta 132 DEFB 133 ENSG00000214643 Defensin, beta 133 Date Recue/Date Received 2022-04-05 DEFB134 ENSG00000205882 Defensin, beta 134 DEFB135 ENS600000205883 Defensin, beta 135 DEFB136 ENS600000205884 Defertsin, beta 136 DEF134A ENSG00000171711 I3efensin, beta 4A
DEFB4B ENSG00000177257 Defensin, beta 4B
DFNA5 ENSG00000105928 Deafness, autosomai dominant 5 DENB31 ENSG00000095397 Deafness, autosomal recessive 31 DGCR2 ENS600000070413 DiGeorge syndrome critical region gene 2 DWI ENS(300000139549 Desett hedgehog DHRS4 ENSG00000157326 Dehydrogenaserreductase (SDR family) member 4 DEIRS41,2 EN SG00000187630 Dehydrogenase/teductase (SDR family) member 4 like 2 DHRS7 EN SG00000100612 Dehydrogenaseireductase (SDR family) member 7 DITRS7C ENS600000184544 Dehydrogerutseireductase (SDR family) member 7C
DHRS9 ENS(3000000717:37 Dehydrogertaselreductase (SDR family) member 9 DHRSX ENS(300000169084 Dehydrogenaseireductase (SDR family) X-linked DEIX29 ENS000000067248 DEAH (Asp-Ght-Ala-ii is) box polypeptide DHX30 EINSG00000132153 DEAH (Asp-Giu-Ala-His) box helicase 30 DI-IX8 ENSG00000067596 DEAH (Asp-Gin-Ala-His) box polypeptide 8 D102 ENS(300000211448 Deiodinase, iodothyronine, type II
DLXDC1 ENSGu0000150764 DIX domain containing 1 DKK1 ENS000000107984 Dickkopf WNT signaling pathway' inhibitor DKK2 ENSG00000155011 Dickkopf WNT signaling pathway inhibitor DKK3 E.NSG00000050165 Dickkopf WNT signaling pathway inhibitor DKK4 ENSG1)0000104371 Dickkopf WNT signaling pathway inhibitor DKKI., I ENSG0000010490 I DicIdcopf4ike 1 D1,64 ENSG00000132535 Discs, large homolog 4 (Drosophila) 131,K I ENSG00000185559 Delta-like 1 hornolog (Drosophila) DLL I ENSG00000198719 Delta-like 1 (Drosophila) DLL3 ENS(3000000)0932 Delia-like 3 (Drosophila) DNIBTI ENSG00000187908 Deleted in malignant brain tumors 1 DN4KN ENSG00000161249 Dermokine NAP! ENSG00000152592 Dentin matrix acidic phosphoprotein I
DMRTA2 ENSG00000142700 UNTRT-like family A2 .....
-15WATNIT------EST- aiiiiiiiiiigliiiii-----6;;;111,-a-X-orteinal, assembly factor 5 DNAHI4 ENSG00000185842 Dynein, axonemal, heavy chain 14 DNAJB Ii ENSG00000090520 Dna] (Hsp40) homolog, subfamily B, member DNAJB9 ENSG00000128590 Dnal (lisp40) homolog, subfamily B, member 9 DNAJC25-GNGIO ENSG00000244115 DNAJC25-GNGIO readdnough DNAJC3 ENS(300000102580 Dual (Ffsp40) homolog. subfamily C.
member 3 DNA SEI ENSG00000213918 Deoxyribonuclease 1 DNASEIL I EN8G00000013563 Deoxyribonuclease I-like 1 DNASE1L2 ENSG00000167968 Deoxyribonuclease I-like 2 DNASEIL3 ENSG00000163687 Deoxyribonuclease I-like 3 DNASE2 ENS600000105612 Deoxyribottuclease II, lysosomal Date Recue/Date Received 2022-04-05 DNASE2B ENSG00000137976 Deoxyribonuclease II beta DPEP I ENSG000000154 13 Dipeptidase 1 (renal) DPEP2 ENSG00000167261 Dipeptidase 2 DPEP3 ENSG00000141096 Dipeptidase 3 DPF3 ENSG00000205683 D4, zinc and double PHD fingers. family 3 DPP4 ENSG00000197633 Dipcptidyl-peptidase 4 DPP7 ENSG00000176978 Dipeptidy i-peptidase 7 DIrf EN8600000143196 Denaratopontin DRAX1N ENSG00000162490 Dorsal inhibitory axon guidance protein DSE ENSG00000111817 Dennatan sulfate epimerase DSG2 ENSG00000046604 DesmogIcin 2 DSPP ENSG00000152591 Dentin sialophosphoprotein DST EN SG00000151914 Dvstonin DUOX1 ENSG00000137857 Dual oxidase 1 DYNLI3 ENSG00000165169 Dynein, light chain, Tctex-type 3 E2F5 ENSG00000133740 E2F transcription factor 5, p130-binding EBAG9 ENSG00000147654 Estrogen receptor binding site associated. antigen, 9 EBI3 ENSG00000105246 Epstein-Barr vinis induced 3 ECHDC I ENSG00000093144 Eihylmalonyl-CoA decarboxy lase 1 ECM! ENSG00000143369 Extracellular matrix protein 1 ECM2 ENSG00000106823 Extracellular matrix protein 2, female organ and adipocyte specific ECSIT ENSG00000130159 ECSIT signalling integrator EDDM3A ENSG00000181562 Epididy ma) protein 3A
EDDM3B ENSG1)0000181552 Epididyrnal protein 38 EDEM2 ENSG00000088298 ER degradation enhancer, mannosidase alpha-like 2 EDEM3 ENSG00000116406 ER degradation enhancer, litallif0SidaSe alpha-like 3 EDIL3 ENSG00000164176 EGF-like repeats and discoidin I-like domains 3 EDN1 ENSG00000078401 Endothelin 1 --ET5ST2-----------ESIS-Ciii0000127129-----fk-n-d¨otTe-EiT----EDN3 ENSG00000124205 Endothelin 3 EDNRB EN SG00000136160 Endothelin receptor type B
EFEMP1 ENSG00000115380 EU': containing fibulin-like extracellular matrix protein 1 EFENff'2 ENSG00000172638 EGF containing fibulin-like extracellular matrix protein 2 EFNA 1 IiNSCR10000169242 Ephrin-Al EFNA2 ENSG00000099617 Ephrin-A2 EFNA4 ENSG00000243364 Ephri n -A4 EGFL6 EN SG00000198759 EGF-like-domain, multiple 6 EGFL7 ENSG00000172889 EGF-like-domain. multiple 7 EGFL8 ENSG00000241404 EGE-like-domain, multiple 8 EGFLAM ENSG00000164318 EGF-like, fibronectin type III and laminin G domains EGER EN SG00000146648 Epidemial growth factor receptor E1413P1 ENSG00000115504 EH domain binding protein 1 EHF ENSG00000135373 Ets homologous factor Eiuvrri ENSG00000181090 Euchromatic histone-lysine N-inethylininsferase 1 Date Recue/Date Received 2022-04-05 DEMANDE OU BREVET VOLUMINEUX
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PLUS D'UN TOME.

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Claims

What is claimed is:

1. A fusion protein comprising the amino acid sequence of SEQ ID NO: 40.
2. A method of producing a modified cell comprising introducing into the cell the fusion protein of claim 1.
3. The method of claim 2, wherein the modified cell is a T cell.
4. The method of claim 3, wherein the modified T cell is a modified stem memory T cell (Tscm) or a modified central memory T cell (Tcm).
5. The method of claim 3, wherein the modified T cell is an allogeneic cell.
6. The method of claim 3, wherein the modified T cell is an autologous cell.
7. A modified T cell comprising a fusion protein of claim 1.
8. A modified T cell produced by the method of claim 2.
9. A composition comprising a population of modified cells comprising the fusion protein of claim 1.
10. A composition comprising a population of modified cells produced by the method of

claim 2.

Date Recue/Date Received 2022-04-05