CA3105873A1

CA3105873A1 - Modified stem cell memory t cells, methods of making and methods of using same

Info

Publication number: CA3105873A1
Application number: CA3105873A
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
Also published as: JP2024096417A; JP2019528760A; AU2020227020A1; IL276907A; CA3154236A1; CA3036926C; WO2018064681A1; IL265157A; JP2021006057A; CA3036926A1; JP2021191312A; KR20230035467A; CN110199015A; IL265157B; AU2020227020B2; SG11201901959YA; KR20190063468A; KR20200108927A; IL287286A; KR102588469B1

Abstract

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.
Date Recue/Date Received 2021-01-15

Description

MODIFIED STEM CELL MEMORY T CELLS, METHODS OF MAKING AND
METHODS OF USING SAME
RELATED APPLICATIONS
[01] This application claims the benefit of provisional applications USSN
62/402,707 filed September 30, 2016, USSN 62/502,508 filed May 5, 2017, USSN 62/553,058 filed August 31, 2017 and USSN 62/556,309 filed September 8, 2017.
FIELD OF THE DISCLOSURE
[03] The disclosure is directed to molecular biology, and more, specifically, to methods of making and using modified stem-cell memory T cells.
BACKGROUND
[04] There has been a long-felt but unmet need in the art for a method of producing modified stem-cell memory T cells for administration to a subject as, for example, an adoptive cell therapy. The disclosure provides a solution to this long-felt but unmet need.
SUMMARY
[05] Unlike traditional biologics and chemotherapeutics, modified-T cells of the disclosure possess the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, modified-T cells of the disclosure must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses. Thus, intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory cells, especially stem cell memory (Tscm).
Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal Date Recue/Date Received 2021-01-15 and multipotent capacity to derive central memory (Tcm), effector memory (Tam) and effector T cells (TE), thereby producing better tumor eradication and long-term modified-T cell engraftment Modified-T cells of the disclosure include, but are not limited to, those cells that express an antigen receptor comprising a protein scaffold of the disclosure.
Modified-T cells of the disclosure include, but are not limited to, those cells that express a chimeric antigen receptor (CAR) (i.e. CAR-T cells of the disclosure). 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 VHF-I, 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).
[061 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 produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a modified stem memory T cell (Tscm). The disclosure provides a method of producing a plurality of modified stem memory T cells (Tscm), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposasc composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or

- 2 -Date Recue/Date Received 2021-01-15 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 (1'scm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm).
In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stern memory T
cells (Tscm). In certain embodiments, the method produces a plurality of modified T
cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and 1L-21213. 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 plasmid DNA
transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two

- 3 -Date Recue/Date Received 2021-01-15 cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac Tm or a Super piggyBacTM
(SPB) transposase.
[081 In certain embodiments of the methods of the disclosure, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac' or a Super piggyBac Tm (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBaclm (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.
[091 In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBacTM (PB) transposase enzyme. The piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:

4).
[0101 In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac Tm (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:

Date Recue/Date Received 2021-01-15 541 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).
[012] in certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBacTm (SPB) transposase enzyme. In certain embodiments, the Super piggyBacTM (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 4 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBacTM (SPB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

- 5 -Date Recue/Date Received 2021-01-15 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
5).
10131 In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacThl or Super piggyBacTm transposase enzyme may further comprise an amino acid substitution at one or more of positions 3,46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ
ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm or Super piggyBaerm 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 swine (5) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 4 or SEQ TD NO: 5 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of

- 6 -Date Recue/Date Received 2021-01-15 SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a phenylalanine (F).
In certain embodiments, the amino acid substitution at position 180 of SEQ ID
NO: 4 or SEQ
ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenylalanine (F).1n certain embodiments, the amino acid substitution at position 207 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a prolinc (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ Ill 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 lcucinc (L). In certain embodiments, the amino acid substitution at position 296 of SEQ Ill

- 7 -Date Recue/Date Received 2021-01-15 NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) fora 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 (1). 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 ty'rosine (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 (S). hi 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 mcthioninc (M). In certain embodiments, the amino acid substitution at position 552 of SEQ

- 8 -Date Recue/Date Received 2021-01-15 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).
[0141 In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super piggyBacTm transposase enzyme may further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ
ID NO: 4 or SEQ ID NO: 5. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at two, three, four. five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super piggyBacTm transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450,509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID
NO: 5. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparaginc (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ 11) NO: 5 is a substitution of a scrim (S) for an

- 9 -Date Recue/Date Received 2021-01-15 asparagine (N). In certain embodiments, the piggyBacTm transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID
NO: 4. In certain embodiments, including those embodiments wherein the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID
NO: 4, the piggyBacTm transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
in certain embodiments, the piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, and a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 4. In certain embodiments, the piggyBacTm transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ
ID NO: 4 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 4.
[015] The disclosure provides a method of producing a modified stem memory T
cell (Tscm), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase: to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a modified stem memory T cell (Tscm). The disclosure provides a method of producing a plurality of modified stem memory T cells (Tscm), comprising introducing into a plurality of primary human T
cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%,

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 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 2021-01-15 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 (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 stein memory T cell (TscA), 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, 1L-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).
10161 In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

- 11 -Date Recue/Date Received 2021-01-15 241 FQMDNDPKHT SKVVAI<WLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL
301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 6).
[0171 In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 7).
[0181 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 stern 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 A, 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 I 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 2021-01-15 61 CCCACCAGCC AATCAGAAGT GACTATGCAA. ATTAAC:CCAA CAAAGATGGC AGTTAAATTT

421 GTCCCACCCC CACA.GAATCA GCCAGAATCA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG

541 TCTAAA.GAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG AAAGATGCCG GCGTTATCGA
601 CAGAAAATGT CTGCA.GAGCA ACGTGCGTCT GATCTTGAAA GAAGGCGGCG CCTGCAACAG

841 CTTCTCAGCA AAAATGGAGT ACA.TGAGGAT GCAATTCTCG AACATAGTTG TGGTGGAATG

1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AG.AATACACG GACAAGTTTA TCACCGTACT

1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA. TCTGAAGCAG CAGCAAAAGG TATTGCTCCC

1621 CAAA_TCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT ATCCTATTCT TTTTCCACAT
1681 GGTGAAAAAG GCTGGGGPAC AGATATTGCA TTAAGACTC.A GAGACAACAG TGTAATCGAC

1981 AATGACA_ATG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA GGGTAGTCCC

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

Date Recue/Date Received 2021-01-15 2821 TATGTGAGCG CT CCT GAGGC TGTTTGGAGA CTTTTTGCAA. TGC GAATG CA TGACCAATCT

3001 TT CTTATT GA ATAGA.GAAGA TTCT GAT GCA CGTAATTATT ATTATTGGGA GATT CCACAG

3121 GGTAGACT GT TCACTGTGAG CTTTAGAGAA. C CAGAAC GAT ATTACCTTAG ACTTTTGCTT
3181 CT GCATGTAA AAGGTGCGA.T AAGTTTT GAG GATCT GC GAA. CT GTAGGAGG TGTAACTTAT
3241 GATAC.ATTTC AT GAAGCT G C TAAACACCGA GGATTATTAC TT GATGACAC TAT CTGGAAA

3541 CATTTCAAAC TT CCG GACT.A TCCTTTATTA ATGAA.TGC.AA ATACAT GT GA TCAA.TTGTAC

3661 CAGACTATAA CTTCA.GCCAT C GAAGAT CAA ACTGTACACC CCAAATGCTT TTTCTTGGAT

3901 AGACTCGATA. TAAAGAGT GA AGTTGCTAAA ACCATTAAAA AGGCCCAACT T CT CATTATT

4321 GAAGCTAC CT TT GGAAATAG TATATCTATA GATAATATTA .AAAATATATC TAAACGTGCA
4381 ATTCTTTGTC CAAAAAAT GA GCAT GTT CAA. AAATTAAATG AAGAAATTTT GGATATACTT

4561 AAATTAAAAT TGAAAGTGGG TGCAATCATC ATGCTATT GA GAAATCTTAA. TAGTAAATGG

Date Recue/Date Received 2021-01-15 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.
[0211 An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:

1081 GRGGTVLPMk STGIAANLLL GGRTFHSQYK LPIPLNETSI SRLDIKSEVA KTIKKAQLLI

Date Recue/Date Received 2021-01-15 1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVINVN TSSQGKLVICi SESVFTLNVV YRE I LE (SEQ ID
NO: 28).
[0221 In Helitron transpositions, a hairpin close to the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5'-TC/CTAG-3' motif. A 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence GTGCACGAAT TTC GT GCACCGGGC CACTAG (SEQ ID NO: 29).
[0231 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 transposasc composition comprising a transposase or a sequence encoding the transposasc; 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 1' 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 Date Recue/Date Received 2021-01-15 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 (1'scm), 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 (TscNi), 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 stern memory T
cells (Tscm). In certain embodiments, the method produces a plurality of modified T
cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L
and CD45RA. In certain embodiments, the cell-surface markers of the CAR-TscNi comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2143. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD45RA, CD95, 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.
(0241 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 lcilobases 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:

Date Recue/Date Received 2021-01-15 FEVLASAMN

SEGCGEGSDG
301 VEFQDASR.VL DQDDGFEFQL PKHQKCACHL LNLVS SVDA.Q KALSNEHYKK LYRSVFGKCQ

RVHLE
541 PLDHKKELAN S SSDDEDFFA SLKPTTHF.AS KELDGYLACV S DT RESLLT F P.AIC S LS I
KT
601 NT P LPASAAC ERLFSTAGLL FS PKRAPLDT NNFENQLLLK LNLRFYN FE (SEQ ID NO: 30).

[025] An exemplary, To12 transposon of the disclosure, including inverted repeats, subterininal sequences and the To12 transposase, is encoded by a nucleic acid sequence comprising the following:

181 TTATTTTTTG GAGA.TCACTT CATTCTATTT TCCCTTGCTA TTACCAAA.CC .AATTGAATTG

361 TTACCACAAT GCACA.GCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAA.TCAGTG

541 AAATAAAGAT TCATTC.AAGA TGAAATGTGT C CTCT GT CTC CC GCTTAATA AAGAAATATC

1021 TTAAATAACC AT GAGCAATA CATTTGTTAC T GTAT CT GTT .AAT CTTTGTT AACGTTAGTT
1081 AATAGAAATA CAGAT GTT CA TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAA.TGTT
1141 AACAAGATAT AAAGTATTAG TAAATGTTGA AA.TTAACATG TATACGTGCA GTTCATTATT

C

1381 TCAA_CAAGTA TTTAACATTA TAAAGTGTGC AATT GGCT GC AAATGTCAGT TTTATTAAAG

Date Recue/Date Received 2021-01-15 2041 GAGTCATTAA. TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA

2461 GATCATGAAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACC,ACAAC

3061 AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT TTAAGGCCAA ACC.AAACGCG

3601 ATGATGAAAC CATCATAAAA CG.AGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC

3901 GATGTAGATG ACTGCACGTA, AATGTAGTTA ATGACAAAAT CCATAAAATT TGTTCCCAGT

Date Recue/Date Received 2021-01-15 4201 GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCA.TC GGCTGCCTGT GAGAGGCTTT

4441 AACCTTGTAT GC.ATTTCATT TAATGTTTTT TGAGATTAAA. AGCTTAAA.CA .AGAATCTCTA

4681 TG (SEQ ID NO: 31).
10261 The disclosure provides a method of producing a modified central memory T-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 central memory T-cell (Tem), thereby producing a modified central memory T-cell (Tem). The disclosure provides a method of producing a plurality of modified central memory 1-cells (Tem), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells; wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a central memory T-cell (Teri), thereby producing a plurality of modified central memory T-cells (Teti). 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 1-cell (Tem), thereby producing a plurality of modified central memory 1-cells (Tem). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tem), thereby producing a plurality of modified central memory 1-cells (Tem). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory T-cell (Tem), thereby producing a plurality of Date Recue/Date Received 2021-01-15 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 (Tem). 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 (Tem). In certain embodiments, the method produces a plurality of modified T
cells, wherein at least 90% of the plurality of modified T cells expresses one or more cell-surface marker(s) of central memory 1-cell (Tem), thereby producing a plurality of modified central memory T-eens (Tem). 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 1-cells (Tem). In certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, CCR7, and CD62L. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBacTM or a Super 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 Beaut 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 1o12 transposon, the transposase is a To12 transposase.

Date Recue/Date Received 2021-01-15 [027] 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 1-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, CD95,11-2RP, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified Tscm and a plurality of modified TCM. In certain embodiments of this method, the modified stem memory T-cells (Tscso) 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 1-cells (Tcm) comprise at least 1%, 2%, 5%, r/i), 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% 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 (Tcm) 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 (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 Date Recue/Date Received 2021-01-15 (Tscm) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (1cm) 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 ntunber of cells of the composition and the modified central memory T-cells (Tem) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory 1-cells (Tsmi) 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 (Tcm) 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 piegyBac transposon, the transposase is a piggyBacTM or a Super piggvBacTM (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 (SBIOOX ). In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a To12 transposon. In certain embodiments, including those embodiments wherein the transposon is a To12 transposon, the transposase is a To12 transposase.
10281 In certain embodiments of the methods of the disclosure, the transposon may be derived or recombined from any species. Alternatively, or in addition, the transposon may be synthetic.
[029] in certain embodiments of the methods of the disclosure, the antigen receptor is a 1-cell receptor. hi 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 T-cell receptor is not naturally-occurring, the T-eell receptor comprises one or more mutation(s) compared to a wild-type 1-cell receptor. In Date Recue/Date Received 2021-01-15 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 CARTynn. 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 further comprises introducing into the primary human T
cell (d) a second transposase composition comprising a transposase or a sequence encoding the transposase. In certain embodiments, the second transposase composition transposes the transposon of (c). In certain embodiments, the transposase composition of (b) transposes the transposon of (a) and the transposase composition of (d) transposes the transposon of (c). In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac Tm 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 Date Recue/Date Received 2021-01-15 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 1-cell, wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a stem memory T
cell (Tscm), thereby producing a modified 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 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 plurality of activated modified T-cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of activated modified 1 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 (fscm), thereby producing a plurality of activated modified stem memory T
cells (Tsai). In Date Recue/Date Received 2021-01-15 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 (1'scm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 85% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T
cells (Tscm). In certain embodiments, the method produces a plurality of activated modified T
cells, wherein at least 95% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated modified stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated modified Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R13. In certain embodiments, the cell-surface markers of the activated modified Tscm comprise one or more of CD45RA, CD95, CR7, and CD62L.
[032] In certain embodiments of the methods of the disclosure of producing a modified stem memory T cell (Tscm), comprising: (a) introducing into a primary human T
cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified 1-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, 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 I-cell and a 'F-cell expansion Date Recue/Date Received 2021-01-15 composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified 1-cells expresses one or more cell-surface marker(s) of a stem memory T
cell (Tscm). In certain embodiments of this method, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface 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 1-cells that express cell-surface marker(s) of a stem memory T cell (Tam). 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 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 1-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 1-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramediy1-5-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 1-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 Date Recue/Date Received 2021-01-15 a concentration of between 0.2 me/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 T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 innol/kg and 640 ttmol/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.7 ttmol/kg and 70 innol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 ttmol/kg and 75 timol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 pniol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 tunol/kg and 25 ttmol/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 timol/kg. palmitic acid at a concentration of about 7 ttmol/kg, linoleic acid at a concentration of about 7.5 gmol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 timol/kg.
10331 The disclosure provides a method of producing a modified central memory T-cell (Tem), comprising: (a) introducing into a primmy 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, wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a central memory T-cell (Tcm), thereby producing a central memory T-cell (Tai). The disclosure provides a method of producing a plurality of modified central memory T-cell (Tcm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified I cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified 1-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospccific tetrameric antibody complex, an anti-human CD28 monospccific Date Recue/Date Received 2021-01-15 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 1-cell (Tcm), thereby producing a plurality of activated modified central memory 1-cell (Tcm). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tem), thereby producing a plurality of activated modified central memory T cell (Tem). in certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 50% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (To ...1), thereby producing a plurality of activated modified central memory T cell (Tem). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T
cell (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 (Toil), thereby producing a plurality of activated modified central memory T cell (Tem). In certain embodiments, the method produces a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a plurality of activated modified central memory T
cell (Tar). 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 950/0 of the plurality of activated modified T cells expresses one or more cell-surface marker(s) of a central memory I cell (Tcm), thereby producing a plurality of activated Date Recue/Date Received 2021-01-15 modified central memory T cell (Tcm). In certain embodiments, the cell-surface markers of the activated modified Tcm comprise one or more of CD45RO, CD95, IL-2R13, CCR7, and CD62L.
[034] In certain embodiments of the methods of the disclosure of producing a modified central memory T cell (Tcm), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, this method further comprises the step of (c) contacting the activated modified 1-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (Tc:m). 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 (Tcm). 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 (Tcm). 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%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified 1-cells that express cell-surface marker(s) of a central memory T cell (Tcm). 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 60% of modified 1-cells that express cell-surface marker(s) of a central memory T cell (Tcm). In certain embodiments of this method, the enriching step comprises isolating modified 1-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the plurality of enriched modified 1-cells.
In certain embodiments of this method, the enriching step further comprises contacting the Date Recue/Date Received 2021-01-15 isolated modified Tcm and a T-cell expansion composition comprising one or more of human senun albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified Tem. 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-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 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 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 ttmol/kg and 70 ttmol/lcg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 ttmol/Icg and 75 tunol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 ttmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 ttmol/kg and 25 timol/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 fAmol/kg, palmitic acid at a concentration of about 7 timol/kg, linoleic acid at a concentration of about 7.5 ttmol/kg, oleic acid at a concentration of about 7.5 1=01/kg and a sterol at a concentration of about 2.5 ttmol/kg.
[0351 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 Date Recue/Date Received 2021-01-15 (Tcm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (Tscm) and a plurality of activated modified central memory T-cells (Tcm), wherein the plurality of activated modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RP and the plurality of activated modified Tcm expresses one or more CD45RO, CD95, 1L-2R(, CCR7, and CD62L, thereby producing a composition comprising a plurality of modified TSCM and a plurality of modified TON. 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 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 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 T-cells (Tscm) comprise at least 20% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 80% of the total number of cells of the composition.
In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 80% of the total number of cells of the composition and the modified central memory I-cells (Tcm) comprise at least 20% of the total number of cells of the composition.
In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells Date Recue/Date Received 2021-01-15 (Tem) comprise at least 70% of the total number of cells of the composition.
In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 70% of the total number of cells of the composition and the modified central memory T-cells (Teti) comprise at least 30% of the total number of cells of the composition.
In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 60% of the total number of cells of the composition.
In certain embodiments of this method, the modified stem memory 1-cells (Ism) comprise at least 60% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 40% of the total number of cells of the composition.
In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 50% of the total number of cells of the composition.
[0361 In certain embodiments of methods of the disclosure of producing a composition comprising a plurality of modified stem memory T-cells (Taw) and a plurality of modified central memory 1-cells (Tem), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T
cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a 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 1-cells (Tsem) and a plurality of activated modified central memory 1-cells (Tem), the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, this method further comprises the step of (c) contacting the composition the plurality of activated modified stem memory 1-cells (Tsem) and the plurality of activated modified central memory T-cells (Teri) with a 1-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoedianol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein the plurality of expanded modified Tsem expresses one or more CD62L, CD45RA, CD28, CCR7, CDI27, CD45RO, CD95, CD95 and EL-2R p and the plurality of expanded modified Tem expresses one or more CD45RO, CD95, 1L-2R(, CCR7, and CD62L, thereby producing a composition Date Recue/Date Received 2021-01-15 comprising a plurality of expanded modified Tsem and a plurality of expanded modified Tem.
In certain embodiments of this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Tsem) from the plurality of enriched modified T-cells or isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tem) from the plurality of enriched modified 1-cells. In certain embodiments of this method, the enriching step comprises isolating modified T-cells that express one or more cell-surface marker(s) of a stem memory T cell (Isem) from the plurality of enriched modified 1-cells and isolating modified T-cells that express one or more cell-surface marker(s) of a central memory T cell (Tem) from the plurality of enriched modified T-cells. In certain embodiments of this method, the enriching step further comprises contacting the composition comprising the isolated modified TSCM
and the isolated modified Tem with a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a composition comprising a plurality of expanded enriched modified Tscm and a plurality of expanded enriched modified Tem. 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 1-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 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 nig/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 compriscs one or more of octanoic acid at a concentration of between 6.4 pmol/kg Date Recue/Date Received 2021-01-15 and 6401/m01/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 iimol/kg and 70 pniol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pinol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 umol/lcg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 mol/kg and 25 i/mol/kg, inclusive of the endpoints.
In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 1/m01/kg, palmitic acid at a concentration of about 7 umol/kg, linoleic acid at a concentration of about 7.5 i/mol/kg, oleic acid at a concentration of about 7.5 umol/kg and a sterol at a concentration of about 2.5 umol/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 (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 (Taw) comprise at least 10% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 90% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory 1-cells (Tscm) comprise at least 90% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 10% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory 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 (Tsem) comprise at least 80% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 20% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory 1-cells (Tsem) comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells (Tcm) comprise at least 70% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory 1-cells (Tam) comprise at least 70% of the total number of cells of the composition and the Date Recue/Date Received 2021-01-15 modified central memory T-cells (Tem) comprise at least 30% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory 1-cells (Tscm) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified 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 (Tem) 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 (Tem) comprise at least 50% of the total number of cells of the composition.
P7] In certain embodiments of the methods of producing an activated modified Tscm or Teri 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 1-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 I cell of the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of a portion of primary T cells of the plurality of T cells. In certain embodiments, the portion of primary T
cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of the total number of primary T cells in the plurality of T cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition contacts a genomic sequence of each primary T cell of the plurality of T
cells. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a single strand break. In certain embodiments of the introduction step comprising a homologous recombination, a genomic editing composition induces a double strand break. In certain embodiments of the introduction step comprising a homologous recombination, the introduction step further comprises a donor sequence Date Recue/Date Received 2021-01-15 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 stein memory T cells (1'scm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsai), thereby producing a plurality of modified stem memory T cells (Tsai).
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 (Tsai). 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 (Tsai), thereby producing a plurality of modified stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L
and CD45RA. In certain embodiments, the cell-surface markers of the CAR-Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RD. In certain embodiments, the cell-surface markers of the CAR-TscNi comprise one or more of CD45RA, CD95, IL-2110, CR7, and CD62L. In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a Helraiser transposon, the transposase is a Helitron transposase.
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 2021-01-15 composition. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding the antigen receptor, a 5' genomic sequence and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 5' to the break point induced by the genomic editing composition and the 3' genomic sequence is homologous or identical to a genomic sequence of the primary T cell that is 3' to the break point induced by the genomic editing composition. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition and donor sequence composition are contacted with the genomic sequence sequentially, and the genomic editing composition is provided first. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introduction step comprising a homologous recombination, the genomic editing composition comprises a DNA
binding domain and a nuclease domain. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a TALEN. In certain embodiments of the genomic editing composition, the DNA
binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genomic editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof.
In certain embodiments of the genomic editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic Acid (D) at position 10 (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 Asparaginc (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a type IIS
endonuclease. In certain embodiments of the genomic editing composition, the type IIS
endonuclease comprises Acil, Mn11, Alw1, Bbvl, Bccl, BecAl, BsmAl, BsmF1, BspCN1, Date Recue/Date Received 2021-01-15 BsrI, BtsCI, Hgal, HphI, HpyAV, Mboll. My II, Pie!, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, Bpml, BpuEI, BsaI, BseRI, Bsgl, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsT, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQl, Sap!, Baei, BsaXI, CspCI, Bfii, Mboll, Acc361, Fokl or Clo051. In certain embodiments, the type IIS endonuclease comprises C1o05 I. 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.
[0381 in certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments, the viral vector comprises one or more sequences isolated, derived, or recombined from an RNA virus. hi 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 'F-cell activator Date Recue/Date Received 2021-01-15 composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, a viral vector comprises the antigen receptor. In certain embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus.
In certain embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus.
[040J In certain embodiments of the methods of producing an activated modified Tscm or 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 tmnsposon, 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 l 0 or AAV 1 I. 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, AAV I 0 or AAV11. In certain embodiments, the AAV comprises a sequence isolated, derived, or recombined from AAV2.
In certain embodiments, including those in which the vector crosses the blood brain barrier Date Recue/Date Received 2021-01-15 (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.
[0431 In certain embodiments of the methods of producing an activated modified Tscm or Tc.µm 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.
10441 In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHH
sequence(s). In certain embodiments, die CAR is a VCAR.

Date Recue/Date Received 2021-01-15 10451 In certain embodiments of the methods of producing an activated modified Tscm or Tcm of the disclosure, including those methods comprising (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement, the method further comprises introducing into the primary human T
cell, a composition comprising a therapeutic protein to produce a modified T
cell capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretable protein and the method produces a modified T cell capable of secreting the therapeutic protein. In certain embodiments, the introducing step comprises a homologous recombination and a donor sequence comprises a sequence encoding the therapeutic protein.
In certain embodiments, the donor sequence that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first donor sequence comprises the antigen receptor and a second donor sequence comprises the therapeutic protein. In certain embodiments, a vector comprises a sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle. In certain embodiments, the vector that comprises the antigen receptor further comprises the therapeutic protein. In certain embodiments, a first vector comprises the antigen receptor and a second vector template comprises the therapeutic protein.
10461 The disclosure provides a method of producing a modified stem memory T
cell (Tscm), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetraineric 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, Date Recue/Date Received 2021-01-15 and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T
cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a modified stem memory T cell (Tscm). In certain embodiments of this method, at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a stern memory T cell (Tscm). In certain embodiments of this method, the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. The disclosure provides a method of producing a modified stem memory T cell (Tscm), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-T cell and (b) contacting the CAR-T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex. an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce an activated CAR-T cell, wherein the activated CAR-T cell expresses one or more cell-surface marker(s) of a stem memory T
cell (Tscm), thereby producing a CAR-expressing stem memory T cell (Tscm) (CAR-Tscm). The disclosure provides a method of producing a plurality of modified stem memory T cells (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 (Tsai), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the methods further comprises the step of: (c) contacting the activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, lscove's MDM, Date Recue/Date Received 2021-01-15 and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-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 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 T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 lumol/kg and 640 ttmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 ttmol/kg and 70 ttmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 tunol/kg and 75 tunol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 !Amol/kg and 75 p.mol/kg, inclusive of the endpoints; and a sterol at a concentration of Date Recue/Date Received 2021-01-15 between 0.25 mol/kg and 25 mol/ke, 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 umol/kg, palmitic acid at a concentration of about 7 timol/kg, linoleic acid at a concentration of about 7.5 umol/lcg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 pinol/ka. In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 tunol/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of about 7.56 mol/kg and a sterol at a concentration of about 2.61 timol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of 7.56 mol/kg and a sterol at a concentration of 2.61 mol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40 /o, 45%, 50%, 600/c, 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 stem memory T cell (Tscm). In certain embodiments, at least 60%
of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T
cell (Tscm). In certain embodiments, the method further comprises the step of:
(d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45olo,õ
50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, the method further comprises the step of: (d) enriching the plurality of expanded modified 1-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 1-cells. In certain embodiments, the enriching step further comprises contacting the isolated modified Tscm and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transfcrrin, 2-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, nicotinamidc, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (1'MDD), diisopropyl adipate Date Recue/Date Received 2021-01-15 (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. in certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g.
cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg =
parts per million). In certain embodiments, the 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 tug/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 timol/Icg and 640 mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 ttmol/kg and 70 ttmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 p.mol/kg and 75 ttmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 tunol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 timol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 mol/kg, palmitic acid at a concentration of about 7 mong, linoleic acid at a concentration of about 7.5 timol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 p.mol/kg. In certain embodiments, the 1-cell expansion Date Recue/Date Received 2021-01-15 composition comprises one or more of octanoic acid at a concentration of about 63.75 palmitic acid at a concentration of about 7.27 p.mol/kg, linoleic acid at a concentration of about 7.57 p.mol/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 ilmol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 vmol/kg, palmitic acid at a concentration of about 7.27 innol/kg, linoleic acid at a concentration of about 7.57 umol/kg, oleic acid at a concentration of 7.56 ttmol/kg and a sterol at a concentration of 2.61 umol/kg.
[047] The disclosure provides a method of producing a modified central memory T cell (Tcm), comprising: (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, wherein the activated modified-T cell expresses one or more cell-surface marker(s) of a central memory T cell (Tem), thereby producing a modified central memory T cell (Tem). The disclosure provides a method of producing a plurality of modified central memory T cells (Tem), comprising:
(a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a plurality of modified T cells, wherein a transposon comprises the antigen receptor, and (b) contacting the plurality of modified T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified -T
cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm), thereby producing a modified central memory T cell (Tcm). In certain embodiments of this method, at least 60% of the plurality of activated modified -T cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm). hi certain embodiments of this method, the T-cell activator composition of (b) further comprises an anti-human CD2 monospccific tetrameric antibody complex. In certain embodiments, the methods further comprises the step of (c) contacting the activated modified T cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, htunan Date Recue/Date Received 2021-01-15 transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of expanded modified 1-cells expresses one or more cell-surface marker(s) of a central memory T cell (Teri). 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-decyri-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonarnide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, the 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 wig/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 ing/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 prnol/kg and 640 timol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 iimol/kg and 70 pinol/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 gmol/kg and 75 Litnol/kg, inclusive of the endpoints; and a Date Recue/Date Received 2021-01-15 sterol at a concentration of between 0.25 1=01/kg and 25 ttmol/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 ttmol/kg, palmitic acid at a concentration of about 7 mol/kg, linoleic acid at a concentration of about 7.5 ttmol/kg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 mol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 ttmol/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 mol/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 pinol/kg, palmitic acid at a concentration of about 7.27 tunol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of 7.56 timol/kg and a sterol at a concentration of 2.61 mol/kg. hi certain embodiments, at least 2%. 5%, 10%, 15%, 20%, 25%, 30 /o, 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 (Teri). In certain embodiments, at least 60% of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, the method further comprises the step of (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified 1-cells that express cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, the method further comprises the step of: (d) enriching the plurality of expanded modified T-cells to produce a composition comprising at least 60% of modified T-cells that express cell-surface marker(s) of a central memory T cell (Tem). In certain embodiments, the enriching step further comprises isolating modified 1-cells that express one or more cell-surface marker(s) of a central memory T cell (Tcm) from the plurality of enriched modified 1-cells.
In certain embodiments, the enriching step further comprises contacting the isolated modified Tem and a 1-cell expansion composition comprising one or more of htunan serum albumin, recombinant human insulin, human transfcrrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded enriched modified Tem.
hi certain embodiments, the 1-cell expansion composition further comprises one or more of octanoic acid, nicotinamidc, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (1'MDD), diisopropyl adipatc Date Recue/Date Received 2021-01-15 (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. in certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g.
cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;
oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg =
parts per million). In certain embodiments, the 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 tug/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 timol/Icg and 640 timol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 tunol/kg and 70 timol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 timol/kg and 75 timol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 tunol/kg and 75 timol/kg, inclusive of the endpoints; and a sterol at a concentration of 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 molikg, linoleic acid at a concentration of about 7.5 timol/kg, oleic acid at a concentration of about 7.5 pmol/kg and a sterol at a concentration of about 2.5 timol/kg. In certain embodiments, the 1-cell expansion Date Recue/Date Received 2021-01-15 composition comprises one or more of oetanoic acid at a concentration of about 63.75 timol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of about 7.56 pmol/kg and a sterol at a concentration of about 2.61 timol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 pmol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 ttmol/kg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 innol/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 (Tem), comprising: (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor to produce a composition comprising a plurality of modified stem memory T-cells (Tscm) and a plurality of modified central memory T-cells (Tcr4).
wherein a transposon comprises the antigen receptor, and (b) contacting the composition and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex. an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T-cells (Tscm) and a plurality of activated modified central memory 1-cells (Tcm), wherein the plurality of activated modified Tscm expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R[3 and the plurality of activated modified Tcm expresses one or more CD45RO, CD95, IL-21213, 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-Mercaptocthanol, Iscovc's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the composition comprising a plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, the methods further comprises the step of: (c) contacting the composition and a 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 Date Recue/Date Received 2021-01-15 supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the composition comprising a plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a central memory T cell (Tcm). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethy1-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonarnide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a steml (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 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 T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 lumol/kg and 640 timol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 uniol/kg and 70 gmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 tunol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 !Amol/kg and 75 gmol/kg, inclusive of the endpoints; and a sterol at a concentration of Date Recue/Date Received 2021-01-15 between 0.25 timol/kg and 25 timol/ke, 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 umol/kg, palmitic acid at a concentration of about 7 timol/kg, linoleic acid at a concentration of about 7.5 tunol/kg, oleic acid at a concentration of about 7.5 mol/kg and a sterol at a concentration of about 2.5 tunolika. hi certain embodiments, the 1-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 timol/kg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of about 7.56 tunol/kg and a sterol at a concentration of about 2.61 timol/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 p.mol/kg, palmitic acid at a concentration of about 7.27 'Among, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of 7.56 tunol/kg and a sterol at a concentration of 2.61 limol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40 /o, 45%, 50%, 600/c, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%
or any percentage in between of cells the composition comprising a plurality of expanded modified Tscm and a plurality of expanded modified Tcm expresses cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 99% or any percentage in between of cells the composition comprising a plurality of expanded modified Tscm and a plurality of expanded modified Tcm expresses cell-surface marker(s) of a central memory T
cell (Tavi). In certain embodiments, the method further comprises the step of:
(d) enriching the composition to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of modified T-cells that express cell-surface marker(s) of a stem memory T cell (Tscm). In certain embodiments, the method further comprises the step of: (d) enriching the composition to produce a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%

or any percentage in between of modified T-cells that express cell-surface marker(s) of a central memory T cell (Tem). 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 (Tsem) from the composition or isolating modified 1-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 I-cells that express one Date Recue/Date Received 2021-01-15 or more cell-surface marker(s) of a stem memory, T cell (Tsai) from the composition and isolating modified 1-cells that express one or more cell-surface marker(s) of a central memoly 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 Tscvi and/or Tcm. 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-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide. 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hOrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg. inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg = parts per million). In certain embodiments, the 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 T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linolcic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 Date Recue/Date Received 2021-01-15 pmol/kg and 640 mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 timol/kg and 70 mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol/kg and 75 tiniolikg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 tunol/kg and 75 tunol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 mol/kg and 25 pmol/kg, inclusive of the endpoints. In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 mol/kg, palmitic acid at a concentration of about 7 timol/lcg, linoleic acid at a concentration of about 7.5 pmol/kg, oleic acid at a concentration of about 7.5 p.mol/kg and a sterol at a concentration of about 2.5 timol/kg. In certain embodiments, the 1-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27 moL/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of about 7.56 timol/lcg 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 timol/kg, palmitic acid at a concentration of about 7.27 inol/kg, linoleic acid at a concentration of about 7.57 pmol/kg. oleic acid at a concentration of 7.56 mol/kg and a sterol at a concentration of 2.61 mol/kg. In certain embodiments of this method, the modified stein 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 1-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 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 1-cells (Tscm) comprise at least 90% of the total number of cells of the composition and the modified central memory 1-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 1-cells (Tcm) comprise at least 80% of the total number of cells of the composition. In certain embodiments of this method, the modified Date Recue/Date Received 2021-01-15 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 (Tem) comprise at least 20% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 30% of the total number of cells of the composition and the modified central memory T-cells (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 (Tem) comprise at least 30% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 40% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 60% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tsem) comprise at least 60% of the total number of cells of the composition and the modified central memory 1-cells (Tcm) comprise at least 40% of the total number of cells of the composition. In certain embodiments of this method, the modified stem memory T-cells (Tscm) comprise at least 50% of the total number of cells of the composition and the modified central memory T-cells (Tem) comprise at least 50% of the total number of cells of the composition.
10491 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 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 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 I cell capable of secreting the therapeutic protein. In certain embodiments, the method further comprises introducing a transposase composition. In certain embodiments, the transposase composition transposes the transposon of (a) and the second transposon. In certain cmbodimcnts, the method comprises introducing a first transposase composition and a Date Recue/Date Received 2021-01-15 second transposase composition. In certain embodiments, including those wherein the method comprises introducing a first transposase composition and a second transposase composition, the first transposase composition transposes the transposon of (a) and the second transposase composition transposes the second transposon. In certain embodiments of this method, the transposon is a plasmid DNA transposon with a sequence encoding the antigen receptor or the therapeutic protein flanked by two cis-regulatory insulator elements.
In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac Tm 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.
10501 In certain embodiments of the methods of the disclosure, including those wherein the method comprises introducing into a primary human T cell (a) introducing into a primary human T cell a composition comprising an antigen receptor to produce a modified T cell, wherein a transposon comprises the antigen receptor, and (b) contacting the modified T cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell, the method further comprises introducing into the primary human T cell a sequence encoding a therapeutic protein, to produce a modified T cell, wherein the modified T cell is capable of expressing the therapeutic protein. In certain embodiments of introducing a sequence encoding a therapeutic protein, the introducing step comprises a homologous recombination.
In certain embodiments of introducing a sequence encoding a therapeutic protein, a vector comprises the sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector, hi certain embodiments, the vector is a nanoparticle.

Date Recue/Date Received 2021-01-15 [051] In certain embodiments of the methods of the disclosure, the introducing step further comprises a composition comprising a genomic editing construct. In certain embodiments, the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CItISPR) associated protein 9 (Cas9) DNA
endonuclease. In certain embodiments, the genomic editing construct comprises a DNA binding domain and a type TIS endonuclease. In certain embodiments, the genomic editing construct encodes a fusion protein. In certain embodiments, the genomic editing construct encodes the DNA
binding domain and the type IIS endonuclease and wherein the expressed DNA
binding domain and the expressed type ITS endonuclease are non-covalently linked. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a Cas9 endonuclease. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a Cas9 endonuclease is the DNA binding domain. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain, the sequence derived from a Cas9 endonuclease encodes an inactive Cas9. In certain embodiments, including those embodiments wherein the sequence derived from a Cas9 endonuclease is the DNA
binding domain, the sequence derived from a Cas9 endonuclease encodes a truncated Cas9. In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D10A).
In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for a Histidine (H) at position 840 (H840A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dCas9 (SEQ ID
NO: 33). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an Alanine (A) for an Asparagine (N) at position 580 (N580A).
In certain embodiments, the sequence derived from a Cas9 endonuclease comprises dSaCas9 (SEQ ID NO: 32). In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS
endonuclease, the gcnomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN). In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS
endonuclease, the sequence derived from a TALEN is the DNA binding domain. In certain embodiments, Date Recue/Date Received 2021-01-15 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 piggyl3acTm (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBacTM (SPB) transposase, the sequence encoding the transposase is an mRNA

sequence. In certain embodiments, the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4. In certain embodiments, the piggyBac transposase is a hyperactive variant and the hyperactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282 and 538 of SEQ ID NO: 4. In certain embodiments, the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine (I) (I30V). In certain embodiments, the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of a serine (S) for a glycine (G) (G1655). In certain embodiments, the amino acid substitution at position 282 of SEQ ID NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V). In certain embodiments, the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for an asparaginc (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 Date Recue/Date Received 2021-01-15 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 ceitain 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.
[0531 In certain embodiments of the methods of the disclosure, the transposon further comprises a selection gene. In certain embodiments, the T-cell expansion composition further comprises a selection agent.
[054] In certain embodiments of the methods of the disclosure, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not naturally-occurring. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments of this method, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR
comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
[0551 In certain embodiments of the methods of the disclosure, the cell-surface markers of the modified Tscm comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the modified Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rii. In certain embodiments, the cell-surface markers of the modified Tscm comprise one or more of CD45RA, CD95, IL-2Ri3, CR7, and CD62L.
[056] In certain embodiments of the methods of the disclosure, the plurality of expanded modified T-cells comprises a naïve T-cell (modified TN) and the cell-surface markers of the CAR-TN comprise one or more of CD45RA, CCR7 and CD62L. In certain embodiments, the plurality of expanded modified T-cells comprises a central memory T-cell (modified Tcm) and the cell-surface markers of the CAR-Tcm comprise one or more of CD45RO, CD95, IL-2R13, CCR7, and CD62L. In certain embodiments, the plurality of expanded modified T-cells comprises an effector memory T-cell (modified TEm) and the cell-surface markers of the CAR-Tim comprise one or more of CD45RO, CD95, and IL-2RO. In certain embodiments, Date Recue/Date Received 2021-01-15 plurality of expanded modified T-cells comprises an effector T-cell (modified TEFF) and the cell-surface markets of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-211.13.
[057] In certain embodiments of the methods of the disclosure, the plurality of expanded modified 1-cells comprises a central memory 1-cell (modified Tem) and the cell-surface markers of the CAR-Tem comprise one or more of CD45RO, CD95, CCR7, and CD62L. In certain embodiments, the most abundant cell in the plurality of expanded modified 1-cells is a central memory 1-cell (modified Tem) 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 T-cells is a central memory T-cell (modified Tcm), the plurality of expanded modified 1-cells comprises a Tsem 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-2RD.
[0581 The disclosure provides a method of producing a modified stem memory I
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 1-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex and an activation supplement to produce 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-Tsem). The disclosure provides a method of producing a plurality of modified stem memory T cells (Isem), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CART
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-1' cells, wherein at Date Recue/Date Received 2021-01-15 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-I cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60%
of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85%
of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). in certain embodiments, the method produces a plurality of activated CAR1' cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2RD. In certain embodiments, the cell-surface markers of the activated CAR Tsai comprise one or more of CD45RA, CD95, IL-2R13, CR7, and CD62L. The disclosure provides a method of producing a modified stem memory T cell (Tsar), comprising: (a) introducing into a primary human T cell a composition comprising a chimeric antigen receptor (CAR) to produce a CAR-I
cell and (b) Date Recue/Date Received 2021-01-15 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).
[0591 The disclosure provides a method of producing a plurality of modified stem memory T cells (Tscm), comprising: (a) introducing into a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T
cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-htunan CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%.
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T
cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 50%
of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory I cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-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 I cells Date Recue/Date Received 2021-01-15 (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stein memory T cells (Tsc.m). hi certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tsar), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95%
of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-212(3. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-2R[3, CR7, and CD62L.
[060] In certain embodiments, this method may further comprise the step of:
(c) contacting the activated CAR-T cell and a T-cell expansion composition comprising one or more of human 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-I 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, linolcic acid, oleic acid and a sterol (e.g.
cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 ing/kg, inclusive of the endpoints;
palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the cndpoints;
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 =

Date Recue/Date Received 2021-01-15 parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 ing/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of about 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 mol/kg and 640 mol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 timol/kg and 70 mol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 gmol/kg and 75 mol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 mol/kg and 75 timol/kg, inclusive of the endpoints; and a sterol at a concentration of 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 ttmol/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 timol/kg, palmitic acid at a concentration of about 7.27 mol/kg, linoleic acid at a concentration of about 7.57 pmol/kg, oleic acid at a concentration of about 7.56 mot/kg and a sterol at a concentration of about 2.61 mot/kg. In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of about 63.75 mol/kg, palmitic acid at a concentration of about 7.27iumol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 tunol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 950/a, 99% or any percentage in between of the plurality of expanded CAR-T cells expresses cell-surface Date Recue/Date Received 2021-01-15 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 Date Recue/Date Received 2021-01-15 comprising a chimeric antigen receptor (CAR) to produce a plurality of CAR-T
cells and (b) contacting the plurality of CAR-T cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR
stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR.-T cells, wherein at least 50% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 80%
of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm), thereby producing a plurality of activated CAR stem memory T cells (Tscm). In certain embodiments, the method produces a plurality of activated CAR-I cells, wherein at least 90% of the plurality of activated CAR-I 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 Date Recue/Date Received 2021-01-15 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-2RD. In certain embodiments, the cell-surface markers of the activated CAR Tscm comprise one or more of CD45RA, CD95, IL-2R13, CR7, and CD62L.
10621 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-Tcm) and the cell-surface markers of the CAR-Tam comprise one or more of CD45RO, CD95, IL-211(3, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector memory T-cell (CAR-Ti) and the cell-surface markers of the CAR-TEm comprise one or more of CD45RO, CD95, and IL-2R13. In certain embodiments, the plurality of expanded CAR-T cells comprises an effector T-cell (CAR-TEFF) and the cell-surface markers of the CAR-TEFF comprise one or more of CD45RA, CD95, and IL-2RD.
10631 In certain embodiments of the methods of the disclosure, a transposon comprises a chimeric antigen receptor (CAR) of the disclosure. The transposon may be a plasmid DNA
transposon with a sequence encoding the CAR flanked by two cis-regulatory insulator elements. In certain preferred embodiments, the transposon is a piggyBac transposon. In certain embodiments, a step introducing a composition comprising a chimeric antigen receptor (CAR) of the disclosure may further a composition comprising an mRNA
sequence encoding a transposase. In certain preferred embodiments, the transposase is a Super piggyBacTm (SPB) transposase.
[064] In certain embodiments, a transposon of the disclosure may further comprise a selection gene. When a transposon of the disclosure comprises a selection gene, the T-cell expansion composition of the methods of the disclosure may further comprise a selection agent to simultaneously select and expand an activated or modified T cell of the disclosure.
[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.

Date Recue/Date Received 2021-01-15 [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-4,7-diol (TMDD), diisopropy-1 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 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 ing/kg, oleic acid at a concentration of about 2.13 ing/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 Date Recue/Date Received 2021-01-15 (wherein rag/kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pinol/kg and 640 pmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol/kg and 70 pinol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pinol/kg and 75 pmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 pinol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 Ltmol/kg and 25 trinol/kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 pmol/kg, palmitic acid at a concentration of about 7 p.mol/kg, linoleic acid at a concentration of about 7.5 gmol/kg. oleic acid at a concentration of about 7.5 timol/kg and a sterol at a concentration of about 2.5 1.tmol/kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 t.imol/kg, palmitic acid at a concentration of about 7.27 pmol/kg, linoleic acid at a concentration of about 7.57 mol/kg, oleic acid at a concentration of about 7.56 trinol/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 mai/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.571.tmol/kg, oleic acid at a concentration of 7.56 pmol/kg and a sterol at a concentration of 2.61 pmol/kg. In certain embodiments of the nucleofection, the transposon composition is a 0.5 pg/til solution comprising nuclease free water and the cuvette comprises 2 t.d of the transposon composition to yield 1 pg of transposon. The transposon composition may comprise a piggyBac transposon. The transposon composition may comprise a Sleeping Beauty transposon. In certain embodiments of the nucleofection, the transposase composition comprises 5 pg of transposase. The transposase composition may comprise a hyperactive piggyBacTm or Super piggyBacTm (SPB) transposase. The transposase composition may comprise a hyperactive Sleeping Beauty (SB100X) transposase.
In certain embodiments, the transposon may comprise a 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.
10671 In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition and a first transposase Date Recue/Date Received 2021-01-15 composition and a composition comprising a plurality of primary human T cells in a cuvette.
In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition, a second transposon composition, a first transposase composition and a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a first transposon composition, a second transposon composition, a first transposase composition, a second transposase composition and a composition comprising a plurality of primary human T cells in a cuvette.
In certain embodiments, the first transposon comprises a sequence encoding an antigen receptor. In certain embodiments, the second transposon comprises a sequence encoding a therapeutic protein. In certain embodiments, the first transposon composition and the second transposon composition are identical. In certain embodiments, the first transposon composition and the second transposon composition are not identical. In certain embodiments, the first transposase mobilizes the first transposon composition and the second transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition but not the second transposon composition. In certain embodiments, the second transposase mobilizes the second transposon composition but not the first transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition and the second transposase mobilizes the second transposon composition. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding an antigen receptor. hi certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments, the first transposon composition comprises a sequence encoding an antigen receptor and the second transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein is a secreted or secretable protein. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection comprises contacting a transposon composition, a first transposase composition, a second transposase composition and a composition comprising a plurality of primary human T cells in a cuvette.
In certain embodiments, the transposon composition comprises a sequence encoding the Date Recue/Date Received 2021-01-15 antigen receptor. In certain embodiments, the transposon composition comprises a sequence encoding the therapeutic protein. In certain embodiments of the methods of the disclosure, including those embodiments wherein the introducing step comprises a nucleofection or an electroporation, the nucleofection further comprises contacting a composition capable of inducing homologous recombination at a specific site in the genome with a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments, the composition capable of inducing homologous recombination comprises an exogenous donor molecule. In certain embodiments, the exogenous donor molecule comprises a sequence encoding the antigen receptor and the transposon comprises a sequence encoding the therapeutic protein. In certain embodiments, the exogenous donor molecule comprises a sequence encoding the therapeutic protein and the transposon comprises a sequence encoding the antigen receptor. In certain embodiments, the composition comprising the transposon, the composition comprising the transposase and the composition capable of inducing homologous recombination at a specific site in the genome are contacted with the composition comprising a plurality of primary human T cells simultaneously. In certain embodiments, the composition comprising the transposon and the composition comprising the transposase are contacted with the composition comprising a plurality of primary human T cells first, and the composition capable of inducing homologous recombination at a specific site in the genome is contacted with the composition comprising a plurality of primary human T cells second. In certain embodiments, the composition capable of inducing homologous recombination at a specific site in the genome is contacted with the composition comprising a plurality of primary human T cells first and the composition comprising the transposon and the composition comprising the transposase are contacted with the composition comprising a plurality of primary human T cells second. In certain embodiments of the methods of producing a modified Tscm of the disclosure, the composition comprising primary human T
cells comprises a buffer that maintains or enhances a level of cell viability and/or a stein-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. hi 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 nucicofcction. In certain embodiments, the buffer comprises a P3 primary cell solution Date Recue/Date Received 2021-01-15 (Lonza). In certain embodiments, the buffer comprises one or more of KC!, MgCl2, ClNa, Glucose and Ca(NO3)2 in any absolute or relative abundance or concentration, and, optionally, the buffer further comprises a supplement selected from the group consisting of HEPES, Tris/HC1, and a phosphate buffer. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCl2, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl2, 90 mM CINa, 10 mM
Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 20 mM HEPES and 75 mM
Tris/HC1. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl2, 90 mM
ClNa, 10 mM Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 40 mM
Na2HPO4/NaH2PO4 at pH 7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 Ill of the buffer and between 5x106 and 25x106 cells.
[0681 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 I.11 of the buffer and between 5x106 and 25x106 cells.
10691 As used herein, the terms "supplemented T-cell expansion composition" or "1-cell expansion composition" may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement at 37 C. Alternatively, or in addition, the terms "supplemented 1-cell expansion composition" or "1-cell expansion composition"
may be used interchangeably with a media comprising one or more of phosphorus, an octanoic fatty acid, a palmitic fatty acid, a linoleic fatty acid and an oleic acid. In certain embodiments, the media comprises an amount of phosphorus that is 10-fold higher than may be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available at 'ThermoFisher Scientific as Catalog number 12440053).
10701 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 '7-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 "1'-cell expansion composition"
may be Date Recue/Date Received 2021-01-15 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 ing/L, potassium at 15 mg/L and calcium at 4 mg/L.
(0711 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 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), 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 tenns "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-dccyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipatc (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 Date Recue/Date Received 2021-01-15 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 -7-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 "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 free amino acids:
histidine, asparagine, serine, glutamate, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine and tryptophan. In certain embodiments, the terms "supplemented T-cell expansion composition" or "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 T-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 .78%), asparagine (about 0.4%), scrinc (about 1.6%), glutamine (about 67.01%), argininc (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valinc (about 3.45%), isoleucine Date Recue/Date Received 2021-01-15 (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 "7-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/1% 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 "T-cell expansion composition"
may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg, and a sterol at a concentration of about 1 mg/kg (wherein mg/kg = parts per million). ). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or mom 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/I%, linoleic acid at a concentration of 2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (wherein mg/kg = parts per million). In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 6.4 gmol/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; linolcic acid at a concentration of between 0.75 Amol/kg Date Recue/Date Received 2021-01-15 and 75 timol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 timol/kg and 75 ttmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 tunol/kg and 25 timol/kg, inclusive of the endpoints. In certain embodiments, the terms "supplemented T-cell expansion composition" or "T-cell expansion composition"
may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 64 gmol/kg, palmitic acid at a concentration of about 7 tunol/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 timol/kg. In certain embodiments, the terms "supplemented 1-cell expansion composition" or "T-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 punol/kg, palmitic acid at a concentration of about 7.27 gmolikg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of about 7.56 timol/kg and a sterol at a concentration of about 2.61 pirnol/kg. In certain embodiments, the terms "supplemented T-cell expansion composition" or "1-cell expansion composition" may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 gmol/kg, palmitic acid at a concentration of about 7.27 timol/kg, linoleic acid at a concentration of about 7.57 timol/kg, oleic acid at a concentration of 7.56 timol/kg and a sterol at a concentration of 2.61 ttmol/kg.
10751 As used herein, the term "P3 buffer" may be used interchangeably with a buffer comprising one or more of KC1, MgCl2, CINa, Glucose and Ca(NO3)2 in any absolute or relative abundance or concentration, and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/HC1, and a phosphate buffer.
The term "P3 buffer" may be used interchangeably with a buffer comprising 5 mM KCl, 15 mM
MgCl2, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2, and, optionally, the further comprising a supplement selected from the group consisting of HEPES, Tris/HC1, and a phosphate buffer. The term "P3 buffer" may be used interchangeably with a buffer comprising 5 mM KC1, 15 m114 MgCl2, 90 mM CINa, 10 mM Glucose and 0.4 mM
Ca(NO3)2 and a supplement comprising 20 mM HEPES and 75 mM Tiis/HC1. The term 'T3 buffer' may be used interchangeably with a buffer comprising 5 mM KC1, 15 mM MgCl2, 90 mM
CINa, 10 mM Glucose and 0.4 mM Ca(NO3)2 and a supplement comprising 40 mM
Na2HPO4/NaH2PO4 at pH 7.2.
[076] As used herein, the terms "supplemented RPMI-1640 media" or "T-cell conditioned media (TCCM)" may be used interchangeably with a media comprising one or more of Date Recue/Date Received 2021-01-15 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 HCI, vitamin B12, D-Glucose, Glutathione (reduced), L-Glutarnine and Mercaptoethanol in any absolute or relative abundance or concentration. The terms "supplemented RPM!-! 640 media" or "T-cell conditioned media (TCCM)" may be used interchangeably with a media comprising water, fetal bovine serum, FIEPES, 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-Thoonine, 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 HCI, vitamin B12, D-Glucose, Glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration.
10771 As used herein, the terms "supplemented AIM-V" or "supplemented 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-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isolcucinc, L-Lcucinc, L-Lysinc HCI, L-Mcthioninc, L-Phcnylalaninc, L-Prolinc, L-Scrinc, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 21-120, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid (hemicalcium), pyridoxine HC1, riboflavin, thiamine HCI, vitamin B12, D-Glucose.

Date Recue/Date Received 2021-01-15 glutathione (reduced), L-Glutamine and 2-Mercaptoethanol in any absolute or relative abundance or concentration. The terms "supplemented AIM-V" or "supplemented AIMV"
media may be used interchangeably with a media comprising water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, a phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HC1, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2Na 21-120, L-Valine, D-Biotin, choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCI, 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 "hnmunoCultrm medium" may be used interchangeably with a medium comprising one or more of water, human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cysteine 2HCI, L-Glutamic acid, L-Glutamine, L-Histidine hydrochloride H20, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-Calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-Inositol, calcium chloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium selenite, D-Glucose, HEPES and Sodium pyttivate in any absolute or relative abundance or concentration. The term "ImmunoCultna medium" may be used interchangeably with a medium comprising water, human serum albumin, recombinant human insulin, human transfenin, 2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cysteine 2HC1, L-Glutamic acid, L-Glutaminc, L-Histidinc hydrochloride H20, L-Isolcucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-Calcium pantothenate, folic acid, niacinamidc, pyridoxal hydrochloride, riboflavin, thiamine Date Recue/Date Received 2021-01-15 hydrochloride, vitamin B12, i-Inositol, calcium chloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in any absolute or relative abundance or concentration.
[079] Modified T-cells of the disclosure, including modified Tsem and/or Tem of the disclosure, may be incubated, cultured, grown, stored, or otherwise, combined at any step in the methods of the procedure with a growth medium comprising one or more inhibitors a component of a PI3K pathway. Exemplary inhibitors a component of a PI3K
pathway include, but are not limited to, an inhibitor of GSK3i3 such as TWS 1 I 9 (also known as GSK
3B inhibitor XII; CAS Number 601514-19-6 having a chemical formula C18H14N402).
Exemplary inhibitors a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIOTm).
[0801 As used herein, the terms "electroporation" and "nucleofection" are meant to describe alternative means to deliver a nucleic acid, transposon, vector or composition of the disclosure to a cell by providing an electric pulse that induces a cell membrane (the cell membrane, nuclear membrane, or both) to become permeable or to become more penneable to the nucleic acid, transposon, vector or composition of the disclosure.
[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. In certain embodiments of the nucleofection, the incubating step comprises incubating the composition comprising the plurality of primary human T cells in a pre-warmed 1-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 nuinber/portion of hours in between. The incubation step may have a period of at least 1, 2, 3, 4, 5, 6 or 7 days or any number/portion of days in between. The incubation step may have a period of at least 1 week. In certain embodiments of the nucleofection, the incubation step has a period of two days. In certain embodiments of the nucleofection, the applying step may comprise applying one or more of the following program(s) El-I 15, El-I 51, El-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, EO-151, E0-148, E0-156, E0-210, EO-213, and FI-156. In certain cmbodimcnts, the applying step may comprise applying one or more of the following Date Recue/Date Received 2021-01-15 program(s) El-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-I51, ES-115, ES-151, E0-151, EO-148, EO-156, E0-210, E0-213, and FI-156, or a program that provides the same number of electrical pulses, each pulse having the same duration and intensity, and a substantially similar interpulse duration of time. In certain embodiments, the applying step may be performed using a known elecuoporation/nucleofection device, including, but not limited to, Lonz.a Amaxa, MaxCyte technology, BTX PulseAgile, and BioRad GenePulser. In certain embodiments of the nucleofection, the applying step may comprise applying at least one electrical pulse. In certain embodiments of the nucleof-ection, 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.
[083] In certain embodiments of the methods of producing a modified T cell (e.g. a Tscm and/or Tcm) of the disclosure, the activation supplement comprises one or more cytokine(s).
The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFT). The one or more cytokine(s) may comprise IL-2.
10841 In certain embodiments of the methods of producing a modified T cell (e.g. a Tscm and/or Tcm) of the disclosure, the expansion supplement comprises one or more cytokine(s).
The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplary lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleulcin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-2 1 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFO. The one or more cytokine(s) may comprise IL-2.
[085] In certain embodiments of the methods of producing a modified T cell (e.g. a Tscm and/or Tcm) of the disclosure, the primary human T cell is a naïve T cell. The naïve 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 2021-01-15 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 Tcm of the disclosure, the primary human T cell is a memory T cell.
10871 In certain embodiments of the methods of producing a modified Tscm and/or Tcm of the disclosure, the primary human T cell expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and 1L-2RD.
[0881 In certain embodiments of the methods of producing a modified Tsai and/or Tem 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 Tem of the disclosure, the primary human T
cell is a modified Tscm a T memory stem cell (modified Tsem) and the modified Tscm expresses one or more of CD45RA, CD95, IL-2R13, CR7, and CD62L. In certain embodiments of the methods of producing a modified Tsem and/or Teri of the disclosure, the primary human T cell is a central memory T-cell (modified Teti) and the modified Tem expresses one or more of CD45RO, CD95, IL-2R13, CCR7, 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 an effector memory T-cell (modified TEm) and the modified TEM
expresses one or more of CD45RO, CD95, and IL-2R. In certain embodiments of the methods of producing a modified Tscm and/or TCM of the disclosure, the primary human T
cell is an effector T-cell (modified TEFF) and the modified TEFF expresses one or more of CD45RA, CD95, and 1L-2RD.
10891 In certain embodiments of the methods of producing a modified Tsem and/or Tem of the disclosure, the primary human T cell may express CD4 and/or CD8. In certain embodiments, the primary human T cell may express CD4 and/or CD8 at various ratios. In certain embodiments, the primary human T cell may express CD4 and/or CD8 at various ratios that are not naturally-occurring. In certain embodiments, the primary human T cells that express CD4 and/or CD8 at various ratios, that may be not naturally occurring, are a heterologous cell population.
[090] In certain embodiments of the methods of producing a modified Tsai and/or Tem of the disclosure, the primary human T cell may be isolated, prepared or derived from for Date Recue/Date Received 2021-01-15 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 Tschr or Tar 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 llS endonuclease and wherein the expressed DNA binding domain and the expressed type IIS
endonuclease are non-covalently linked. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a Cas9 endonuclease. hi certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type LIS
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 Date Recue/Date Received 2021-01-15 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 Histidine (H) at position 840 (H840A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an inactivated Cas9 (dCas9) (SEQ ID NO: 33). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an alanine (A) for an Asparagine (N) at position 580 (N580A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises a truncated and inactivated Cas9 (dSaCas9) (SEQ ID NO:
32). In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).
In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from a TALEN is the DNA binding domain. In certain embodiments, the genomic editing construct comprises a TALEN. In certain embodiments, including those embodiments wherein the genomic editing construct comprises a DNA binding domain and a type IIS
endonuclease, the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZEN).
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-fmaer nuclease (ZEN).
10931 The methods of making modified Tscm and/or Tcm cells of the disclosure may be optimized to produce a greater number or greater proportion of modified Tscm and/or Tcm cells. For example, the population of cells subjected to the methods of the disclosure may be enriched to contain an increased number or greater proportion of naive T
cells. As the number and/or proportion of naive T cells increases in the population of T
cells subjected to the methods of the disclosure, the number and/or proportion of modified Tscm and/or Tem cells of the disclosure produced also increases. Alternatively, or in addition, as the length of time or duration required for a method of disclosure to precede decreases, the number and/or proportion of modified Tscm and/or 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 Date Recue/Date Received 2021-01-15 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-2RD. In certain embodiments, the primary human T
cell is a naïve 1-cell (TN) and the IN expresses one or more of CD45RA, CCR7 and CD62L.
In certain embodiments, the primary human T cell is a T memory stem cell (Tscm) and the Tscm expresses one or more of CD45RA, CD95, IL-2R13, CR7, and CD62L. In certain embodiments, the primary human I 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 (TEm) and the EM
expresses one or more of CD45RO, CD95, and IL-2R13. In certain embodiments, the primary human T cell is an effector T-cell (TEFF) and the TEFF expresses one or more of CD45RA, CD95, and IL-2R. In certain embodiments, the primary human T cell expresses CD4 and/or CD8.
[095] The disclosure provides a composition comprising a modified Tscm produced a method of the disclosure. The disclosure provides a composition comprising a modified Tcr,i produced a method of the disclosure. The disclosure provides a composition comprising a modified TSCM and a modified Tavi 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 TS0/1 may comprise at least 1%, 2%, 5%, 10%, 15 A, 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.
[0961 The disclosure provides a use of a composition comprising a modified Tscm and/or TcNi 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 Tscm and/or Tcm is allogeneic.
In certain embodiments, the antigen receptor is a T-cell receptor. In certain embodiments, the T-cell receptor is naturally-occurring. In certain embodiments, the T-cell receptor is not Date Recue/Date Received 2021-01-15 naturally-occurring. In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor comprises one or more mutation(s) compared to a wild-type T-cell receptor. In certain embodiments, and, in particular, in those embodiments wherein the T-cell receptor is not naturally-occurring, the T-cell receptor is a recombinant T-cell receptor. In certain embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. in certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR.
[097] The disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a modified Tali and/or Tem produced a method of the disclosure. In certain embodiments of this method, the modified Tscm and/or Tem 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 T-cell receptor is a recombinant T-cell receptor. In certain embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequence(s). In certain embodiments, the CAR is a VCAR. In certain embodiments of this method, the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen. In certain embodiments of this method, the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antigen. In certain embodiments, the disease or disorder is a disease or disorder caused by a lack of an activity or an insufficient amount of a secretory protein. In certain embodiments, the disease or disorder is a disease or disorder treated by a replacement of an activity of a therapeutic protein or by an increase in an amount of the therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein. In certain embodiments, the secretory protein is lacking an activity or a sufficient amount within a local area of a body. In certain embodiments, the local area of a Date Recue/Date Received 2021-01-15 body is accessible by a native T-cell or a modified T-cell. In certain embodiments, the modified T-cell is produced in vivo, ex vivo, in vitro or in situ.
BRIEF DESCRIPTION OF THE DRAWINGS
[098] Figure us 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 I at day 19. The population of CAR-Tscm cells produced by the method described in Example 1 at day 19 contained no B cells or lymphocytes. The majority of the cells are CD3+ T-cells. Only 1.1% are Natural Killer cells and 1.7% are Natural Killer T-cells.
[01.00] 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 I. Cells were nucleofected with a surrogate CARTyrin plasmid. CAR-Tscm cells express CD62L and CD45RA as shown in the bottom two plots.
[0102] Figure 5 is a series of plots depicting the absence of T-cell exhaustion at Day 19 of the method described in Example 1. At Day 19, the cell population produced by this method does not express PD1, which is a marker for T cell activation and exhaustion.
These cells expressing the CARTyrin have almost successfully reached a resting state post-manufacture.
They do not exhibit signs of antigen-independent (tonic) signaling which would otherwise drive higher levels of PD1 expression. Tonic signaling is hypothesized to be caused by some CAR molecules that lead to early exhaustion and reduced efficacy of a CAR T-cell therapy.
[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 piggyBac transposase.

Date Recue/Date Received 2021-01-15 [01041 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.
[01051 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.
[01061 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.
[01071 Figure 7 is a graph showing a phenotypic difference between piggyBac-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 (MO1 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-), Tem (CD62L-CD45RA-) and Teff (CD62L-CD45RA+). All CAR+ T cells were CD95+.
[01081 Figure 8A-B is a pair of graphs showing that piggyBac' preferentially transposes naive T cells. Human pan T cells were sorted (using a BD FACSAria 11 flow cytometer) into naïve (CD62L+CD45RA+), Tern (CD62L+CD45RA-), Tern (CD62L-CD45RA-), and Tcff (CD62L-CD45RA+) subsets. The sorted subsets were each either transposed with piggyBac-GFP or transduced with lentivirus-GFP. For the former, each sorted subset was transposed with PiggyBac-GFP, stimulated with anti-CD3/CD28 beads at day 2 post-transposition, expanded, and examined on day 19 post-transposition. For the latter, the sorted subsets were stimulated with aCD3/CD28 beads, transduced with lentivirus encoding GFP (MO1 5), expanded, and examined on day 19 post-stimulation. n=3 donors.

Date Recue/Date Received 2021-01-15 [01091 Figure 9 is a pair of graphs showing that the piggyBacTM manufacturing process yields high levels of Tscm in samples from multiple myeloma (MM) patients even when naive T cells are rare. T cells from MM patients (triangles) and healthy donors (circles) were characterized for memory marker expression by flow cytometry before (left) and after (right) the Poseida manufacturing process. Expression of CD45RA and CD62L was assessed by FACS and plots are shown for the MM patients and a healthy donor. It is known that T cells from MM patients generally have lower frequencies of naive and Tscm cells, but higher frequencies of Teff unlike those from healthy normal donors which are the opposite.
Regardless of the input frequency of naive and 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).
[01.101 Figure 10 is a series of Fluorescence Activated Cell Sorting (FACs) plots characterizing T and Tscm cell markers in human pan T cells transformed with the Sleeping Beauty (SB100x) transposition system and the methods of the disclosure.
Sleeping Beauty (SB100x) Transposition yields predominately Tscm phenotype using Poseida manufacture process. Human pan T cells were transposed using I fag of either a Sleeping Beauty or piggyBac transposon plasmid and SB100x or SPB mRNA, respectively as shown.
Following transposition, cells were expanded ex vivo and all non-transposed cells were depleted using the Poseida manufacture drug selection system. Following 18 days in culture, cells were stained with the phenotypic markers CD4, CD8, CD45RA, and CD62L. Stem cell memory phenotype (Tscm) is defined by CD45RA and CD62L double positive cells and make up >65% of the cells in all of samples. All panels in a column share common x-axis and y-axis parameters. In each row, from top to bottom, are shown data from T cells transposed with (top), 2.5 microgram (f.tg) of the Sleeping Beauty transposon SB100x, (second from top) 5 fig of SB100x, (3rd from top) 10 fig of SB/00x, (second from bottom) 5 fig of the piggyBac transposon P-BCMA-101 and at bottom, an unstained control. The x-axis, in order from left to right, in the first and second columns shows Forward Scatter (FSC), units from 0 to 250 thousand (abbreviated "k"), in increments of 50k. The x axis of the third column from the left shows CD8 expression, with markings reading from 0 to 103 incrementing by powers of 10.
The final right hand column shows CD62L expression, with markings reading from 0 to 103 incrementing by powers of 10. The y-axis, in the first column, shows Side Scatter (SSC), in units from 0 to 250k in increments of 50k. The y-axis in the second coltunn from the left Date Recue/Date Received 2021-01-15 shows expression of the cell viability marker 7 aminoactinomycin D (7AAD), from 0 to 105 incrementing by powers of 10. The y-axis of the third column from the left shows the expression of the marker CD4, from 0 to 105 incrementing by powers of 10. The y-axis in the right hand column show expression of the marker CD45RA, from 0 to 105 incrementing by powers of 10.
[0111] Figure 11 is a schematic diagram showing the human coagulation pathway leading to blood clotting. Contact activation, for example by damaging an endothelium, activates an intrinsic clotting pathway. Tissue factors activate an extrinsic clotting pathway, for example following trauma. Both pathways converge onto the conversion of Prothrombin into Thrombin, which catalyzes the conversion of fibrinogen into fibrin.
Polymerized fibrin together with platelets forms a clot. In the absence of Factor IX (circled), clotting is defective. Factor VIII (FVIII) deficiency leads to development of Hemophilia A. Factor IX
(FIX) deficiency leads to development of Hemophilia B. Hemophilia B is a rare disease, occurring with a frequency of about one in between 25,000 and 30,000. Sixty percent of hemophilia B cases are severe. Fewer than one percent of individuals with Hemophilia B
have normal FIX levels. Prior to the compositions and methods of the disclosure, the standard treatment for hemophilia B involved an infusion of recombinant FIX every 2 to 3 days, at an expense of approximately $250,000 per year. In sharp contrast to this standard treatment option, Tscm cells of the disclosure are maintained in humans for several decades.
[01.12] Figure 12 is a series of Fluorescence-Activated Cell Sorting (FACS
plots) depicting FIX-secreting T cells. T cells encoding a human Factor IX transgene showed a TSCM
phenotype in approximately 80% of cells. The 6 panels are described in order from left to right. (1) Forward scatter (FSC) on the x-axis versus side scatter (SSC) on the y-axis. The x-axis is from 0 to 250 thousand (abbreviated k) in increments of 50k, the y-axis is for 0 to 250k, in increments of 50k. (2) FSC on the x-axis versus the cell viability marker 7 aminoactinomycin D (7AAD). The x-axis is labeled from 0 to 250k in increments of 50k.The y-axis reads, from top to bottom, -103, 0, 103, 104, 105. (3) On the x-axis is shown anti-CD56-APC conjugated to a Cy7 dye (CDC56-APC-Cy7), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown anti-CD3 conjugated to phycoerythrin (PE), units from 0 to 105 incrementing in powers of 10. (4) On the x-axis is shown anti-CD8 conjugated to fluomseein isothiocyanate (FITC), units from 0 to 105 incrementing in powers of 10. On the y-axis is shown anti-CD4 conjugated to Brilliant Violet 650 dye (BV650), units from 0 to 105 incrementing in powers of 10. (5) On the x-axis is shown an anti CD62L
antibody conjugated Date Recue/Date Received 2021-01-15 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.
[01131 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
[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.
(01151 Figures 14A-E are a series of plasmid maps for site-specific integration into the AAVS1 site using either HR or MMEJ and corresponding sequences. Donor plasmids for testing stable integration into the genome of human pan T cells via A) site-specific (AAVS1) homologous recombination (HR), B) site-specific (AAVS1) microhomology-mediated end-joining (MMEJ) recombination and C) TTAA-specific piggyBacTm transposition.
For HR and MMEJ donor plasmids, GFP-2A-DHFR gene expression cassettes were flanked by CRISPRiCas9 targeting sites and homology aims for AAVS1 site integration; for piggvBacTM donor plasmid, GFP-2A-DHFR gene expression cassette is flanked by piggyBaerm transposon elements. The homology arms for the HR and MMEJ plasmids are 500 bp and 25 bp, respectively. Panels D and E, and F depict SEQ ID NOs 41 and respectively.
[0116] Figure 15 is a graph showing transgene (GFP) expression in primary human pan T
cells 3 days post-nucleofection. HR or MMEJ donor plasmids were co-delivered with or without CRISPR ribonucleoprotein (RNP) targeting reagents into pan T cells via nucleofection. T cells receiving donor plasmids alone were included as controls. Pan T cells were also modified using the piggyBacTm transposon delivery system. T cells were activated via TCR stimulation on Day 0 and GFP-1- 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-nucleofcction and selection. Activated T cells with stably integrated Date Recue/Date Received 2021-01-15 transgenes were selected by methotrexate addition using the DHFR selection gene encoded in the bi-cistronic GFP-2A-DHFR integration cassettes. GFP+ cell percentage was assessed at Day 11 post-nucleofection by flow cytometiy and data are summarized in bar graph. GFP-F
cells were highly enriched via selection in pan T cells receiving transposition reagents, RNP
plus HR or MIVIEJ donor plasmids, but not in T cells receiving donor plasmids alone.
[0118] Figure 17A-C is a series of graphs showing the phenotype of primary human pan T
cells modified by HR and MMEJ at the AAVS I site. The phenotype of GFP+ CD8+
pan T
cells was analyzed at Day 11 post-nucleofection by flow cytometry. A) Cells were stained with 7AAD (cell viability), CD4, CD8, CD45RA and CD62L, and FACS plots show gating strategy. CD8+ T cell subsets were defined by expression of CD45RA+CD62L+
(stem cell memory T cells (Tscm)), CD45RA-CD62L+ (central memory T cells (Tem)), CD45RA-CD62L- (effector memory T cells (Tern)), and CD45RA+CD62L- (T effectors (Tell)). 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-nucleofeetion and data are summarized in bar graph.
[0119] Figure 18A-B is a pair of photographs of gel electrophoresis results showing site-specific integration into the AAVS I site. Selected cells from each group were harvested and genomic DNA was extracted and used as template for PCR to confirm site-specific integration into the AAVS1 site for A) HR and B) MMEJ. Two pairs of primers individually airnplify 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 arm 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-1r GTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38) and the other priming the AAVS1 region beyond the 500 bp 5'-homologue arm AAVS-2f CTGGGGACTCTTTAAGGAAAGAAG (SEQ ID NO: 39), yielding a 0.76 kb DNA
fragment for HR or MMEJ) of the AAVSI target site. PCR products were displayed on Agarosc gel. Non-specific bands in HR samples arc the result of only a single round of PCR
and would likely have been resolved given additional rounds.
DETAILED DESCRIPTION

Date Recue/Date Received 2021-01-15 [0120] The disclosure provides a method for producing human chimeric antigen receptor (CAR) expressing-T cells using the piggyBacTM Transposon System under conditions that preserve or induce stem-cell memory T cells (Tscm) with potent CAR activity (referred to herein as a CAR-1'scm. Compositions comprising CAR-Tscm produced using the methods of the disclosure comprise > 60% CAR-Tscm and exhibit a distinct functional profile that is consistent with this T cell subset. Other T cell subsets found in the compositions of the disclosure include, but are not limited to, central memory CAR-T cells (CAR-Tcm), effector memory CAR-T cells (CAR-TEm), effector CAR-T cells (CAR-TE), and terminally-differentiated effector CAR-T cells (CAR-TIE). A linear pathway of differentiation may be responsible for generating these cells: Naïve T cells (TN) > Tscm > Tcm > Tam > TE > TIE, whereby TN is the parent precursor cell that directly gives rise to Tscm, which then, in turn, directly gives rise to Tem, etc. Compositions comprising CAR-Tsc:m, CARTyrin-Tsem and/or VCAR-Tscm of the disclosure may comprise one or more of each parental CAR-T
cell subset with CAR-Tscm being the most abundant (e.g. Tscm > Tcm > Tam > TE > TrE).
While, the absolute quantities/abundances and relative proportions of each parental T
cell subset may vary among samples of patient blood and naturally-occurring cell populations, and naturally-occurring cell populations may have a high abundance and/or proportion of Tscm, compositions of the disclosure comprising non-naturally occurring CAR-Tscm are more potent and efficacious in treating patients against diseases and cancers.
[0121] Tmmunotherapy using chimeric-antigen receptor (CAR)-T cells is emerging as an exciting therapeutic approach for cancer therapies. Autologous CAR-modified T
cells targeting a tumor-associated antigen (Ag) can result in robust tumor killing, in some cases resulting in complete remission of CD19+ hematological malignancies. Unlike traditional biologics and chemotherapeutics, CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments. To achieve this, CAR-T cells must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses.
Thus, intensive efforts have been focused on the development of CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as of a CAR-T
product containing early memory cells, especially stem cell memory (Tscm). A
stein cell-like CAR-T would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (Tcm), effector memory (Tad) and effector T cells (TE), thereby producing better tumor eradication and long-term CAR-T engraftment.

Date Recue/Date Received 2021-01-15 [01221 CAR-Tscm of the disclosure may comprise a Centyrin-based CAR, ref-erred 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.
[01231 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.
[01241 In certain embodiments of the methods of the disclosure, the piggyBacTM
(PB) Transposon System may be used for stable integration of antigen-specific (including cancer antigen-specific) CARTyrin or VCAR into resting pan T cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme (although the transposon and transposase would be comprised in separate compositions until they were introduced into a cell), called Super piggyBacTm (SPB), in a single electroporation reaction.
Delivery of piggyBacTm transposon into untouched, resting primary human pan T cells resulted in 20-30% of cells with stable integration and expression of PB-delivered genes.
Unexpectedly, a majority of these modified CARTyrin-expressing T cells were positive for expression of CD62L and CD45RA, markers commonly associated with stem memory T-cells (Tscm cells).
To confirm that this phenotype was retained upon CAR-T cell stimulation and expansion, the modified CARTyrin-expressing T cells positive for expression of CD62L and CD45RA were activated via stimulation of CD3 and CD28. As a result of stimulation of CD3 and CD28, >
60% of CARTy-rin+ 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.
[01251 To determine whether or not the PB system directly contributed to enhancing the expression of stem-like markers, the phenotype of CAR-T cells generated either by PB
transposition or lentiviral (LV) transduction was compared. To do this, a new vector was constructed by subcloning the CARTyrin transgcnc into a common LV construct for Date Recue/Date Received 2021-01-15 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+ T cells, the LV-transduced CARTyrin+ T cells did not exhibit an augmented memory phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of target tumor cells.
Together, these data demonstrate that CAR-T cells produced by PB transposition are predominantly Tscm cells, a highly desirable product phenotype in the CAR-T field. Furthermore, these CARTyrin+ T
cells exhibit strong anti-tumor activity and may give rise to cells that persist longer in vivo due to the use of a Centyrin-based CAR, which may be less prone to tonic signaling and functional exhaustion.
Chimeric Antigen Receptors [01261 The disclosure provides a chimeric antigen receptor (CAR) comprising:
(a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises one or more sequences that each specifically bind an antigen; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region may comprise two sequences that each specifically bind an antigen to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three sequences that each specifically bind an antigen to produce a tri-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain. Sequences that each specifically bind an antigen may include, but not limited to, a single chain antibody (e.g. a scFv), a sequence comprising one or more fragments of an antibody (e.g. a VHH, referred to in the context of a CAR as a VCAR), an antibody mimic, and a Centyrin (referred to in the context of a CAR as a CARTyrin).
[01271 In certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD38, CD3s, CD37, 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 Date Recue/Date Received 2021-01-15 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 atggeactgccagtcaccgccctgetgctgcctctggctctgctgctgcacgcagetagacca (SEQ ID NO:
9).
[01281 In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD35, CD3E, CD37, CD3Cõ CD4, CD8a, CD! 9. CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. The CD8a transmembrane domain may comprise an amino acid sequence comprising IYTWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IY1WAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8a transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatttsggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ
ID NO:
11).
[0129] In certain embodiments of the CARS of the disclosure, the endodomain may comprise a human CD3i; endodomain.
[01301 In certain embodiments of the CARS of the disclosure, the at least one costimulatory domain may comprise a htunan 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CARS of the disclosure, the at least one costimulatory domain may comprise a CD28 and/or a 4-1BB
costimulatory domain. The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSE1GMKGERRRGKGHDGLYQGLSTATKDTYDALHM.QALP
PR (SEQ ID NO: 12) or a sequence having at least 70%, 800%, 90%, 95%, or 99%
identity to the amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA.LHMQALP
PR (SEQ ID NO: 12). The CD28 costimulatory domain may be encoded by the nucleic acid sequence comprising Date Recue/Date Received 2021-01-15 cgcgtgaagtrtagicgatcagcagatgccccagcrtacanacaeggacaganccagctgtataacgagctgantaggg ccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcnanAaccctca,g gaagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggc aaaeg gcacgatgggctgtaccagggactgagcaccgccacapageacacctatgatgctctgcatatgcaggcactgcctcca agg (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLINIFKQPFIVIRPVQTTQEEDGCSCRFPEEEEGGCEI., (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 aagagaggeaggaagaaaetgctgtatattttcaaacagccettcatgcgccccgtgcagactacccaggaggaagacg ggtgctce tgtcgattecctgaggaagaggaaggegggtgtgagctg (SEQ ID NO: 15). The 4-1BB
costimulatory domain may be located between the transmembmne domain and the CD28 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 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID
NO: 16). The human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcct gcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).
[0132] 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 Date Recue/Date Received 2021-01-15 encode any gene product (e.g. transcript, protein, enzyme) essential for cell viability and survival. Exemplary selection genes encode any gene product (e.g. transcript, protein, enzyme) essential for conferring resistance to a drug challenge against which the cell is sensitive (or which could be lethal to the cell) in the absence of the gene product encoded by the selection gene. Exemplary selection genes encode any gene product (e.g.
transcript, protein, enzyme) essential for viability and/or survival in a cell media lacking one or more nutrients essential for cell viability and/or survival in the absence of the selection gene.
Exemplary selection genes include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylate Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).
[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%, 900/, 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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggasanccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ 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 Date Recue/Date Received 2021-01-15 comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQ'TLNFDLLKLAGDVESNPGP (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 GSGVKQTLNFDLILICIAGDVESNPGP (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 second self-cleaving peptide located, for example, downstream of the one or more of a sequence that specifically binds an antigen of the disclosure. The first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO:
18)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ
ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A
peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCT'NYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an Date Recue/Date Received 2021-01-15 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 GSGQC'TNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTLNF'DLLICLAGDVESNPGP (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 GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[0137] The disclosure provides a composition comprising the transposon the disclosure. In certain embodiments, a method introducing the composition may further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme.
The sequence encoding a transposase enzyme may be an mRNA sequence.
[01381 Transposons of the disclosure may comprise piggyBac transposons.
Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
[0139] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.
[0140] Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lenti virus, an adenovirus, an adeno-associated virus or any combination thereof The viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (1TR) 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).

Date Recue/Date Received 2021-01-15 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.
[01411 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 Siithetase), MGNIT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidnig 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. hi some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a self-cleaving peptide is located between a CAR and a selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR. The self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a 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 ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaceaaaacccaggacca (SEQ ID NO:
20). An Date Recue/Date Received 2021-01-15 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 GSGAINFSLLKQAGDVEENPGP (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).
[01431 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.
[01441 The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is a nanoparticle. Exemplary nanoparticic 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 Date Recue/Date Received 2021-01-15 combination thereof. A nanoparticle vector may be passively or actively transported across a cell membrane.
[01451 Nanoparticle vectors of the disclosure may comprise a selection gene.
The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound. Exemplary selection genes of the disclosure may include, but are not limited to, nen (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Mediotrexate), TYMS (encoding Thymidylate Synthetase), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase I family, member Al), FRANCF, RADS IC
(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 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 EGRGSLLTCGDVEEN POP (SEQ Ill 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 Ill NO: 19) or a Date Recue/Date Received 2021-01-15 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 ggatctggagagggaagggeaagcctgctgacctgtggagacgtggaggaaaacceaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QC'TNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID
NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
(0147] The disclosure provides a composition comprising a vector of the disclosure.
CAR7'yrins 101481 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 cmbodimcnts, the antigen recognition region may comprise three Ccntyrins to Date Recue/Date Received 2021-01-15 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.
[01491 The disclosure provides a chimeric antigen receptor (CAR) comprising:
(a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one protein scaffold or antibody mimetic; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region may comprise two scaffold proteins or antibody mimetics to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three protein scaffolds or antibody mimetics to produce a tri-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
[01501 in certain embodiments of the CARS of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD35, CD3c, CD37, CD3C CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments of the CARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD8a signal peptide. The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID
NO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagetagacca (SEQ ID NO:
9).
[01511 In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD2, CD35, CD3e, CD31, CD3C CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments of the CARS of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. The CD8a transmembrane domain may comprise an amino acid sequence comprising IYIW'APLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAG1'CGVLLLSLVI1LYC (SEQ ID NO: 10). The CD8a Date Recue/Date Received 2021-01-15 transmembrane domain may be encoded by the nucleic acid sequence comprising atctacatugggcaccactggccgggacctgtggaggctgctgagagcctggtcatcacactgtactgc (SEQ ID
NO:
11).
[01521 In certain embodiments of the CARS of the disclosure, the endodomain may comprise a human CD3 endodomain.
[0153] 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
EGLYNELQKDICMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
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 cgcgtgaagtttagtcgatcagcag,atgccccagettacaaacagggacag,aaccagctgtataacgagagaatctg ggccgccga gaggaatatgacgtgctggataageggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcagg aagg cctgtataacgagctgcagaaggaraaliatggcagaagcctattctgagatcggcatgaagggggagcgacggagagg caaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctetgcatatgcaggcactgcctcca age (SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID
NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-IBB costimulatory domain may be encoded by the nucleic acid sequence comprising aagagaggcaggangaaactgctgtatattucaaacagccettcatgcgccccgtgcagactacccaggaggaagacgg gtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BB
costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

Date Recue/Date Received 2021-01-15 [01541 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 ITTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising 1-1-1PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (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).
[0155] Centyrins of the disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding an antigen. Centyrins of the disclosure may comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, wherein the scaffold is capable of specifically binding an antigen.
The at least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be Tenascin-C. The consensus sequence may comprise LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDL
TGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) or MLPAPKNLVVSEV'TEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYD
LTGLKPGTEYTVSIYGVKGGIIRSNPLSAEFIT (SEQ ID NO: 2). The consensus sequence may encoded by a nucleic acid sequence comprising atgctgcctgcaccaaagaacctggtggtgtcteatgtgacagaggatagtgccagactgtcatggactgeteccgacg cagccttcg atagattatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtectgacagtgccagggtccgaacgctct tatgacctg acagatctgaageccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagettccactgtecgc aatcttcac caca (SEQ ID NO: 3). The consensus sequence may be modified at one or more positions within (a) a A-B loop comprising or consisting of the amino acid residues TEDS
at positions 13-16 of the consensus sequence; (b) a B-C loop comprising or consisting of the amino acid residues TAPDAAF at positions 22-28 of the consensus sequence; (c) a C-D loop comprising or consisting of the amino acid residues SEKVGE at positions 38-43 of the consensus sequence; (d) a D-E loop comprising or consisting of the amino acid residues GSER at positions 51-54 of the consensus sequence; (e) a E-F loop comprising or consisting of the amino acid residues GLKPG at positions 60-64 of the consensus sequence; (f) a F-G loop Date Recue/Date Received 2021-01-15 comprising or consisting of the amino acid residues KGGHRSN at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type III (FN3) domains. The scaffold may bind an antigen with at least one affinity selected from a KD of less than or equal to 1 0-9M, less than or equal to 10-wM, less than or equal to 10-"M, less than or equal to 10-12M, less than or equal to 10-13M, less than or equal to 10-14M, and less than or equal to 10-15M. The Kll may be determined by surface plasmon resonance.
[01561 The disclosure provides a composition comprising the CAR of the disclosure and at least one pharmaceutically acceptable carrier.
[01571 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 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), ALDH I

(encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), and NIOC2.2 (encoding NK2 Homcobox 2).
[01591 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 Date Recue/Date Received 2021-01-15 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 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 ggatctggagagggaaggggaagcetgctgacctgtggagacgtggaggaaaacecaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ NO:
23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A
peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[0160] Transposons of the disclosure may comprise a first and a second self-cleaving peptide, the first self-cleaving peptide located, for example, upstream of one or more of a Date Recue/Date Received 2021-01-15 protein scaffold, Centyrin or CARTyrin of the disclosure the second self-cleaving peptide located, for example, downstream of the one or more of a protein scaffold, Centyrin or CARTyrin of the disclosure. The first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A
peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A
peptide may comprise an amino acid sequence comprising EGRGSIITCGDVEENPGP (SEQ ID
NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A
peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP
(SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99%
identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A
GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatetggagagggaaggggaagcctgctgacctgtggagacgtggaggninneccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO:21). A GSG-E2A peptide may comprise an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID
NO: 23) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide may comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

Date Recue/Date Received 2021-01-15 [01611 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.
[01621 Transposons of the disclosure may comprise piggyBae transposons.
Transposase enzymes of the disclosure may include piggyBac transposases or compatible enzymes.
[01631 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.
[01641 Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof.
The viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (ITR) sequences located in cis next to a sequence encoding a protein scaffold, Centyrin or CARTyrin of the disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to all seroty, pes (e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV
(scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g.
AAV2/5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.
[01651 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 fvlethotrexate), TYMS (encoding Thymidylate Synthetasc), MGMT ( encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR I), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANC F, RAD5 IC

Date Recue/Date Received 2021-01-15 (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.
[0166] Viral vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a self-cleaving peptide is located between a CAR and a selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide and wherein a first self-cleaving peptide is located upstream of a CAR and a second self-cleaving peptide is located downstream of a CAR. The self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A
peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO:
20). An E2A peptide may comprise an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 900/0, 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 VKQTLNFDLLICLAGDVESNPGP (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 GSGVKQILNFDLLKLAGDVESNPGP (SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGVKQTLNFDLLICLAGDVESNPGP (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 2021-01-15 sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID
NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).
[0167] The disclosure provides a vector comprising the CAR of the disclosure.
In certain embodiments, the vector is an mRNA vector. The vector may be a recombinant mRNA
vector. T cells of the disclosure may be expanded prior to contacting the 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.
[0169] Nanoparticle vectors of the disclosure may comprise a selection gene.
The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound. Exemplary selection genes of the disclosure may include, but are not limited to, neo (conferring resistance to neomycin), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), TYMS (encoding Thymidylatc Synthetase), MGMT
(encoding 0(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family, member Al), FRANCF, RAD51C
(encoding RAD51 Paralog C), GCS (encoding glucosylceramidc synthase), NKX2.2 (encoding NK2 Homeobox 2) or any combination thereof.
[0170] Nanoparticle vectors of the disclosure may comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self-Date Recue/Date Received 2021-01-15 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-P2.A 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 g,gatctggagagggaaggggaagcctgctgacctgtggagacgtggaggasaacccaggacca (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 2021-01-15 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 ATNFSLLKQAGDVEENF'GP (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).
[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 Ill (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, hi 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 Fc 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.
[01751 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 2021-01-15 [0177] The disclosure provides a method of generating libraries of a protein scaffold based on a fibronectin type ill (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 eukaryotic 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 III (FN3) repeat protein, including, without limitation, mammalian-derived scaffold, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding protein scaffold based on the consensus FN3 sequence. The disclosure further includes, but is not limited to, methods of making and using such nucleic acids and protein scaffolds, including diagnostic and therapeutic compositions, methods and devices.
[0180] The protein scaffolds of the disclosure offer advantages over conventional therapeutics, such as ability to administer locally, orally, or cross the blood-brain barrier, ability to express in E. Coli allowing for increased expression of protein as a function of resources versus mammalian cell expression ability to be engineered into bispecific or tandem molecules that bind to multiple targets or multiple epitopes of the same target, ability to be conjugated to drugs, polymers, and probes, ability to be formulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues and tumors.

Date Recue/Date Received 2021-01-15 [0181] Moreover, the protein scaffolds possess many of the properties of antibodies in relation to their fold that mimics the variable region of an antibody. This orientation enables the FN3 loops to be exposed similar to antibody complementarity determining regions (CDRs). They should be able to bind to cellular targets and the loops can be altered, e.g., affinity matured, to improve certain binding or related properties.
[0182] Three of the six loops of the protein scaffold of the disclosure correspond topologically to the complementarity determining regions (CDRs 1-3), i.e., antigen-binding regions, of an antibody, while the remaining three loops are surface exposed in a manner similar to antibody CDRs. These loops span at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO: 1. Preferably, the loop regions at or about residues 22-28, 51-54, and 75-81 are altered for binding specificity and affinity. One or more of these loop regions are randomized with other loop regions and/or other strands maintaining their sequence as backbone portions to populate a library and potent binders can be selected from the library having high affinity for a particular protein target. One or more of the loop regions can interact with a target protein similar to an antibody CDR interaction with the protein.
[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 VHH). 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 2021-01-15 certain embodiments, the sequence comprises two heavy chain variable regions of an antibody, wherein the complementarity-determining regions (CDRs) of the VHH
are human sequences. Scaffolds of the disclosure may comprise a sequence comprising one or more fragments of an antibody (e.g. a VHH). The disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one a sequence comprising one or more fragments of an antibody (e.g. a VHH); (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region may comprise two sequences comprising one or more fragments of an antibody (e.g. two VHHs) to produce a bi-specific or tandem CAR. In certain embodiments, the antigen recognition region may comprise three sequences comprising one or more fragments of an antibody (e.g. three VHHs) to produce a tri-specific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide.
Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
[0185] Scaffolds of the disclosure may comprise an antibody mimetic.
[01861 The term "antibody mimetic" is intended to describe an organic compound that specifically binds a target sequence and has a structure distinct from a naturally-occurring antibody. Antibody mimetics may comprise a protein, a nucleic acid, or a small molecule.
The target sequence to which an antibody mimetic of the disclosure specifically binds may be an antigen. Antibody mimetics may provide superior properties over antibodies including, but not limited to, superior solubility, tissue penetration, stability towards heat and enzymes (e.g. resistance to enzymatic degradation), and lower production costs.
Exemplary antibody mimetics include, but are not limited to, an affibody, an afflilin, an af'fimer, an affitin, an alphabody, an anticalin, and avimer (also known as avidity multimer), a DARPin (Designed Ankyrin Repeat Protein), a Fynomer, a Kunitz domain peptide, and a monobody.
[01871 Affibody molecules of the disclosure comprise a protein scaffold comprising or consisting of one or more alpha helix without any disulfide bridges.
Preferably, affibody molecules of the disclosure comprise or consist of three alpha helices. For example, an affibody molecule of the disclosure may comprise an immunoglobulin binding domain. An affibody molecule of the disclosure may comprise the Z domain of protein A.
[01881 Affilin molecules of the disclosure comprise a protein scaffold produced by modification of exposed amino acids of, for example, either gamma-B
c*,'stallin or ubiquitin.

Date Recue/Date Received 2021-01-15 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-I-bided protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.
[0189] Affimer molecules of the disclosure comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide a high affinity binding site for a specific target sequence. Exemplary affimer molecules of the disclosure comprise a protein scaffold based upon a cystatin protein or tertiary structure thereof.
Exemplary affimer molecules of the disclosure may share a common tertiary structure of comprising an alpha-helix lying on top of an anti-parallel beta-sheet.
[0190] Affitin molecules of the disclosure comprise an artificial protein scaffold, the structure of which may be derived, for example, from a DNA binding protein (e.g. the DNA
binding protein Sac7d). Affitins of the disclosure selectively bind a target sequence, which may be the entirety or part of an antigen. Exemplary affitins of the disclosure are manufactured by randomizing one or more amino acid sequences on the binding surface of a DNA binding protein and subjecting the resultant protein to ribosome display and selection.
Target sequences of affitins of the disclosure may be found, for example, in the genome or on the surface of a peptide, protein, virus, or bacteria. In certain embodiments of the disclosure, an affitin molecule may be used as a specific inhibitor of an enzyme. Affitin molecules of the disclosure may include heat-resistant proteins or derivatives thereof.
101911 Alphabody molecules of the disclosure may also be referred to as Cell-Penetrating Alphabodies (CPAB). Alphabody molecules of the disclosure comprise small proteins (typically of less than 10 kDa) that bind to a variety of target sequences (including antigens).
Alphabody molecules are capable of reaching and binding to intracellular target sequences.
Structurally, alphabody molecules of the disclosure comprise an artificial sequence fonning 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 2021-01-15 may comprise an artificial protein derived from a human lipocalin. Anticalin molecules of the disclosure may be used in place of, for example, monoclonal antibodies or fragments thereof. Anticalin molecules may demonstrate superior tissue penetration and thermostability than monoclonal antibodies or fragments thereof. Exemplary anticalin molecules of the disclosure may comprise about 180 amino acids, having a mass of approximately 20 kDa.
Structurally, anticalin molecules of the disclosure comprise a barrel structure comprising antiparallel beta-strands pairwise connected by loops and an attached alpha helix. In preferred embodiments, anticalin molecules of the disclosure comprise a barrel structure comprising eight antiparallel beta-strands pairwise connected by loops and an attached alpha helix.
[0193] Aylmer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). Avimers of the disclosure may recognize multiple binding sites within the same target or within distinct targets. When an avimer of the disclosure recognize more than one target, the avimer mimics function of a bi-specific antibody. The artificial protein avimer may comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. Amino acid sequences of one or more of the peptides of the avimer may be derived from an A domain of a membrane receptor. Avimers have a rigid structure that may optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure may demonstrate greater heat stability compared to an antibody.
[01941 DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high affinity for a target sequence. In certain embodiments, DARPins of the disclosure are derived from ankyrin proteins and, optionally, comprise at least three repeat motifs (also referred to as repetitive structural units) of the ankyrin protein. Ankyrin proteins mediate high-affinity protein-protein interactions. DARPins of the disclosure comprise a large target interaction surface.
[01951 Fynomers of the disclosure comprise small binding proteins (about 7 kDa) derived from the human Fyn SH3 domain and engineered to bind to target sequences and molecules with equal affinity and equal specificity as an antibody.
[0196] Kunitz domain peptides of the disclosure comprise a protein scaffold comprising a Kunitz domain. Kunitz domains comprise an active site for inhibiting protease activity.
Structurally. Kunitz domains of the disclosure comprise a disulfide-rich alpha+beta fold. This Date Recue/Date Received 2021-01-15 structure is exemplified by the bovine pancreatic try, psin inhibitor. Kunitz domain peptides recognize specific protein structures and serve as competitive protease inhibitors. Kunitz domains of the disclosure may comprise Ecallantide (derived from a human lipoprotein-associated coagulation inhibitor (LAC)).
[0197] Monobodies of the disclosure are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable in size to a single chain antibody. These genetically engineered proteins specifically bind target sequences including antigens.
Monobodies of the disclosure may specifically target one or more distinct proteins or target sequences. in preferred embodiments, monobodies of the disclosure comprise a protein scaffold mimicking the structure of human fibronectin, and more preferably, mimicking the structure of the tenth extracellular type III domain of fibronectin. The tenth extracellular type III domain of fibronectin, as well as a monobody mimetic thereof, contains seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three complementarity determining regions (CDRs) of an antibody. In contrast to the structure of the variable domain of an antibody, a monobody lacks any binding site for metal ions as well as a central disulfide bond. Multispecific monobodies may be optimized by modifying the loops BC and 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).
101991 Amino acids from a scaffold protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.
[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 2021-01-15 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 inununogenicity (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 [02011 The methods of the disclosure produce a modified Tscm of the disclosure regardless of the method used for introducing an antigen receptor into a primary human T
cell of the disclosure. The methods of the disclosure produce a modified Tscm of the disclosure with greater efficacy and/or a greater abundance, proportion, yield of modified -Tscm of the disclosure when the antigen receptor or the therapeutic protein of the disclosure is introduced to the primary human T cell using the piggyBac transposon system. A piggyBac transposon system of the disclosure may comprise a piggyBac transposon comprising an antigen receptor of the disclosure. Preferably, the primary human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a transposase of the disclosure simultaneously (or in very close temporal proximity, e.g. the primary human T
cell, the transposon and the transposase are contained in the same container (such as a cuvette) prior to introduction of the transposon and transposase into the cell ¨ however they would not be permitted to interact in the absence of the cell. Preferably, the primary human T cell contacts a piggyBac transposon comprising an antigen receptor of the disclosure and a Super piggyBacTM (SPB) transposase of the disclosure simultaneously prior to introduction of the transposon and transposase into the cell. In certain preferred embodiments, the Super piggyBacTM (SPB) transpognse is an mRNA sequence encoding the Super piggyBacTm (SPB) transposasc.
102021 Additional disclosure regarding piggyBac transposons and Super piggyBac n4 (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 2021-01-15 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 2021-01-15 541 PGTSDDSTEE PVMKKRTYCT YCPSKrRRKA. 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 pigK,,BacTm (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 2021-01-15 an amino acid sequence at feast 75%. 80%, 85%, 90%, 95%, 99%orany percentage in betweenidenticalto:

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO:
5).
[0209] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTM or Super 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 piggyBacTm or Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328. 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO:
4 or SEQ ID
NO: 5 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a thmonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a ttyptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid Date Recue/Date Received 2021-01-15 substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a phenylalanine (F).
In certain embodiments, the amino acid substitution at position 180 of SEQ ID
NO: 4 or SEQ
ID NO: 5 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a glycine (G) for an alanine (A). In certain embodiments. the amino acid substitution at position 200 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a phenylalanine (F),In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a proline (P) for a valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ
ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO:
4 or SEQ
ID NO: 5 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at Date Recue/Date Received 2021-01-15 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) 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 (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 lcucinc (L) for a methionine (M). In certain embodiments, the amino acid substitution at Date Recue/Date Received 2021-01-15 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 TD NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for a glutamine (Q).
[0210] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTM transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ
ID NO: 4 or SEQ ID NO: 5. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30. 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super piggyBacTM transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBacTm transposase enzyme may comprise or the Super pigk,,BacTm 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 valinc (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID
NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for a lysinc (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 2021-01-15 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 piggyBacTM transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID
NO: 4, the piggyBacTm transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
In certain embodiments, the piggyBacTm transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, and a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 4. In certain embodiments, the piggyBacTM
transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ
ID NO: 4 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 4.
[0211] By "introducing" is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell. The methods of the invention do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
[02121 As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.
[02131 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 polynucleotide construct introduced into the host does not integrate into the genome of the host Date Recue/Date Received 2021-01-15 [02151 In preferred embodiments, the piggyBac transposon system is used to introduce exogenous sequences into a primary human T cell by stable transformation to generate a modified Tscm or Tom.
Additional 7'ransposon Systems [02161 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).
[02171 The disclosure provides a method of producing a modified stem memory 1-cell (Tscm) or a modified central memory T-cell (Tem) , comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T
cell expresses one or more cell-surface marker(s) of a modified stem memory 1-cell (Tscm) or a modified central memory 1-cell (Tcm), thereby producing a modified stem memory T-een (Tscm) or a modified central memory T-cell (Tcm). The disclosure provides a method of producing a plurality of modified stem memory 1-cells (Tscm) or a plurality of modified central memory 1-cells (Tcm), comprising introducing into a plurality of primary human T
cells (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%

or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory 1-cell (Tscm) or a central memory 1-cell (To), thereby producing a plurality of modified stem memory 1-cells (Tscm) or a plurality of modified central memory 1-cells (Tcm).
[02181 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 transposasc or a hyperactive Sleeping Beauty transposasc (SB100X).
102191 In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

Date Recue/Date Received 2021-01-15 241 FQMDNDPKHT SKVVAI<WLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL
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:

241 FQHDNDPKHT SKVVA.KWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR 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 Helraiser transposon of the disclosure includes Helibatl, which comprises a nucleic acid sequence comprising:
1 TCCTATATAA TAAAAGAGAA ACATGCAAA1".CGACCATCCC TCCGCTACGC TCAAGCCACG

121 GCATACGCAG GTGTCAAGCG CCCC.AGGAGG CAACGGCGGC CGCGGGCTCC CAGGACCTTC

361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA. CTGACAGCAG GGAGGAGGAA

541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG .AAAGATGCCG GCGTTATCGA
601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAA.A. GAAGGCGGCG CCTGCAACAG
6E1 AATGTATCTG AAGAGC.AGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA ACAGCGGCGT

781 CGACAGAATA TGTCTAGA.GA ACAGTCATCA ACAAGTA.CTA CCAATACCGG TAGGAA.CTGC

961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCCAA. ATGATATACA TTTTCCAGAT
1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGTAA AAA.TTTCATG

Date Recue/Date Received 2021-01-15 1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAA. ATTTCATCAA AGCAAACCAA
1921 TCTAAGTTGA GAGTTGAAAA ATATAGTGGT TTGATGGATT ATCTCAAATC TAGATCTGAA.
1981 AATGACAA.TG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA GGGTAGTCCC

2221 AATGCTCTTT TAAA.TGATAT ATGTAAATTC CATTTATTTG GCAAAGTAAT AGCTAAAATT

2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA. ATTGTAAAAT CAAATATGGT ACATGGACCA

2521 CCAAAAGAAT TTCAAAATGC GACCATTGGA AATATTGATG GATATCCCAA. ATACAAACGA

2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA TGCGAA.TGCA TGACCAATCT
2881 CATGCAkTCP. CAAGATTAGC TATTCATTTG CCAAATGATC AGAATTTGTA TTTTCATACC

3241 GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC TTGATGACAC TATCTGGAAA.

Date Recue/Date Received 2021-01-15 3481 GAAATGCATG CC CTTAAC GA AATTCAGGAG GTATTC:ACAT TGCATGGAAT GAAATGTTCA

3601 GAGCAACAAC AG GCAGAGGT TTTG.ATAAAT TCTCTGAATG AT GAACAGTT GGCAGCCTTT

3841 GGAAGAAC CT TT CA.TT CC CA ATATAAATTA C C-AATTC CAT TAAATGAAAC TTCAATTTCT

4201 GATT CTGCTT ATAGTGAATG GTTAGTAAAA CTIGGAGA.TG GCAAACTT GA TAGC.AGTTTT
4261 CATTT.AGGAA TGGATATT.AT T GAAATC CC C CATGAAAT GA TTTGTAACGG ATCTATTATT

4681 GAAGTATTAA CAGGA.T CT GC AGAGGGAGAG GTTGTTCT GA TT C CAAGAAT TGATTTGTCC
4741 CCATCTGACA CT GGC CTC C C ATTTAAATTA ATTCGAA.GAC AGTTTC CC GT GAT GCCAGCA

4921 TGTGACGTTA. AAGTTAAAGT TGTAAATACT T CAT CACAAG GGAAATTAGT CAAGCACTCT

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 superfamily of nucleases.
[0223] An exemplary Helitron transposase of the disclosure comprises an amino acid sequence comprising:

Date Recue/Date Received 2021-01-15 1441 LPEPVTAHGQ LYVAFSRVRRACDVKVKVVN TSSQGKLWAH SESVFTLNVV YREILE (SEW") NO: 28) .
[0224] In Helitron transpositions, a hairpin close to the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase.
resulting in the transduction of flanking sequences. 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).
[0225] in certain embodiments of the methods of the disclosure, the transposase is a To12 transposase. To12 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT flimily. Exemplary To12 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene Date Recue/Date Received 2021-01-15 encoding the Tol2transposase, whichcontainsfourexons. An exemplary Tol2transposaseof the disclosure comprises an amino acid sequence comprising the following:

601 NTPLRASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEQIDNO:30).
[0226] An exemplary To12 transposon of the disclosure, including inverted repeats, subterminal sequences and the To12 transposase, is encoded by a nucleic acid sequence comprising the following:
1 CAGAGGTGTA. AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG
61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATC.AATACT TTTACTTTTA CTT.AATTACA
121 TTTTTTTAGA AAAAAAAGT.A CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC

421 GAAGAAA.ATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA

781 GACAATATAA GGCTCA.CGT.A ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT
841 CCA.CGGGAAA. TTTGGCGCCT ATTGCAGCTT TGAA.TAATCA. TTATC.ATTCC GTGCTCTCAT
901 TGTGTTTGAA TTCATGCA.AA ACACAAGAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT

Date Recue/Date Received 2021-01-15 2401 GCCTGGC.ATT TCTGTCATTA CAAGGCCTAC TTTACGCTCC AAGATAGCTG AAGCTGCTCT

3001 CTACAGATCT GTCTTTGGCA, AATGCCAAGC TTTATGGAAT AAAAGCAGCC GATCGGCTCT

3241 TATCGATGTA AACAAATGTG GGTTGTTTTT GTITA,ANACT CTTTGATTAT GCTGATTTCT

3421 TGCCTAGTGT CCATCAGTTA, AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGGTACT

Date Recue/Date Received 2021-01-15 3721 TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATC:TCAA TATGAATATT GATGTAAAGT
3781 ATTCTTGCAG CA.GGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT CATATGTATC

4081 ATGATGAAGA ITTTTTCGCT TCTTTGAAAC CGACAACACA. TGAAGCCAGC AAAGAGTTGG

4501 GTTTTCTTTC TTGCTTTT.AC TTTT.ACTTCC TTAATACTCA AGTACAATTT TAATGG.AGTA
4561 CTTTTTTACT TTTACTCAAG TAAGATTCTA GCCAGATACT TTTACTTTTA ATTGAGTAAA.

4681 TG (SEQ ID NO: 31).
Homologous Recombination [0227] In certain embodiments of the methods of the disclosure, a modified CAR-Tscm or CAR-Tcm of the disclosure is produced by introducing an antigen receptor into a primary human T cell of the disclosure by homologous recombination. In certain embodiments of the disclosure, the homologous recombination is induced by a single or double strand break induced by a genomic editing composition or construct of the disclosure.
Homologous recombination methods of the disclosure comprise contacting a genomic editing composition or construct of the disclosure to a genomic sequence to induce at least one break in the sequence and to provide an entry point in the genomic sequence for an exogenous donor sequence composition. Donor sequence compositions of the disclosure are integrated into the genomic sequence at the induced entry point by the cell's native DNA repair machinery.
102281 In certain embodiments of the methods of the disclosure, homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a "genomic safe harbor" site. In certain embodiments, a mammalian genomic sequence comprises the genomic safe harbor site. In certain embodiments, a primate genomic sequence comprises the genomic safe harbor site. hi certain embodiments, a human genomic sequence comprises the genomic safe harbor site.
102291 Gcnomic 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 2021-01-15 (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.
[02301 In certain embodiments of the methods of the disclosure, homologous recombination introduces a sequence encoding an antigen receptor and/or a donor sequence composition of the disclosure into a sequence encoding one or more components of an endogenous T-cell receptor or a major histocompatibility complex (MHC). In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous T-cell receptor or the MI-IC disrupts the endogenous gene, and optionally, replaces part of the coding sequence of the endogenous gene with a donor sequence composition of the disclosure. In certain embodiments, inducing homologous recombination within a genomic sequence encoding the endogenous 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 element. In certain embodiments of the methods of the disclosure, introduction of a sequence encoding an antigen receptor or a donor sequence composition of the disclosure by homologous recombination operably links the antigen receptor or the therapeutic protein to a transcriptional regulatory clement. In certain embodiments, the transcriptional regulatory element comprises an endogenous T cell 5' U'TR.
[02311 In certain embodiments of the introduction step comprising a homologous recombination, a gcnomic 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 2021-01-15 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 rccombination, the genomic editing composition comprises a DNA
binding Date Recue/Date Received 2021-01-15 domain and a nuclease domain. In certain embodiments of the genomic editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genomic editing composition, the DNA binding domain comprises a DNA-binding domain of a TALEN. in certain embodiments of the genomic editing composition, the DNA
binding domain comprises a DNA-binding domain of a ZFN. In certain embodiments of the genomic editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof.
In certain embodiments of the genomic editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of a Alanine (A) for Aspartic Acid (D) at position 10 (Dl OA) and a substitution of Alanine (A) for Histidine (H) at position 840 (H840A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32, comprising a substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 (D10A) and a substitution of an Alanine (A) for an Aspamgine (N) at position 540 (N540A)). In certain embodiments of the genomic editing composition, the nuclease domain comprises or further comprises a type IIS
endonuclease. In certain embodiments of the genomic editing composition, the type IIS
endonuclease comprises AciI, Mn1I, AlwI, BbvI, Bed, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, MbolI, My II, Pie!, SfaNI, Acid, BciVT, BfuAI, BmgBI, BmrI, BpmI, BpuEl, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Btsl, Ear!, EciI, MmeI, NmeAIII, Bbv-CI, Bpul0I, BspQI, SapI, Bad, BsaXI, CspCI, BfiI, Acc36I, FokI or 0o051. In certain embodiments, the type US 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.
102321 In certain embodiments of the methods of homologous recombination of the disclosure, the nuclease domain of a genoinic 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 2021-01-15 and, furthermore, may comprise, consist essentially oforconsistot ahomodimerora heterodimer. In certain embodiments, the nuclease is an endonuclease. Effector molecules, including those effector molecules comprising ahomodimer oraheterodimer, may comprise, consist essentially oforconsistof, a Cas9, aCas9nuclease domainorafragmentthereof In certain embodiments, the Cas9 isacatalytically inactive or "inactivated" Cas9 (dCas9). In certain embodiments, the Cas9 is acatalytically inactive or "inactivated"
nuclease domain of Cas9. In certain embodiments, the dCas9 is encoded byashortersequencethat is derived fromafull length, catalytically inactivated, Cas9, referred to herein as a"small"dCas9or dSaCas9.
[0233] Incertainembodiments,the inactivated, small, Cas9 (dSaCas9)operatively-linkedto an active nuclease. In certain embodiments, the disclosure provides afusionprotein comprising, consisting essentiallyoforconsistingofaDNAbindingdomain and molecule nuclease, wherein the nuclease comprisesasmall, inactivated Cas9 (dSaCas9). In certain embodiments, the dSaCas9 ofthedisclosure comprises the mutations D1OAandN580A
(underlined and bolded) which inactivate the catalytic site. In certain embodiments, the dSaCas9 ofthedisclosure comprises the amino acid sequence of:

1021 ASKTQSIKKY STDILGNLYE VKSKKHPQII KKG (SEQ IDNO: 32).
[02341 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 2021-01-15 dCas9 with substitutions at positions 10 and 840 of the amino acid sequence of the dCas9 which inactivate the catalytic site. In certain embodiments, these substitutions are D 10A and H840A. In certain embodiments, the amino acid sequence of the dCas9 comprises the sequence of:

1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD (SEQIEONO:
33).
[0235] In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dCas9 or a dSaCas9 and a type IIS
endonuclease. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and a type ilS endonuclease, including, but not limited to, AciI, Mn1I, AlwI, BbvI, Bed, BceAI, BsmAI, BsmFI, BspCNI, Bsrl, BtsCI, HgaI, HphI, HpyAV, Mbo II, My II, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, Bmrl, Bpmi. BpuEI, Bsa, BseRI, Bsgl, Bsml, BspMI, BsrBI, BsrB1, BsrD1, BtgZI, Btsl, Earl, Ecil, Mmel, NmeAIII, BbvCI, Bpul OI, BspQI, SapI, Bael, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051.
In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or Date Recue/Date Received 2021-01-15 consist of a dSaCas9 and Clo051.An exemplary Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:
EGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLAINEYGFKGRH
LGGSRKPDGI'VY STILEDNFGHVDTKA YSEGYSLPISQADEMERY V RENSNRDEEVN
PNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE
KIRSGEMTIEELERAMFNNSEFILKY (SEQ ID NO: 34).
[02361 An exemplary dCas9-Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of (Clo051 sequence underlined, linker bold italics, dCas9 sequence in italics):
MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKONRLFEMKVLELL

ENSNRDEEVNPNKWVENFSEEVKKYYFVFISGSFKGKFEEQLR.RLSMTFGVNGSAV
NVVNLLLGAEK I RSG EMTIEELERAMFNNSEFILKY GGGGSDKKYSIGLAIG .77,KSVGWA
VITDEYKVPSKKFKVLGATDRHSIKKNLIGALLFDS'GETAE4TRLKRTARRRYTRRICNRICY
LQEIFSNEMAKVDDSFFIIRLEESFLVEEDKKHERKPIEGNIVDEVAYHEKYPTIYHLRKKL
VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDATSDVDKLFIQLVQTYNQLFEENPINA
S'GVDAKA1LSARLSICSRRLETVLIAQLPGEKKNGIFGNIJAISI.GLTPNFKS1vFDLAEDAL7,Q
LSKDTYDDDLDNLLAQIGDQYADLFLAAICNLS'DAILLSDILRVNTEITKAPLS'ASMIKRY DE
HHQUEILLKALVRQQLPEKYKEIFFDQS1aG YAGYIDGGAVELTYKFIKPILEKVIDGTE
ELLVKI-NREDLIRKQRTFDNGSIPHQINI,GELHAILRRQEDFYPFLKDNREKIEKILTFRIP
YYVGPLARGNSREAWMIRKSEETITPWNFEEVVDKGASAQSFIERMTATDKATIPNEKVLP
KHSLLYEY1,7VYNELIKVKYVTEGAIRKPAFLSGEQKKAIVDLLEKTNRKVTVKQLKEDY
KIGECEDSVELSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLIITLFEDREVI
EERIXTYAFILFDDKFMKQIXRRRYTG WG RLS'RKLINGIRDKOGKT1I,DFLICS'DGFANR
NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKLM
GRHKPENIV1EMARENQ7TQKGQKNSRERMKRIEEGIKE'LG SQILKEHP VENTQLQNEKL

SEEVVKKAIKNYWRQLINAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLTETRQTIKHV
AOLDSRAINTKYDENDKLIREVKVITLKSKLVSDFRKDIVEYKVREINNYHHAIIDAYLNAV
VGIALIKKYPKLESEPVYGDY KVY DV RKMIA KSEQLIGKATAKY SNIMNPIXTETILAN
GEIRKRPLIE .TNGETG EIVWDKGRDFA TVRKVISMPQVMVKKTEVQTGGFSKESILPK RN
SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGICSICKLKSVKELLGITIMERSSFEK
NPIDFLEAKGY KETKKDLIIKLPKY SLIELENGRKRAILASAGELQKGNELALPSKYVNELY

Date Recue/Date Received 2021-01-15 DICPIREQAENIIHLFTLTIVLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
SQLGGDGSPKKKRKVSS (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 novo creation of synthetic transcription factors that bind to DNA sequences of interest and, if desirable, also allowing a second domain present on the protein or polypeptide to perform an activity related to DNA. TAL proteins have been derived from the organisms Xanthomonas and Ralstonia.
[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 IIS
endonuclease. In certain embodiments of the disclosure, the type IIS endonuclease may comprise, consist essentially of or consist of AciI, Mn 11, Alvd, BbvI, Bed, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, Hgal, Hphl, HpyAV, Mbol I, My 11, PleI, SfaNI, AcuI, BciVI, BfiiAl, BmeBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQI, SapI, Bad, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the type IIS
endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID NO: 34).
[02391 In certain embodiments of the disclosure, the nuclease domain of the genomic editing composition or construct may comprise, consist essentially of or consist of a nuclease domain isolated, derived or recombined from a zinc finger nuclease (ZFN) and a type IIS
endonuclease. In certain embodiments of the disclosure, the type IIS
endonuclease may comprise, consist essentially of or consist of AciI, Mn H, AlwI, BbvI, Bed, BceAT, BsmAI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, MbolI, My II, PleI, SfaNI, AcuI, BciVI, BfuAl, BmgBI, Bmrl, Bpm1, BpuEl, Bsal, BscRI, Bsgl, Bsml, BspM1, BsrBI, BsrBI, BsrD1, Date Recue/Date Received 2021-01-15 BtgZI, BtsI, Earl, EciI, MmeI, NmeAIII, BbvCI, Bpul0I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments of the disclosure, the type ITS
endonuclease may comprise, consist essentially of or consist of Clo051 (SEQ ID
NO: 34).
[0240] In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be covalently linked. For example, a fusion protein may comprise the DNA binding domain and the nuclease domain.
In certain embodiments of the genomic editing compositions or constructs of the disclosure, the DNA binding domain and the nuclease domain may be operably linked through a non-covalent linkage.
Secreted Proteins from Modified T Cells [0241] In certain embodiments of the composition and methods of the disclosure, modified T-cells express therapeutic proteins. Therapeutic proteins of the disclosure include secreted proteins. Preferably, in a therapeutic context, the therapeutic protein is a human protein, including a secreted human protein. When expressed or secreted by CAR-T cells of the disclosure, the combination comprising the CAR-T cell and the therapeutic protein secreted therefrom may be considered a monotherapy. However, the CAR-T cells of the disclosure may be administered as a combination therapy with a second agent. A database of human secreted proteins that may be expressed or secreted by modified T-cell of the disclosure can be found at proteinatlas.org/search/protein class:Predicted%20secreted%20proteins.
Exemplary human secreted proteins are provided, but are not limited to the human secreted proteins, in Table 1.
[0242] TABLE 1. Exemplary Human Secreted Proteins Gene Ensembl ID Gene description AlBG ENSG00000121410 Alpha- 1-B glycoprotein A2M ENSG00000175899 Alpha-2-macroglobulin A2ML1 ENSG00000166535 Alpha-2-macroglobulin-like 1 A4GNT ENSG00000118017 Alpha-1,4-N-acetylglucosaminyltransferase AADACL2 ENSG00000197953 Arylacetamide deacetylase-like AANAT ENSG00000129673 Aralkylamine N-acetyltransferase ABCG1 ENSG00000160179 ATP-binding cassette, sub-family G (WHITE), member 1 ABHD1 ENSG00000143994 Abhydrolase domain containing ABHD10 ENSG00000144827 Abhydrolase domain containing ABHD14A ENSG00000248487 Abhydrolase domain containing ABHD15 ENSG00000168792 Abhydrolase domain containing ABI3BP ENSG00000154175 ABI family, member 3 (NESH) binding protein AC008641.1 ENSG00000279109 Date Recue/Date Received 2021-01-15 AC009133.22 ENSG00000277669 AC009491.2 ENSG00000279664 AC011513.3 ENSG00000267881 AC136352.5 ENSG00000277666 AC145212.4 ENSG00000277400 MaFF-i nteracting protein AC233755.1 ENSG00000275063 ACACB ENSG00000076555 Acetyl-CoA catboxy lase beta ACAN ENSG00000157766 Aggrecan ACE ENSG00000159640 Angioicusin 1 converting enzyme ACHE ENSG00000087085 Acetylcholinestenise (Yt blood group) ACP2 ENSG00000 131575 Acid phosphatase 2. lysosomal ACP3 ENSCi00000102575 Acid phosphatase 5, tartrate resistant ACP6 ENSG00000162836 Acid phosphatase 6, lysophosphatidic ACPP ENSG00000014257 Acid phosphatasc, prostate ACR ENSG00000100312 Acrosin ACRBP ENSG00000 1 11644 Acrosin binding protein ACRV1 ENSG00000134940 Acrosomal vesicle protein 1 ACSF2 ENSG00000167107 Acyl-CoA synthetase family member 2 A CTLIO ENSG00000182584 Actin-like 10 ACVR1 ENSG00000I 15170 Act ivin A receptor, type!
ACVR IC EN SG00000123612 Activin A tumor, type IC
ACVRL I ENSCi00000139567 Activin A receptor type 11-like 1 ACYP I ENSG00000 119640 Acylphosphatase 1, elythrocyle (common) type ACYP2 ENSG00000170634 Acylphosphatasc 2, muscle type ADAM10 ENSG00000137845 ADAM metallopeptidase domain 10 ADAM12 ENSG00000 I 48848 ADAM metallopeptidase domain 12 ADAM15 EN SG00000143537 ADAM metallopeptidase domain 15 ADAM 17 ENSG00000151694 ADAM metallopeptidase domain 17 ADAM 18 EN SG00000168619 ADAM metallopeptidase domain 18 ADAM22 ENSG00000008277 ADAM metallopeptidase domain 22 A DAM28 ENSG00000042980 ADAM rnetallopeptidase domain 28 ADAM29 ENSG00000168594 ADAM metallopeptidase domain 29 ADAM32 ENSG00000197140 ADAM metallopeptidase domain 32 ADAM33 ENSG00000149451 ADAM metallopeptidase domain 33 ADAM7 ENSG00000069206 ADAM metallopeptidase domain 7 ADAM8 EN SG00000151651 ADAM metallopeptidase domain 8 ADAM9 ENSG00000 168615 ADAM metallopeptidase domain 9 ADAMDEC I ENSG00000134028 ADAM-like, decysin 1 A DAMISI ENSG00000154734 ADAM metallopeptidase with thivinbospondin type 1 motif, 1 ADAMTS 10 EN SG00000 I 42303 ADAM inetallopeptidase with thrombospondin type I motif, 10 ADAMTS12 ENSG00000151388 ADAM metallopeptidase with thromhospondin type 1 motif, 12 ADAMTS 13 ENSG00000 I 60323 ADAM metallopeptidase with thronthospondin type I motif, 13 ADAMTS14 ENSG00000138316 ADAM metallopeptidase with thrombospondin type 1 motif, 14 ADAMTS15 ENSG00000166106 ADAM metallopeptidase with thrombospondin type 1 motif, 15 Date Recue/Date Received 2021-01-15 ADANITs16 ENSG00000 I 45536 ADAM metallopeptidase with thrombospondin type I motif, 16 ADAMTS17 ENSG00000140470 ADAM metallopeptidase with thrombospondin type 1 motif, 17 ADAMTS18 ENSG00000140873 ADAM metallopeptidase with lhnmbospondin type I motif, 18 ADAMTS19 ENS600000145808 ADAM metallopeptidase with thrombospondin type 1 motif. 19 ADAMTS2 EN SG0000008'7116 ADAM metallopeptidase with thrombospondin type I motif, 2 ADA.MTS20 ENSG00000173 157 ADAM metallopeptidase with throntbospondirt type I motif, 20 ADAMTS3 ENSG00000156140 ADAM metallopeptidase with thrombospondin type 1 motif, 3 ADAM'FS5 ENS(300000154736 ADAM metallopeptidase with ituumbospondin type 1 motif, 5 ADAMTS6 ENSG00000049192 ADAM metallopeptidase with thrombospondin type 1 motif, 6 ADAMTS7 ENSG00000 136378 ADAM metallopeptidase with thrombospondin type I motif, 7 A DAMIS8 EN SG000001349 i 7 ADAM metallopeptidase with thrombospondin type 1 motif, 8 ADAMTS9 ENSCi00000163638 ADAM metallopeptidase with thrombospondin type 1 motif, 9 ADAMTSL1 ENSG0000017803 I ADAMTS-like I
ADAMISL2 ENS600000197859 ADAMTS-like 2 ADAMTSL3 ENSG00000156218 ADAMTS-like 3 ADAm-rsu EN SG00000143382 ADAWS-like 4 ADAMTSL5 ENSG00000185761 ADAMTS-like 5 ADCK1 ENSG00000063761 AarF domain containing kinase I
ADCYAP I ENSG00000141433 Adenv1atc cyclase activating poiypeptide 1 (pituitary) ADCYAP I R I ENSG00000078549 Aden y late cyclase activating polypeptide I (pituitary) receptor type ADGR A3 ENSG00000 I 52990 Adhesion G protein-coupled receptor A3 ADGRB2 ENSG00000121753 Adhesion G protein-coupled receptor B2 ADGRD1 ENS(300000111452 Adhesion G protein-coupled receptor DI
ADGRE3 ENSG00000131355 Adhesion G protein-coupled receptor E3 ADGRE5 ENSG00000123146 Adhesion 6 protein-coupled receptor E5 ADGRE I ENSG00000153292 Adhesion G protein-coupled receptor Fl ADGRG1 ENSG00000205336 Adhesion G protein-coupled receptor G1 ADGRG5 ENS(300000159618 Adhesion G protein-coupled receptor 65 ADGRG6 ENSG00000112414 Adhesion G protein-coupled receptor (36 ADGRV I EN SG00000 16.1199 Adhesion G protein-coupled receptor VI
ADII ENSG00000182551 Acireductone dioxygenase 1.
ADIG ENS600000182035 Adipogenin ADIPOQ ENSG00000181092 Adiponectin. C IQ and collagen domain containing ADM ENSG00000148926 Adrenomeduilin ADM2 ENSG00000128165 Adrenomedidlin 2 A DM5 ENS600000224420 Adrenomedullin 5 (ptilative) ADPGK ENSG00000159322 ADP-dependent glucokinase ADPRHL2 ENSG00000116863 ADP-ribosylhydrolase like 2 AEBP1 ENSG00000106624 AE binding protein I
AFM ENS600000079557 Afamin APP ENSG00000081051 Alpha-letoprotein AGA. ENSG00000038002 Asparty Iglucosaminidase AGER ENS600000204305 Advanced glycosylation end product-specific receptor AGK ENSG00000006530 AgIglycerol kinase Date Recue/Date Received 2021-01-15 ACiPS ENSG00000018510 Alkylglycerone phosphate sy:nthase AGR2 ENSG0000010654 I Anterior gradient 2, protein disulphide isometase family member AGR3 ENSG00000173467 Anterior gradient 3, protein disulphide isomerase family member ACiRN ENSG00000188157 Agrin AGRP ENSG00000159723 Agouti related neuropeptide AGT ENSG00000135744 Angiotensinogen (serpin peptidase inhibitor, clade A. member 8) AGTPBP1 ENSG00000135049 ATP/GTP binding protein 1 AGTRAP ENSG00000177674 Angiotensin 11 receptor-associated protein AHCYL2 ENSC100000158467 Adetiosylhoinocy mein:Ise-like 2 AHSG ENSG00000145192 Alpha-2-HS-glycoprotein AIG I ENSG00000146416 Androgen-induced I
AK4 ENSG00000162433 Adeny late kinase 4 AKAN() EN SG00000 I 08599 A kinase (PRKA) anchor protein 10 AKR ICI ENSG00000187134 Aldo-keto reductase family I, member CI
AL356289. I ENSG00000279096 AL589743.1 ENSG00000279508 ALA52 ENSG00000158578 5'-aminolevulinate synthase 2 ALB ENSG0000016363 I Albumin ALD119A1 ENSG00000143149 Aldehyde dehydmgenase 9 tinily, member Al ALDOA ENSG00000149925 Aldolase A. fructose-bisphosphate ALGI ENSG00000033011 .ALGL chitobiosyldiphosphodolichol beia-mannosyltransferase ALG5 ENSG00000120697 ALG5, dolichyl-phosphate bcta-glueosv II
nmsferase ALG9 ENSG00000086848 ALG9, alpha-1,2-mannosy It ransferase ALKBH I EN SG00000 I 01)601 AlkB homolog 1, historic H2A
dioxygenase ALKBH5 ENSG00000091542 AlkB homolog 5, RNA demethylase ALP! ENSCi00000163295 Alkaline phosphatase. intestinal ALPL ENSG00000162551 Alkaline phosphatase, liver/bone/kidney ALPP ENSG00000163283 Alkaline phosphatase, placental ALPPL2 ENSG00000163286 Alkaline phosphatase, placental-like 2 AMBN ENSG00000178522 Ameloblastin (enamel matrix protein) AMBP ENSG00000106927 Alpha-l-microglobulinflaikunin precursor AMELX ENSG00000 125363 Amelogenin. X-linked AMELY ENSG0000009972 I Amelogenin, AMH ENSG00000104899 Anti-Mullerian hormone AM1CA I ENSG00000160593 Adhesion molecule, interacts with CXADR
antigen I
AMPD1 ENSCi00000116748 Adenosine monophosphate deaminase 1 AN1TN ENSG00000 187689 Amelotin AMY IA ENS(300000237763 Amylase, alpha IA (salivary) AMY I B ENSG00000174876 Amylase, alpha 1B (salivary) AMY IC EN SG00000 I 87733 Amylase, alpha IC (salivary) AMY2A ENSG00000243480 Amylase, alpha 2A (pancreatic) AMY2B ENSG00000240038 Amylase, alpha 2B (pancreatic) ANG ENSG00000214274 Angiogenin, riboanclease, RNase A family, Date Recue/Date Received 2021-01-15 ANGELI ENSG00000013523 Angel hornolog 1 (Drosophila) ANGPT1 EN SG00000154188 Angiopoietin 1 ANGPT2 ENSG0000009 1879 Angiopoidin 2 ANGPT4 EN SG000001012S0 Angiopoictin 4 ANGPTL1 ENSG00000 I 16194 Angiopoietin-like I
ANGPTL2 ENSG00000136859 Angiopoietin-like 2 ANGPTL3 ENSG00000132855 Angiopoietin-like 3 ANGPTL4 ENSG00000167772 Angiopoietin-like 4 ANGPTL5 ENSG00000187151 Angiopoietin-like 3 ANGPTL6 ENSG00000130812 Angiopoictin-like 6 A NGPTL7 ENSG000001718 19 Angiopoietin-like 7 ANKI ENSCi00000029534 Ankyrin 1, erytluocytic ANKDD IA ENSG00000166839 Ankyrin repeat and death domain containing IA
ANKRD54 ENSG00000100124 Ankyrin repeat domain 54 ANKRD60 ENSG00000124227 Ankyrin repeal domain 60 ANO7 ENSG0000014624)5 Anoctarnin 7 ANOS1 ENSG00000011201 Anosmin 1 ANTXR I ENSG00000169604 Anthrax toxin receptor 1 A0All ENSG00000136250 Acyloxyacyl hydrolase (neutrophil) AOC I ENSG00000002726 Amine oxidase, copper containing 1 A0C2 EN SG00000 131480 Amine oxidase, copper containing 2 (relina-specific) A0C3 ENSCi00000131471 Amine oxidase. copper containing 3 AP000721.4 ENSG00000256100 AP000866.1 ENSG00000279342 A PBB I EN SG00000166313 Ainyloid beta (A4) precursor protein-binding, family B, member 1 (Fe65) APCDD1 ENSG00000154856 Adenomatosis polyposis cob down-regulated APCS ENSG00000132703 Amyloid P component. serum APELA ENSG00000248329 Apel in receptor early endogenous ligand APLN ENSG00000171388 Apelin APLP2 ENSG00000084234 Amyloid beta (A4) precursor-like protein APOA I EN SG00000 I 18 137 Apolipoptotein A4 APOAIBP ENSG00000163382 Apolipoprotein A4 binding protein AP0A2 ENSG00000158874 Apolipoprotein A41 AP0A4 EN SG00000110244 Apolipoprotein A-1V
AF'0A5 ENSG00000 110243 Apolipoptotein A-V
APOB ENSG00000084674 Apolipoptotein B
APOCI ENSG00000130208 Apolipoprotein C-I
APOC2 ENSG00000234906 Apolipoprotein C4I
APOC3 ENSCi00000110245 Apolipoprotein C-Ill APOC4 ENSG00000267467 Apolipoprotein C-IV
APOC4-APOC2 ENSG00000224916 APOC4-APOC2 teadthrough (NMD candidate) APOD ENSG00000189058 Apolipoprotein D
APOE ENSG00000130203 Apolipoprolein E
APOF ENSG00000175336 Apolipoprotein F

Date Recue/Date Received 2021-01-15 APOH ENSG00000091583 Apolipoprotein H (beta-2-glycoptotein 1) APOL I ENSG00000100342 Apolipoprotein L, 1 APOL3 ENSG00000128284 Apolipoprotein L, 3 APOM ENSG00000204444 Apolipoprotein M
APOOL EN SG00000155008 Apolipoptotein 0-like ARCN1 ENSG00000095139 Arcliain 1 ARF1P2 ENSG00000132254 ADP-ribosylation factor interacting protein 2 ARHGAP36 ENSG00000147256 Rho GTPase activating protein .36 ARFIGAP6 ENSG00000047648 Rho GTPase activating protein 6 ARHGEF4 ENSG00000 1 36002 Rho guanine nucleotide exchange factor (GEF) 4 ARL16 ENSG00000214087 ADP-ribosylation factor-like 16 ARMC5 ENSCi00000140691 Armadillo repeat containing 5 ARNTL ENSG00000133794 Atyl hydrocarbon receptor nuclear translocator-like ARSA ENSG00000100299 AiyIsulfatase A
ARSB ENSG00000 1 13273 Alylsulfatase B
ARSE ENSG00000157399 Atylsullatase E (choncliodysplasta punctata 1) ARSG ENSG00000141337 Arylsulfatase ARSI ENSG00000183876 Arylsulfatase family, member I
ARSK EN S000000164291 ArylsuLfatase family, member K
ART3 ENSG00000156219 ADP-ribosyltransferase 3 ART4 EN SG00000111339 ADP-ribosy Itransferase 4 (Dombrock blood group) ARTS ENSCi00000167311 ADP-ribosyltransferase 5 ARTN ENSG00000117407 An.emin ASAH1 ENSG00000104763 N-acylsphingosinc amidohydrolase Meld ceramidase) 1 ASAH2 EN SG0000018861 1 N-acylsphingosine antidohydrolase (non-lysosomal ceramidase) ASCLI ENSG00000139352 Achaete-scute faintly bHI.H transcription factor I
ASIP ENSG00000101440 Agouti signaling protein ASPN ENSG00000106819 Asporin =
ASTI, ENSG00000188886 Astacin-like metallo-endopeptidase (M12 family) ATAD5 ENSG00000176208 ATPase family, AAA domain containing 3 ATAT1 ENSG00000137343 Alpha tubidin acetyltransfetase 1 ATG2A ENSG00000110046 Autophagy related 2A
ATG5 ENS000000057663 Autophagy related 5 ATmiN ENSG00000166454 ATM inienictor ATP13A 1 EN S000000105726 ATPase type 13A1 ATP5F1 EN SG00000116459 ATP synthase, H+ transporting, mitochondnal Fo complex.
subunit 131 ATP6AP 1 ENSG00000071553 ATPase, H+ transporting. lysosomal accessory _protein 1 protein¨i ATPAF I ENSG00000123472 ATP s-ynthase mitochondria' Fl complex assembly factor 1 AUH ENSG00000148090 AU RNA binding protein/enoyl-CoA
hydratase AVP ENSG00000101200 Argi nine vasopressin AXIN2 ENS000000168646 Axin 2 --A-2-631--------ECigiiiiiiiiiii-6-6i/E----AVG:2-glycoiCTein 1, zinc-binding Date Recue/Date Received 2021-01-15 AZUL ENSG00000 I 72232 Azurocidin 1 Et2M ENSG00000166710 Beta-2-microglobulin B3GALNT1 ENS(300000169255 Beta-1,3-N-acetylgalactosaminyltransferase 1 tgloboside blood group) B3GALNT2 ENSG00000162885 Beta-1,3-N-acetylgalactosaminy It ransfentse 2 B3GALT1 ENSG00000172318 1.1DP-Gal:beirtGlcNAc beta I ,3-galactosy It ninsferase, poly peptide 1 B3GALT4 ENSG00000235863 LIDP-GalteraGicNAc beta 1,3-galactosyltransferase, poly peptide 4 B3GALT5 ENSG00000183778 UDP-Gal:beraGIcNAc beta 1.3-galactosy It ransferase, poly peptide 5 B3GALT6 ENSG00000176022 1.1DP-Gal:betaGal beta 1,3-galactosyliransfemse polypeptide 6 B3GAT3 ENSG00000149541 Beta-1,3-gluctirony It ransferase 3 B3GLCT ENSG00000187676 Beta 3-glucosy It ransferase B3GNT4 ENSG00000176383 131)P-GIcNAc:betaGal beta-1,3 -N-acetylglitcosaminvItransferase 4 133GNT6 ENSG00000198488 UDP-GleNAc:netaGal beta-1,3 -N-acetylgiucosanainvitransferase 6 B3ONT7 ENS(300000156966 UDP-01cNAc:betaGal beta-1,3-N-acety Igiticosamittyltransfetase 7 B3GNT8 ENSG0000017719 I LIDP-GleNAc: betaGal beta-1.3-N-acetylalucosaminylt ransferase 8 B3(iNT9 ENSG00000237172 1.1DP-GIcNAc:netaGal beta-1,3 -N-acetylgiucosarninvitransferase 9 B4GALNT I ENSG00000135454 Beta-1,4-N-acetyl-galactosaminy I
transferase 1 B4GALNT3 ENSG00000139044 Beta-1.4-N-acetyl-galactosatninyltransferase 3 B4GALNT4 ENSG00000182272 Beta-1,4-N-acetyl-galactosaminyl transferase 4 B4GALT4 ENSG00000121578 IlDP-GatbetaGIcNAc beta 1,4-galactosyltransferase, poly peptide 4 B4GALT5 ENSG00000158470 UDP-Gal:betaGIcNAc beta 1.4-gaiactosyltransferase, poly peptide 5 B4GALT6 EN SG00000118276 UDP-Gal:betaGlcNAc beta 1,4-galactosyltmnsferase, poly peptide 6 B4GA. T1 EN SG00000174684 Bet a-1,4-glucurony it ransferase 1 B9D I ENSG00000108641 139 protein domain 1 BACE2 ENSG00000182240 Beta-site APP-cleaving enzyme 2 BAGE5 ENS(300000279973 B melanoma antigen Jaunty, member 5 BCAN1 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 Buty ry lc hot' neste rase BCKDHB ENSG00000083 123 Branched chain keto acid dehvdrogenase El, beta polypeptide BDNF EN SG00000176697 Brain-derived nettrotrophic factor BGLAP ENSG00000242252 Bone prunnt-carboxyglutarnate (gia) protein BGN ENSCi00000182492 Biglycan BLVRB ENSG00000090013 Biliveidin teductasc B
BMP1 ENS(300000168487 Bone rnorphogenetic protein 1 Date Recue/Date Received 2021-01-15 BM.P10 EN SG00000163217 Bone morphogenetic protein 10 EtIVP15 ENSG00000130385 Bone morphogenetic protein 15 BMP2 ENSG00000125845 Bone morphogenetic protein 2 BMP3 ENSG00000152785 Bone morphogenetic protein 3 BMP4 ENSG00000125378 Bone morphogenetic protein 4 BMP6 EN8G00000153162 Bone morphogenetic protein 6 BMP7 ENSG0000010 1144 Bone morphogenetic protein 7 BMP8A ENSG00000183682 Bone morphogenetic protein 8a BMP8B ENSG00000116985 Bone morphogenetic protein 8b BMPER EN SC100000164619 BMP binding endothelial regulator BNC1 EN S000000169594 Basonuclin 1 BOC ENSCi00000144857 BOC cell adhesion associated. oncogene regulated BOD1 ENSG000001459 19 Biorientat ion of chromosomes in cell division 1 BOLA1 ENS000000178096 BolA family member I
BPI ENSG00000101425 Bacterieidal/penneability-increasing protein BPEFA1 EN S00000019818:; BPI fold containing family A, member 1 BPEFA2 ENSG00000131050 BPI fold containing family A. member 2 BPIFA3 ENSG00000131059 BPI fold containing family A. member 3 BPIFB I ENSG00000125999 BPI fold containing family B. member 1 BPIEB2 ENSCi00000078898 BPI fold containing family B, member 2 BPEFB3 EN SG00000186190 BPI fold containing family B, member 3 BPEFB4 EN SG00000186191 BPI fold containing family B, member 4 BPIFB6 ENSG00000167104 BPI fold containing family B, member 6 BPIFC ENSG00000184459 BPI fold containing family C
BRE! EN SG00000185024 BRF1, RNA polyinerase HI transcription initiation factor 90 kDa subunit BRINP I ENSG00000078725 Bone morphogenetic protein/retinoic acid inducible neural-specific I
BRINP2 ENSG00000198797 Bone morphogenctic protcin/retinoic acid inducible neural-specific 2 =
BRINP3 EN SGt M000162670 Bone morphogenetic protein/retinoic acid inducible neural-specific 3 BSG ENSG00000172270 Basigin (Ok blood group) BSPHI ENSG00000188334 Binder of sperm protein homolog 1 BST1 ENSG00000109743 Bone marrow stromal cell antigen 1 BIBD1.7 ENSG00000204347 BTB (P02) domain containing 17 BTD ENSG00000169814 Biotinidase BTN2A2 EN S(100000124508 Butyrophilin, subfamily 2, member A2 13IN3A1 ENSG00000026950 Butyrophilin. subfamily 3, member Al BTN3A2 ENSG00000186470 Butyrophilin, subfamily 3, member A2 ENSC100000111801 Butyrophilin, sitblamily 3, member A3 Cl0orf10 ENSCi00000165507 Chromosome 10 open reading frame 10 Cl 0or199 ENSG00000188373 Chromosome 10 open reading frame 99 CI lorfl EN SG00000137720 Chromosome 11 open reading frame 1 C I lorf24 ENSG00000171067 Chromosome 11 open reading frame 24 Cl 1or145 ENS600000174370 Chromosome 11 open reading frame 45 Date Recue/Date Received 2021-01-15 Cl lot-f94 ENSG00000234776 Chromosome 11 open reading frame 94 C12orf10 ENSG00000139637 Chromosome 12 open reading frame 10 C12 049 ENSG00000111412 Chromosome 12 open reading frame 49 C12orf73 ENSG00000204954 Chromosome 12 open reading frame 73 Cl 2orf76 ENSG00000174436 Chromosome 12 open reading frame 76 C I 4orf110 ENSG00000185347 Chromosome 14 open reading frame 80 C14orf93 ENSG00000100802 Chromosome 14 open reading frame 93 Cl6or189 ENSG00000153446 Chromosome 16 open reading frame 89 CI6or190 ENSG00000215131 Chromosome 16 open reading frame 90 C I 7orf67 ENSG00000214226 Climmosome r open reading frame 67 Cl 7otf75 ENSG00000108666 Chromosome 17 open reading frame 75 C17orf99 ENSCi00000187997 Chromosome 17 open reading frame 99 C 1 800154 ENSG00000166845 Chromosome 18 open reading frame 54 C I 9or147 ENSG00000160392 Chromosome 19 open reading frame 47 C I 9orf70 EN SG00000174917 Chromosome 19 open reading frame 70 Cl 90rf80 EN SG00000130173 Chromosome 1.9 open reading frame 80 CIGALTI ENSG00000106392 Core 1 synthase, glycoprotein-N-acetylgalactosarnine 3-beta-galactosy1transfemse 1 Clorf127 ENSG00000175262 Chromosome I open reading frame 127 C I orl 159 ENSG00000131591 Chromosome (open reading frame 159 Clorf198 EN SG00000119280 Chromosome 1 open reading frame 198 C 1 orf234 ENSG00000227868 Chromosome I open reading frame 234 CI or154 ENSG00000118292 Chromosome 1 open reading frame 54 Cl orf56 ENSG00000143443 Chromosome I. open reading frame 56 C I QA ENSG00000173372 Complement component I. q subcomponent, A
chain CIQB ENSG00000173369 Complement component I. q subcomponent, B
chain C1QC ENSG00000159189 Complement component I, q subcomponent. C
chain Cl QL1 EN SG00000131094 Complement component 1, q subcomponent-like I
C I QL2 EN SG00000144119 Complement component 1, q subcomponent-like 2 C1QL3 ENSCi00000165985 Complement component 1, q subcomponent-like 3 CIQL4 ENSG00000186897 Complement component I. q subcomponent-like 4 C1QTNF I EN SG00000173918 Clq and tumor necrosis factor related protein 1 Cl Q1NE2 EN SG0000014586 I Clq and tumor necrosis factor related protein 2 Cl QTNF3 ENSG00000082196 Clq and tumor necrosis factor related protein 3 C1QTNF4 ENSG00000172247 Clq and tumor necrosis factor related protein 4 CIQTNF5 ENSG00000223953 Clq and tumor necrosis factor related protein 5 Cl QMIF7 EN SG00000163 145 C It] and tumor necrosis factor related protein 7 C I QTNF8 EN S(10000018447 I Cfq and tumor necrosis factor related protein 8 CI WM:9 ENSG00000240654 Clq and rumor necrosis factor related protein 9 C1QTNF9B ENSG00000205863 Clq and tumor necrosis factor related protein 9B
CIR ENSG00000159403 Complement component I. r subcomponent C1 RI, ENSG00000139178 Complement component 1, r subcomponent-like CIS ENSG00000182326 Complement component 1, s subcomponent C2 ENSG00000166278 Complement component 2 Date Recue/Date Received 2021-01-15 C2 lorf33 EN SG00000 I 60221 Chromosome 21 open reading frame 33 C2lorf62 ENSG00000205929 Chromosome 21 open reading frame 62 C22orll 5 ENSG00000169314 Chromosome 22 open reading frame 15 C22orf46 ENSG00000184208 Chromosome 22 open reading frame 46 C2CD2 EN SG0000015'7617 C2 calcium-dependent domain containing C2orf40 ENSG00000 I 19147 Chromosome 2 open reading frame 40 C2orf66 ENSG00000187944 Chromosome 2 open reading frame 66 C2orf69 ENSG00000178074 Chromosome 2 open reading frame 69 C2orf78 EN SG00000187833 Chromosome 2 open reading frame 78 C3 EN SC100000125730 Complement component 3 C3orf33 ENSG00000174928 Chromosome 3 open reading frame 33 C3orf58 EN SCi00000181744 Chromosome 3 open reading frame 58 C4A EN SG00000244731 Complement component 4A (Rodgers blood group) C4B ENSG00000224389 Complement component 48 (Chido blood group) C4BPA ENSG00000123838 Complement component 4 binding protein, alpha 0113P8 EN SG00000123843 Compleinetit component 4 binding protein, beta C4orf26 ENSG00000174792 Chromosome 4 open reading frame 26 C4o1148 ENSG00000243449 Chromosome 4 open reading frame 48 C5 ENSG00000106804 Complement component 5 C5orf46 EN SG00000178776 Chromosome 5 open reading frame 46 C6 ENSG00000039537 Complement component 6 C6orf120 ENSCi00000185127 Chromosome 6 open reading frame 120 Ctiotf15 ENSG00000204542 Chromosome 6 open reading frame 15 C6orf25 ENSG00000204420 Chromosome 6 open reading frame 25 C6orf58 ENSG00000184530 Chromosome 6 open reading frame 58 C7 EN SG00000112936 Compleinetit component 7 C7orf57 EN SG00000164746 Chromosome 7 open reading frame 57 C7orl73 ENSG00000243317 Chromosome 7 open reading frame 73 C8A EN SG00000157131 Complement component 8, alpha polypeptide C88 ENSG00000021852 Complement component 8, beta polypeptide C8G ENSG00000176919 Complement component 8, gamma polypeptide C9 ENSG00000113600 Complement component 9 C9otf47 EN SG00000186354 Chromosome 9 open reading frame 47 CA10 ENSG00000154975 Carbonic anhydrase X
CA I I ENSG00000063180 Carbonic anhydiase XI
CA6 EN SG00000131686 Carbonic anhydrase VI
CA9 EN SG00000107159 Carbonic anhydrase IX
CABLES1 ENSG00000134508 Cdk5 and Abl enzyme substrate 1 CAB?! ENSG00000157782 Calcium binding protein 1 CACNA2DI ENSG00000153956 Calcium channel. voltage-dependent, alpha 2/delta subunit 1 CACNA2D4 ENSG00000151062 Calcium channel, voltage-dependent, alpha 2/delta subunit 4 CADM3 ENSG004)00162706 Cell adhesion molecule 3 CALCA ENSG00000110680 Calcitonin-related polypeptide alpha CALCB EN SG00000175868 Calcitonin-telated polypeptide beta Date Recue/Date Received 2021-01-15 CALCR ENSG00000004948 Calcitonin receptor CALCRL ENSG00000064989 Calcitonin receptor-like CALR ENSG00000179218 Calreticulin CALR3 ENSG00000269058 Calreticulin 3 CALU ENSG00000128595 Calumenin CAMK21) ENSG00000145349 Calciumicaltnodulin-dependent protein kinase 11 delta CAMP ENSG00000164047 Cathelicidin antimicrobial peptide CANX ENS(300000127022 Cal nexin CARKD ENSG00000213995 Carbohydrate kinase domain containing CARM1 ENSG00000142451 Coact i ator-associated argi nine met hyltransferase 1 CARNS1 EN SG00000172508 Carnosine synthase I
CARTPT ENSCi00000164326 CART prepropeptide CASQI ENSG00000143318 Calsequestrin I (fast-twitch skeletal muscle) CASQ2 ENSG00000118729 Calsequestrin 2 (cardiac muscle) CATSPERG ENSG00000099338 Camper channel auxiliary subunit gamma CBLN1 ENSG00000102924 Cerebellin I precursor CBLN2 ENSG00000141668 Cerebellin 2 precursor CBLN3 ENSG00000139899 Cerebellin 3 precursor CBLN4 ENSG00000054803 Cerebellin 4 precursor CCBE I ENSG00000183287 Collagen and calcium binding EGE domains I
CCDC108 ENSG00000181378 Coiled-coil domain containing 108 CCDC112 ENSCi00000164221 Coiled-coil domain containing 112 CCDC129 ENSG00000180347 Coiled-coil domain containing 129 CCDC134 ENSG00000100147 Coiled-coil domain containing 134 CCDC149 ENSG00000181982 Coiled-coil domain containing 149 CCDC3 ENSG00000151468 Coital-cod domain containing 3 CCDC80 ENSG00000091986 Coiled-coil domain containing 80 CCDC85A ENSG00000055813 Coiled-coil domain containing 85A
CCDC88B ENS(300000168071 Coiled-coil domain containing 88B
CCER2 ENSG00000262484 Coiled-coil glutamate-rich protein 2 CCK ENSG00000187094 Cholecystokinin CCL1. ENSG00000108702 Chemokine (C-C motif) ligand CCL11. ENSG00000172156 Chemokine (C-C motif) ligand II
CC113 ENS(300000181374 Chemokine (C-C motif) ligand 13 CCLI4 ENSG00000276409 Chemokine (C-C motif) ligand 14 CCL15 EN SG000002757 18 Chemokine (C-C motif) ligand 15 CCL16 ENSG00000275152 Chemokine (C-C motif) ligand 16 CCL17 ENSG00000102970 Chemokine (C-C motif) ligand 17 CCL18 ENSG00000275385 Chemokine (C-C motif) ligand 18 (pulmonary and act rvation-tegulated) CCL19 ENSG00000172724 Chemokine (C-C motif) ligand 19 CCL2 ENSG00000108691 Chemokine (C-C motif) ligand 2 CCL20 ENSG00000115009 Chemokine (C-C motif) ligand 20 CCL21. ENSG00000137077 Chemokine (C-C motif) ligand 21 CCL22 EN SG00000102962 Chemokine (C-C motif) ligand 22 Date Recue/Date Received 2021-01-15 CCL23 ENSG00000274736 Chemokine (C-C motif) ligand 23 CCL24 ENSG00000106178 Chemokine (C-C motif) ligand 24 CCL25 EN SG000001:31142 Chemokine (C-C motif) ligand 25 CCL26 ENSG00000006606 Chemokine (C-C motif) ligand 26 CCL27 ENSG00000213927 Chemokine (C-C motif) ligand 27 CCL28 ENSG00000151882 Chemokine (C-C motif) ligand 28 CCL3 ENSG00000277632 Chemokine (C-C motif) ligand 3 (7CL31,3 ENSG00000276085 Chemokine (C-C moll ligand 3-like 3 CCL4 ENSG00000275302 Chemokine (C-C motif) ligand 4 CCIAL2 ENSG00000276070 Chemokine (C-C motif) ligand 4-like 2 C(1..5 EN SG00000271503 Chemokine (C-C motif) ligand 5 CCL? ENSCi00000108688 Chemokine (C-C motif) ligand 7 CCL8 EN S(30000010870() Chemokine (C-C motif) ligand 8 CCNB 1 I.P1 EN SG00000100814 Cy:chit Ell interacting protein 1, E3 ubiquitin protein ligase CCNI1 EN SG00000163660 Cycl in L
ENSG00000221978 Cycl in L2 CD14 ENSG00000170458 CD14 molecule CD160 EN SG00000117281 CD160 molecule CD164 ENSG00000135535 CD164 molecule. sialomucin CD177 ENSG00000204936 CD177 molecule CD1E ENSG00000158488 (Die molecule CD2 ENSCi00000116824 C1)2 molecule CD200 ENSG00000091972 CD200 molecule CD200R 1 ENSG00000163606 CD200 receptor 1 CD22 EN SG00000012124 CD22 molecule CD226 ENSG00000150637 CD226 molecule CD24 ENSG00000272398 C1)24 molecule CD276 ENSG00000103855 CD276 molecule (7D300A ENSG00000167851 CD300a molecule CD300LB ENSG00000178789 CD300 molecule-like family member b CD300LF EN SG00000186074 C1)300 molecule-like family member f CD300LG ENSG00000161649 C1)300 molecule-like family member g CD3D ENSG00000167286 CD3d molecule, delta (CD3-TCR complex) (1)4 EN SG00000010610 CD4 molecule CD40 ENSG00000101017 CD40 molecule, TNE receptor superfamily member 5 CD44 ENSG00000026508 C1)44 molecule (Indian blood group) CD48 EN SG00000117091 C1)48 molecule CD5 ENSG00000110448 C1)5 molecule C1)55 ENSG00000196352 CD55 molecule, decay accelerating factor for complement (Cromer blood group) C1)59 ENSG00000085063 C1)59 molecule, complement regulatory protein C1)5L ENSG00000073754 CD5 molecule-like CD6 EN S(10000001:3725 C1)6 molecule CD68 ENSG00000129226 C1)68 molecule Date Recue/Date Received 2021-01-15 CD7 ENSG00000173762 CD7 molecule CD79A ENSG00000105369 CD79a molecule, immunoglobulin-associated alpha (1)80 ENSG00000121594 CD80 molecule CD86 ENSG00000114013 CD86 molecule CD8A ENSCi00000153563 CD8a molecule CD8B ENSG00000172116 CD8b molecule CD99 ENSG00000002586 CD99 molecule CDC23 ENSG00000094880 Cell division cycle 23 CDC40 ENSG00000168438 Cell division cycle 40 CDC45 ENSG00000093009 Cell division cycle 45 CDCP1 EN SG0000016:3814 CUB domain containing protein 1 CDCP2 ENSCi00000157211 CUB domain containing protein 2 CDH1 ENSG00000039068 Cadherin 1, type 1 CDH 11 ENSG00000140937 Cadherin 11, type 2. OB-cadherin (osteoblast) CDH13 ENSG00000140945 Cadherin 13 0)1417 EN SG00000079112 Cadherin 17, LE cadherin (liver-intestine) CDH18 ENSG00000145526 Cadherin 18, type 2 CDH19 ENSG00000071991 Cadherin 19, type 2 CD1123 ENSG00000107736 Cadherin-related 23 CDH5 ENSG00000179776 Cadherin 5, type 2 (vascular endothelium) CDHR1 ENSG00000148600 Cadherin-related family member 1 CDHR4 ENSCi00000187492 Cadherin-related family member 4 CDHR5 ENSG00000099834 Cadherin-related family member 5 CDKN2A ENSG00000147889 Cychn-dependent kinase inhibitor 2A
CDNF ENSG00000183267 Cerebral dopamine netimirophic factor CDON ENSG00000064309 Cell adhesion associated, oncogene regulated CDSN ENSG00000204539 Corneodesrnosin CEACAM16 EN SG00000213892 Carcinoembryonic antigen-related cell adhesion molecule 16 CE.ACAM18 ENSG00000213822 Carcinoembryonic antigen-related cell adhesion molecule 18 CEACAM19 ENSG00000186567 Carcinoettiblyonic antigen-related cell adhesion molecule 19 CEACAM5 ENSG00000105388 Carcinoembryonic antigen-related cell adhesion molecule 5 CEACAM7 ENSG00000007306 Carcinoembryonic antigen-related cell adhesion molecule 7 CEACAM8 ENSG00000124469 Carcinoemblyonic antigen-related cell adhesion molecule 8 CECR1 ENSG00000093072 Cat eye syndrome chromosome region, candidate 1 CECR5 ENSG00000069998 Cat eye syndrome chromosome region, candidate 3 CEL ENSG00000170835 Carboxyl ester lipase CELA2A ENSG00000142615 Chymotrypsin-like elastase family, member CELA2B ENSG00000215704 Chymotrypsin-like elastase family, member CELA3A ENSG00000142789 Chyminry psin-like elastase family, member 3A
CELA3I3 ENSCi00000219073 Chy mot rypsin-like elastase family, member 3B
CEMIP ENSG00000103888 Cell migration inducing protein.
hyaluronan binding CEP89 ENSG00000121289 Centrosoinal protein 89kDa CER1 ENSG00000147869 Cerbents 1, DAN family BM? antagonist CERCAM ENSG00000167123 Cerebral endothelial cell adhesion molecule Date Recue/Date Received 2021-01-15 CERS1 ENSG00000223802 Ceramide synthase 1 CES1 ENSG00000198848 Carboxylesterase 1 CES3 ENSG00000172828 Carboxylesierase 3 CES4A ENSG00000172824 Carbox-ylestcrase 4A
CES5A ENSG00000159398 Carboxylesteiase 5A
CETP ENSG00000087237 Cholesteryl ester transfer protein, plasma CFB ENSG00000243649 Complement factor B
CFC I ENSG00000136698 Cripto, FRL-1, cryptic family 1 CFCIB ENSG00000152093 Cripto, FRI,-1, cryptic family 1B
CFD ENSG00000197766 Complement factor D (adipsin) CFDP1 ENSG00000153774 Craniolacial development ptotein 1 CFH ENSCi00000000971 Complement factor H
CFHR1 ENSG00000244414 Complement factor H-related 1 CFHR2 EN SG00000080910 Complement factor H-related 2 CFHR3 ENSG00000116785 Complement factor H-related 3 CFHR4 ENSG00000134365 Complement factor H-rekned 4 CFEER5 ENSG00000134389 Complement factor H-related 5 CFI ENSG00000205403 Complement factor I
CFP ENSG00000126759 Complement factor properdin CGA ENSG00000135346 Glycoprolein hormones, alpha polypeptide CGB ENSG00000104827 Chorionic gonadotropin, beta poly peptide CGB1 ENSCi00000267631 Chorionic gonadotropin. beta polypeptide CGB2 ENSG00000104818 Chorionic gonadotropin, beta polypeptide CGB5 ENSG00000189052 Chorionic gonadotropin, beta polypeptidc CGB7 ENSG00000196337 Chorionic gonadotropin, beta polypeptide CGB8 ENSG00000213030 Chotionic gonadoiropin, beta polypeptide COREY' I ENSG00000138028 Cell growth regulator with EF-hand domain I
CH507-9B2.3 ENSG00000280071 CHAD ENSG00000136457 Chondroadherin CHADL ENSG00000100399 Chondroadlierin-like CHEK2 ENSG00000183765 Checkpoint ki wise 2 CHGA ENSG00000100604 Chromogranin A
CHGB ENSG00000089199 Chromogranin B
0113I, I ENSG00000133048 anti inase 3-like I (cartilage glycoprotein-39) CHI3L2 ENSG00000064886 Chitinase 3-like 2 CHIA ENSG0000013-1216 Chitinase, acidic CHID! ENSG00000177830 Chitinase domain containing I
CHIT! ENSG00000133063 Chitinase 1 (chitouiosidase) CHL I ENSG0000013412 I Cell adhesion molecule L I-like CHN I ENSG00000128656 Chimerin 1 CHPF ENSG00000123989 Chondroitin polymerizing factor CHPF2 ENSG00000033100 Cliondroitin polymerizing factor 2 CHRD ENSG00000090539 Chordirt CHRDL1 ENSG00000101938 anordin-like 1 Date Recue/Date Received 2021-01-15 CHRDL2 ENSG00000054938 Chordin-like 2 CHRNA2 ENSG00000120903 Cholinergic receptor, nicotinic, alpha 2 (neuronal) CHRNA5 ENSG00000169684 Cholinergic receptor, nicotinic, alpha 5 (neuronal) CHRNB I ENSG00000170175 Cholinergic receptor, nicotinic, beta I
(muscle) CHRND ENSG00000135902 Cholinergic receptor, nicotinic, delta (muscle) CHST1. ENSG00000175264 Carbohydrate (keratan sulfate (ial-6) sulfotransferase CHSTIO ENSG00000115526 Carbohydrate sulfotransferase 10 oisT t I ENSG00000171.3 H) Carbohydrate (chondroit in 4) sulfotransferase 11 CHST13 EN SG00000180767 Carbohydrate (chondroitin 4) sulfotninsferase 13 CHST4 ENSG00000140835 Carbohydrate (N-acetylgincosamine 6-0) sulfotransferase 4 CHST5 ENSG00000135702 Carbohydrate (N-acetylglucosarnine 6-0) sullotransfetase 5 CHST6 ENSCi00000183196 Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 6 CtisT7 ENSG00000147119 Carbohydrate (N-acetylglitcosamine 6-0) sulfotransferase 7 C1IsT8 ENSG00000124302 Carbohydrate (N-aectyigalactostimine 4-0) sulfotransfcrasc 8 CHSY1 ENSG00000131873 Chondmitin sulfate synthase 1 CHSY3 EN SG00000198108 Chondmitin suUale 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 ENS000000141076 Cirrhosis, autosomal recessive IA
(cirhin) CKLF ENSG00000217555 Chemokine-like factor CKMT I A ENSG00000223572 Creatine kinase, mitochondria! IA
CKIVIT1 B NS(300000237289 eremitic kinase, mitochondria! 1B
CLCA1 ENSG00000016490 Chloride channel accessory I
CLCF1 ENSG00000175505 Caidionophin-like cytokine factor I
CLDN15 ENSG00000106404 Claudin 15 CLDN7 ENSG00000181885 Claudin '7 CLDND I ENS(300000080822 Claudin domain containing I
CLEC I I A ENSG00000105472 C-type lectin domain flintily 11, member A
CLEC I 6A ENSG00000038532 C-type lectin domain family 16, member A
CLEC I 8A ENSG00000157322 C-type lectin domain family 18, member A
CLEC1813 ENSG00000140839 C-type lectin domain family 18, member B
CLEC I8C ENSG00000157335 C-type lectin domain family IS. member C
CLEC I9A ENSG00000261210 C-type lectin domain flintily 19, member A
CLEC2B ENSG00000 110832 C-type lectin domain family 2, member B
CLEC3A ENSG00000166509 C-type lectin domain family 3, member A
CLEC3B ENSG00000163815 C-type lectin domain family 3, member B
CLGN ENSG00000153132 Calmegin CLN5 ENSG00000102805 Ceroid-lipoluscinosis, neuronal 5 CLPS ENSG00000137392 Colipase, pancreatic CLPSL1 ENSG00000204140 Colipase-hke I
CLPSL2 ENSG00000 196748 Colipase-like 2 CLPX EN SC700000166855 Caseinolytic mitochondria' matrix peptidase chaperone subunit CLSTN3 ENSG00000139182 Calsyntenin 3 Date Recue/Date Received 2021-01-15 CLU ENSG00000120885 Clusterin CLUL1 ENSG0000007910 I Clusterin-like 1 (retinal) CMA1 ENSG00000092009 Chyrnase I, mast cell CMPK I ENSG00000162368 Cytidinc monophosphate (UMP-CMP) kinasc 1, cytosolic CNBD1 ENSG00000 176511 Cyclic nucleotide binding domain containing 1 CNDPI EN sG00000150656 Carnosine dipeptidase I
tmetallopeptidase M20 family) CNPY2 ENSG00000257727 Canopy FGF signaling regulator 2 CNPY3 ENS(300000137161 Canopy FGE signaling regulator 3 CNPY4 ENSG00000166997 Canopy Rir, signaling regulator 4 (NM ENSG00000122756 Ciliar:v nettrotrophic factor receptor CNTN I ENSG0000001.8236 Contact in 1 CNTN2 ENSCi00000184144 Contactin 2 (axonal) CNTN3 ENsG000001 13805 Contactin 3 (plasmacy lima associated) CNTN4 ENSG00000144619 Contact in 4 CNTN5 ENSG00000149972 Contact in 5 CNTNAP2 ENSG00000174469 Contact in associated protein-like 2 CNTNAP3 ENSG00000106714 Contactin associated protein-like 3 CNTNAP3B ENSG00000154529 Contactin associated protein-like 3B
COASY ENSG00000068120 CoA synthasc COCH ENS(300000100473 Cochlin COG3 ENSG00000136152 Component of oligomeric golgi complex 3 COLIOA1 ENSCi00000123500 Collagen, type X, alpha 1 COL I IA I ENSG00000060718 Collagen, type XI, alpha 1 COLI1A2 ENS000000204248 Collagen, type Xi, alpha 2 COL12A1 ENSG00000111799 Collagen. type XII, alpha I
COL14A I ENSG00000187955 Collagen. type XIV, alpha 1 COL15A1 ENSG0000020429 I Collagen, type XV, alpha COL16A I ENSG00000084636 Collagen, type X NIL alpha 1 COL18A1 ENS(300000182871 Collagen, type XVIII, alpha 1 COL 19A I ENSG00000082293 Collagen, type XIX, alpha 1 COL 1A1 ENSG0000010882 I Collagen. type I, alpha 1 COL1A2 ENSG00000164692 Collagen, type I, alpha 2 COL.20A I ENSG00000101203 Collagen, type XX, alpha 1 COL21. AI ENS(300000124749 Collagen, type XXI, alpha 1 COL22AI ENSG00000169436 Collagen. type XXII, alpha 1 COL24A1 ENSG00000171502 Collagen. type XXIV, alpha 1 C0L26A1 ENSG00000160963 Collagen, type XXVI. alpha 1 COL27A1 ENSG00000196739 Collagen, type XXVII, alpha 1 COL28A1 ENS(300000215018 Collagen, type XXVIII, alpha 1 COL2A 1 ENSG00000139219 Collagen, type II, alpha 1 COL3A1 ENSG00000168542 Collagen. type III, alpha 1 COL4A1 ENSG00000187498 Collagen. type IV. alpha 1 COL4A2 ENSG00000134871 Collagen, type IV, alpha 2 COL4A3 ENS(300000169031 Collagen, type IV, alpha 3 (Goodpasture antigen) Date Recue/Date Received 2021-01-15 COL4A4 ENSG00000081052 Collagen, type IV, alpha 4 COL4A5 ENSG00000188153 Collagen, type IV, alpha 5 COL4A6 ENSG00000197565 Collagen, type IV, alpha 6 COL5A1 ENSG00000130635 Collagen. type V. alpha 1 COL5A2 ENS(100000204262 Collagen. type V. alpha 2 COL5A3 ENSG00000080573 Collagen, type V. alpha 3 COL6A1 ENSG00000142156 Collagen, type VI, alpha 1 COL6A2 ENS(300000142173 Collagen, type VI, alpha 2 COL6A3 ENSG00000163359 Collagen, type VI, alpha 3 COL6A5 ENSG00000172752 Collagen. type VI, alpha 5 COL6A6 ENS000000206384 Collagen. type VI, alpha 6 COL7A1 ENSCi00000114270 Collagen, type VII, alpha 1 COL8A1 EN S00000014481() Collagen, type VIII, alpha 1 COL8A2 EN SG00000171812 Collagen, type VIII, alpha 2 COL9A1 ENSG00000112280 Collagen. type TX, alpha 1 COL9A2 ENS000000049089 Collagen. type IX, alpha 2 COL9A3 ENSG00000092758 Collagen, type IX, alpha 3 COLECIO ENSG00000184374 CoIlectin sub-family member 10 (C-type lectin) COLEC 11 EN SG00000118004 Colleen n sub-family member 11 COLGALT1 ENSG00000130309 Collagen beta( I -0)galactosyltransfenise COLGALT2 ENSG00000198756 Collagen heta(l-0)galaclosyltrtnsferase 2 COLQ ENSCi00000206561 Collagen-like tail subunit (single strand of homotrimer) of asy =nevi c acetylcholinesterase COMP ENSG00000105664 Cartilage oligonteric matrix protein COPS6 ENSG00000168090 COP9 signalosome subunit 6 C006 ENSG00000119723 Cocitiy me Qb monooxygenase CORT ENSG00000241563 Cortistatin Cl' EN S0000000'47457 Ceruloplasmin (termxidase) CPA1 ENSG00000091704 Carboxypeptidase Al (pancreatic) CPA2 ENSCi00000158516 Carboxypeptidase A2 (pancieatic) CPA3 ENS00000016375 Carboxypeptidase A3 (mast cell) CPA4 ENSG00000128510 Calbox-ypeptidase A4 CPA6 ENSG00000165078 Carboxypeptidase A6 CPAMD8 ENS00000016011 I C3 and PZP-Iike, alpha-2-mac toglobulin domain containing 8 CPB1 ENSG00000153002 Carboxypeptidase B1 (tissue) CPB2 ENSG00000080618 Carboxypeptidase B2 (plasma) CPI EN SG00000109472 Carboxypeptidase CPM EN SG00000135678 Carboxypeptidase M
CPN I ENSG00000120054 Calboxypeptidase N, txdypeptide 1 CPN2 ENSG00000178772 Carboxypeptidase N. polypeptide 2 CP0 ENS(300000144410 Carboxypeptidase 0 CPQ ENS(300000104324 Carboxypeptidase Q
CPVL ENSG00000106066 Carboxypeptidase, vitellogenic-like cpxml ENSG00000088882 Carboxypeptidase X (M14 ('amily), member Date Recue/Date Received 2021-01-15 CPX.M2 EN SG00000121898 Car'ooxypeptidase X (M14 family), member CPZ ENSG00000109625 Qtrboxypeptidase Z
CR IL ENSG00000197721 Complement component (3b/4b) receptor 1-like CRB2 ENSG00000148204 Crumbs family member 2 CREG1 ENSG00000143162 Cellular repressor of E1A-st mulled genes CREW EN sG00000175874 Cellular repressor of E1A-stimulated genes 2 CRELD I ENSG00000163703 Cysteine-rich with EGF-like domains I
CRELD2 ENSG00000184164 C)isteirie-rich with EGF-like domains 2 CRH ENSG00000147571 Corticotropin releasing hormone CRHI3P ENSG00000145708 Con icotropin releasing hormone binding protein CRHR I EN SG00000120088 Corticotropin releasing hormone receptor CRHR2 ENSCi00000106113 Corticotropin releasing hormone receptor CRISP1 ENSG00000124812 Cysteirie-rich secretory protein 1 CRISP2 ENSG00000124490 C'ysteine-rich secretory protein 2 CRISP3 ENSG00000096006 Cysteine-rich secretory protein 3 CRISPLD2 EN SG00000103196 Cysteine-rich secretory protein LCCL
domain containing 2 CM:Ft ENSG00000006016 Cytokine receptor-like factor 1 CRP ENSG00000132693 C-reactive ptotein, pent raxin-related CRTAC I EN SG00000095713 Cartilage acidic protein 1 CRTAP ENSG00000170275 Cartilage associated protein CRY2 EN SG0000012167 I Ciyptochrome circadian clock 2 CSAD ENSCi00000139631 Cysteine sunlit acid decarboxy lase CSF I EN SG00000184371 Colony stimulating factor 1 (macrophage) CSFIR ENSG00000182578 Colony stimulating factor 1 receptor CSF2 EN SG00000 I 64400 Colony stimulating factor 2 (granulocyte-macrophage) CSF2R A EN SG00000198223 Colony StiflIlliaiing factor 2 receptor, alpha, low-aliinny (grantilocyte-mactophage) CSF3 EN SG00000108342 Colony stimulating factor 3 (granuiocyle) CSGALNACT1 ENSG00000147408 Chondroitin sulfate N-acetylgalactosittriinyltransferase 1 CSH1 ENSCi00000136488 Chorionic somatomanunotropin hormone 1 (placental lactogen) CSI-12 ENSG00000213218 Chorionic somatomammotropin hormone 2 CSEILI ENSG00000204414 Chorionic somatomammotropin hormone-like CSNISI EN SG00000126545 Casein alpha s I
CSN2 EN SG00000135222 Casein beta CSN3 ENSG00000171209 Casein kappa CST1 ENSG00000170373 (.'statin SN
CST I 1 ENSG00000125811 Cystatiti II
CST2 EN SG00000170369 Cystatin SA
CST3 EN SG00000101439 Cystatin C
CST4 EN SCi00000101441 Cystatin S
CST5 ENSG00000170367 (.'stat in D
CST6 ENSG00000175115 Cystatin E.1%.
CST7 EN SG00000077984 Cystatin F (leukocystatin) EN SG00000125815 Cysiatin 8 (cystatin-related epididytnal specific) Date Recue/Date Received 2021-01-15 CST9 ENSG00000173335 Cystatin 9 (testatin) CST9L ENSGn0000101435 Cystatin 9-like CSTI, I ENSGoo000125823 CYstatin-like I
CT55 ENSGo00001.69551 Cancer/testis antigen 35 CTB-60B18.6 ENSG00000267335 CTBS EN SG00000117151 Chitobiase, di-N-acetyl-CTD-2313N118.7 ENSG00000225805 CID-2370N5.3 ENSG00000265118 CI-GE ENSG00000118523 Connective tissue growth factor CTHRC I ENSG00000164932 Col lagen triple helix repeat containing I
CFLA4 ENSG00000163599 Cytotoxic T-Iymphocyte-associated protein CTNS ENSCi00000040531 Cystinosin, lysosotnal cystine transporter CTRB I ENSG00000168925 01yr:tot ry psinogen 13I
CIR132 ENSG00000168928 Chymotrypsinogen 132 CTRC ENSG00000162438 Ch y motrypsin C (caldecrin) CTRL EN SG00000141086 Chy mot r' psi n-I ike CTSA ENSG00000064601 Cathepsin A
CTSB ENSG00000164733 Cat hcpsin B
CISC ENSG0000010986I Cathepsin C
CTSD ENSG00000117984 Cathepsin D
CFSE ENSG00000196188 Cathepsin E
CTSF ENSG00000174080 Cathepsin F
--eTSG ENSG00000100448 Cathepsin G
CTSH 1-TNSG110000103811 Cathepsin H
CTSK ENS600000 I 4" Cathepsin K
("1"-SL EN SG00000 135047 Cathepsin L
crso EN SG00000256043 Cathepsin 0 CTSS ENSG00000163131 Cathepsin S
CTSV ENSGo)oo0136943 Cathepsin V
CTSW ENSO000001725-I3 Cathepsin 'W
CTSZ EN SC500000101160 Cathepsin CUBN EN SG00000107611 Cubilin (intrinsic factor-cobalamin receptor) CUTA ENSG00000112514 CutA divalent cation tolerance homolog E.
coli) CX3CL1 ENSG00000006210 Chemokine (C-X3-C motif) ligand I
CXADR ENSG00000154639 Coxsackie virus and adenovints receptor CXCL 1 ENSG00000163739 Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) CXCLIO EN SG00000169245 Chemokine (C-X-C motif) ligand 10 CXCL11 EN SG00000169248 Chemokine (C-X-C motif) ligand 11 CXCL12 ENSG00000107562 Chemokine (C-X-C motif) ligand 12 CXCI.,13 ENSG00000156234 Chemokine (C-X-C motif) ligand 13 CXCL 4 ENS(300000145824 Chemokine (C-X-C motif) ligand 14 CXCL17 ENSG00000189377 Chemokine (C-X-C motif) ligand 17 CXCL2 EN SG0000008104 I Chemokine (C-X-C motif) ligand 2 Date Recue/Date Received 2021-01-15 CXCL3 ENSG00000163734 Chemokine (C-X-C motif) ligand 3 CXCL5 ENSG00000163735 Chemokine (C-X-C motif) ligand 5 CXCL6 ENSG00000124875 Chemokine (C-X-C motif) ligand 6 CXCL8 ENSG00000169429 Chemokine (C-X-C motif) ligand 8 CXCL9 ENSG00000138755 Chernokine (C-X-C motif) ligand 9 CXorf36 EN SG00000147113 Chromosome X open reading frame 36 CYB5D2 ENSG00000167740 Cytochrome b5 domain containing 2 CYHR1 ENS(300000187954 Cysteinethistidine-rich 1 CYP17A1 ENSG00000148795 Cytochromc P450. family 17, subfamily A.
polypeptide 1 CYP20A1 ENSG00000119004 Cytochrome P450, family 20, subfamily A.
polypeptide 1 CYP21A2 EN SG00000231852 Cytochrome P450, family 21, subfamily A, poly peptide 2 CYP26B1 ENSG00000003137 Crochroine P450, family 26, subfamily B, polypeptide 1 CYP2A6 ENSG00000255974 Cytochrome P450. family 2, subfamily A, polypeptide 6 CYP2A7 ENSG00000198077 Cytochrome P450. family 2, subfamily A, polypeptide 7 CYP2B6 ENSG00000197408 Cytochrome P450, family 2, subfamily B, polypeptide 6 CYP2C18 ENSG00000108242 Cytochrome P450, flintily 2, subfamily C, polypeptide 18 CYP2C19 ENSG00000165841 Cytochrome P450, family 2, subfamily C.
polypeptide 19 CYP2C8 EN SG00000138115 Cytochrome P450. family 2, subfamily C.
polypeptide 8 CYP2C9 ENSG00000138109 Cytochromc P450. family 2, subfamily C, polypeptide 9 CYP2E1 ENSG00000130649 Cytochrome P450, family 2, subfamily E, polypeptide 1 CYP2F1 EN SG00000197446 Cytochrome P450, family 2, subfamily F.
polypeptide 1 CYP2.12 ENSCi00000134716 Cytochrome P450, family 2, subfamily J.
polypeptide 2 CYP2R1 ENSG00000186104 Cytochrome P450. family 2, subfamily R.
polypeptide 1 CYP2S1 ENSG00000167600 Cytochrome P450. family 2, subfamily S.
polypeptide 1 CYP2W1 ENSG00000073067 Cytochrome P450. family 2, subfamily W, polypeptide 1 CYP46A I ENSG00000036530 Cytochrome P450, family 46, subfamily A.
polypeptide 1 CYP4F1.1 ENSG00000171903 Cytochrome P450, family 4. subfamily F, polypeptide 11 CYP4F2 ENSG00000186115 Cytochrome P450. family 4, subfamily F, po13,:pemide 2 CYR61 EN S(300000142871 angiogenic inducer, 61 CYTLI ENSG00000170891 Cytokine-like 1 D2HGDE1 EN SG000001M902 D-2-hydroxyglutarate dehydrogenase DAG" ENSG00000173402 Dystroglycan 1 (dystropltin-associated glycoprotein 1) DAND5 ENSG00000179284 DAN domain family member 5. BMP
antagonist IMO ENS(300000110887 D-amino-acid oxidase DAZAP2 ENSG00000183283 DAZ associated protein 2 DBH ENSG00000123454 Dopamine beta-hydroxylase (dopamine beta-monooxygenase) DBNI, ENSG00000136279 Drebrin-like DCD ENSG00000161634 Dermcidin DCN ENS(300000011465 Decorin DDIAS ENSG00000165490 DNA damage-induced apoptosis suppressor DDOST ENSG00000244038 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit (non-catalytic) DDR1 ENSG00000204580 Discoidin domain receptor tyrosine kinase DDR2 ENSG00000162733 Discoidin domain receptor tyrosine kinase Date Recue/Date Received 2021-01-15 DDT EN SG00000099977 D-doptichrome tantome rase DDX17 ENSG00000100201 DEAD (Asp-Glu-Ala-Asp) box helicase 17 DDX20 ENS(300000064703 DEAD (Asp-Glu-Ala-Asp) box polypeptide 20 DDX25 ENSG00000109832 DEAD (Asp-Glu-Ala-Asp) box hclicasc 23 DDX28 ENSG00000182810 DEAD (Asp-G1 it-Ala-Asp) box polypeptide 28 DI-EAF1 SG00000177030 DEA1:1 transcription factor DEF8 ENSG00000140995 Differentially expressed in FDCP 8 homolog (mouse) DEFA1 ENSG00000206047 Defensin, alpha 1 DEFA I B ENSG00000240247 DeibilSi31, alpha 113 DEFA3 ENSG00000239839 Defensin. alpha 3, neutrophil-specific DEFA4 EN SG00000164821 Defensin. alpha 4, corticostal in DEFA5 ENSCi00000164816 Defensin, alpha 5, Paneth cell-specific DEFA6 ENSG00000164822 Delensin, alpha 6, Paneth cell-specific DEFB1 ENSG00000164825 Dcfertsirt, beta 1 DEFB1n3 A ENSG00000176797 Defensin. beta 103A
DEE:13103B EN SG00000177243 Defensin. beta 103B
DEFB104A ENSG00000176782 Defensin, beta 104A
DEFB104B ENSG00000177023 Defensin, beta 1048 DEFB105A. ENSG00000186562 Defensin. beta 105A.
DE/13105B ENSG00000186599 Defensin, beta 10513 DEE:8106A EN SG00000186579 Defensin. beta 106A
DEFB106B ENSCi00000187082 Defensin, beta 106B
DEFB107A ENSG00000186572 Defensin, beta 107A
DEFB107B ENSG00000198129 Defensin, beta 1078 DE17131088 ENSG00000184276 Defensin. beta 1088 DEFB 110 EN SC100000203970 Defensin. beta 110 DEFI3113 EN SG00000214642 Defensin, beta 113 DEFB114 ENSG00000177684 Defensin, beta 114 DEFB115 ENSG00000215547 Defensin, beta 115 DEFB116 ENSG00000215545 Defensin. beta 116 DEE:8118 EN SG00000131068 Defensin. beta 118 DE113119 EN SG00000 I 80483 Defensin, beta 119 DEFB121 ENSG00000204548 Defensin, beta 121 DEFB123 ENSG00000180424 Defensin, beta 123 DER3124 ENSG0000018038:1 Defensin. beta 124 DEFB 125 ENSG00000178591 Defensin. beta 125 DEFI3126 EN SG00000 I 25788 Defensin, beta 126 DEFB127 ENSG00000088782 Defensin, beta 127 DEFB128 ENSG00000185982 Defensin, beta 128 DEFB129 ENSG00000 I 25903 Defensin. beta 129 DEFB 130 ENSG00000232948 Defensin. beta 130 DEFB131 EN SG00000186146 Defensin. beta 131 DEFB132 ENSG00000186458 Defensin, beta 132 DEFB133 ENSG00000214643 Defensin, beta 133 Date Recue/Date Received 2021-01-15 D I:1E13134 ENSG00000205882 Defensin, beta 134 DEFI3135 ENSG00000205883 Defensin, beta 135 DEFB136 ENSG00000205884 Delensin, beta 136 DEFF34A ENSG00000171711 Defensin. beta 4A
DEFB4B ENSG00000177237 Defensin, beta 4B
DFNA5 ENsG00000105928 Deafness, autosomal dominant 5 DFNB31 ENSG00000095397 Deafness, autosomal recessive 31 DGC12 ENS(300000070413 DiGeorge syndrome critical region gene 2 DKR ENSG00000139549 Desert hedgehog DHRS4 ENSG00000157326 Dehydrogenaseiteductase (SDR family) member 4 DFIRS4L2 ENSG00000187630 Dehydrogenase/teductase (SDR family) member 4 like 2 DHRS7 ENSCi00000100612 Dehydrogenasereductase (SDR family) member 7 DHRS7C ENSG00000184544 Dehydrogenase1reductase (SDR family) member 7C
DHRS9 ENSG00000073737 Dehydrogenase/reductase (SDR family) member 9 DHRSX ENSG00000169084 Dehydrogenaseireductase (SDR family) X-linked DFIX29 ENSG00000067248 DEAH (Asp-Ght-Ala-His) box polypeptide 29 DHX30 EN SG00000132153 DEAH (Asp-Gin-Ala-His) box helicase 30 DHX8 ENSG00000067596 DEAH (Asp-Gin-Ala-His) box polypeptide 8 1)IO2 ENSG00000211448 Dciodinase, iodothyroninc, type 11 DIXDC1 ENSCi00000150764 DIX domain containing 1 DKK1 ENSG00000107984 Dickkopf WT signaling pathway inhibitor 1 DKK2 ENSCi00000155011 Dickkopf WNT signaling pathway inhibitor DKK3 ENSG00000050165 Dickkopf WNT signaling pathway inhibitor DKK4 ENSG00000104371 Dickkopfl,vNT signaling pathway inhibitor DKKL I EN SG00000104901 Dickkopf-like I
D1,64 ENSG00000132535 Discs, large homolog 4 (Drosophila) DLK1 ENSG00000185559 Delta-like 1 homolog (Drosophila) DLL1 ENSG00000198719 Delta-like 1 (Drosophila) DLL3 ENSG00000090932 Delta-like 3 (Drosophila) DMT3T1 ENSG00000187908 Deleted in malignant brain tumors 1 DMKN ENSG00000161249 Dermokine DMPI ENSG00000152592 Dentin matrix acidic phosphoprotein 1 DNRTA2 ENSG00000142700 DIVIRT-like family A2 DNAAF5 EN S(300000164818 Dynein, axonemai, assembly factor 5 DNAH14 ENSG00000185842 Dy nein, axonemal, heavy chain 14 DNAJT3 1! ENSG00000090520 Dnai (Hsp40) homolog, subfamily B. member DNA.1139 ENSG00000128590 Dnai (lisp40) homolog, subfamily 13, member 9 DNAJC25-GNGIO ENSG00000244115 DNAJC25-GNGIO readthrough DNAJC3 ENSG00000102580 Dria,1 (Ffsp40) homolog. subfamily C.
member 3 DNASE I ENSG00000213918 Deoxy ribonuclease DNASE IL! ENSG00000013563 Deoxyribonuclease 1-like 1 DNASEIL2 ENSG00000167968 Deoxyribonuclease Hike 2 DNASE 1L3 ENSG00000163687 Deox-yribonuclease I-like 3 DNASE2 EN S(300000105612 Deoxyriborinclease II, lysosomal Date Recue/Date Received 2021-01-15 DNASE2B ENSG00000137976 Deoxyribonuelease 11 beta DPEP1 EN SG00000015413 Dipeptidase 1 (renal) DPEP2 ENSG00000167261 Di pept idase 2 DPEP3 ENSG00000141096 Dipcptidase 3 DPF3 ENSG00000205683 04, zinc and double PHI) fingers, family DPP4 EN sG00000197635 Dipeptidyi-peptidase 4 DPP7 ENSG00000176978 Dipeptidyl-peptidase 7 DPT ENSG00000143196 De !Immo po tit in DRAXIN ENSG00000162490 Dorsal inhibitory axon guidance protein DSE ENSG000001 I 1817 Dermaian sulfate epirnerase DSG2 ENSG00000046604 Desmoglein 2 DSPP ENSCi00000152591 Dentin sialophosphoprotein DST ENSG00000151914 Dystonin DUOX 1 ENSG00000137857 Dual oxidase 1 DYNLT3 EN SG00000165169 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 ECIIDC1 ENSG00000093144 Ethylmalonyl-CoA decarbox-ylase 1 ECM I ENSG00000143369 Extracellular matrix protein 1 ECM2 ENSG00000106823 Extracelltdar matrix protein 2, female organ and aclipocyte specific ECSIT ENSG00000130159 ECSIT signalling integrator EDDM3A EN SG00000181562 Epididymal protein 3A
EDDM3B EN SG00000181552 Epididy,.mill protein 313 EDEM2 ENSG00000088298 ER degradation enhancer. mannosidase alpha-like 2 EDE,M3 ENSG00000116406 ER degradation enhancer, mannosidase alpha-like 3 EDIL3 ENSG00000164176 EGF-like repeats and discoidin I-like domains 3 EON) ENSG00000078401 Endothelin 1 EDN2 ENSCi00000127129 Endothelin 2 EDN3 ENSG00000124205 Endothelin 3 EDNRB EN SG00000136160 Endothelin receptor type B
=WI ENSG00000115380 EU' containing fibulin-like extracellular matrix protein 1 EFEMP2 ENSG00000172638 EGF containing 11MM-like extracellular matrix protein 2 EFN A I EN SG00000169242 Ephrin-A I
EFNA2 ENSG00000099617 Ephrin-A2 EFNA4 ENSG00000243364 Ephrin-A4 EGFL6 ENSG00000198759 EGF-like-domain. multiple 6 EGFL7 ENSG00000172889 EGF-like-domain. multiple 7 EGFL8 EN SCi00000241404 EGF-tike-domain, multiple 8 EGFLAM EN SG00000164318 EGF-like, fibronectin type III and laminin G domains EGER ENSG00000146648 Epidermal growth factor receptor EHBP1 ENSG00000115504 EH domain binding protein 1 EFT ENSG00000135373 Ets homologous factor EHMTI EN SG00000181090 Euchromatic histone-lysine N-tnethyltransferase 1 Date Recue/Date Received 2021-01-15 EHMT2 EN SG00000204371 Etichromatic histone-lysine N-methyltransfenise 2 EIF2 AK I ENSG00000086232 Etikalyotic translation initiation factor 2-alpha kinase EL ANE ENSG00000197561 Elastase. nettirophil expressed ELN ENSG00000049540 Elastin ELP2 ENSG00000134739 Elongator acety It ransferase complex subunit 2 ELSPBP I EN sG00000169393 Epididytnal sperm binding protein 1 EMC I EN SG00000127463 ER membrane protein complex subunit 1 EMCIO ENSG00000161671 ER membrane protein complex subunit 10 EMC) ENSG00000100908 ER membrane protein complex subunit 9 FMCN ENSG00000164015 Endomucin EMID1 EN S000000186998 EMI domain containing 1 EMILIN I ENSCi00000138080 Elastin microfibfil interfacer 1 EMIL1N2 ENSG00000132205 Elastin microlibril interfacer 2 EMIL1N3 ENSG00000183798 Elastin inicrofibri1 interfacer 3 ENAM EN SG00000132464 Enamelin ENDOG ENSG00000167136 Endonuclease G
ENDOLT ENSG00000111405 Endonuclease, polyU-specific ENHO EN SG00000168913 Energy homeostasis associated EN04 EN SG00000188316 Enolase family member 4 ENPP6 EN SG00000164303 Ectonucleotide pyrophosphataseiphosphodiesierase 6 ENPP7 EN SG00000182156 Ectonucleolide pyrophosphalase/phosphodiesterase 7 ENTPD5 ENSCi00000187097 Ectonucleoside triphosphate diphosphohydrolase 5 ENTPD8 ENSG00000188833 Ectonucleoside uiphosphate diphosphohydrolase 8 EOGT EN SG00000163378 EGF domain-specific 0-linked N-acetylgIticosamine (G1cNAc) transferase EPCAM EN SG00000119888 Epithelial cell adhesion molecule EPDR 1 ENSG00000086289 Ependy min related 1 EPGN EN SG00000182585 Epithelial mitogen EPH A 10 EN S000000183317 EPH receptor MO
EPHA3 ENSCi00000044524 EPH receptor A3 EPH A4 EN SG00000116106 EPH receptor A4 EPHA7 EN SG00000135333 EPH receptor A7 EPHA8 ENSG00000070886 EPH receptor A8 EPHB2 EN SG00000133216 EPH receptor 82 EPHB4 EN SG00000196411 EPH receptor B4 EPH X3 EN SG0000010513I Epoxide hydrolase 3 FPO ENSG00000130427 Erythropoietin EPP1N ENSG00000101448 Epididymal peptidase inhibitor EPP1N-WFDC6 EN SG00000249139 EPPIN-WFDC6 readilatough EPS15 ENSG00000085832 Epidermal growth factor receptor pathway substrate 13 EPS8L1 EN SG00000131037 EPS8-like 1 FPX EN SC100000121053 Risinopitilperoxidase EPYC ENSG00000083782 Epiphycan EQTN EN SG00000120160 Ectuatorin, sperm actosome associated Date Recue/Date Received 2021-01-15 ER AP 1. ENSG00000164307 Endoplasmic retiCUIUM aminopeptidase 1 ERAP2 ENSG00000164308 Endoplasmic reticulum aminopeptidase 2 ER13133 ENSG0000006536 I Erb-h2 receptor tyrosine kinase 3 ERLIN I ENSG00000107566 ER lipid raft associated 1 ERLIN2 ENSG00000147475 ER lipid raft associated 2 ERNI ENSG00000178607 Endoplasmic reticulum to nucleus signaling 1 ERN2 ENSG00000134398 Endoplasmic reticulum to nucleus signaling 2 ERO I A ENSG00000197930 Endoplasmic reticulum oxidoteductase alpha ERO I B ENSG00000086619 Endoplasmic reticulum oxidoreductase beta ERP27 ENSG00000139055 Endoplasmic reticulum protein 27 ERP29 EN SG00000089248 Endoplasmic reticulum protein 29 ERP44 EN SCi00000023318 Endoplasmic reticulum protein 44 ERv3. I ENSG00000213462 Endogenous retrovirus group 3, member I
ESMI, ENSG00000164283 Endothelial cell-specific molecule 1.
ESRP1 EN SG00000104413 Epithelial splicing regulatory protein 1 EXOG ENSG00000157036 Endolexonuclease endonuclease G-like EXIT 1 ENSG00000158008 Exostosin-like glycosyltransferase 1 EXTL2 ENSG00000162694 Exostosin-like glycosyltransferase 2 FIO ENSG00000126218 Coagulation factor X
Fl I ENSG00000088926 Coagulation factor Xi F12 EN SG00000131187 Coagulation factor XII (Hagman factor) 1713B ENSG00000143278 Coagulation factor XIII. B polypeptide F2 ENSG000001802 10 Coagulation factor H (thrombin) F2R EN SG00000181104 Coagulation factor II (thrombin) receptor F2RL3 ENSG00000127533 Coagulation factor II (thrombin) receptor-like 3 F5 ENSG00000198734 Coagulation factor V (proaccelerin, labile factor) 177 ENSG00000057593 Coagulation factor VII (serum prothrombin conversion accelerator) ENSG00000185010 Coagulation factor VIII, procoagulant component F9 ENSCi0000010198 I Coagulation factor LX
FABP6 ENSG00000170231 Fatty acid binding piotein 6, ilea!
F.A1V110713 ENSG00000065809 Family with sequence similarity 107, member B
FAM131 A ENSG00000175182 Family with sequence similarity 131, member A
FAM132 A ENSG00000184163 Family with sequence similarity 132.
member A
FAM132B ENSG00000178752 Family with sequence similarity 132, member B
FAM150 A ENSG0000019671 I Family with sequence similarity 150, member A
FAIN/1150B ENSG00000189292 Family with sequence similarity 150, member B
F.kM171A1 ENSG00000148468 Family with sequence similarity 171, member Al FAM171B ENSG00000144369 Family with sequence similarity 171.
member B
FAM172A ENSG0000011339 I Family with sequence similarity 172, member A
FAM175A ENSG00000163322 Family with sequence similarity 173, member A
F.A1V1177A1 EN S(100000151327 Family with sequence similarity 177, member AI
FAM1.7913 ENSG00000198718 Family with sequence similarity 179, member B
FAM180A ENSG00000189320 Family with sequence similarity 180.
member A
FAM189A1 ENSG00000104059 Family with sequence similarity 189, member Al Date Recue/Date Received 2021-01-15 FAM198A ENSG00000144649 Family with sequence similarity 198, member A
FAM19A1 ENSG00000183662 Family with sequence similarity 19 (chemokine (C-C motif)-like), member Al FAM19A2 ENSG00000198673 Family with sequence similarity 19 (chemokine (C-C motif)-like), member A2 FAM19A3 ENSG000001114599 Family with sequence similarity 19 (chemokine (C-C motif)-like), member A3 FAMI9A4 ENSG00000163377 Family with sequence similarity 19 (chemokine (C-C motif)-like), member A4 FAM1.9A5 ENSG00000219438 Family with sequence similarity 19 (chemokine. (C-C motif).-like), member A5 FAM20A ENSG00000108950 Family with sequence similarity 20, member A
FAM20C ENSG00000177706 Family with sequence similarity 20, member C
FAM213A ENSG00000122378 Family with sequence similarity 213, member A
FAM26D ENSG00000164451 Family with sequence similarity 26, member D
FAM46B ENSG00000 158246 Family with sequence similarity 46.
member B
FAM57A ENSG00000167695 Family with sequence similarity 57. member A
FA1478A ENSG00000126882 Family with sequence similarity 78, member A
FAM96A ENSG00000166797 Family with sequence similarity 96, member A
FAM9B ENSG00000177138 Family with sequence sinnlanty 9, member B
FAP ENSG00000078098 Fibroblast activation protein, alpha FAS ENSG00000026103 Fas cell surface death receptor FAT 1 ENSG00000083857 FAT atypical cadherin 1 FBLN1 ENSG00000077942 Fibulin FBLN2 EN SG00000163520 Fibulin 2 FBLN5 ENSG00000140092 Fibulin 5 FBNI ENSG00000166147 Fibrillin 1 FBN2 ENSG00000138829 Fibrillin 2 FBN3 ENSG00000142449 Fibrillin 3 FBXW7 ENSG00000109670 F-box and WD repeat domain containing 7, E3 tibiquitin protein ligase FCAR EN SG00000186431 Fe fragment of IgA receptor FCGBP ENSG4.10000275395 Fe fragment of IgG binding protein FCGR1B ENSG00000198019 Fc fragment of IgG, high affinity lb, receptor (CD64) FCGR3A ENSG00000203747 Fc fragment of IgG, low affinity ha, receptor (CD16a) FCGRT ENSG00000104870 Fe fragment of IgG, receptor, transporter, alpha FCMR ENSG00000162894 Fe fragment of IgM receptor FCN1 ENSG00000085265 Ficolin (collagen/fibrinogen domain containing) 1 FCN2 ENSG00000160339 Ficolin (collagen/fibrinogen domain containing lectin) 2 FCN3 ENSG00000142748 Ficolin (collagen/librinogen domain containing) 3 FCRL I ENSG00000163534 Fe receptor-like I
FCRL3 ENSG00000160856 Fe receptor-like 3 FCRL5 EN SC:410000143297 Fe receptor-like 5 Date Recue/Date Received 2021-01-15 FCRLA EN SG00000132185 Fc receptor-like A
FCRLB ENSG00000162746 Fc receptor-like B
FDCSP EN SG00000181617 Follicular dendritic cell secreted protein FETUB EN SG00000090512 Fetuin B
FGA EN S(100000171560 Fibrinogen alpha chain FGB EN SG00000171564 Fibrinogen beta chain FGFIO ENSG00000070193 Fibroblast growth factor 10 FGF17 ENSG00000158815 Fibroblast growth factor 17 FGFI8 ENSG00000156427 Fibroblast growth factor 18 FGF19 ENSG00000162344 Fibroblast growth factor 19 FGF21 ENSG00000105550 Fibroblast growth factor 21 FGF22 ENSCi00000070388 Fibroblast growth factor 22 FGF23 ENSG00000118972 Fibroblast growth factor 23 FGF3 ENSG00000186895 Fibroblast growth factor 3 FGF4 ENS(100000075388 Fibroblast growth factor 4 FGF5 ENSG00000138675 Fibroblast growth factor 5 FGF7 ENSG00000140285 Fibroblast growth factor 7 FGF8 EN SG0000010783 I Fibroblast growth factor 8 (androgen-induced) FGFBP1 ENSG00000137440 Fibroblast growth factor binding protein FGFBP2 EN SG00000137441 Fibroblast growth factor binding protein FGFBP3 ENSG0000017472 I Fibroblast growth factor binding protein FGFR1 ENSCi00000077782 Fibroblast growth factor receptor 1 FGFR2 ENSG00000066468 Fibroblast growth factor receptor 2 FGFR3 ENSG00000068078 Fibroblast growth factor receptor 3 FGFR4 ENSG00000160867 Fibroblast growth factor receptor 4 FGFRL1 EN SG00000127418 Fibroblast growth factor receptor-like 1 FGO EN SG00000171557 Fibrinogen gamma chain FGL I ENSG00000104760 Fibrinogen-like I
FGL2 EN SG0000012795 I Fibrinogen-like 2 FITL1 ENSCi00000022267 Four and a half LIM domains I
MOW ENSG00000134775 Formin homology 2 domain containing 3 FIBIN EN SG00000176971 Fin bud initiation factor hornolog (zebrafish) FICD ENSG00000198855 FIC domain containing FIGF ENSG00000165197 C-los induced growth factor (vascular endothelial growth factor D) F.IX1 ENSG00000179431 Four jointed box I
FKBP10 EN SG00000141756 FK506 binding protein 10.65 kDa FICBP11 ENSG00000134285 FK506 binding protein 11, 19 kDa FKBP14 ENSG00000106080 FK506 binding protein 14. 22 kDa FKBP2 ENSG00000173486 FK506 binding protein 2, 13kDa FKBP7 ENSG00000079150 FK506 binding protein 7 FKBP9 ENSG00000122642 FK506 binding protein 9. 63 k,Da FLT I EN SG00000102755 Fms-relatcd tyrosine kinasc FLT4 ENSG00000037280 Fins-mimed tyrosine kinase 4 FM01 ENSG00000010932 Flavin containing monooxygenase 1 Date Recue/Date Received 2021-01-15 FM02 ENSG00000094963 Flavin containing monooxygenase 2 (non-functional) FM03 ENSG00000007933 Flavin containing monooxygenase 3 FM05 ENSG00000131"781 Flavin containing monooxygenase 5 FMOD ENSG00000122176 Fibromodulin FN I ENS000000 I 15414 Fibronectin I
ENDO EN8G00000164694 Fibronectin type 11.1 domain containing 1 FNDC7 ENSG00000143107 Fibronectin type III domain containing 7 FOCAL) ENS(300000188352 Focadhesin FOLR2 ENSG00000165457 Folate receptor 2 (fetal) FOLR3 EN SGOOoo0110203 Folate receptor 3 (gamma) FOXRE D2 ENSG00000100350 FAD-dependent oxidoreductase domain containing 2 FP325331.1 ENSG00000278881 Uncharacterized protein UNQ6126/PR020091 FPGS ENSG00000136877 Folylpolyglutamate sy inhase MASI ENSG00000138759 Fraser extracelittlar matrix complex subunit 1 FREM I EN SG00000 I 64946 MASI related extracellular matrix 1 FREM3 ENSG00000183090 ERAS! related extracellular matrix 3 FIIMPD2 ENSG00000170324 FERM and PDZ domain containing 2 FRZB ENSG00000162998 Ftizzled-related piotein FRO ENSG00000131808 Follicle stimulating hormone, beta polypeptide FSHR ENSG00000170820 Follicle stimulating hormone receptor FST ENSG00000134363 Follistatin ENSG00000163430 Follistatin-like 1 FSTL3 ENSG00000070404 Follistatin-like 3 (secreted glycoprotein) FSTL4 ENSG00000053108 Follistatin-like 4 FSTL5 ENSG00000168843 Follistatin-like 5 FTCDNL1 EN SG00000226124 Formintinotransferase cyclodeatninase N-terminal like FUCA1 ENSG00000179163 Fucosidase, alpha-L- 1, tissue FUCA2 ENSG00000001036 Fucosidase, alpha-L- 2, plasma FURIN EN SG00000140564 Furin (paired basic amino acid cleaving enzyme) FUT 10 ENSG00000 I 72728 Fticosyltransferase 10 (alpha (1,3) .fttcosy It mitsferase) FUT11 ENSG00000196968 Fucosyltransferase 11 (alpha (1,3) fucosy It ransferase) FXN ENSG00000165060 Frataxin FXR I ENSG00000114416 Fragile X mental retaniation, autosomal homolog 1 FXYD3 ENSG00000089356 FXYD domain containing ion transporl regulator 3 GABBR I ENSG00000204681 Garnina-antinobutyric acid (GABA) 13 receptor. 1 GABRA I EN SGO0o00022355 Gamma-aminobtaync acid (GABA) A
receptor, alpha 1 0A13RA2 ENSG00000151834 Gamma-aminobutync acid (GABA) A receptor, alpha 2 G.kBRA5 ENSG00000186297 Gamma-aminobutyric acid (GABA) A
receptor, alpha 5 GABRG3 ENS(300000182256 Gamma-aminobutyric acid (GABA) A
receptor, gamma 3 GABRP ENSG00000094755 Ganitna-timinobittyric acid (GABA) A
receptor, pi GAL ENS000000069482 Galanin/GMAP prepropeptide GAL3ST1 ENSG00000 I 28242 Galactose-3-0-sulfotransferase G.kL3ST2 ENSG00000154252 Galactose-3-0-sulfotransferase 2 GAL3ST3 ENS(300000175229 Galactose-3-0-sulfoiransferase 3 Date Recue/Date Received 2021-01-15 GALC ENSG00000054983 Galactosylceramidase GALNS ENSG00000141012 Galactosamine (N-acety1)-6-sulfatase GALNT1O ENSG00000164574 Poly peptide N-acetylgalactosaminyltransferase 10 GALNT12 ENSG00000119514 Polypeptidc N-acetylgalactosaminyltransfenise 12 GALNT15 EN SG00000131386 Polypeptide N-acetylgalactosaminyltransferase 15 GALNT2 EN 5G0000014364 I Polypeptide N-acetylgalactosaminylinmsfemse 2 GALNT6 ENSG00000139629 Polypeptide N-acetylgalactosaminyltransferase 6 GALN`Di ENSG00000130035 Poly peptide N -acetylgalactosaminy It ransferase 8 GALNIL6 ENSG00000174473 Poll/peptide N-acetylgalactosaminyltransfenise-like 6 GALP EN SG00000197487 Galanin-like peptide GANAB EN SG00000089597 Glucosidase. alpha; neutral AB
GARS ENSCi00000106105 Glycyl-tRNA synthetase GAS1 ENSG00000180447 Growth arrest-specific I
GAS6 ENSG00000183087 Growth arrest-specific 6 GAST ENSG00000184502 Gastrin GBA ENSG00000177628 Glucosidase. beta, acid GBGT1 ENSG00000148288 Globoside alpha-1,3-N-acetylgalactosaminyltransferase 1 GC ENSG00000145321 Group-specific component (vitamin D
binding protein) GCG ENSG00000115263 Glucagon GCGR ENSG00000215644 Glucagon receptor GCNI7 EN SG00000124091 (ilucosarairtyl(N-acety,.1) transferase family member 7 GCSH ENSCi00000140905 Glycine cleavage system protein H
(aminomethyl carrier) GDF I ENSG00000130283 Growth differentiation factor 1 GDF I 0 ENSG00000266524 Growth differentiation factor 10 GDF11 ENSG00000135414 Growth differentiation factor 11 GDF15 ENSG00000130513 Growth differentiation factor 15 GDF2 EN SG0000026376 I Growth differentiation factor 2 GDF3 ENSG00000184344 Growth differentiation factor 3 GDF5 ENSG00000125965 Growth differentiation factor 5 GDF6 ENSG00000156466 Growth differentiation factor 6 GDF7 EN SG0000014:3869 Growth differentiation factor 7 GDF9 ENSG00000164404 Growth difkrentiation factor 9 GDNF ENSG00000168621 Glial cell derived neurottophic factor GFOD2 ENSG00000141098 Glucose-fructose oxidoteductase domain containing 2 GFPT2 ENSG00000131459 Glittarnine-fnictose-6-phosphate transaininase 2 GFRA2 ENSG00000168546 GDNF family receptor alpha 2 GFRA4 ENSG00000 I 2586 I GDNF family receptor alpha 4 GGA2 ENSG00000103365 Golgi-associated, gamma adaptin ear containing, ARF binding protein 2 GGH ENSCi00000137563 Gamma-glutamy,.1 hydrolase (conjugase, folylpolygammaglutainy I hydrolase) GGT1 EN SG00000100031 Gamma-glutamyltransferase I.
GGT5 ENSG00000099998 Gamma-glutamyltransferase 5 G1-11 ENSG00000259384 Growth hormone I
G1.12 ENSG00000136487 Growth hormone 2 Date Recue/Date Received 2021-01-15 GHDC ENSG00000167925 GH.3 domain containing GHRH ENSG00000118702 Growth hormone releasing hormone GHRHR ENSG00000106128 Growth botmone releasing hormone =coot GIRL EN S000000157017 Glutlin/obcstatin prepropeptide OW ENSG00000134812 Gastric intrinsic factor (vitamin B
synthesis) GIP ENS000000 I 59224 Gastric inhibitory polypeptide GKN I ENSG00000169605 Gastrokine 1 GKN2 ENS000000183607 Gastroki tie 2 GLA, ENSG00000102393 Galactosidasc, alpha GLB1 ENSG00000170266 Cialactosidase, beta 1 GLB IL ENSG00000163521 Galactosidase, beta 1-like GLBIL2 ENSCi00000149328 Galactosiclase, beta 1-like 2 GLCE ENSG00000138604 Glucuronic acid epitnerase GLG1 ENS000000090863 Golgi glycoproteirt I.
GLIPR1 ENS(100000139278 CiLi pathogenesis-related 1 GLIPR IL 1 ENS000000173401 GLI pathogenesis-related I like I
GLIS3 ENSG00000107249 GLIS family zinc finger 3 GLIAP ENSG00000198715 Glycosylated lysosomal membrane protein GLRB ENSG00000109738 Glycinc receptor, beta GLS ENSG00000115419 Glittaminase GLT6D1 ENS000000204007 Glycosy lininsfe rase 6 domain containing I
GLTPD2 ENSCi00000182327 Glycolipid transfer protein domain containing 2 GLUT)! ENSG00000148672 Glutamate dehydrogenase 1 GM2A ENSG00000196743 GM2 ganghoside activator GML ENSG00000104499 Glycosylpliospliatidylinositol anchored molecule like GNAS ENS000000087460 GNAS complex locus ONLY ENS000000115523 Gnmulysin GNPTG ENSG00000090581 N-acetylglucosamine-l-phosphate transferase, gamma subunit GNRH I ENSG00000147437 Gonadottopin-releasing hormone 1 (luteinizing-releasing hormone) GNRH2 ENSG00000125787 Gonadoimpin-releasing hormone 2 GNS ENSG00000135677 Glucosarnine (N-acety1)-6-su11ttase GOLME ENSG00000135052 Golg,i membrane protein 1 GORAB ENS000000120370 Golgin, RAB6-inteincting GOT2 ENS000000125166 Glutamic-oxaloacetic transaminase 2, mitochondria( GP2 ENSG00000169347 Glycoptotein 2 (zy mogen granule membrane) GP6 ENS000000088053 Glycoptotein VI {platelet) GPC2 ENSG00000213420 Glypican 2 GPC5 ENSG00000179399 Glypican 5 GPC6 ENS000000183098 Glypican 6 GPD2 ENS000000115159 Glycerol-3-phosphate dehydrogenase 2 (nitochondrial) GPER I ENS000000164850 G protein-coupled estrogen receptor 1 GPHA2 ENSG00000149733 Glycoprotcin hormone alpha?
GPHB5 ENSG00000179600 Glycoprotein hormone beta 5 Date Recue/Date Received 2021-01-15 GPIFIBPI ENSG00000277494 Glycosy 1phosphatidy,.linositol anchored high density lipoprotein binding protein 1 GPLDI EN SG00000112293 Glycosylphosphatidylinositol specific phospholipase 1)1 GPNMB ENSG00000136235 Glycoprotein (tratismembrane) timb GPR 162 EN SG00000250510 G protein-coupled receptor 162 GPX3 ENSG00000211445 Gluttithionc peroxidase 3 GPX4 ENSG00000167468 Glutathione peroxidase 4 GPX5 ENSG00000224586 Glutathione peroxidase 5 GPX6 EN SG00000198704 Glutathione peroxidase 6 GPX7 ENSG00000116157 Glutathione peroxidase 7 GREMl. EN SG00000166923 Gremlin I. DAN farrtily BM{ antagonist GREM2 ENSG00000180875 Gremlin 2. DAN family BMP antagonist GRHL3 ENSG00000158055 Grainy head-like transcription factor 3 GRIA2 ENSG00000120251 Glutamate receptor, ionotropic, AMPA 2 GRIA3 ENSG00000125675 Glutamate receptor. ionotropic. AMPA 3 GRIA4 ENSG00000152578 Glutamate receptor. ionotropic. AMPA 4 GRIK2 ENSG00000164418 Glutamate receptor. ionotropie, kainate 2 GR1N2B ENSG00000273079 Glutamate receptor, ionotropic, N-methyl D-aspartate 2E3 GRM2 EN SG00000164082 Glutamate receptor, inetabotropic 2 GRM3 ENSG00000198822 Glutamate receptor. membotropic 3 GRM5 ENSG00000168959 Glutamate receptor, metabotropic 5 GRN ENSG000000305132 Granulin GRP EN SG00000 I 34443 Gastrin-releasing peptide G SG I ENSG00000111305 Germ cell associated 1 GSN EN SG00000148180 Gelsolin GTDC 1 ENSG00000121964 Glycosyltiansferase-like domain containing 1 GTPEIPIO ENSG00000105793 GTP-binding protein 10 (putative) GUCA2A ENSG00000197273 Gnarly late cyclase activator 2A
(guanylin) G'UCA2B ENSG00000044012 finny late cyclase activator 213 (uroguanylin) GUSB ENSCi00000169919 Glucuronidase, beta GVQW1 EN SG00000241043 (NOW motif containing 1 GXYLTI ENSG00000151233 Glucoside xylosyltransferase 1 GXYLT2 ENSG00000172986 Glucoside xylosyltransferase 2 GYLTL113 EN SG00000165905 Glycosyltransfelase-like 1/3 GYPB ENSG00000250361 Glycophorin B (MNS blood group) Ga4A ENSG00000145649 Granzyrac A (granzyme I. cytotoxic T-Iymphocyte-associated serine esterase 3) GZMB ENSG00000100453 Granzymc B (granzymc 2, cytotoxic T-Iymphocyte-associated serine esterase 1) GTh11-1 EN SG00000100450 Gmnzyme H (cathepsin (i-like 2. protein h-CCPX) GZIAK NSG00000113088 Grarizyme K (granzy me 31 try ptase II) GZIAN4 ENSG00000197540 Granzyme M (lymphocyte met-ase 1) H6PD ENSG00000049239 Hexose-6-phosp1uite dellydrogenase (glucose 1-dehydrogenase) HABP2 ENSG00000148702 Hyaluronan binding protein 2 Date Recue/Date Received 2021-01-15 HADHB ENSCi00000138029 Hydroxyacy I-Co A deltydrogenase/3-ketoacyl-CoA
thiolase/enoyl-C'oA hydratase (triliinctional protein), beta subunit HAMP ENSG00000105697 Hepcidin antimicrobial peptide HAPLN I ENSG0000014568 I Hyaluronan and proteoglycan link protein HAPLN2 ENSG00000132702 Hyaluronan and proteoglycan link protein HAPLN3 ENSG00000140511 Hyaluronan and proteoglycan link protein HAPLN4 ENS(300000187664 Hyaluronan and proteoglycan link protein HARS2 ENSG00000112855 Histidyl-tRNA synthetase 2, mitochondria' HAVCR1 ENSG00000113249 Hepatitis A virus cellular receptor 1 HCCS ENSG00000004961 Holocytochrome c synthase Ft CRT ENSG00000161610 livpoctetin (orexin) neuropeptide precursor HEATP5A ENSG00000129493 HEAT repeat containing 5A
HEPH ENSG00000089472 Heph.aestin HEXA ENSG00000213614 Flexosaminidase A (alpha polypeptide) HEXB ENSG00000049860 Hexosaminidase B (beta polypeptide) HFE2 ENSG00000168509 Hemochromatosis type 2 (juvenile) HOF EN S(300000019991 Hepatocyte growth factor (hepapoieti it A; scatter ractor) HGFAC ENSCi00000109758 HGF activator HHIP ENSG0000016416 I Hedgehog interacting protein HHIPLE ENSG00000182218 HHIP-like 1 HHIPL2 ENSG00000143512 HHIP-Iike 2 HHLA1 ENS(300000132297 FIE.RV-F1 LTR-associating 1 HH1A2 ENSG00000114455 HERV-H LTR-associating 2 HIBADH ENSG00000106049 3-hy div xy isobitty rate dehydrogenase HINT2 ENSG00000137133 Histidine triad nucleotide binding protein 2 HLA-A ENSG00000206503 Major histocompai ibility complex, class I. A
F1LA-C ENS(300000204525 Major lustocoinpatibility complex, class I. C
HLA-DOA ENSG00000204252 Major histocompatibility complex, class II, DO alpha ENSG00000231389 Major hismcornpatibility complex, class Ii.
DP alpha I
ENSG00000 I 967 3 3 Major histocompatibility complex, class II, DO alpha 1 HLA-DQB1 ENSG00000179344 Major histocompatibility complex, class H, DO beta 1 HLA-DQB2 ENS(300000232629 Major Instocompatibility complex, class Ii, IX) beta 2 HMCNI ENSG00000143341 Hemicentin 1 HMCN2 ENSG00000148357 Hcmicentin 2 HMGCL ENSG00000117305 3-hydroxy met hy1-3-niethylglutaryl-CoA
lyase HMHA I ENSG00000180448 Histocompatibility (minor) HA-1 FIMSD ENS(300000221887 Histocompatibility (minor) semi n domain containing HP ENSCi00000257017 Haptoglobin HPR ENSG0000026170 I Haptoglobin-related protein HPSE ENSG00000173083 Heparanase HPSE2 ENSG00000172987 Heparanase 2 (inactive) HPX ENSG00000110169 Hemopexin F1RC ENSG00000130528 Histidine rich calcium binding protein HRG ENSG00000113905 Histidine-rich glycoprotcin Date Recue/Date Received 2021-01-15 HRSP12 ENSG0000013254 I Heat-responsive protein 12 HS2STE ENSG00000153936 Heparan sulfate 2-0-sulfotransferase 1 HS3ST1 ENS(300000002587 Heparan sulfate (glitcosamitie) 3-0-sulfottansferase 1 HS6ST1 ENSG00000136720 Heparan sulfate 6-0-sulfoiransferase 1 FIS6ST3 ENS(100000185352 Heparin sulfate 6-0-sulfotransferase 3 MD 118 IL EN8G00000167733 Hydroxy steroid (11-beta) dehydrogenase 1-like HSD171311 ENSG00000198189 Hydroxysteroid (17-beta) dehydrogenase 11 FISD1787 ENS(300000132196 Hydroxysteroid (17-beta) dehydrogenase 7 H8P9013 ENSG00000166598 Heat shock protein 90k0a beta (Grp94), member 1 FISPA13 EN SG00000155304 Heat shock protein 70kDa family, member HSPA5 ENSG00000044574 Heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) HSPG2 ENSCi00000142798 Heparan sulfate proteoglycan 2 HT,ATIP2 ENS(300000109854 HIV-I Tat interactive protein 2, 30kDa frrN I. ENSG00000126550 Histatin 1 FITN3 ENSG00000205649 Histatin HTRA I ENSG00000166033 HirA serine peptidase 1 HTRA3 EN SG00000170801 HtrA senile peptidase 3 HTRA4 ENSG00000169495 HtrA senile peptidase 4 HYAL1 ENSG00000114378 Hyaltironogiucosaminidase 1 HYAL2 ENSG0000006800 I Hyaltironoghicosaminidase 2 HYAL3 ENSG00000186792 Hyalaronogincosatni nidase 3 HYOU1 ENSCi00000149428 Hypoxia up-regulated 1 LAPP ENSG00000121351 Islet amyloid poly peptide IBSP ENSG00000029559 Integrin-binding sirdoprotein ICAMI ENSG00000090339 Intercellular adhesion molecule 1 ICAM2 ENSG00000108622 intercellular adhesion molecule 2 ICAM4 EN SG0000010537I intercellular adhesion molecule 4 (Landsteiner-Wiener blood group) !DI ENSG00000125968 inhibitor of DNA binding 1, dominant negative helix-loop-helix protein IDE ENSG00000119912 Insulin-degrading enzyme IDNK ENSG00000148057 idtiK, gluconokinase liontolog (E. coil) IDS ENSG00000010404 Iduronate 2-sullatase !DIM ENSG00000127415 Iduronidase, alpha-L-IF127L2 ENS(300000119632 interferon, alpha-inducible protein 27-like 2 11:130 ENSG00000216490 interferon, gamma-inducible protein 30 IFNA 1 EN SG0000019'7919 interferon, alpha 1 If-NA10 ENSG00000 I 86803 Interferon, alpha 10 IFNA13 ENSG00000233816 Interferon, alpha 13 IFNAI4 ENS(300000228083 Interferon, alpha 14 ENSG00000147885 Interferon, alpha 16 IFNA17 ENS000000234829 interferon, alpha 17 IFNA2 ENSCi00000188379 Interferon, alpha 2 IFNA21 ENSCi00000137080 Interferon, alpha 21 Date Recue/Date Received 2021-01-15 IFNA5 ENSG00000 147873 interferon, alpha 5 IFNA6 ENSG00000120235 Interferon, alpha 6 IFNA7 ENSG00000214042 Interferon, alpha 7 IFNA8 ENSG00000120242 interferon, alpha 8 IFNAR1 ENSG00000142166 Interferon (alpha. beta and omega) receptor 1 IFNB1 ENSG00000 I 71855 interferon, beta I, fibroblast IFNE ENSG00000184995 Interferon, epsilon IFN(3 ENSG00000111537 Interferon, gamma IFNGR1 EN SG00000027697 Interferon gamma receptor 1 ENSG00000182391 Interferon, lambda 1 IFNL2 ENSG00000183709 interferon, lambda 2 IFNL3 ENSG00000197110 Interferon, lambda 3 IFNLR1 ENSG00000185436 Interferon, lambda receptor 1 ir:Nwi ENSG00000177047 Interferon, omega 1 iCiF I ENSG00000017427 Insulin-like growth factor I
(sornatomedin C) ICiF2 ENSG00000167244 Insulin-like growth factor 2 IGFALS ENSG00000099769 Insulin-like growth factor binding protein, acid labile subunit IGFBP I ENSG00000146678 Insulin-like growth factor binding protein I
IGFBP2 ENSG00000115457 Insulin-like growth factor binding protein 2, 36kDa Ic3FBP3 ENSG00000146674 Insulin-like growth factor binding protein 3 IGFBP4 EN SG00000141'753 insulin-like growth factor binding protein 4 IGFBP5 ENSCi00000115461 Insulin-like growth factor binding protein 5 IGFBP6 ENSG00000167779 Insulin-like growth factor binding protein 6 IGFBP7 ENSG00000163433 Insulin-like growth factor binding protein 7 IGFI3PLI ENSG00000137142 Insulin-like growth factor binding protein-like 1 ICiFL1 ENSG00000188293 IGF-like family member 1 IGFL2 ENSG00000204866 IGF-like family member 2 TGFL3 ENSG00000188624 IGF-like family member 3 10FLRI ENSG00000126246 IGF-like family receptor 1 !GIP ENSCi00000182700 IgA-inducing protein IGLON5 EN SG00000142549 IgLON family member 5 IGSF1. ENSG00000147253 immunoglobulin supetfamily. member 1 IGSFIO ENSG00000152580 Immunoglobulin superfamily, member 10 IGSF11 ENSG00000144847 Immunoglobtilin superfamily, member 11 IGSF2 I ENSG00000117154 Imintutoglobin superfamily, member 21 IGSF8 ENSG00000162729 Immunoglobtilin supetfamily, member 8 IGSF9 ENSG00000085552 immunoglobulin superfamily. member 9 11-111 ENSG00000163501 Indian hedgehog IL 10 ENSG00000136634 Inlet-led in 10 IL II ENSCi00000095752 Interletikin II
IL I IRA ENSG00000137070 interieukin 11 receptor, alpha IL I2B ENSG00000113302 Interieukin 1211 IL I2RB I ENSG00000096996 Interieulcin 12 receptor, beta I
IL12RB2 ENSG00000081.985 Inierlettkin 12 receptor, beta 2 Date Recue/Date Received 2021-01-15 IL 13 ENSG00000169194 Interleukin 13 ILI3RA1 ENSG00000131724 Interleukin 13 receptor, alpha 1 IL I 5RA ENSG00000134470 Interleukin 15 receptor, alpha IL17A ENSG00000112115 IntcrIctikin 17A
IL17B ENSG00000127743 Interleukin 17B
IL 17C EN SG00000124391 Interleukin 17C
IL I7D ENSG00000172458 Interleukin I7D
IL I 7F ENSG00000112116 'wetted n I 7F
IL17RA. ENSG00000177663 Interleukin 17 receptor A
I7RC ENSG00000163702 Interieukin 17 receptor C
IL I 7RE ENSG0000016:3701 interieuk in 17 fecepior E
IL I8BP ENSG00000137496 Interleukin 18 binding protein IL 18k 1 ENSG00000115604 Interleukin 18 receptor 1 IL 18RAP ENSG00000115607 Interieukin 18 receptor accessory protein IL 19 ENSG00000142224 Interleukin 19 IL IR I EN SG00000115594 Interleukin 1 receptor, type 1 IL IR2 ENSG00000115590 Interleukin 1 receptor, type II
IL IRA? ENSG00000196083 Ituerleukin 1 receptor accessory protein EARL' ENSG00000115602 Interleukin 1 receptor-like I
IL1RL2 ENS(300000115598 inierietik in 1 receplor-like 2 IL IRN EN SG00000136689 interieukin 1 receptor antagonist IL2 ENSG00000109471 Interleukin 2 IL20 ENSG00000162891 Itnerleukin 20 IL2ORA. ENSG00000016402 Interleukin 20 receptor, alpha IL21 ENSG00000138684 inierleukin 21 IL22 ENSG00000127:318 Interleukin 22 11,22RA2 EN SG00000164485 interieukin 22 receptor, alpha 2 IL23A ENSG00000110944 Ituerleukin 23, alpha subunit p19 1L24 ENSG00000162892 I nierletik in 24 IL25 ENSG00000166090 litierletikin 25 11,26 ENSG00000 111536 Interleukin 26 IL27 ENSG00000197272 Interleukin 27 IL2RB ENSG00000100385 Itnerleukin 2 receptor, beta 11,3 ENSG00000164399 Inierletik in 3 IL31 ENSG00000204671 inierleukin 31 11.3 IRA ENSG00000164509 Interleukin 31 receptor A
11,32 ENSG00000008517 Interleukin 32 IL34 ENSG00000157368 Interleukin 34 ILIR A ENSG00000185291 'metier& in 3 receptor, alpha (low affinity) IL4 ENSG00000113520 inierietik in 4 11.4I1 ENSG0000010495 I Interieukin 4 induced IL4R EN SG00000077238 interIeukin 4 receptor IL5 ENSG00000113525 Interleukin 5 1L5R A EN SG00000091181 Inierletikin 5 receptor, alpha Date Recue/Date Received 2021-01-15 11,6 ENSG00000136244 interleukin 6 IL6R ENSG00000160712 Interleukin 6 receptor 1L6ST ENSG00000134352 Interleukin 6 signal transducer 11,7 ENSG00000104432 Intcrictikin 7 IL7R ENSCi00000168685 interlenkin 7 receptor 1L9 ENSG00000145839 Interleukin 9 ILDR1 ENSG00000145103 Immunoglobulin-like domain containing receptor 1 II.DR2 ENSG00000143195 Immunoglobtilin-like domain containing receptor 2 IMP4 ENSG00000136718 IMP4, U3 small nucleolar ribonucleopmicin IMPG1 ENSG00000112706 Intaphotoreceptor matrix pmteoglycan 1 INHA ENSG00000123999 inhibin, alpha INHBA ENSCi00000122641 Inhibim beta A
NUMB ENS600000163083 Inhibit:. beta B
INE1BC ENSG00000175189 Inhibit!, beta C
INHBE ENSG00000139269 inhibin, beta E
INPP5A ENSG00000068383 Inositoi polyphosphate-5-phosphatase A
INS ENSG00000254647 Insulin INS-IGF2 ENSG00000129965 INS-IGF2 readthrough INSI,3 ENS600000248099 Insulin-like 3 (Lcydig cell) EN SG00000120211 Insulin-like 4 (placenta) INSL5 ENSG00000172410 insulin-like 5 INSL6 ENSG00000120210 Insulin-like 6 INTS3 ENSG00000143624 Integrator complex subunit 3 IPOI 1 ENSG00000086200 Itnportin 11 IP09 ENSG00000198700 Importin 9 IQCF6 ENSG00000214686 IQ motif containing 1-76 IRAK3 ENSG00000090376 interieukin-1 receptor-associated kinase IRS4 ENSG00000133124 Insulin receptor substrate 4 !SLR ENSG00000129009 harnunoglobtilin superfamily containing leucine-rich repeat ISLR2 ENSG00000167178 immunoglobtilin stiperfainily containing leucine-rich repeat 2 ISM!. ENSG00000101230 1st hmin 1, artgiogenesis inhibitor ISM2 ENSG00000100593 Isthmin 2 ITGA4 ENSG00000115232 Integiin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) ITGA9 ENS600000144668 integrin, alpha 9 ITGAL ENSG00000005844 integrin. alpha L (antigen CD11 A (p180), lymphocyte function-associated antigen 1; alpha polypeptide) ITGAX ENSG00000140678 integrin, alpha X (complement component 3 receptor 4 subunit) ITGB1 EN SG00000150093 integrin, beta 1 (libronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12) rt6132 ENSG00000160255 !Megrim beta 2 (complement component 3 receptor 3 and 4 subunit) ITGB3 ENSG00000259207 Integiin, beta 3 (platelet glycoprotein IIIa, antigen CD61) ITGB7 ENSG00000139626 Imegfin, beta 7 ITGB LI ENSG00000198542 integrin, beta-like 1 (with EGF-like repeat domains) Date Recue/Date Received 2021-01-15 ENSG00000055957 inter-alpha-trypsin inhibitor heavy chain I
ENSG00000151655 Inter-alpha-tinsin inhibitor heavy chain 2 11IH3 ENSG00000162267 Inter-alpha-try psi ti inhibitor heavy chain 3 IT1114 ENSG00000055955 inter-alpha-trypsin inhibitor heavy chain family, member 4 ITTH5 ENSG00000123243 Inter-alpha-trypsin inhibitor heavy chain family, member 5 1T1146 EN SG00000102313 Inter-alpha-trypsin inhibitor heavy chain family, member 6 ITLN1 ENSG00000179914 Intelectin 1 (galactofuranose binding) rn.N2 ENSG000001:58764 linelectin 2 IZUNIOIR ENSG00000183560 IZUMOI receptor, JUNO
TZUM04 ENSG00000099840 IZUMO family member 4 JCHAIN EN SG00000132465 Joining chain of multimeric IgA and IgM
JMJD8 ENSCi00000161999 Jumonji domain containing 8 iSRPI ENSG00000167476 Junctional sarcoplasinic reticulum protein I
KANSL2 ENSG00000139620 KAM regulatory NSL complex subunit 2 KAZALDI ENSG00000 10'782 I Kaial-type serine peptidase inhibitor domain 1 KCNIP3 ENS00000011504 1 KN. channel interacting protein 3, calseniiin KCNK7 ENSG00000173338 Potassium channel, two pore domain subfamily K, member 7 KCNN4 ENSG00000104783 Potassium channel, calcium activated intermediate/small conductance subfamily N alpha, member 4 KCNU I ENSG00000215262 Potassium channel. subfamily U. member I
KCP ENSG00000135253 Kielinichordin-like protein KDELC1 ENSG00000134901 KDEL (Lys-Asp-Gin-Len) containing 1 KDELC2 ENS000000 I 78202 KDEL (Lys-Asp-Cilti-Len) containing 2 KDM1A ENSG00000004487 Lysine (K)-specific deinethylase IA
KDM3B ENSG00000120733 Lysine (K)-specific demethylase 3B
KDM6A ENSG00000147050 Lysine (K)-specific dcmethylase 6A
KDM7A ENSG00000006459 Lysine (K)-specific demethylase 7A
KDSR EN SG00000119537 3-koodihydrosphingosine re,cluctase K ERA ENSG00000139330 Keratocan K IFC2 ENSG00000167702 Kinesin family member C2 KIR2DI4 ENSG00000189013 Killer cell inuntinoglobitlin-like receptor, two domains, long cytoplasmic tail, 4 KIR3DX1 ENSG00000 I 04970 Killer cell i Hiatt inoglobulin-like receptor, three domains, XI
KIRREL2 ENSG00000126259 Kin of IRRE like 2 (Drosophila) KISS! ENSG00000170498 KiSS-1 metastasis-suppressor KLHLI 1 ENSG00000178502 Ketch-like family member 11 KLHL22 ENSG00000099910 Ketch-like family member 22 KLK I ENSG00000167748 Kallikrein 1 KLKIO EN SC100000129451 Kallikrein-related peptidase 10 KLK I ENSG00000167757 Kallikrein-related peptidase 11 KLK12 ENSG00000186474 Kallikrein-related peptidase 12 Date Recue/Date Received 2021-01-15 KL.K13 ENSG00000167759 Kallikrein-related peptidase 13 KLK14 ENSG00000129437 Kallikrein-related peptidase 14 KLK15 ENSG00000174562 Kallikrzin-related peptidase 15 KLK2 ENSG00000167751 Ka1Hk/tin-related peptidase 2 KLK3 EN SG00000142515 Kallikrein-related peptidase.
KL.K4 ENSG00000167749 Kallikrein-related peptidase 4 KLK5 ENSG00000167754 Kallikrein-related peptidase 5 KLK6 ENSG00000167755 Ka111kt-6n-related peptidase 6 KLK.7 ENSG00000169035 Kallikrein-related peptidase 7 KLK8 ENSG00000129455 Kallikrein-related peptidase KLK9 ENSG00000213022 Kallikrein-related peptidase 9 KLICB1 ENSCi00000164344 Kallikrein B. plasma (Fletcher factor) 1 KNDC I ENS(300000171798 Kinase non-catalytic C-lobe domain (KIND) containing 1 KNG1 ENSG00000113889 Kininogen I
KRBA2 ENSG00000184619 KR AB-A domain containing 2 KREMEN2 EN SG00000131650 Kringie containing transmembrane protein KRMAP ENSG00000188508 Kenitinocyte differentiation-associated protein L I CAM ENSG00000198910 L I cell adhesion molecule L3MBTL2 ENSG00000100395 1.(3)mbt-Like 2 (Drosophila) LA16c-3801-15.3 ENSG00000270168 LACE!. ENSG00000135537 Lactation devated I
LACRT ENSCi00000135413 Lacritin LACTB ENSG00000103642 Lactamase, beta LAG3 ENSG00000089692 Lymphocyte-activation gene 3 LA1R2 ENSG00000167618 Letikoeyte-associated iinntimoglobiti in-like receptor 2 LALBA ENSG00000167531 Lactalburni rt. alpha-LAMA I ENSG00000101680 Laminin, alpha I
LAMA2 ENSG00000196569 Laminin, alpha 2 LAMA3 ENSG00000053747 Laminin, alpha 3 LAMA4 ENSG00000112769 Larni inn, alpha 4 LAMAS EN SG00000130702 Latninin, alpha 5 LAMB I ENSG00000091136 Laminin, beta 1 LAM132 ENSG00000172037 Laminin, beta 2 (laminin S) LAMB3 ENSG00000196878 Laminin, beta 3 L AMB4 ENSG00000091128 Laminin. beta 4 LAMC I ENSG00000135862 Laminin, gamma I (formerly LAMB2) LAMC2 ENSG00000058085 Laminin, gamma 2 LAMC3 ENSG00000050555 Laminin, gamma 3 LAMP3 ENSG0000007808 I Lysosoinal-associated membrane protein 3 LAT ENSG00000213658 Linker for activation of T cells LAT2 ENSG00000086730 Linker for activation of T cells family, member 2 LBP EN SG00000129988 Lipopolysaccharide binding protein LCAT ENSG00000213398 Lecithin-cholesterol acyltransferase LCN1 ENSG00000160349 Lipocalin 1.

Date Recue/Date Received 2021-01-15 LCNIO EN SG00000187922 Lipocalin 10 LCN12 ENSG00000184925 Lipocalin 12 LCN15 EN S(300000177984 Lipocalin 15 =
LCN2 ENSG00000148346 Lipocalin 2 LCN6 ENSG00000267206 Lipocalin 6 LCN8 ENSG00000204001 Lipocalin 8 LCN9 ENSG00000148386 Lipocalin 9 LCORI. ENSG00000178177 Ligand dependent nuclear receptor corepressor-like =
LDLR ENSG00000130164 Low density' lipoprotein receptor I.DLRAD2 ENSG00000187942 Low density lipoprotein receptor class A
domain containing 2 LEA.P2 ENSG00000164406 Liver expressed antimicrobial peptide 2 LECT2 ENSCi00000145826 Leukocyte cell-derived chemotaxin 2 LEFTY! ENSG00000243709 Left-right determination factor 1 =
LEFTY2 ENSG00000143768 Left-right determination f.tictor 2 LEP EN SC100000174697 Leptin I.FNG ENSG00000106003 LENG O-fucosylpeptide 3-beta-N-acetylghicosaminvItnins1er45e LGALS3BP ENSG00000108679 Lectin, galactoside-binding, soluble, 3 binding protein EN SG00000153012 Leucine-rich repeat LGI family, member 2 LGI3 EN SG00000168481 LetiGine-dell repeat LGI family, member LGI4 ENSG00000153902 Leucine-rich repeat LW family, member 4 LGAN ENSG00000100600 Legumain LGR4 ENSG000002052 13 Leucine-rich repeal containing G protein-coupled receptor 4 LHB ENSG00000104826 Luteinizing hormone beta polypeptide LHCGR ENSG00000138039 Luteiniting hormonelchoriogonadottopin receptor LW ENSO00000128342 Leukemia inhibitory factor LIFR ENSG00000113594 Leukemia inhibitory factor receptor alpha LILRA1 ENS(300000104974 Leukocyte inuntinoglobulin-like receptor, subfamily A (with TM domain). member 1 LILRA2 ENSG00000239998 Leukocyte immunoglobulin-like receptor.
subfamily A (with TM domain). member 2 LILRB3 ENSG00000204577 Leukocyte immunoglobulin-like receptor.
subfamily B (with TM and ITIM domains), member 3 LIME I ENSG00000203896 Lck interacting transmembrane adaptor 1 LING01 ENSG00000169783 Leucine rich repeat and Ig domain containing 1 LIPA ENSG00000107798 Lipase A. lysosomal acid, cholesterol esterase LIPC ENSG00000166035 Lipase, hepatic LIPF ENSG00000182333 Lipase, gastric LIPG ENSG00000101670 Lipase, endothelial LIN{ EN SG00000163898 Lipase, member) LIPK ENSG00000204021 Lipase, family member K
LIPM ENSG00000173239 Lipase, family member M
URN ENSG00000204020 Lipase, family member N
LMAN2 ENSCi00000169223 Lectin, mannose-binding 2 Date Recue/Date Received 2021-01-15 LM NTD I ENSG00000152936 Ltunin tail domain containing 1 LNX I ENSG0000007220 I Ligand of numb-protein X 1. E3 ubiquitin protein ligase LOX ENSG00000113083 Lysy I oxidase LOXL I ENSG00000129038 Lysyl oxidasc-like LOXI2 ENSG00000134013 Lysyl oxidase-like 2 LOXL3 EN sG00000 I 15318 Lysyl oxidase-like 3 LOXL4 ENSG00000138131 Lysyl oxidase-like 4 LPA EN SG00000198670 Lipoprotein, Lp(a) LPL ENSG00000175445 Lipoprotein lipase LPG ENSG00000167410 Lactoperoxidase LRAT EN SG00000121207 Lecithin retinol acylttansferase (phosphatidy1choline¨retinol 0-acy itransferase) LRCH3 EN SG00000186001 Letter ne-tich repeats and calponin homology (CH) domain containing 3 LRCOL1 ENSG00000204583 Leucine rich colipase-like 1 LRFN4 EN SG00000173621 Leucine rich repeat and fibrottectin type III domain containing 4 LRFN5 ENSG00000165379 Leucine rich repeat and fibronectin type III domain containing 5 LRG1 EN SG00000171236 Lend ne-rich alpha-2-glycoprotein 1 LRP I ENSG00000123384 Low density lipoprotein receptor-related protein 1 LRPII ENSG00000120256 Low density lipoprotein receptor-related protein 11 LRP1B NSG00000168702 Low density lipoprotein receptor-related protein IB
LRP2 ENSG00000081479 Low density lipoprotein reccptor-related protein 2 LRP4 ENSG00000134569 Low density lipoprotein receptor-related protein 4 LRPAPI ENSG00000163956 Low density lipoprotein receptor-related protein associated protein 1 LRRC17 ENSG00000128606 Leucine rich repeat containing 17 LRRC32 ENSG00000137507 Leucine rich repeat containing 32 LRRC3B ENSG00000179796 Leucine rich repeat containing 3B
LRRC4B EN SG00000131409 Lent:ine rich repeal containing 4B
LRRC70 ENSG00000186103 Leucine rich repeat containing 70 LRRN3 EN SG00000173114 Leucine rich repeat neuronal 3 LRRTM1 ENS600000162951 Leucine rich repeat transmenthrane neuronal 1 LRRTM2 ENSG00000146006 Leucine rich repeat transmembrane neuronal 2 LRRTM4 ENSG00000176204 Leucine rich repeat transmembrane neuronal 4 LR.I1V12 ENSG00000166159 Leucine-rich repeats and transmembnine domains 2 LSR ENSG00000105699 Lipolysis stimulated lipoprotein receptor LST I ENSG00000204482 Leukocyte specific transcript 1 LTA ENSG00000226979 Lymphotoxin alpha LTBP1 ENSG00000049323 Latent transforming growth factor beta binding protein LTBP2 ENSG00000119681 Latent transforming growth 1:11ctor beta binding protein 2 LTBP3 ENSG00000168056 Latent transforming growth factor beta binding protein 3 LTBP4 NSG00000090006 Latent transforming growth factor beta binding protein 4 LTBR EN SG00000111321 Lymphotoxin beta receptor (TNFR
superfamily, member 3) LTF ENSG00000012223 Lactotransferrin urK. ENSG00000062524 Leukocyte receptor tyrosine kinase Date Recue/Date Received 2021-01-15 LUM ENSG00000139329 Lumican LUZP2 ENSG00000187398 Leucine zipper protein 2 LVRN ENSG00000172901 Laeverin LY6E ENSG00000160932 Lymphocyte antigen 6 complex. locus E
LY6G5B ENSG00000240053 Lymphocyte antigen 6 complex, locus G5B
LY6661) EN8G00000244355 Lymphocyte antigen 6 complex, locus G61) LY6G6E ENSG00000255552 Lymphocyte antigen 6 complex, locus G6E
(pseudogene) LY6H ENSG00000176956 Lymphocyte antigen 6 complex, locus H
LY6K ENSG00000160886 Lymphocyte antigen 6 complex, locus K
1Y86 ENSG00000112790 Lymphocyte antigen 86 LY96 ENSG00000154589 Lymphocyte antigen 96 LYGI ENSCi00000144214 Lysozyme (3-like LYG2 ENSG00000185674 Lysozyme (3-like 2 LYNX! EN SG00000180155 Ly6/neurotoxin I
LYPDI EN SG0000015055 I LY6/PLAUR domain containing 1 LYPD2 ENSG00000 19735:; LY6IPLA UR domain containing 2 LYPD4 ENSG00000273 Ill LY6/PLAUR domain containing 4 LYPD6 ENSG00000187123 LY6/PLAUR domain containing 6 LYPD6B ENSG00000150556 LY6/PLAUR domain containing 613 LYPD8 ENSG00000259823 LY6/PLAUR domain containing 8 LYZ EN SG00000090382 Lysozy me LYZL4 ENSCi00000157093 Lysozyme-like 4 LYZL6 ENSG00000275722 Lysozyme-like 6 M6PR ENSG00000003056 Mannose-6-phosphate receptor (cation dependent) MADIL1 ENSG00000002822 MAD1 mitotic arrest deficient-like I
(yeast) MAG ENSG00000105695 Myelin associated glycoprotein MAGTI ENSG00000102158 Magnesium transporter I
MALSUI ENSG00000156928 Mitochondriai assembly of nbosomal large subunit 1 MAMDC2 ENSG00000165072 MAM domain containing 2 MAN2B1 ENSG00000 104774 Mannosidase, alpha, class 2B, member 1 MAN2B2 EN SG00000013288 Mannosidase, alpha, class 28. member 2 MANB A ENSG00000109323 Mannosidase, beta A, lysosomal MANEAL ENSG00000185090 Mannosidase, endo-alpha-like MANF ENSG00000145050 Mesencephalic astrocyte-derived neurotrophic factor MANSCI ENSG00000IIl26I MANSC domain containing 1 MAP3K9 EN SG00000006432 Mitogen-activated protein kinase 9 MASP I ENSG00000 I 27241 Marman-binding lectin serine peptidase I (C4/C2 activating component of Ra-reactive factor) MASP2 EN SG00000009724 Marman-binding lectin serine peptidase 2 MATN1 ENSCi00000162510 Matrilin I, cartilage matrix protein MATN2 ENSG00000132561 Matrilin 2 MATN3 ENSG0000013203 I Matrilin 3 MATN4 EN SG00000124159 Matrilin 4 MAIR3 ENSG00000015479 Main n Date Recue/Date Received 2021-01-15 MA1R3 ENSG00000280987 Mairin 3 MAU2 ENSG00000129933 MAU2 sister chromatid cohesion factor MAZ ENSG00000103495 MAT-associated zinc finger protein (purine-binding transcription factor) MBD6 ENSG00000166987 Methyl-CpG binding domain protein 6 MBL2 EN SG0000016547 I Manuose-hinding Win (protein C) 2.
soluble MBNL I EN SG00000152601 Muscleblind-like splicing regulator 1 MCCCI ENSG00000078070 Methylcmtonoyl-CoA carboxylase I (alpha) MCCD1 ENSG00000204511 Mitochondria) coiled-coil domain 1 MCEE ENSG00000124370 Methy !malonylCoA epimerase MCF2L EN SG00000126217 MCF.2 cell line derived transfo m ling sequence-like MCFD2 ENSG00000180398 Multiple coagulation factor deficiency 2 MDFIC ENSG00000135272 MyoD family inhibitor domain containing MDGA1 ENSG00000112139 MAM domain containing glycosylphosphatidylinosnol anchor 1 MDK ENSG00000110492 Midkine (neurite growth-promoting factor 2) MED20 ENSG00000124641 Mediator complex subunit 20 MEGFIO EN SG00000145794 Multiple EGF-like-domains 10 MEGF6 ENSG00000162591 Multiple EGF-like-domains 6 ---1Cifil¨ENV.iiiiiiiii6i-6-7.7.--------geTO-11-c-a-j;;;It-;-;;;;trde¨d-1-;ra7forniation protein 1 ME14 ENSG00000269964 Meiotic double-stranded bleak formation protein 4 MEIS1 ENSG00000143995 Meis homeobox 1 ME1S3 EN SG00000105419 Meis horneobox 3 MEPE ENSCi00000152595 Matrix extracellular phosphoglycoprotein MESDC2 ENSG00000117899 Mesoderm development candidate 2 MEST ENSG00000106484 Mesoderm specific transcript MET ENSG00000105976 MET proto-oncogene. receptor tyrosine kinase METRN ENSG00000103260 Meteorin, ghat cell differentiation regulator METRNL ENSG00000176845 Meteorin. glial cell differentiation regulator-like METTL17 ENSG00000165792 Methy It ransferase like 17 METTL24 ENSG00000053328 Methyliransfemse like 24 METTL7B ENSG00000170439 McthlLransferase like 7B
ENSG00000197006 Methyltransferase like 9 MEX3C ENSG00000176624 Mex-3 RNA binding family member C
MFAP2 ENSG00000117122 Microfibrillar-associated protein 2 MFAP3 ENS000000037749 Microfibrillar-associated protein 3 MFAP3L ENSG00000198948 Microfibrillar-associated protein 3-like MFA.P4 ENSG00000166482 Microfibnliar-associated protein 4 MFAP5 ENSG00000197614 Microfibrillar associated protein 5 MFGE8 ENSG00000140545 Milk fat globule-EGF factor 8 protein M1712 ENSG00000163975 Antigen p97 (melanoma associated) identified by monoclonal antibodies 133.2 and 96.5 MFNG EN SG00000100060 MFNG 0-filcosylpeptide 3-beta-N-acetylgiticosaininyltransferase MGA EN SG00000174197 MGA. MAX dimenzation protein Date Recue/Date Received 2021-01-15 muAT2 ENSG00000168282 Marmosy I (alpha- 1.6-)-glycoprotein beta-1,2-N-acetylglitcoiminyltuinsfentse MOAT3 EN S000000128268 Mannosy I (oda- 1,4-)-glycoprotei n beta-I .4-N-acetylglucosaminylt tansfentse MGAT4A ENSG00000071073 Mannosyl(alpha-13-)-glycoprotein beta-1,4-N-cetylglucosamirtyhtartsferase, isozy me A
IMGAT4B EN SG00000161013 Mannosy I (alpha-1.3-)-glycoprot ci n beta-1,4-N-acetylgiucosaminyhtansfenise, isozy rue B
MGAT4D EN SG00000205301 MGAT4 family, member D
MOLL EN SG00000074416 Monoglyceride lipase MOP EN SG00000111341 Matrix Gla potent MGST2 ENS(300000085871 Microsomal glutatbione S-ttansferase 2 MIA EN SG00000261857 Melanoma inhibitory activity MIA2 ENSG00000150526 Melanoma inhibitory activity 2 MIA3 ENS000000 I 54305 Melanoma inhibitory activity family, member 3 MICU1 ENSG00000107745 Mitochondrial calcium uptake 1 MIER!. EN S(300000198160 Mesoderm induction early response 1.
ttanscriptional regulatot MINOS I -NBLI ENS000000270136 MINOS1-:NBL I readthrough MINPP1 EN SG00000107789 Multiple inositol-polyphosphate phosphatasc 1 MLEC ENS000000110917 Malect in MLN ENSG00000096395 Motilin MLXIP ENS(300000175727 Mi.,X interacting pioteitt MLXIPI, ENS000000009950 MIX interacting protein-like KEMP! EN SG00000196611 Matrix inctallopeptidase 1 MMP 10 ENSG00000166670 Matrix inetallopeptniase 10 MMP II ENS000000099953 Matrix metallopeptidase 11 MMP12 ENSG00000262406 Mattix metallopeptidase 12 MMP13 ENSG00000137745 Matrix ruetallopeptidase 13 IVEvIP14 ENSG00000157227 Matrix metallopeptidase 14 (membrane-inserted) ENS000000123342 Matrix metallopeptidase 19 MMP2 ENSG00000087245 Matrix metallopeptidase 2 MMP20 EN S000000137674 Matrix metallopeptidase 20 AMP21 ENSG00000154485 Matrix mctallopeptidase 21 IVIMP25 ENSG00000008516 Matrix inetallopeptniase 25 MMP26 EN SG00000 I 67346 Matrix metallopeptidase 26 MMP27 ENSG00000137675 Matrix metallopeptidase 27 MMP28 ENSG00000271447 Matrix metallopeptidase 28 AMP3 ENSG00000149968 Matrix metallopeptidase 3 MMP7 ENSG00000137673 Matrix inetallopeptidase 7 IMMP8 ENSG00000118113 Matrix metallopeptidase 8 MMP9 ENSG00000100985 Matrix metallopeptidase 9 MMRN I EN S(300000138722 Multi:net-in 1 AMRN2 ENSG00000173269 Multimerin 2 Date Recue/Date Received 2021-01-15 MOXD1 ENSG0000007993 I Monooxygenase, .01314-like 1 MPG ENSG00000005381 Myeloperoxidase MPPED1 ENSG00000186732 Metallophosphoestetase domain containing MPZL1 ENSG00000197965 Myelin protein zero-like 1 MR I ENSG00000153029 Major hisiocompatibility complex. class 1-te1ated MKPL2 ENSG00000112651 Mitochondrial ribosomal protein L2 NIRPL21 ENSG00000197345 Mitochondrial ribosomal protein L21 NIRPL22 ENSG00000082515 Mitochondrial ribosomal protein L22 MRPL24 ENSG00000143314 Mitochondria" ribosomal protein L24 MRPL27 ENSG00000108826 Mitochondriai ribosomal protein L27 MRPL32 EN SG00000106591 Mitochondriai ribosomal protein L32 MRPL34 ENSG00000130312 Mitochondrial ribosomal protein L34 MRPL35 ENSG00000132313 Mitochondrial nbosotnal protein L35 NIRPL52 ENSG00000172590 Mitochondrial ribosomal protein L52 MRPL55 ENSG00000162910 Mitochondrial ribosomal protein L55 MRPS14 ENSG00000120333 Mitochondrial ribosomal protein S14 NIRPS22 ENSG00000175110 Mitochondrial ribosomal protein S22 MRPS28 ENSG00000147586 Mitochondrial 6bosomal protein S28 MS4A1.4 ENSG00000166928 Membrane-spanning 4-domains, subfamily A, member 14 MS4A3 ENSG00000149516 Membrane-spanning 4-domains, subfamily A, member 3 (hematopoietic cell-specific) MSH3 EN SG00000113318 MutS homolog 3 MSH5 ENSG00000204410 MutS homolog 5 MSLN ENSG00000102854 Mesothelin MSMB ENSG00000263639 MiCIDSCminoprotein, beta-MSRA EN S000000175806 Methionine sulfoxide rcductase A
MSRB2 ENSG00000148450 Methionine sulfoxide reductase B2 NISRB3 ENSG00000174099 Methionine sulfoxide teductase B3 MSTI EN SG00000173531 Macrophage stimulating 1 MSTN ENSCi00000138379 Myostatin Niri0 ENSG00000125144 Metallothionein 1G
NITHI:D2 ENSG00000065911 Methylenetetrahydrofolate dehydrogertase (NADP+ dependent) 2, methenyiteirahydmfolate cyclohydrolase NcrmR14 ENSG00000163719 Myonibularin related protein 14 MTRNR2L11 ENSG00000270188 MT-RNR2-like 11 (pseudogene) MTRR EN SG000001242'15 5-met hylietrahydio101aie-hoinocy sieine methyltralisferase reductase MTTP ENSG00000138823 Microsomal ttiglyceride transfer protein N1TX2 ENSG00000128654 Metaxin 2 MUC1 EN SGt i0000185499 Mucin 1, cell surface associated MUG13 ENSG00000173702 Wein 13, cell surface associated MUC20 ENSG00000176945 Mucin 20, cell surface associated MUC3A EN SG00000169894 Mucin 3A, cell surface associated MUC5AC ENSG00000215182 Mucin SAC. oligomeric milcusigel-fonning MUC5B ENSG00000117983 Mucin 5B, oligomeric mucus/gel-forming Date Recue/Date Received 2021-01-15 MUC6 EN SG00000184956 Muciti 6, oligomeric mucus/gel-forming 1\TLIC7 ENSG00000171195 Mucin 7, secreted MUCL1 EN SG00000172551 Mucin-like 1 =
MXRA5 EN SG00000101825 Matrix-remodelling associated 5 MXRA7 ENSG00000182534 Matrix-remodelling associated 7 MYDGF EN5G00000074842 Myeloid-derived growth factor MYLI ENSG00000168530 Myosin, light chain 1. alkali. skeletal.
fast MYOC ENSG00000034971 Mvocilin. trabectilar meshwork inducible glucocorticoid it:sponse MYRFL ENSG00000166268 Myelin regulatory factor-like MZB1 ENSG00000170476 Marginal zone B and BI cell-specific protein N4BP21,2 ENSG00000244754 NEDD4 binding protein 2-like 2 NAA38 ENSG00000183011 N(alpha)-acetyliransferase 38, NatC
auxiliary subunit.
NAAA ENSG00000138744 N-acyleitianolamine acid arnidase NAGA ENSG00000198951 N-acetylgalactosaminidase, alpha-NAGLU ENSG00000108784 N-acety iglucosaminidase. alpha NAGS ENSG00000161653 N-acety !glutamate synthase NAF'SA ENSG00000131400 Napsin A aspartic peptidase --1-41.7:1[1.--------ESIViliiiii-tioi.-570-477------;Cleitro¨Rt-sTrTrUTIT:liWitTiTi-gKiica-nTa-g¨oZt---------NCAM 1 ENSG00000149294 Neural cell adhesion molecule 1 NCAN ENSG00000130287 Neurocan NCBP2-AS2 ENSG00000270170 NCBP2 antisense RNA 2 (head to head) NCSTN ENSG00000162736 Nic,astrin NDNF ENSG00000173376 Neuron-derived neurotrophic factor ND.P ENSG00000124479 Norrie disease (pseudoglioma) NDUFAIO ENSG00000130414 NADH dehydrogenase (ubiquinone) I alpha subcomplex, 10, 421(Da NDUFB5 EN SG00000136521 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa NDUFS8 ENSG00000110717 NADH dchydrogenase (ubiquinonc) Fe-S
protein 8. 23kDa iNADH-coenvme Q reductase) NDUFNI I ENSG00000167792 NADH dchydrogenase (ubiquinortc) flavoprotein 1, 51kDa NECAB3 ENSG00000125967 N-terminal EF-Itand calcium binding protein 3 NELL1 ENSG00000165973 Neural EGFL like 1 NELL2 EN S000000184613 Neural EGFL like 2 NENF ENSCi00000117691 Neudesin neurotrophic factor NET01 ENSG00000166342 Neuropilin (NRP) and tolloid (TLL)-Iike 1 ENSG0000008264 I Nuclear factor, erythroid 2-like I
NFE2L3 ENSO00000050344 Nuclear factor, etythroid 2-like 3 NGEF ENSG00000066248 Neuronal guanine nucleotide exchange factor NGF ENSG00000134259 Nerve growth factor (beta polypeptide) N GLY 1 EN SG00000151092 N-glycartase 1 NGRN ENSG00000182768 Neugrin, neurite outgrowth associated NHLRC3 EN S0000001888 ii NHL neat containing 3 NIDI ENSG00000116962 Nidogen 1 Date Recue/Date Received 2021-01-15 ND, EN SG00000087303 Nidogen 2 (osteortidogen) NKG7 ENSG00000105374 Natural killer cell granule protein 7 NLGN3 ENSG00000196338 Neuroligin 3 NLGN4Y ENSG00000165246 Ncuroligin 4, Y-linked NLRP5 ENSG00000171487 NLR family. pyrin domain containing 5 N MB EN sG00000197696 Neurotnedin 13 NME I ENSG00000239672 NMENM23 nucleoside diphosphate kinase 1 NME1-NME2 ENS000000011052 NME I -NIME2 readi Itio ugh NME3 ENSG00000103024 NMEINM23 nucleoside diphosphate kinase NMS ENS000000204640 Neuromedin S
N MU EN SG00000109255 Neurotnedin U
NOA1 ENSCi00000084092 Nitric oxide associated 1 NODAL ENS000000156574 Nodal growth differentiation factor NOG EN S000000183691 Noggin NOM03 ENSG00000103226 NODAL modulator 3 NOS LAP EN SG00000198929 Nitric oxide sy nthase 1 (neuronai) adaptor protein NOTCH3 ENSG00000074181 Notch 3 NO'TUM ENSG00000185269 Noium pectinacetylesterase homolog (Drosophila) NOV ENSG00000136999 Nephroblastoina ovcrexpressed NPB ENSG00000183979 Neuropeptide B
NPC2 EN SG00000119655 Nietnann-Pick disease, type C2 NPFF ENSCi00000139574 Neuropeptide FF-amide peptide precursor NPFFR2 ENSG00000056291 Neuropeptide FF receptor 2 NPHSI EN SG00000161270 Ncphrosis I, congenital, Finnish type (nephrin) NPNT EN SG00000168743 Nepliroitectiti NPPA EN SG00000175206 Natriuretic peptide A
NPPB EN SG00000120937 Natriuretic peptide 13 NPPC ENSG00000163273 Natriuretic peptide C
NPS ENSG00000214285 Neuropeptide S
NPTX I EN SG00000 I 71246 Neuronal pentraxiii I
NPIX2 EN SG00000106236 Neutonal pentiaxin II
N vrxR EN SG00000221890 Neuronal pentraxin receptor NPVF ENSG00000105954 Neuropeptide VF precursor NPW EN S000000183971 Neuropeptide W
NPY ENSG00000122585 Nett ropept ide Y
NQ02 ENSG0000012-1588 NAD(P)H dehydrogenase, quinone 2 NRCAM EN SG00000091129 Neuronal cell adhesion molecule NRG1 ENSG00000157168 Neuregulin I
NRN IL ENSG00000188038 Neuritin 1-like NRP1 ENSG00000099250 Neuropilin I
NRP2 ENSG00000118257 Ncuropilin 2 NRTN EN SG00000171119 Neurturin NRXN1 ENSG00000179915 Neurexin 1 NRXN2 ENSG00000110076 Nettrexin 2 Date Recue/Date Received 2021-01-15 NT5C3A ENSG00000122643 5'-nocleotidase, eytosolic 111A
NT5DC3 ENSG00000111696 5'-nucleotidase domain containing 3 NT5E ENSG00000135318 5'-nucleotidase, ecto (CD73) ENSG00000185652 Neurotrophin 3 NTF4 ENSG00000225930 Neurotrophin 4 NTM EN sG00000182667 Neurotrinnn NTN I ENS000000065320 Netrin I
NTN3 ENS000000162068 Nevin 3 NTN4 ENSG00000074527 Nevin 4 NTN3 ENSG00000142211 Net rin 3 NINCil ENS000000162631 Netrin 01 NTN02 ENS000000196358 Netrin 02 NTs ENSG00000133636 Neurotensin NUBPL ENSG00000151413 Nucleotide binding protein-like NUCBI EN SC100000104805 Nucleobindin 1 NiUCB2 ENS000000070081 Nucleobindin 2 NUDT19 ENS000000213965 Nudix (nucleoside diphosphate linked moiety X)-type motif 19 NITDT9 ENSG00000170502 Nudix (nucleoside diphosphate linked moiety X)-type motif 9 NUP155 EN SG00000113569 Nucleoporin 155kDa NUP214 ENSCi00000126883 Nucleoporin 214kDa NiiP85 ENS000000125450 Nucleoporin 85kDa NXPE3 ENSCi00000144815 Neurexophilin and PC-esterase domain family, member 3 NXTE4 ENS000000137634 Neurexophilin and PC-esterase domain family, member 4 NXPH1 ENS000000122584 Neurexophilin 1 NXPH2 ENSG00000144227 Nettrexophilin 2 NXPE13 ENS000000182575 Neurexophilin 3 NXPE14 ENS000000182379 Neurexophil in 4 NYX ENS000000188937 Nyctalopin OAF ENS000000184232 Out at first homolog OBP2A EN SG00000122136 Odorant binding protein 2A
OBP2B ENS000000171102 Odorant binding protein 2B
0C90 ENSG00000253117 Otoconin 90 OCLN ENS000000197822 Occludin ODAM ENSG00000109205 Odontogenic, ameloblast asssociated OGG] ENSG00000114026 8-oxoguanine DNA gly cosy lase OGN ENSG00000106809 Osteoglycin 0rr3 EN S000000138315 Oncoprotein induced transcript 3 OLFM1 ENS000000130558 Olfactomedin 1 OLFM2 ENSG00000105088 Olfactomedin 2 OLFM3 ENSG00000118733 Olfactomedin 3 OLFM4 ENSG00000102837 Olfactomedin 4 OLFML1 EN S000000183801 011actomedin-like 1 OLFML2A ENS000000185585 Olfactomedin-like 2A
OLFML2B ENSG00000162745 Olfactomedin-like 2B

Date Recue/Date Received 2021-01-15 01,1-111.3 ENSG00000116774 Olfactomedin-like 3 OMD EN SG00000127083 Osteomodulin OMG EN SG00000126861 Oligodendrocyte myelin glycoptotein 00SP2 ENSG00000149507 Oocyte secreted protein 2 OPCML ENSG00000183715 Opioid binding protein/cell adhesion molecule-like ovrc ENSG00000188770 Opticin ORAII ENSG00000276045 ORAI calcium release-activated calcium modulator 1 ORM I EN SG00000229314 Otosomucoid 1 ORM2 ENSG00000228278 Orosomucoid 2 ORMDL2 ENSG00000123351 ORMDL sphingolipicl biosynthesis regulator 2 0S9 ENSG00000135506 Osteosarcoma amplified 9, endoplasmic reticulttin lectin OSCAR EN SCi00000170909 Osteoclast associated, immunoglobulin-like receptor OSM ENSG00000099985 Oncostatin M
OSMR EN SG00000145623 Oucostatin M receptor OSTN ENS(100000188729 Osteocrin OTOA ENSG00000155719 Otoanconn OTOG EN SG00000188162 Otogelin OTOGL ENSG00000165899 Otogelin-like OTOL1 EN SG00000182447 Otolin 1 OTOR ENSG00000I 25879 Otoraplin OTOS ENSG00000178602 Otospiralin OVCH1 ENSCi00000187950 Ovochymase OVCH2 EN SG00000183378 Ovochymase 2 (gene/psendogene) OVGP I EN SG'00000085465 Oviductal glycoprotein 1, 120kDa OXCTI ENSG00000083720 3-oxoacid CoA tninsferase oxcr2 ENSG00000198754 3-oxoacid CoA transferase 2 OXNAD1 EN SG00000154814 Oxidoreductase NAD-binding domain containing 1 OXT ENSG00000101405 Oxytocinineurophysin I prepropepfide P3F11 ENSG00000117385 Pmly13-hydroxylase P3H2 ENSG00000090530 Prolyl 3-hydroxylase 2 P3I-13 EN SG0000011081 I Proly13*droxy lase 3 P31-14 ENSG00000141696 Proly I 3-hydroxy lase family member 4 (non-enzymatic) P4HA1 ENSG00000122884 Proly14-hydroxy lase, alpha poly peptide I
P4F1A2 ENSG00000072682 Proly14-hydroxylase, alpha polypeptide ii P41-IA3 ENSG00000 149380 Proly14-hydroxylase, alpha polypeptide P4HB ENSG00000185624 Proly14-hydroxylase. beta poly peptide PAEP ENSG00000122133 Pmgestagen-associated endotnetrial protein PAM ENSG00000145730 Peptidylglycine alpha-amidating monooxygenase PAMR I ENSG00000149090 Peptidase domain containing associated with muscle regeneration 1 PAPL ENSG00000183760 iron/zinc purple acid phosphatase-like protein P.APLN ENSG00000100767 Papilin, proteoglycan-like sulfated glycoprotein PAPPA ENSG00000182752 Pregnancy-associated plasma protein A.
pappalysin 1 PAPPA2 EN SG00000116183 Pappaly sin 2 PAR PIS ENSG00000173200 Poly (ADP-ribose) polytnerase family, member 15 Date Recue/Date Received 2021-01-15 PAR VB ENSG00000188677 Pary in, beta PATE1 ENSG00000171053 Prostate and testis expressed 1 PATE2 ENSG00000196844 Prostate and testis expressed 2 PATE3 ENSG00000236027 Prostate and testis expressed 3 PATFA ENS(100000237353 Prostate and testis expressed 4 PAIL2 EN sG00000229474 Protein associated with topoisomerase II
homolog 2 (yeast) PAX2 ENSG00000075891 Paired box 2 PAX4 ENSG0000010633 I Paired box 4 PCCB ENSG00000114054 Propionyl CoA carboxy lase, beta POtYpeptidc PCDH I ENSG00000156453 Protocadherin 1 PCD1112 ENSG00000 I 1:3555 Protocadherin 12 PCDH15 ENSG00000150275 Protocadherin-related 13 PCDHA I ENSG00000204970 Protocadhe tin alpha 1 PCDHA 10 ENSG00000250120 Protocadherin alpha 10 PCDHA1 I ENS(100000249158 Protocadherin alpha 11 PCDH.A6 EN SG00000081842 Protocadherin alpha 6 PCDHB12 ENSG00000120328 Protocadherin beta 12 PCDHGA11 ENSG00000253873 Protocadherin gamma subfamily A. 11 PCF11 ENSG00000165494 PCF 11 cleavage and polyadeny 'talon factor subunit PCOLCE ENSG00000106333 Procollagen C-endopeptidase enhancer PCOLCE2 EN SG00000163710 Procoilagen C-endopeptidase enhancer 2 PCSK1 ENSG00000175426 Proprotein convertase subtilisialexin type 1 PCSK IN ENSG00000102109 Proprotein convertase subtilisinikexin type 1 inhibitor PCSK2 ENSG00000125851 Proprotein convertase subtilisinikexin type 2 PCSK4 ENSG00000115257 Proprotein convertase subtilisinikexin type 4 PCSK5 ENSG00000099139 Proprotein convertase subtilist tilkex in type 5 PCSK9 ENSG00000169174 Proprotein convertase subtilisialexin type 9 PCYOX I ENSG00000116005 Pmnyleysteine oxidase 1 PCYOX IL ENSG00000145882 Pienylcysteine oxidase 1 like PDDC1 ENSG00000177225 Parkinson disease 7 domain containing 1 PDE I IA ENSG00000128655 Phosphodiesterase I IA
PDE2A ENSG00000186642 Phosphodiesterase 2A, cOMP-stimulated PDE7A ENSG00000205268 Phosphodiestemse 7A
PDF ENSG00000258429 Peptide deformy lase (mitochondria') PDGFA ENSG00000197461 Platelet-derived growth factor alpha polypeptide PDGFB ENSG00000100311 Platelet-derived growth factor beta polypeptide PDOFC ENSG0000014543 I Platelet derived growth factor C
PDGFD ENSG00000170962 Platelet derived growth factor D
PDGFRA ENSG00000134853 Platelet-detived growth factor receptor, alpha poly peptide PDGFRB ENSG00000113721 Platelet-derived growth factor receptor, beta poly-peptide PDGFRL EN SG0000010421:3 Platelet-derived growth factor receptor-like PDH AI EN SG00000131828 Pymvate delnydrogenase (lipoamide) alpha PDIA2 ENSG00000185615 Protein disulfide isomerase family A, member 2 PDIA3 EN S(300000167004 Protein disulfide isomerase family A, member 3 Date Recue/Date Received 2021-01-15 PD1A4 ENSG00000 1 55660 Protein disulfide isomerase family A, member 4 PDIA5 ENSG00000065485 Protein disulfide isomerase family A, member 5 PDIA6 ENSG00000143870 Protein disulfide isomerase family A, member 6 PDILT ENSG00000169340 Protein disulfide isomcnise-like, testis expressed PDYN ENSG00000101127 Prodynoiphin PDZD8 ENSG00000165650 PDZ domain containing 8 PDZRN4 ENSG00000165966 PDZ domain containing ring finger 4 PEAR1 ENSG00000187800 Platelet endothelial aggregation teceptor I
PEBP4 ENSG00000134020 Phosphatidy lethanolamine-binding protein PECAM I ENSG00000261371 Platelet/endothelial cell adhesion molecule 1 PENK EN SG00000181195 Proenkephal in PETE 17 ENSCi00000232838 PETE 17 homolog PF4 ENSG00000163737 Platelet factor 4 PF4V1 EN SG00000109272 Platelet factor 4 variant 1 PFKP ENSG00000067057 Phosphofnictokinase, platelet PFN I EN SG00000108518 Profilin 1 PGA3 ENSG00000229859 Pepsinogen 3, group I (pepsinogen A) PGA4 ENSG00000229183 Pepsinogen 4. group I (pepsinogen A) PGA5 EN SG00000256713 Pepsinogen 5, group I (pepsinogen A) PGAM5 ENSG00000247077 PGAM family member 5, serine/threonine protein phosphatase, mitochondria!
PGAP3 ENSG00000161395 Post-GPI attachment to proteins 3 PGC EN SG00000096088 Progastricsin (pepsirtogen C) PGF ENSG0000011.9630 Placental growth factor PGLYRP I ENSG00000008438 Peptidoglycan recognition protein I
PGLYRP2 EN SG0000016103I Peptidoglycan recognition protein 2 PGLYRP3 ENSG00000159527 Peptidoglycan recognition protein 3 PGL YRP4 EN SG00000163218 Peptidoglycan recognition protein 4 PHACTR1 ENSG00000112137 Phosphatase and actin regulator 1 PHB ENSCi00000167085 Prohibitin P115 ENSG00000137558 Peptidase inhibitor 15 PI3 ENSG00000124102 Peptidase inhibitor 3, skin-derived PIANP ENSG00000 139200 P1LR alpha associated neural protein PIGK ENSG00000142892 Phosphatidylinositol glycan anchor biosynthesis, class K
PIGL ENSG00000108474 Phosphatidylinositol glycan anchor biosynthesis, class L
P1G1 ENSG00000124155 Phosphatidylinositol glycan anchor biosynthesis, class 1' PIGZ EN SG00000119227 Phosphatidylinositol glycan anchor biosynthesis, class Z
PIK3AP I EN SG00000 155629 Phosphoinositide-3-kinase adaptor protein PIK31P1 EN SG000001(X)100 Phosphoi nositide-3-kinase interacting protein I
PILRA ENSG00000085514 Paired immunoglobin-like type 2 receptor alpha P1LRB ENSG0000012 1716 Paired immutioglobin-like type 2 receptor beta PINLYP ENSG00000234465 Phospholipase A2 inhibitor and LY6/PLAUR
domain containing PIP ENSG00000159763 Prolactin-induced protein PIWIL4 ENSG00000134627 Piwi-like RNA-mediated gene silencing 4 Date Recue/Date Received 2021-01-15 PKDCC ENSG00000162878 Protein kinase domain containing, cytoplasmic PKHD I ENSG00000170927 Polycystic kidney and hepatic disease I
(autosomal recessive) PLA IA ENSG00000144837 Phospholipase AI member A
PLA2G10 ENSG00000069764 Phospholipase A2. group X
PLA2G12A ENSG00000123739 Phospholipase A2. group MIA
PLA261213 ENSG00000138308 Phospholipase A2, group XI1B
PLA2GI5 ENSG00000103066 Phospholipase A2, group XV
PLA2G1B ENSG00000170890 Phospholipase A2, group IB (pancreas) PLA2G2A ENS000000188257 Phospholipase A2. group 1IA (platelets, synovial fluid) PLA2G2C ENSG00000187980 Phospholipase A2, group ITC
PLA2G2D ENS000000117215 Phospholipase A2, group HD
PLA2G2E ENSCi00000188784 Phospholipase A2, group IIE
PLA2G3 ENSG00000100078 Phospholipase A2, group 111 PLA2G5 ENSG00000127472 Phospholipase A2. group V
PLA2G7 ENSG00000146070 Phospholipase A2, group VII (platelet-activating factor acetylhydrolasc, plasma) PLA2R1 ENSG00000153246 Phospholipase A2 receptor 1, 180IcDa "'LAC] EN SG00000170965 Placenta-specific I
PLAC9 ENS000000189129 Placenta-specific 9 PLAT ENSG00000104368 Plasminogen activator, tissue PLAU ENSG00000122861 Plasminogen activator, urokinase PLAUR ENSG00000011422 Plasminogen activator, urokinase receptor PLBD1 EN SG00000121316 Phospholipase B domain containing 1 PLBD2 ENS000000151176 Phospholipase B domain containing 2 PLG EN SG00000122194 Plasminogen PLOLB I EN S000000183281 Plasminogen-like BI
PLGLB2 EN SG0000012555 I Plasminogen-like B2 PLOD1 EN S(10000008:3444 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 PLOD? ENS000000152952 Procollagen-lysine, 2-oxoglutarate 5-droxygenase 2 PLOD3 ENSG00000106397 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 PLTP ENS00000014X19 79 Phospholipid transfer protein PLXNA4 ENSG00000221866 Plexin A4 PLXNB2 ENSG00000196576 Plexin B2 PM20D1 EN SG00000162877 Peptidase M20 domain containing 1 PMCH ENSG00000183395 Pro-melanin-concentrating hormone PMEL ENSG04)000185664 Premelanosome protein PMEPA I EN SG00000124225 Prostate transmembrane protein, androgen induced 1 PNLIP EN S(i00000175535 Pancreatic lipase PNLIPRP1 ENSG00000187021 Pancreatic lipase-related protein 1 PNLIPRP3 ENSG00000203837 Pancreatic lipase-related protein 3 PNOC ENSG000001684)81 Prepronociceptin PNP EN SG00000198805 Purine nucleoside phosphorylase PNPLA4 ENSG00000006757 Patatin-like phospholipase domain containing 4 PODNL1 ENSG00000132000 Podocan-like 1 Date Recue/Date Received 2021-01-15 POFUT I ENSG00000 I 01346 Protein 0-fucosyltrans1erase 1.
POFUT2 ENSG00000186866 Protein 0-fucosyltransferase 2 POGLIITI ENSG00000163389 Protein 0-glucosy ltransferase 1 =
POLL EN SG00000166169 Poly merasc (DNA directed). lambda POMC EN Sci00000115138 Proopionelanocoriin POM.CiNT2 EN SG00000144647 Protein 0-linked inannose N-acetylglucosaminyltransferase 2 (beta 1,4-) PON I ENSG00000005421 Paraoxonase I
PON2 ENSG00000105854 Paraoxonase 2 PON3 ENSG00000105852 Paraoxonase 3 POSTN EN SG00000133 I 10 Pe riost itt, osteoblast specific factor PPBP ENSG00000163736 Pro-platelet basic protein (chernokine (C-X-C motif) ligand 7) PPIB EN SG00000166794 Peptidylprolyl isomerase B (cyclophilin B) PPIC N S(300000168938 Peptidy Iproly1 isornerase C (cyclophil in C) PPDX ENSG00000143224 Proloporphyrinogen oxidase PPP1CA ENSG0000017253 I Protein phosphatase 1, catalytic subunit, alpha isozyme PPTI EN SG00000131238 Palmitoyl-protein thioesterase 1 PPT2 ENSG00000221988 Palmitoy.1-protein thioesterase 2 Pancreatic poly peptide PRAC2 ENSG00000229637 Prostate cancer susceptibility candidate PRADC1 ENSG00000135617 Protease-associated domain containing PRAP1 ENSG00000165828 Proline-rich acidic protein 1 PRB I ENSG00000251655 Proline-rich protein BstNI subfamily 1 PRB2 EN SG00000121335 Ptoline-rich protein BstNI subfamily 2 PR.B3 ENSG00000197870 Proline-rich protein BstNl subfamily 3 PRB4 ENSG00000230657 Proline-rich protein BstNI subfamily 4 PRCD EN SC0)000214140 Progressive rod-cone degeneration PRCP EN SG00000137509 Prolylcarbox-ypeptidase (angiotensinase PRDM 12 ENS(30000013071 I PR domain containing 12 PRDX4 EN SCi0000012313 I Peroxiredoxin 4 PRELP ENSG00000188783 Prolineiarginine-rich end leucine-rich repeat protein PRFI ENSG00000180644 Perforin 1 (pore forming protein) PRG2 ENSG00000186652 Proteoglycan 2. bone marrow (natural killer cell activator, eosinophil granule major basic protein) PRG3 ENSG00000156575 Proteoglycan 3 116690 ---F1>otcoilycan-1------------------PRH I ENSG00000231887 Prolinc-rich protein linen' subfamily I
PR.H2 ENSG00000 I 3455 I Proline-rich protein HaeTII subfamily PRKAG1 ENSG00000181929 Protein kinase, AMP-activated, gamma 1 non-catalytic subunit PRK.CSH EN SG00000 I 30175 Protein kinase C substrate 80K-H
Protein kinase D1 PRL ENSG00000172179 Prolactin PRLH ENSG00000071677 Prolact in releasing hormone PRLR EN SG00000113494 Prolacti it receptor PRNP ENSG00000 I 71867 Priori protein Date Recue/Date Received 2021-01-15 PRNT ENSG00000180259 Prion protein (testis specific) PROC ENSG00000115718 Protein C (inactivator of coagulation factors Va and Villa) PROK I ENsG00000143125 Piokinencin 1 PROK2 ENSG00000163421 Prokineticin 2 PROL I ENSG00000171199 Proline rich, lacrimal 1 PROM). ENSG00000007062 Proniinin 1 PROS I ENSG00000184500 Protein S (alpha) PROZ ENSG0000012623 I Protein Z, vitamin K-dependent plasma glycoprotei ri PR.R27 ENSG00000187533 Proline rich 27 PR R4 ENSG00000 I 14215 Proline rich 4 (lacrimal) PRRG2 ENSG00000126460 Proline rich Gla (G-carboxygliitamic acid) 2 PRRT3 ENSCi00000163704 Proline-rich transmembrane protein 3 PRRT4 ENSG00000224940 Proline-rich transmembrane protein 4 PR.SS I ENSG00000204983 Protease, scrim, I (trypsin I) PRSSI2 ENS(100000164099 Protease. senile, 12 (nenrot iy psi n, molopsin) PRSSI6 ENSG00000 112812 Protease, senile, 16 (thymus) PRSS2 ENSG00000275896 Protease, serine, 2 (trypsin 2) PRSS21 ENSG00000007038 Pnatcase; serinc, 21 (iestisin) PR.SS22 ENSG00000005001 Protease, scrim, 22 PR.SS23 ENSC100000150687 Protease, senile, 23 PRSS27 ENSG00000172382 Protease, serine 27 PRSS3 ENSCi00000010438 Protease, serine, 3 PRSS33 ENSG00000103355 Protease; serinc, 33 PR.SS35 ENSG00000146250 Protease, scrinc, 35 PRSS36 ENSG00000178226 Protease, senile, 36 PRSS37 ENSG00000165076 Protease, senile, 37 PRSS38 ENSG00000185888 Protease, serine, 38 PRSS42 ENSG00000178055 Pnatcase; serinc, 42 PRSS48 ENSG00000189099 Protease, senile, 48 PR.SS50 ENSG00000206549 Protease, senile, 50 PRSS53 ENSG00000151006 Protease, senile, 53 PRSS54 ENSG00000103023 Protease, mine. 54 PRSS55 ENSG00000184647 Protease, serinc, 55 PRSS56 ENSG00000237412 Protease, senile, 56 PRSS57 ENSG00000185198 Protease, senile, 57 PRSS58 ENS000000258223 Protease. scrim, 58 PRSS8 ENSG00000052344 Protease, serine, 8 PRTG ENSG00000166450 Protogenin PRTN3 EN SG00000196415 Pivieinase 3 PSAP ENSG00000 497746 Prosaposin PSAPL I ENSG00000178597 Prosaposin-like I (geneipseudogene) PSG1 ENSG00000231924 Pregnancy specific beta- 1-glycoprotein 1 PSG II ENSG00000243130 Pregnancy specific beta-1-glycoprotein 11 PSG2 ENSG0000024222 I Pregnancy specific beta-l-glycopmiein 2 Date Recue/Date Received 2021-01-15 PS63 ENSG00000221826 Pregnancy specific beta- 1-glycoprotein 3 PSG4 ENSG00000243137 Pregnancy specific beta-l-glycopiotein 4 PSG5 ENSG00000204941 Pregnancy specific beta-l-glycomotein 5 PSG6 ENSG00000170848 Pregnancy specific beta-l-glycopmtein 6 PSG7 ENSG00000221878 Pregnancy specific beta-l-glycoprotein 7 (genelpseudogene) PSG8 EN sG00000124467 Pregnancy specific beta- 1-glycopmtein 8 PSG9 ENSG00000183668 Pregnancy specific beta- 1-glycopiotein 9 PSMD1 ENSG00000173692 Ploteasome 26S subunit, non-ATP:Ise 1 PSORS I C2 ENSG00000204538 Psoriasis susceptibility 1 candidate 2 PSPN ENSG00000125650 Persephin PTGDS ENSG00000107317 Prostaglandin 1)2 synthase 21kDa (brain) PTG1R ENSCi00000160013 Prostaglandin 12 (prostacyclin) receptor (IP) Frost ENSG00000095303 Piostaglandin-endoperoxide synthase 1 (prostaglandin GM
synthase and cyclooxygenasef PTGS2 ENSG00000073756 Prostaglandin-endoperoxide synthase 2 (prostaglandin (i/F1 synthase and cyclooxygenase) PTH ENSG00000152266 Parathyroid hormone PIM ENSG00000142538 Parathyroid hormone 2 PTHLH ENSG00000087494 Parathyroid hormone-like hormone PTK7 ENSG00000 112655 Protein tyrosine kinase 7 (inactive) PM ENSG00000105894 Pie lot 10 phi n PTPRA ENSG00000132670 Protein tyrosine phosphatase. receptor type, A
PTPRB ENSG00000127329 Protein tyrosine phosphatase. receptor type, B
PTPRC ENSG00000081237 Protein tyrosine phosphatase, receptor type, C
PTPRCAP ENSG00000213402 Protein tyrosine phosphatase, receptor type. C-associated pmtein PTPRD ENSG00000 I 53707 Protein tyrosine phosphatase, receptor type, D
PTPRF ENSG00000142949 Protein tyrosine phosphatase, receptor type, F
PIM.' ENSG00000149177 Protein tyrosine phosphatase. receptor type, j PTPRO ENSG00000151490 Protein tyrosine phosphatase, receptor type, 0 PTPRS ENSG00000105426 Protein tyrosine phosphatase, receptor type. S
PTTG I IP ENSG00000183255 Pituitary tumor-transforming I
interacting protein PTX3 ENSG00000163661 Pentraxin 3. long PIX4 ENSG00000251692 Pentnixin 4. long PVR ENSG00000073008 Poliov inks receptor PVRL I ENSG00000 110400 Poliovinis receptor-related 1 (herpesvirus ently mexhator C) PXDN ENSG00000130508 Peroxidasin PXDNL ENSG00000147485 Pemxidasin-like PXYLP I ENSG00000155893 2-phosphoxylosc phosphatasc 1 PYY EN SG00000131096 Peptide YY
PZP ENSG00000126838 Pregnancy-zone protein QPCT ENSG00000 I 15828 Glutaminyl-peptide cyciotrattsferase QPRT ENSG00000103485 Quinolinate phosphoribosyltninsferase QRFP ENSG00000188710 Pyroglutamylatcd RFarnidc peptide QSOX l EN SG00000116260 Quiescin 06 sulchydryloxidase I

Date Recue/Date Received 2021-01-15 R31-1DML. ENSG0000010 1074 R3H domain containing-like RAB26 ENSG00000167964 RAB26, member RAS oncogene family RAB36 ENSG00000100228 RAB36, member RAS oncagene family RAB9B ENSG00000123570 RAB9B. member RAS oncogene family RAETIE ENSG00000164520 Retinoic acid early transcript 1E
RAETIG EN 5G00000203722 Retinoic acid early transcript IG
RAMP2 ENSG00000131477 Receptor (G protein-coupled) activity modifying protein 2 RAPGEF5 ENSG00000136237 Rap guanine 'nucleotide exchange factor (GEF) RARRES I ENSG00000118849 Retinoic acid receptor responder (taz.arotene induced) 1 RARRES2 ENSG00000106538 Retinoic acid receptor responder (tazamiene induced) 2 RASA2 ENSG00000155903 RAS p21 protein activator 2 RBM3 ENSCi00000102317 RNA binding motif (RNP I. FtRM) protein RBP3 ENSG00000265203 Retinal binding protein 3, interstitial RBP4 ENSG00000138207 Retinal binding protein 4, plasma RCN! EN5G00000049449 Ret iculocalbin I, EF-hand calcium binding domain RC.N2 ENSG00000117906 Reticulocalbin 2, EF-hand calcium binding domain RCN3 ENSG00000142552 Reticulocalbin 3, EF-hand calcium binding domain RCOR I ENSG00000089902 REST corepressor I
RDH 1 I ENSG00000072042 Retinal dehydrogenase II (all-trans/9-cis/I 1-cis) RDH12 ENSG00000139988 Retinol dehydrogenase 12 (all-trans/9-cis/1 1-cis) RDH13 ENSG00000160439 Retinal dehydrogenase 13 (a1l-trans/9-cis) RDH5 ENSCi00000135437 Retinal dehydrogenase 5 (11-cis19-cis) RDH8 ENSG00000080511 Retinal dehydrogenase 8 (all-trans) REG I A ENSG00000115386 Regenerating islet-derived 1 alpha REG1B ENSG00000172023 Regenerating islet-derived 1 beta REG3A EN SG00000172016 Regenerating islet-derived 3 alpha REG3G EN SG00000143954 Regenerating islet-derived 3 gamma REG4 ENSG00000134193 Regenerating islet-derived family, member RELN ENSG00000189056 Reelin RELT ENSG00000054967 RELT tumor necrosis factor receptor REN EN SG00000143839 Renin REPIN1 ENSG00000214022 Replication initiator I
REPS2 ENSG00000169891 RALBP1 associated Eps domain containing 2 RET EN SG00000165731 Ret proto-ancagene RETN EN SG00000 I (149 I 8 Resist in RETNLB ENSG00000163515 Resisiin like beta RI-EssAT ENSG00000042445 Retinal saturase (all-trans-retinal 13.14-reductase) RI:NG ENSG00000169733 RFNG 0-fucosy 'peptide 3-beta-N-acetylglucosaminy Itransferase RGCC ENSCi00000102760 Regulator of cell cycle RGL4 ENSG00000159496 Ral guanine nucleotide dissociation stimulator-like 4 RGMA EN SG00000182 I ?5 Repulsive guidance molecule family member a RGMB ENSG00000174136 Repulsive guidance molecule family member b RHOQ ENSG00000119729 Ras homolog family member Q
RIC3 ENSG00000166405 11.1C3 acetylcholine receptor chaperone Date Recue/Date Received 2021-01-15 RIMS! ENSG00000079841 Regulating synaptic membrane exocytosis 1 RIPPLYI ENSG00000147223 Ripply transcriptional repressor 1 RLN1 ENSG00000107018 Relaxin 1 =
RLN2 ENSG00000107014 Relaxin 2 RLN3 ENSG00000171136 Relaxin 3 RMDN1 ENSG00000176623 Regulator of microtubule dynamics RNASE I ENSG00000129538 Ribonuclease, RNase A family, I
(pancreatic) RNASE10 ENSG00000182545 Ribonuclease, RNase A family, 10 (non-active) =
RNASE 11 ENSG00000173464 Ribonuclease, RNase A family, 11 (non-active) RNASE12 ENSG00000258436 Ribonuclease, RNase A family, 12 (non-active) RNASE13 EN SG00000206150 Ribonuclease, RNase A family, 13 (non-active) RNASE2 ENSCi00000169385 Ribonuclease. RNase A family, 2 (liver, eosinophil-denved neurotoxin) RNASE3 ENSG00000169397 Ribonuclease. RNase A family, 3 RNA SE4 ENSG00000258818 Ribonuclease, RNase A family, 4 RNASE6 ENSG00000169413 Ribonuclease, RNase A family, k6 RNA SE7 ENSG00000165799 Ribonuclease, RNase A family, 7 RNASE8 EN SG00000173431 Ribonuclease. RNase A family: 8_ family, 9 (non-active) RNASEHI ENSG00000171865 Ribonuclease HI
RNASET2 ENSG00000026297 Ribonuclease T2 RNE146 ENSG00000118518 Ring finger protein 146 RNF148 EN SG00000235631 Ring finger protein 148 RNF 150 ENSG00000170153 Ring finger protein 150 RNF167 ENSG00000108523 Ring finger protein 167 RNF220 ENSG00000187147 Ring finger protein 220 RNF34 ENsG00000170633 Ring finger protein 34, E3 ubiquitin protein ligase RNLS ENSG00000184719 Renalase, FAD-dependent amine oxidase RNPEP ENSG00000176393 Arginyl arninopeptidase (aminopeptidase B) RORI ENSCi00000185483 Receptor tyrosine kinase-like orphan receptor 1 RP1 1-1236K 1.1 ENSG00000233050 RP11-1437.7 ENSG00000259060 RPII-196G11.1 ENSG00000255439 RP11-350014. 18 ENSG00000261793 RP11-520P18.5 ENSG00000261667 RP11-812E19.9 ENSG00000259680 RP11-903H12.5 ENSG00000259171 RP I I-977G19.10 ENSG00000144785 RP4-576H24.4 ENSG00000260861 RP4-608015.3 ENSG000002769 11 Complement factor H-related protein 2 RPL3 EN SG00000100316 Ribosomal protein L3 RPLP2 ENSG00000177600 Ribosomal protein, large. P2 RPN2 ENSG00000118705 Ribophorin II
RPS27L ENSG00000185088 Ribosomal protein S27-like RQCD 1 ENSG00000144580 RCD1 required for cell differentiation I
hornolog (S. pombe) Date Recue/Date Received 2021-01-15 RS I ENSG00000102104 Retinoschisin 1 RSII ENSG00000048649 Remodeling and spacing factor 1 RSPOI. ENSG00000169218 R-spondin I
RSPO2 ENSG00000147635 R-spondin 2 RSPO3 ENS(100000146374 R-spondin 3 RSPO4 ENSG00000 I 01282 R-spondin 4 RSPRY1 ENSG00000159579 Ring finger and SPRY domain containing I
RTBDN ENSG00000132026 Retbinditi RIN4RI, I ENSG00000185924 Reticulon 4 receptor-like 1 RTN4R1,2 ENSG00000186907 Reticulon 4 receptor-like 2 SAM EN SG00000173432 Serum arnyloid Al SAA2 ENSCi00000134339 Serum amy lord A2 SAA4 ENSG00000148965 Serum amy loid A4, constitutive SAP30 ENSG00000164105 Sin3A-associated protein, 30kDa SAR I A EN5G00000079332 Secretion associated, Ras related GTPase IA
SARAF ENSG0000013:3872 Store-operated calcium entry-associated regulatory factor SARM I ENSG00000004139 Sterile alpha and Tilt motif containing I
SATB1 ENSG00000182568 SATB homeobox 1 SAX02 ENSG00000188659 Stabilizer of axonemai microttibules 2 SBSN ENSG00000189001 Suprabasin SBSPON EN SG00000164764 Somatornedin B and thrombospondin, type 1 domain containing SCARF I ENSCi00000074660 Scavenger receptor class F, member 1 SCG2 ENSG00000171951 Secretogranin 11 SCG3 ENSG00000104112 Secretogranin III
SCG5 ENSG00000166922 Secretogranin V
SCGB 1 AI ENSG0000014902 I Secretoglobin, family IA, member 1 (uteroglobin) SCGB IC I ENSG00000188076 Secretoglobin, family IC, member 1 SCGB1C2 ENSG00000268320 Secretoglobin, family IC, member 2 SCGB101 ENSG00000168515 Secretoglobin, family ID, member SCGB I D2 ENSG00000124935 Secretoglobin, family ID, member 2 SCGBID4 ENSG00000197745 Secretoglobin, family ID, member 4 SCGB2A1 ENSG00000124939 Secretoglobin, family 2A, member I
SCGB2A2 ENSG00000110484 Secretoglobin, family 2A, member 2 SCGB2B2 ENSG00000205209 Secretoglobin, family 2B, member 2 SCGB3A1 ENSG00000161055 Secretoglobin, family 3A, member 1 SCGB3A2 ENSG00000164265 Secretoglobin, family 3A, member 2 SCN1.B ENSG0000010571 I Sodium channel, voltage gated, type 1 beta subunit SCN3B ENSG00000166257 Sodium channel, voltage gated, type III
beta submit SCPEP1 ENSG00000121064 Serine calboxypeptidase I
SCRG I ENSG00000164106 Stimulator of chondrogenesis 1 SCT ENSG0000007003 I Secretin SCUBE I ENSG00000159307 Signal peptide, CUB domain. EGF-like 1 SCUBE2 ENSG00000175356 Signal peptide, CUB domain, EGF-like 2 SCUBE3 ENSG00000146197 Signal peptide, CUB domain, EGF-like 3 Date Recue/Date Received 2021-01-15 SDC1 ENS000000 I 15884 Sy tidecan I
SDF2 ENS000000132581 Stromal cell-derived factor 2 SDF2L1 ENSG00000128228 Stromal cell-derived factor 2-like 1 SDF4 ENS000000078808 Stromal cell derived factor 4 SDHAF2 ENSCi00000167985 Succinate dehydrogenase complex assembly factor 2 SDHAE4 ENSG00000154079 Succinate detrydrogenase complex assembly factor 4 SDHB ENS000000117118 Succinate dehydrogenase complex, subunit B, iron sulfur (Ip) SDHD ENS(300000204370 Succinate dehydrogenase complex. subunit D, integral membrane protein SEC14L3 ENSG00000100012 SEC14-iike lipid binding 3 SEC16A ENSG00000148396 SEC16 homolog A. endoplasmic reticultint export factor SEC 168 EN S000000120341 SEC16 homolog B, endoplasmic reticulum export factor SEC22C ENS000000093183 SEC22 homolog C, vesicle trafficking protein SEC3 IA ENSG00000138674 SEC731 homolog A, COPII coat complex component SECISBP2 ENS000000187742 SECIS binding protein 2 SECTM1 ENS000000141574 Secreted and transmembrane 1 SEL IL ENS000000071537 Se!- I suppressor of lin-12-like (C.
elegans) SELM ENS000000198832 Selenoprotein M
SELO ENSG00000073169 Selenoprotein 0 SEMA3A ENSG00000075213 Sema domain, immunoglobulin domain (Ig), short basic domain.
secreted, (semaphorin) 3A
SEMA3B EN SG00000012171 Scma domain, immunogiobulin domain (Ig), short basic domain.
secreted, (semaphorin) 38 SEMA3C ENSG'00000075223 Sema domain, immunogiobulin domain (Ig), short basic domain.
secreted, (semaphorin) 3C
SEMA3E ENSG00000170381 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3E
SEMA3F ENSCi00000001617 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F
SEMA3G ENSG00000010319 Seam domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 36 SEMA4A EN 5C100000196189 Sema domain, immunoglobulin domain (Ig), transtnembrane domain (TM) and short cytoplasmic domain. (seinaphorin) 4A
SEMA4B ENSG00000185033 Sema domain, immunoglobulin domain (ig), transinembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4B
SEM.A4C ENSG00000168758 Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4C
SEMA4D ENSG00000187764 Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain. (semaphorin) 40 SEMA4F ENS000000135622 Sema domain, immunoglobulin domain ag), transmembrane domain (TM) and short cytoplasmic domain. (semaphorin) 4F
SEMA40 ENSCi00000095539 Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain. (semaphorin) 40 Date Recue/Date Received 2021-01-15 SEMA5A ENSG00000112902 Sema domain, seven thrombospondin repeats (type I and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A
SEMA6A ENSG00000092421 Sema domain. transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A
SEMA6C EN SG00000143434 Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6C
SEMA6D ENSG00000137872 Sema domain. transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D
SEMG1 ENSG00000124233 Semenogelin SEMG2 EN SG00000124157 Semenogelin 11 15-Sep EN SG00000183291 15 kDa selenoprotein SEPN1 ENSG00000162430 Selenoprotein N, 1 SEPP1 ENSG00000250722 Selenoprotein P. plasma, I
=
9-Sep ENSG00000184640 Septin 9 SERPINA I ENSG00000197249 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase.
antittypsin), member 1 SERPINA 10 ENSG00000140093 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitiy psin), member 10 SERPINA11 ENSG00000186910 Serpin peptidase inhibitor, chide A (alpha-1 antiproteinase, antitiy psin), member 11 SERPINA12 ENSG00000165953 Serpin peptidase inhibitor, chide A (alpha-1 antiproteinase, antittypsin), member 12 SERPINA3 ENSG00000196136 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antinypsin). member 3 SERPINA3 ENSG00000273259 Seipin peptidase inhibitor, clade A (alpha-1 antiproteinase, antittypsin), member 3 SERPINA4 ENSG00000100665 Serpin peptidase inhibitor, chide A (alpha-1 antiproteinase, antittypsin), member 4 SERPINA5 ENSG00000188488 Serpin pepiidase inhibitor, clade A (alpha-1 antiproteinase, antiuypsin), member 5 SERP1NA6 ENSG00000170099 Serpin pepiidase inhibitor, chide A (alpha-1 antiproteinase, antitrypsin), member 6 SERPINA7 ENSG00000123561 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 SERPINA9 ENSG00000170054 Serpin peptidase inhibitor, chide A (alpha-1 antiproteinase, antitrypsin), member 9 SERPINB2 ENSG00000197632 Serpin peptidase inhibitor, chicle B (ovaibumin). member 2 SERPINC1 ENSG00000117601 Serpin peptidase inhibitor, chide C (antithrombin), member 1 SERPIND I EN 5G00000099937 Serpin peptidase inhibitor, clade (hepaiin cofactor), member 1 SERPINE1 EN SG00000106366 Serpin peptidase inhibitor, clade E (nexi It, plasminogen activator inhibitor type 1), member I
SERPINE2 ENSG00000135919 Sergio peptidase inhibitor, chide E Ole xi plasminogen activator inhibitor type 1), member 2 Date Recue/Date Received 2021-01-15 SERP1NE3 ENSG00000253309 Serpin peptidase inhibitor, chide E
(nexin, plasminogen activator inhibitor type 1), member 3 SE/MINE! EN SG00000132386 Serpin peptidase inhibitor. clade F
(alpha-2 antiplasmin, pigment epithelium derived factor), member 1 SERPINF2 ENS600000167711 Serpin peptidase inhibitor. clade F
(alpha-2 antiplasmin, pigment epithelium derived factor), member 2 SERPING1 EN SG00000149131 Serpin peptidase inhibitor. clade Ci (Cl inhibitor), member 1 SERP1NH1 EN SG00000149257 Serpin peptidase inhibitor, chide H
(heat shock protein 47), member 1, (collagen binding protein 1) SERNNI1 ENSG00000163536 Serpin peptidase inhibitor, chide I
(nettroserpin), member 1 SERPIN12 ENSCi00000114204 Semin peptidase inhibitor, clade I
(pancpin), member 2 SETD8 ENS(300000183955 SET domain containing (lysine nick ransferase) 8 SEZ6L2 ENSG00000174938 Seizure related 6 homolog (mouse)-like 2 SERPI ENSG00000104332 Secreted frizzled-related protein 1 SFRP2 ENSG00000145423 Secreted frizzled-related protein 2 SFRP4 ENSG00000106483 Secreted frizzled-related protein 4 SFRP5 ENS(300000120057 Secreted frizzled-tailed protein 5 SFFA2 ENsG00000196 260 Surfactant associated 2 SFTPA1 ENSG00000122852 Sutfaciant protein Al SFRA2 ENSG00000185303 Surfactant protein A2 SFFPB ENSG00000168878 Surfactant protein B
SFTPD EN S(300000133661 Surfactant protein 1) SFXN5 ENSG00000144040 Siderollexin 5 SGCA ENSG00000108823 Sarcogly can. alpha (50kDa dystrophin-associated glycoprotein) SGS11 ENSG00000181523 N-sitlfogli1COSil in i ne sulfohydrolase SH3RF3 ENSG00000172985 5H3 domain containing ring finger 3 SHBG EN SG00000129214 Sex hormone-binding globulin SHE ENsG00000169291 Src homology 2 domain containing E
514H ENSG00000164690 Sonic hedgehog SIAE ENSCi00000110013 Sialic acid acetylesterase SIDT2 ENSG00000149577 SIDI transmembrane family, member 2 SIGLEC I 0 ENSG00000142512 Sialic acid binding Ig-tike lectin 10 SIGLEC6 ENSG00000105492 Sialic acid binding 1g-like lectin 6 SIG1..EC7 ENSG00000168993 Sialic acid binding ig-like Win 7 S1GLECLI ENSG00000179213 S1GLEC family like 1 SIGMAR1 ENSG00000147955 Sigma non-opioid intracellular receptor 1 SIL1 ENsG00000 1 2 0 .7 2 5 S1L I nucleotide exchange factor SIRPB1 ENSG00000101307 Signal-regulatory protein beta 1 silo ENSG00000125900 Signal-regulatory protein delta SLAMF1 ENsG00000117090 Signaling lymphocytic activation molecule family member 1 SLAMF7 ENSG00000026751 SLAM family member 7 SLC10A3 ENS(300000126903 Solute carrier family 10, member 3 SLC15A3 ENSG00000110446 Solute carrier family 13 (oligopeptidc transporter), member 3 Date Recue/Date Received 2021-01-15 SLC25A14 ENSG00000102078 Solute carrier family 25 (mitochondrial, carrier, brain), member

14 SLC25A25 ENSG00000148339 Solute carrier family 25 (mitochondrial carrier. phosphate carrier), member 25 SLC2A5 ENSG00000142583 Solute carrier family 2 (facilitated glucose/fructose transporter).
member 5 SLC35E3 ENSG00000175782 Solute carrier family 35, member E3 SLC:39A10 ENSG00000196950 Sohue carrier family 39 (zinc transporter), member 10 SLC39A14 ENSG00000104635 Solute carrier family 39 (zinc transporter). member 14 SLC39A4 EN SG00000147804 Solute carrier family 39 (zinc transporter), member 4 SLC39A5 ENSG00000139540 Solute carrier family 39 (zinc transporter), member 3 SLC3A1 ENSG00000138079 Solute carrier family 3 (amino acid transporter heavy chain), member I
SLC5 IA ENSG00000163959 Solute carrier family 51. alpha subunit SLC52A2 ENSG00000185803 Solute carrier family 52 (riboflavin transporter). member 2 SLC5A6 ENSG00000138074 Solute carrier family 5 (sodium/multivitamin and iodide cotransporter). member 6 SLC6A9 EN SG00000196517 Solute carrier family 6 (neurotransmitter transporter, glycine), member 9 SLC8A1 EN SG00000183023 Solute carrier family 8 (sodium/calcium exchanger), member 1 SLC8B1 ENSG00000089060 Solute carrier family 8 (sodium/lithium/calcium exchanger), member B1 SLC9A6 ENSG00000198689 Solute carrier family 9, subfamily A
(NHE6, cation proton antiporter 6), member 6 SLCO1 A2 ENSG00000084453 Solute carrier organic anion transporter family, member 1A2 suTi EN SG00000187122 Slit guidance ligand I
SLIT2 EN SG00000145147 Slit guidance ligand 2 SLIT3 ENSG00000184347 Slit guidance ligand 3 SLITRK3 ENSG00000121871 SLIT and MIRK-like family, member 3 SLPI ENSG00000124107 Secretory leukocyte peptidase inhibitor sum ENSG00000137776 SAFB-like, transcription modulator EN5G00000126233 Secreted LY6iPLAUR domain containing 1 SMARCA2 ENSG00000080503 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 SMG6 ENSCi00000070366 SMG6 nonsense mediated mRNA decay fitctor SMIM7 ENSG00000214046 Small integral membrane protein 7 SMOC I ENSG00000198732 SPARC related modular calcium binding 1 SMOC2 ENSG00000112562 SPARC related modular calcium binding 2 SMPDL3A ENSG00000172594 Sphingornyelin phosphodiesterase. acid-like 3A
SMPDL3B ENSCi00000130768 Sphingomyelin phosphodiesterase, acid-like 3B
SIVER3A ENSG00000109208 Submaxillary gland androgen regulated protein 3A
SIMR3B ENSG00000171201 Submaxilltuy gland androgen regulated protein 313 SNED I ENSG00000162804 Sushi, nidogen and EGF-like domains I
sNTBI ENSG00000172164 Sy rurophin, beta 1 (dystrophin-associated protein Al. 39kDa.
basic component 1) Date Re:cue/Date Received 2021-01-15 SNTB2 ENSG00000 I 68807 Sy ntrophi n, beta 2 (dystrophin-associated protein Al, 59kDa, basic component 2) SNX14 EN SG00000135317 Sorting nexin 14 SOD3 ENSG00000109610 Superoxide dismutase 3, extracellular SOST ENSG00000167941 Selerostin SOSIDC I ENSG00000171243 Sclerostin domain containing 1 SOWAHA ENSG00000198944 Sosondowah ankyrin repeat domain family member A
SPACA3 ENSG00000141316 Sperm acrosoine associated 3 SPACA4 ENSG00000177202 Sperm acrosome associated 4 SPACA5 ENSG00000171489 Sperm acrosome associated 5 SPACA5B ENSG00000171478 Sperm acrosome associated 51.3 SPACA7 ENSG00000153498 Sperm acrosome associated 7 SPAG1 IA ENSG00000 I 78287 Sperm associated antigen I IA
SPAGI1B ENSG00000164871 Sperm associated antigen 11B
SPARC ENSG00000113140 Secreted protein, acidic, cysteine-rich (osteonectin) SPARCL1 ENSG00000152583 SPARC-like 1 (hevin) SPATA20 ENSG00000006282 Spermatogenesis associated 20 SPESP I EN SG00000258484 Sperm equatorial segment protein I
SPINK1 ENSG00000164266 Serine peptidase inhibitor, Kazal type 1 SPINK13 ENSG00000214510 Scrim peptidase inhibitor, Kazal type 13 (putative) SPINK14 ENSG00000196800 Serine peptidase inhibitor. Kazal type 14 (putative) SPINK2 ENSG00000128040 Serine peptidase inhibitor, Kazal type 2 (acrosin-ttypsin inhibitor) 5PINK4 EN SG0000012271 I Serine peptidase inhibitor. Kazal type SPINK5 ENSG00000133710 Scrim peptidase inhibitor. Kazal type 5 SP1NK6 ENS(300000178172 Serine peptidase inhibitor, Kazal type 6 SPINK7 ENSG00000145879 Senile peptidase inhibitor, Kazal type 7 (putative) SPINK8 ENSG00000229453 Serine peptidase inhibitor, Kazal type 8 (putative) SPINK9 EN SG00000204 909 Serine peptidase inhibitor. Kant type 9 SPINT1 EN SG00000166145 Serine peptidase inhibitor, Kunitz type I
SPINT2 ENSG00000167642 Serine peptidase inhibitor, Kunitz type.

SPIN'I'3 ENSG00000101446 Senile peptidase inhibitor, Kunitz type, SPINT4 ENSG00000 149651 Serine pepi idase inhibitor, Kunitz type SPOCK I ENSG00000152377 Sparclosteonectin, cwcv and kazal-like domains prciteoglycan (test Iran) 1 SPOCK2 ENSG00000107742 Sparc/osteonectin, CWC% and kazal-like domains proteoglycan (Iestican).2 S¨parclosteortectin, cwcv domains proteoilyeati itcsticant 3 SPON I ENSG00000262655 Spondin 1, extracellular matrix protein SPON2 ENSG00000159674 Spondin 2, extracellular matrix protein SPP I ENSG00000118785 Secreted phosphoprotein I
SPP2 ENSG00000072080 Secreted phosphoprotein 2. 24kDri SPRN ENSG00000203772 Shadow of prion protein homolog (zebnifish) SPRYD3 ENSG00000167778 SPRY domain containing 3 SPRYD4 ENSG00000176422 SPRY domain containing 4 Date Recue/Date Received 2021-01-15 SPTY2D1-ASI ENSG00000247595 SPTY2D1 antisense RNA 1 SPX ENSG00000134548 Spexin hormone SRGN ENSG00000122862 Serglycin SRL ENSG00000185739 Sarcalumenin SRPI4 ENSG00000140319 Signal recognition panicle 14kDa (homologous Mu RNA
binding protein) SRPX ENSG00000101955 Sushi-repeat containing pmtein, X-litiked SRPX2 ENSG00000102359 Sushi-repeat comaining piotein. X-linked SSC4D EN SG00000146700 Scavenger receptor cysteine rich family, 4 domains SSC5D ENSG00000179954 Scavenger receptor cysteine rich family, 5 domains SSPO ENSG00000197558 SCO-spoildin SSR2 ENSG00000163479 Signal sequence receptor, beta (translocon-associated protein beia) SST ENSG00000157005 Sornatostat sT3GALI. EN SG00000008513 ST3 beta-galactoside alpha-2,3-sialyitransferase 1 ST3GAL4 ENSG00000110080 ST3 beta-galactoside alpha-2,3-sialyltinnsfemse 4 ST6GAL 1 ENS000000073849 ST6 beta-galactosamide alpha-2,6-sialyltiatifenise ST6GALNAC2 ENSG00000070731 ST6 (alpha-N-acetyl-neuraminy1-2,3-beta-galactosy1-1,3)-N-acetylgalactoskuninide alpha-2,6-sialyltransferase 2 ST6GALNAC5 ENSG00000117069 ST6 (alpha-N-acetyl-neuraminy1-2,3-beta-galactosy1-1,3)-N-acetylgalactosaini nide a1pha-2,6-sialyltransferase 5 ST6GALNAC6 ENSG00000160408 sor6 (alpha-N-acetyl-neuraminy1-2,3-beta-galactosy1-1,3)-N-acetylvilactosami nide alphit-2,6-sialyltransferase 6 ST8S1A2 ENSG00000140557 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltmnsierase 2 ST8SIA4 ENSG000001 13532 STS alpha-N-acetyl-netimminide alpha-2,8-sialy Itransferase 4 sTssiA6 ENSG00000148488 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 STARD7 ENSG00000084090 StAR-related lipid transfer (START) domain containing 7 STATH ENSG00000126549 Statherin STC1 ENSG00000159167 Stanniocalcin 1 STC2 ENSG00000113739 Stanniocalcin 2 STMND I ENS(300000230873 Stall:min domain containing I
sTomi.2 ENSG00000165283 Stomatin (EPB72)-like 2 STOX I ENSG00000163730 Storkhead box I
STRC ENSG00000242866 Stereocilin SUCLG1 ENSCi00000163541 Succinate-CoA ligase, alpha subunit SUDS3 ENSG0000011 I 707 SDS3 homolog, SIN3A compressor complex component SULF I EN5G00000137573 Sulfatase 1 SULF2 ENSG00000196562 Sulfatase 2 SUMF1 EN SG00000144455 Sullatase modify mg factor 1 SUMF2 ENSG00000129103 Sulfatase modifying factor 2 SUSDI ENSG00000106868 Sushi domain containing 1 SUSD5 ENSG00000173705 Sushi domain containing 5 SVEP I ENSG00000163124 Sushi, von Willebrand factor ly pe A. EGF
and pent mxin domain containing 1 SWSAP I ENSG00000173928 SWIM-type zinc finger 7 associated protein 1 Date Recue/Date Received 2021-01-15 SYAP1 ENSG00000169895 Synapse associated protein I
SYCN ENSG00000179751 Syncollin TAC1 ENSG00000006128 Tachykinin. precursor 1 =
TAC3 ENSG00000166863 Tachykinin 3 TAC4 ENSG00000 176358 Tachykinin 4 (hernokinin) TAGLN2 SG00000158710 Transgel in 2 TAPBP ENSG00000231925 TAP binding protein (tapasin) TAPBPL ENSG00000139192 TAP binding protein-like =
TBL2 ENSG00000106638 Transducin (bcta)-like 2 TBX10 ENSG00000167800 T-box 10 TCF12 ENSG00000 140262 Transcription factor 12 TCN1 ENSCi00000134827 Transcobalamin I (vitamin 1312 binding protein, R binder family) TCN2 NS(300000185339 Transcobahimin H
TCTN I ENSG00000204852 Tectonic family member 1 TCTN3 ENSG00000119977 Tectonic family member 3 mp2 ENSG00000 It 1802 Tyrosyl-DNA
phosphodiesterase 2 TEK ENSG00000120156 TEK tyrosine kinase, endothelial TEPP ENSG00000 159648 Testis, prostate and placenta expressed TEX 101 EN SG00000131126 Testis expressed 101 TEX264 ENSG00000164081 Testis expressed 264 TT' ENSG00000091513 Transfernn TFAM ENSG00000108064 Transcription factor A.
mitochondria!
TETI EN SG00000160182 Trefoil factor 1 TFF2 ENSG00000160181 Trefoil filctor 2 TFF3 ENSG00000160180 Trefoil factor 3 (intestinal) Trpt ENSG00000003436 Tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) TFP12 EN SG00000105825 Tissue factor pathway inhibitor 2 TG ENSG00000042832 Thyroglobulin TGFB1 ENSG00000105329 Transforming growth factor, beta 1 TGFB2 ENSG00000092969 Transforming growth factor, beta 2 TGFB3 ENSG00000119699 Transforming growth factor.
beta 3 TGFB1 ENSG00000 120708 Transforming growth factor, beta-indaced, 68k1)a TGFBR1 ENSG00000106799 Transforming growth factor, beta receptor 1 TGFBR3 ENS600000069702 Transforming growth factoi, beta receptor III
THBS1 ENSG00000137801 Thrombospondin 1 THBS2 ENSG00000 186340 Thmtnbospondi it 2 THBS3 ENSG0000016923 I Thrombospondin 3 THBS4 ENSG00000113296 Thrombospondin 4 TUFO ENSG00000090534 Thrombopoietin THSD4 ENSG00000187720 Throinbospondin, type I, domain containing 4 THY1 ENSG00000154096 Thy-1 cell surface antigen Date Recue/Date Received 2021-01-15 TIE I ENSCi00000066056 Tyrosine kinase with immunoglobulin-like and EGE-like domains 1 TINEvIDC1 ENSG00000113845 Translocase of inner mitochondrial membrane domain containing 1 TIMP I EN SG00000102265 T1MP metallopeptidase inhibitor 1 TIMP2 ENS(100000035862 TIM? metallopeptidase inhibitor 2 TIMP3 ENSG00000100234 T1MP ntetallopepticlase inhibitor 3 TIMP4 ENSG00000157150 TIMP metallopeptidase inhibitor 4 TINAGL1 EN SG00000142910 Tubtilointerstitial nephritis antigen-like 1 TINF2 ENSG00000092330 TERF I (TRE1)-interacting nuclear factor TLL2 ENSG00000095587 Tolloid-like 2 TI.R I EN SG00000174125 Toll-like receptor 1 TLR3 ENSG00000164342 Toll4ike receptor 3 "FM2D2 ENSG00000169490 '11\42 domain containing 2 TM2D3 ENSG00000184277 TM2 domain containing 3 TM7SF3 ENS(i00000064115 Transmembrane 7 superfamily member 3 TIM9SF1 EN SG00000100926 Tianstnembrane 9 superfilmily member 1 TMC06 ENSG00000113119 Transmembrane and coiled-coil domains 6 -n4E DI ENSG00000099203 Transmembratie p24 trafficking protein 1 ENSG00000166557 Transmembrane p24 trafficking protein 3 "FMED4 ENSG00000158604 Ttansmentbrane p24 trafficking protein 4 TMED5 ENSG000001 17500 Transmembrane p24 trafficking protein 5 TMED7 ENSG00000134970 Transmcmbrane p24 trafficking protein 7 TMEM108 ENSG00000144868 Transmembrane protein 108 TIMEM116 ENSG00000198270 Ttansmentbrane protein 116 TMEM119 ENSG00000183160 Transmembrane protein 119 TMEM155 EN SG00000164112 Transmcmbrane protein 155 mlEm168 ENSG00000146802 Transmembratte protein 168 TMEM178A EN SG00000 152154 Transmembrane protein 178A
TMEM179 ENSG00000258986 Transmembrane protein 179 TMEM196 ENSG00000173452 Transmembnine protein 196 TMEM199 ENSG00000244045 Transmcmbrane protein 199 TMEM205 EN SG00000105518 Transmembrane protein 205 TMEM213 ENSG00000214128 Transmembrane protein 213 TMEM25 EN SG00000149582 Ttansmembiane protein 25 TIMEM30C ENSG00000235156 Transmembrane protein 30C
TMEM38B ENSG00000095209 Transmcmbrane protein 38B
TMEM52 EN SG00000178821 Transmembrane protein 52 TMEM52B ENSG00000165685 Transmembrane protein 52B
TMEM59 EN SG00000116209 Transtnembnine protein 59 TIMEM67 ENSCi00000164953 Transmcmbrane protein 67 --1-W.V177-5--------151-gi0i0000l7-5-6-0-4-----Tr;;;t7i-JninTit-t-t;i37;i(Tri715------Date Recue/Date Received 2021-01-15 TMEM.87A ENSG00000103978 Transmembrane protein 87A
TMEN494 ENSG00000177728 Transmembrane protein 94 TMEM95 ENSG00000182896 Transmembrane protein 95 TMIGD I ENSG00000182271 Transmembrane and immunoglobulin domain containing 1 TMPRSSI2 ENSCi00000186452 Transmernbrane (C-temirnal) protease, serine 12 TMPRSS5 ENS600000166682 Transmernbrane protease, serine 5 TM1,113 I ENSG00000164897 Transmembrtme and ubiquitin-like domain containing 1 TMX2 EN SG00000213593 Thioredoxin-related vat:membrane protein TMX3 ENSG00000166479 Thioredoxin-related transmembrane protein TNC ENSG00000041982 Tenascin C
TNFAIP6 ENSG00000123610 Tumor necrosis bctor, alpha-induced protein 6 TNFRSF I IA ENSCi00000141655 Tumor necrosis factor receptor superfamily, member (la, NFKB activator TNTRSF 11B ENSG0000016476 I Tumor necrosis factor receptor superfamily, member (lb TNTRSF12A ENSG00000006327 Tumor necrosis factor receptor superfamily, member 12A
TNFRS1:14 ENSG00000157873 Tumor necrosis factor receptor superfamily, member 14 TNTR SF18 EN SG00000186891 Tumor necrosis factor receptor superfamily, member 18 TM:R.9:1A ENSG00000067182 Tumor necrosis (actor receptor superfamily, member IA
TNTR SEIB ENSG00000028137 Tumor necrosis factor receptor superfamily, member 1B
TNERSF25 ENSG00000215788 Tumor necrosis factor receptor superfamily, member 25 TNTR SF6B ENSG00000243509 Tumor necrosis factor receptor superfamily, member 61a. decoy TNFSF11 ENSG00000120659 Tumor necrosis factor (ligand) superfamily, member 11 TNESE12 ENSG00000239697 Tumor necrosis (actor (ligand) superfarnrly. member 12 TNFSF I. 2-TN FS1:13 ENSG0000024887 I INFSF12-TNFSF13 readthrough TI\ITSF15 EN SG00000181634 Tumor necrosis factor (ligand) superfamily, member 15 TNN ENSG00000 120332 Tenascin N
"1NR EN SG00000116147 Tenascin R
TNXB EN SG00000168477 Tenascin X.E3 TOMM7 ENSG00000196683 Translocase of outer mitochondrial membrane 7 homolog (yeast) TOP (MT ENSG00000184428 Topoisomerase (DNA) I, mitochondria' TOR I A ENSG00000136827 Torsin family 1. member A (torsin A) TOR I B EN SG00000136816 Torsin family 1, member B (torsin B) TOR2A ENSG00000160404 Torsin family 2, member A
TOR3A ENSG00000186283 Torsin family 3, member A
TPD52 ENSG00000076554 Tumor protein D52 TPO ENSG00000115705 Thyroid peroxidase TPP I ENSG00000166340 Tripeptidyl peptidase '17SAB I ENSG00000172236 Tryptase alpha/beta I
TPSB2 ENSG00000197253 Tryptase beta 2 (geneipseudogene) TPSDI ENSG00000095917 Tryptase delta 1 TPST I ENSG00000169902 Tyrosylprotein strifotransferase 1 TPST2 ENSG00000 128294 Ty rosy Iprotei n Sillfotransfenkse 2 TRABD2A EN SG00000186854 TtaB domain containing 2A
TRABD2B ENSG00000269113 TraB domain containing 213 Date Recue/Date Received 2021-01-15 TREH ENSG00000118094 Trehalase (brush-border membrane glycoprotein) TREM1 ENSG00000124731 Triggering receptor expressed on myeloid cells 1 TREM2 ENSG00000095970 Triggering receptor expressed on myeloid cells 2 TRH ENSG00000170893 Thyrotropin-releasing hormone TRIM24 ENSG00000122779 Tripartite motif contai ning 24 num28 ENsG00000130726 Tripartite motif containing 28 TRIO ENSG00000038382 Trio Rho guanine nucleotide exchange factor TR.NP I ENSG00000253368 TMF I-regulated nuclear protein 1 TSC22D4 ENSG00000166925 ISC22 domain family, member 4 TSHB ENSG00000134200 Thyroid stimulating hormone. beta TSHR ENSG00000165409 Thyroid stimulating hotnione receptor TSKU ENSCi00000182704 Tsukushi, small leucine rich proteoglycan TSLP ENS000000145777 Thymic snort:al lyrnphopoietin TSPAN3 ENSG0000014039 I Tetraspanin 3 TSPAN31 ENS000000135452 Tetraspanin 31 TSPEAR ENSG00000175894 Thrombospondin-type laminin 0 domain and EAR repeats TTC13 ENSG00000143643 Tetratricopeptide repeat domain 13 TIC19 ENSG00000011295 Tetratricopeptide repeat domain 19 TTC9B EN S000000174521 Tetratricopeptide repeat domain 913 TILL11 ENSG00000175764 Tubtilin tyrosine ligase-like family member 11 '17R ENS000000118271 Transthytetin TWSG I EN SCi00000128791 Twisted gastrulation BMP signaling modulator 1 TXNT)C12 ENS000000117862 Thioredoxin domain containing 12 (endoplasmic retie:dm) TXNDC15 ENS00000011362 I Thioredoxin domain containing 15 TXNDC5 ENS000000239264 Thioredoxin domain containing 5 (endoplasinic reticul um) TXNRD2 ENSG00000184470 Thioredoxin reductase 2 TYRPI. ENSG00000107165 Tyrosinase-related protein UBAC2 ENSG00000134882 UBA domain containing 2 UBALD1 ENS000000153443 UBA-like domain containing I
UBAP2 ENSG00000137073 Llb iquit in associated protein 2 U13XN8 EN SG000001()469 I UBX domain protein 8 UCMA, EN S000000165623 Upper zone of growth plate and cartilage matrix associated UCN ENS000000163794 Uroconin UCN2 ENSG00000145040 Urocortin 2 UCN3 ENS000000178473 Urocon in 3 UGGT2 ENSG00000102595 UDP-glucose glycoprotein glucosyltransferase 2 UGTIA10 ENSG00000242515 UDP glucuronosyltransfentse I family, polypeptide A10 UGT2A1 ENSG00000173610 UDP glucuronosykransfentse 2 family, polypeptide Al, complex locus UGT21311 ENS000000213759 UDP giticuronosyltransferase 2 family, polypeptide 1311 UGT2B28 ENS000000135226 UDP glucumnosyltransferase 2 family, polypeptide B28 UCiT2134 ENSG00000156096 UDP glucuronosyltransfentse 2 family, polypeptide 84 UGT2B7 ENS000000171234 UDP glucuronosyitransfentsc 2 family, polypeptidc 137 UGT3 Al ENS000000145626 UDP glycosy hransferase 3 family, polypeptide Al LiGT3A2 ENS000000168671 UDP glycosyltransferase 3 family, polypeptide A2 Date Recue/Date Received 2021-01-15 UCiT8 ENSG00000174607 UDP glycosyltransferase ULDP3 ENSG00000131019 UL16 binding protein 3 UMOD ENSG00000169344 Urotnodul in UNC5C ENSG00000182168 Unc-5 netrin receptor C
UPK 3B ENSG00000243566 Uroplaki 3B
USP I I EN sG00000102226 Ubiquitin specific peptidase II
USP14 EN SG00000101557 Ubiquitin specific peptidase 14 (tRNA-guanine transglycosylase) USP3 ENSG00000140455 Ubiquitin specific peptidase 3 UTS2 ENSG00000049247 Urotensin 2 UTS2B ENSG00000188958 Urotensin 2B
up( ENSG00000183878 Ubiquitously transcribed tetratricopeptide repeat containing, Y-I inked UXS1 ENSG00000115652 UDP-glucuionate decatboxylase I
VASH1 ENSG00000071246 Vasohibin 1 VCAN ENSG00000038427 Vcrsican VEGFA ENSG00000112715 Vascular endothelial growth factor A
VEGF13 ENSG00000173511 Vascular endothelial growth factor B
VEGFC ENSG00000150630 Vascular endothelial growth factor C
VIP ENSG00000146469 Vasoactive intestinal peptide VIPR2 ENSG00000106018 VasoactiVC intestinal peptide receptor 2 VIT ENSG00000205221 Vitrin VKORCi ENSG00000167397 Vitamin K epoxide reductase complex, subunit I
VLDLR, ENSG00000147852 Very low density lipoprorein receptor VMOI ENSG00000182853 Vitelline membrane outer layer 1 homolog (chicken) VNN1 ENSG00000112299 Valli!) I
VNN2 ENSG00000112303 Vanin 2 VNN3 ENSG00000093134 Vanin 3 VOPP I ENSG00000154978 Vesicular, overexpressed in cancer, prosurvival protein 1 VPREB I ENSG00000169575 Pre-B lymphocyte 1 VPREB3 EN SG00000128218 Pre-3 lymphocyte 3 VPS3713 ENSG00000139722 Vacuolar protein sorting 37 homolog B (S. cerevisiae) VPS51 ENSG00000149823 Vacuolar protein sorting 51 homolog (S. ceievisiae) vsiG I ENS(300000 i 01842 V-set and immunoglobulin domain containing 1 VSIGIO ENSG00000176834 V-set and immunoglobulin domain containing 10 VSTM I EN SG00000189068 V-set and ttansmembrane domain containing 1 vsn4.2A ENSG00000 I 70419 V-set and transmembrane domain containing 2A
VSTIVI2B ENSG00000187135 V-set and transmembrane domain containing 2B
i i TiTif ENSG00000165633 V-set and transmembrane domain containing 4 VTN ENSG00000109072 Vittonectin VWA1 ENSG00000179403 Von Willebnind factor A domain containing 1 VWA2 ENSG00000165816 Von Willcbrand factor A domain containing 2 --c:VA-3112------------ECiki0i000i0i4-5-14-8-7(7171-tiViiiTb-r-Triii factor A
domain containing 5B2 Date Recue/Date Received 2021-01-15 N/WA7 ENSG00000204396 Von Willebrand factor A domain containing N.TWC2 ENSG00000188730 Von Willebrand factor C domain containing VWC21. ENSG00000174453 Von Willebtand factor C domain containing protein 2-like VWCE ENSG00000167992 Von Willebrand factor C and EGF domains VWDE ENSG00000146530 Von Willebrand factor D and EGF domains VWF ENSG00000110799 Von Willebrand factor VvDR25 ENSG00000176473 WD repeat domain 25 WD R8 I ENSG00000167716 WD repeat domain 81 WDR90 EN SG00000161996 WD repeal domain 90 WFDC1 ENSG00000103175 WAR four-disulfide core domain I
WFDC 10A ENSG00000180305 WAP four-disulfide core domain 10A
VvTDCIOB EN SCi00000182931 WAP four-disulfide core domain 10B
WFDC I 1 ENSG00000180083 WAR four-disulfide core domain II.
WFDC1.2 ENSG00000168703 WAP four-disulfide core domain 12 WFDC13 ENSG00000168634 WAP four-disulfide core domain 13 WFDC2 ENSG00000101443 WAP four-disulfide core domain 2 VvTDC3 ENSG00000124116 WAP four-disulfide core domain 3 WFDC5 ENSG00000175121 WAR four-disulfide core domain 5 WFDC6 ENSG00000243543 WAP four-disulfide core domain 6 WFDC8 ENSG00000158901 WAP four-disulfide core domain 8 WFIK KN 1 ENSG00000127578 WAP, follistatitilkazal, immunogiobulin, kunilz. and nein!) domain containing I
WFIKKN2 EN SG00000173714 WAP, follistatitilkazal, immunoglobulin, kuniti and netrin domain containing 2 WI! ENSG00000156076 WNT inhibitory factor I
WISP 1 ENSG00000104413 WNT1 inducible signaling pathway protein WISP2 ENSG00000064205 WNTE inducible signaling pathway protein WISP3 ENSG00000112761 NVNT1 inducible signaling pathway protein WNK I ENSG00000060237 WNK. lysine deficient protein kittase 1 WNT I ENSG00000125084 Wingless-type MMTV integration site family, member WNTIOB ENSG00000169884 Wingless-type MMTV integration site family, member 108 WNT 11 ENSG00000085741 Wingless-type i\IVITV integration site family, member 11 WNT16 ENSG00000002745 Wingless-type MMTV integration site family. member 16 WNT2 ENSG00000105989 Wingless-type mNrry integration site family member 2 WNT3 ENSG00000108379 Wingless-type IVIMTV integration site family, member 3 WNI3A EN SG00000 154342 Wingless-type MMTV integration site family, member 3A
WNT5A ENSG00000114251 Wingless-type NEVITV integration site family, member 5A
WNT5B ENSG00000111186 Wingless-type MMTV integration site family. member 5B
WNT6 ENSG00000115596 Wingless-type Ml\fry integration site family, member 6 WNT7A ENSG00000154764 Wingless-type MMTV integration site family, member 7A
WNI7B ENSG00000188064 Wingless-I) pe 1\11\4TV integration site family, member 78 WNT8A ENSG00000061492 Wingless-type MMTV integration site family, member 8A
WNT8B ENSG00000075290 Wingless-type MMTV integration site family, member 8B
WNT9A ENSG00000143816 Wingless-type mNrry integration site family, member 9A

Date Recue/Date Received 2021-01-15 WNT9B ENSC00000I58955 Wingless-type MMTV integration site family, member 9B
WSB I EN SG0000010904.6 WD repeat and SOCS box containing 1 WSCDI ENSG00000179314 WSC domain containing I
WSCD2 ENSG00000075035 WSC domain containing 2 XCI.1 ENSG00000143184 Chemokine (C motif) ligand I
XCI..2 ENSG00000143 185 Chernokine (C motif) ligand 2 XPNPEP2 ENSG00000122121 X-proly I aminopeptidase (aminopeptidase P) 2, membrane-bound XXbac- ENSG00000244255 BPG116M5.17 XXbac- ENSG00000248993 BPG181M17.5 XXbac-BPG32J3.20 ENSG00000204422 XXYLT1 ENSG00000173950 Xyloside xylosyltransferase 1 XYLT1 ENSG00000103489 Xylosyltransfentsc 1 XYLT2 ENSG00000015532 Xylosyltransferase ZEN' VE21 EN SG0000010071 I Zinc finger, FY VE domain containing 21 ZG16 ENSG00000174992 Zymogen granule protein 16 ZG16B ENSG00000162078 Zymogen granule protein 16B
Z1C4 ENSG00000174963 Zic family member 4 ZNI7207 ENSG00000010244 Zinc finger protein 207 ZNF'26 EN SG00000198393 Zinc linger protein 26 ENSG00000196378 Zinc finger protein 34 ZNF4I9 ENSG00000105136 Zinc finger protein 419 ZNF433 ENSG00000197647 Zinc finger protein 433 7.NT449 ENSG00000173275 Zinc finger protein 449 ZNF488 ENSG00000265763 Zinc finger protein 488 ZN1-'511 ENSGI)0000198546 Zinc finger protein 511 ZNTF570 ENSG00000171827 Zinc finger protein 570 ZNF69 I ENSG00000164011 Zinc finger polein 691 ZNF98 ENSG00000197360 Zinc finger protein 98 ZPBP ENSG00000042813 Zona pellucida binding protein ZP13P2 ENSG00000186075 Zona pellucida binding protein 2 ZSCAN29 ENSG00000140265 Zinc finger and SCAN domain containing 29 102431 In some embodiments of the disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the methods of the disorder, compositions comprising CAR-T cells modified to express or to secrete a human protein are used to treat a clotting disorder. Blood clotting occurs through a multistep process known as the coagulation cascade. In the extrinsic pathway, Tissue Factor (also known as factor Ill or thromboplastin) comes into contact with factor VII to form an activated Vila complex. This initiates a coagulation protease cascade, converting the inactive Factor X to an active protease Factor Xa, which, with activated Factor V, produces thrombin Date Recue/Date Received 2021-01-15 (ha) from Prothrombin (II). In the intrinsic pathway, collagen forms a complex with high-molecular-weight-kininogen, prekallikrein and Factor XII, leading to the conversion of Factor XII into Factor Mk. Factor XIla converts Factor XI into Factor Xia, and Factor XIa activates Factor IX to produce Factor 1Xa, which, together with FVIlla form the tenase complex, which activates Factor X, which helps convert Prothrombin (II) into Thrombin (Ha). Thrombin in turn leads to the conversion of Fibrinogen (I) into Fibrin, which together with Factor XIIIa forms a cross-linked fibrin clot. Many clotting disorders are the result of low levels of secreted proteins in the blood that are involved in the coagulation cascade.
Clotting disorders can drastically increase the amount of blood leaving the body upon injury, or cause bleeding to occur under the skin or in vital organs. These disorders are frequently genetic. Exemplary, but non-limiting diseases caused by deficiencies in clotting factors include Hemophilias, von Willebrand disease and deficiencies in Antithrombin III, protein C
or protein S. Hemophila A and B are X-linked, and are caused by insufficient levels of clotting factor VIII and factor IX (FIX) respectively. Hemophila C is caused by insufficient factor XL Factor IL VII, X or XII deficiencies can also cause bleeding disorders. Von Willebrand disease is due to a low level of the von Willebrand clotting factor in the blood. In some cases, deficiencies in blood proteins that regulate clotting lead can lead to too much clotting. Factor V Leiden is a genetic disorder, where the factor V Leiden protein overreacts, causing the blood to clot too often or too much. Deficiencies in Antithrombin III, protein C or protein S, which help regulate bleeding, can also cause excessive clotting.
Currently, clotting disorders such as Hemophilia are treated with blood transfusions or infusions of the missing clotting factor (replacement therapy). However, complications of replacement therapy include developing antibodies to the clotting factor, contracting viral infections from blood derived products and damage to joints. There thus exists a need for additional therapies.
(02441 In some embodiments of the disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the methods of the disorder, compositions comprising CAR-T cells modified to express or to secrete a human protein are used for enzyme replacement therapy. Enzyme replacement therapy typically involves intravenous infusions of therapeutically effective amounts of compositions comprising enzymes that balance underlying enzyme deficiencies that cause the symptoms of the disease. The missing enzyme activity is thus supplied exogenously in this manner. Exemplary diseases that can be treated by modified T cells of the disclosure include, but are not limited to, lysosomal storage diseases Gauchcr's disease (glucocerebrosidase Date Recue/Date Received 2021-01-15 enzyme), Fabry disease, mucopolysaccharidosis I (MPS I), mucopolysaccharidosis I (MPS II, or Hunter syndrome, caused by iduronate-2-sulfatase deficiency), mucopolysaccharidosis VI
(MPS VI, caused by arylsulfatase B deficiency) and Pompe disease (or glycogen stoarage disease type II, caused by a deficiency in acid alpha-glucosidase). Additional diseases treatable with enzyme replacement therapy include but are not limited to Adenosine deaminase (ADA) deficiency, Hyperammonemia due to the deficiency of the hepatic enzyme N-acetylglutamate synthetase (NAGS), Hypophosphatasia, Lysosomal acid lipase deficiency, Morquio Syndrome A, Wolman LAL Lysosomal Acid Lipase deficiency, A1AT (Alphal-Antitrypsin) deficiency and Urea cycle disorder. Enzymes supplied to patients during enzyme replacement therapy include, but are not limited to Alphal-Antitrypsin, Glucocerebrosidase, Adenosine Deaminase, Alpha-Galactosidase A, a-L-Iduronidase, Iduronate-2-Sulfatase, N-Acetylgalactosamine-6 Sulfatase, -Acetylgalactosamine-4 Sulfatase and Lysosomal Acid Lipase.
[02451 In some embodiments of the disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the methods of the disorder, compositions comprising CAR-T cells modified to express or to secrete a human protein are used to produce human antibodies. In some embodiments, the disease to be treated by modified T cells expressing secreted proteins is a disease that can be treated through the intravenous infusion or injection of an antibody or an antibody fragment.
Antibody based therapies are used in the treatment of many types of diseases in addition to cancer, including immune-based diseases such as arthritis and asthma, and infections, as well as other diseases. Exemplary, but non-limiting list of diseases that can be treated with the modified T cells of the disclosure include platelet aggregation, Clostridium difficile infection, Rheumatoid arthritis, Crohn's Disease, Plaque Psoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis, Alzheimer's disease, sepsis, Multiple Sclerosis, hypercholesterolemia, systemic lupus erythematosus, prevention of organ transplant rejections, viral infections, asthma, severe allergic disorders, retinopathy, osteoporosis, inflammatory bowel diseases, inflammatory diseases, influenza A, paroxysmal nocturnal hemoglobinuria, sepsis caused by Grain-negative bacteria, psoriasis, invasive Candida infection, ulcerative colitis, hy-pocholesterolemia, respiratory sy-ncytial virus infection, focal segmental glomerulosclerosis, graft versus host disease, ankylosing spondylitis, HIV
infection, ulcerative colitis, autoimmune diseases, chronic asthma, reduction of scarring after glaucoma surgery, hypercholesterolemia, white blood cell diseases, systemic scleroderma, Date Recue/Date Received 2021-01-15 respiratory syncytial virus (prevention), lupus erythematosus, diabetes mellitus type 1, inflammation, Pseudomonas aeruginosa infection, macular degeneration, anthrax, cytomegalovirus infection, inflammations of the airways, skin and gastrointestinal tract, systemic lupus erythematosus, rheumatic diseases, uveitis, cytomegalovinis infection, dermatomyositis, polymyositis, fibrosis, choroidal and retinal neovascularization, muscular dystrophy, Staphylococcus aureus infection, lupus nephritis, follicular lymphoma, chronic hepatitis B and ulcerative colitis.
Infusion of Mod4fled Cells as Adoptive Cell Therapy [02461 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 2x105 and 5x108 cells per kg of body weight of the patient per administration, or any range, value or fraction thereof.
[02471 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 0.2x106 to 20x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.2x 1 06 cells per kg of body weight of the patient per administration, 2x106 cells per kg of body weight of the patient per administration, 20x106 cells per kg of body weight of the patient per administration, or any cells per kg of body weight of the patient per administration in between.
[02481 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1x106 cells or about 1x106 cells per kg of body weight of the patient per administration.
[02491 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 3x106 cells or about 3x106 cells per kg of body weight of the patient per administration.

Date Recue/Date Received 2021-01-15 [02501 In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 0.7x106 to 6.7x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x106cells per kg of body weight of the patient per administration, 6.7x106 cells per kg of body weight of the patient per administration or any cells per kg of body weight of the patient per administration in between.
[0251] In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 0.7x106 to 16x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x10 cells per kg of body weight of the patient per administration, 2x106 cells per kg of body weight of the patient per administration, 6x106 cells per kg of body weight of the patient per administration, 10.7x106 cells per kg of body weight of the patient per administration, 16x106 cells per kg of body weight of the patient per administration or any cells per kg of body weight of the patient per administration in between.
[02521 in certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1.2x106 to 7.1x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1.2x106 cells per kg of body weight of the patient per administration, 7.1x106 cells per kg of body weight of the patient per administration or any number of cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between 2x106 to 3x106 cells per kg of body weight of the patient per administration.

Date Recue/Date Received 2021-01-15 [0253] In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1106x106 to 2106x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 1106x106 cells per kg of body weight of the patient per administration, 2106x106 cells per kg of body weight of the patient per administration or any number of cells per kg of body weight of the patient per administration in between. In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x106 to 1.3x106 cells per kg of body weight of the patient per administration. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises 0.7x106 cells per kg of body weight of the patient per administration.
1.3x106cells per kg of body weight of the patient per administration or any number of cells per kg of body weight of the patient per administration in between.
[0254] In certain embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises a single or multiple doses. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises a split dose. In certain embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises an initial dose and a maintenance dose.
[02551 In certain embodiments of the disclosure, the modified cells are T
cells and the T
cells may be sorted according to T cell markers prior to either in vitro expansion or formulation with a pharmaceutically acceptable carrier. In some embodiments, modified T
cells may be sorted on using CDR+ and/or CD4+ markers.
Nucleic Acid Molecules Date Recue/Date Received 2021-01-15 [0256] Nucleic acid molecules of the disclosure encoding protein scaffolds can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA
or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
[02571 Isolated nucleic acid molecules of the disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally, with one or more introns, e.g., but not limited to, at least one specified portion of at least one protein scaffold;
nucleic acid molecules comprising the coding sequence for a protein scaffold or loop region that binds to the target protein; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific protein scaffolds of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.
[0258] As indicated herein, nucleic acid molecules of the disclosure which comprise a nucleic acid encoding a protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase can include, but are not limited to, those encoding the amino acid sequence of a protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase fragment, by itself; the coding sequence for the entire protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase or a portion thereof; the coding sequence for a protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional; non-coding sequences, including but not limited to, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example, ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the Date Recue/Date Received 2021-01-15 sequence encoding a protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein scaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase comprising a protein scaffold fragment or portion.
Construction of Nucleic Acids [0259] The isolated nucleic acids of the disclosure can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as well-known in the art.
[0260] The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the disclosure. The nucleic acid of the disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the disclosure.
[0261] Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra;
or Sambrook, supra).
Recombinant Methods for Constructing Nucleic Acids [0262] The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize, wider stringent conditions, to the polynucicotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA
and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).
Vectors and Host Cells [0263] The disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and Date Recue/Date Received 2021-01-15 the production of at least one protein scaffold by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra.
[0264] For example, the PB-EFla vector may be used. The vector comprises the following nucleotide sequence:
tgtacatagattaaccctagaaagataatcatattgtgacgtacgttaaagataatcatgcgtaaaattgacgcatgtg ttttatcggtctgt atatcgaggtttatttattaatttgaatagatattaagttttattatatttacacttacatactaataataaattcaac aaacaatttatttatgtttatt tatttattaaaaaaaaacaaaaactcaaaatttcactataaagtaacaaaacttttatcgaatacctgcagcccggggg atgcagaggga cagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgagccgcccggggctcc gctcc ggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgc tac ctctgaacgcttctcgctgctctttg agcctgcagacacctggggggatacggggaaaagttgactgtgcctttcg atcgaacc atgg a cagttagctttgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaag gagtgggaattg gctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagaggggggaggggtcggcaattgaacc ggtg cctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggaga accgtata taagtgcagtagtcgccgtgaacgactttacgcaacgggtagccgccagaacacaggtaagtgccgtgtgtggacccgc gggcct ggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagc ttcgggttggaag tgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctgg ggccgccg cgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacct gctgcgacgcttttt actggcaagatagtcagtaaatgcgggccaagatctgcacactggtatttcggtttaggggccgcgggcggcgacgggg cccgtg cgtcccagcgcacatgacggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggc cggc ctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgc gtgagcg gaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagt caccc acacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacc tcgattagt tctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggt ggagactgaag ttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcc tcagacagtggttcaa agtttttttcttccatttcaggtgtcgtgagaattctaatacgactcactatagggtgtgctgtctcatcattttggca aagattggccaccaa gcttgtcctgcaggagggtcgacgcctctagacgggcggccgctccggatccacgggtaccgatcacatatgcctttaa ttaaacact agttctatagtgtcacctaaattccctttagtgagggttaatggccgtaggccgccagaattgggtccagacatgataa gatacattgatg agtaggacaaaccacaactagaatgcagtgaaaaaaatgctttatagtgaaatagtgatgctattgctttatagtaacc attataagctg caataaacaagttaacaacaacaattgcattcatatatgtacaggacagggggaggtgtgggaggattacggactctag gacctgcg catgcgcttggcgtaatcatggtcatagctgtttcctgttttccccgtatccccccaggtgtctgcaggctcaaagagc agcgagaagcg ttcagaggaaagcgatcccgtgccaccttccccgtgcccgggctgtccccgcacgctgccggctcggggatgcgggggg agcgcc ggaccggagcggagccccgggcggctcgctgctgccccctagcgggggagggacgtaattacatccctgggggctaggg gggg Date Recue/Date Received 2021-01-15 ggctgtccctctcaccgcggtggagctccagcttttgttcgaattggggccccccctcgagggtatceatgatatctat aacaagaaaat atatatataataagttatcacgtaagtagaacatgaaataacaatataattatcgtatgagttaaatcttaaaagtcac gtaanagataatcat gcgtcattttgactcacgcggtcgttatagttcaaaatcagtgacacttaccgcattgacaagcacgcctcacgggagc tccaagcg,gc gactgagatgtcctaaatgcacagcgacggattcgcgctatttag;wigagagagcaatatttcaagaatgcatgcgtc aattttacgca gactatctttctagggttaatctauctagccttaagggcgcctattgcgttgcgctcactgcccgctttccagtcggga aacctgtcgtgc cagctgcattaatgaatcggccaacgcgeggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg actcgctgc gctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataa cgcaggaa agaacatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatatct tgagatcatt ttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagcta ccaactctttttc cgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaa gaactctgtag caccgcctacatacctcgctagctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtettaccgagttg gactcaagac gatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgaccta caccga actgagatacctacagcgtgagctatgagana cgccacgcttcccgaagggagvangcggacaggtatccgglancggcagg gtcggaacaggagagcgcacgagggagettccagg,gggaaacgcctggtatctttatagtcctgtegggtttcgccac ctctgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaanaargccagcaacgcggcctttttacggttcct ggccttttgct ggccttttgctcacatgagattatconanngatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatc taangtatatatga gtaaacttggtctgacagtcagaagaactcgtcaagaaggcgatagaaggegatgcgctgcgaategggagcggcgata ccgtaaa gcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcg gtecgcca cacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatg ggtcacea cgagatcctcgccgtcgggcatgctcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcticgtc cagatcatc ctgatcgacaagaccucttccatccgagtacgtgctcgctcgatgcgatgatcgcttggtggtegaatgggcaggtagc cggatca agcgtatgcagccgccgcattgcatcagccatgatggatactttetcggcaggagcaaggtgagatgacaggagatcct gccccggc acttcgcccaatagcagccagtccatcccgcttcagtgacaacgtegagcacagctgcgcaaggaacgcccgtcgtggc cagcca cgatagccgcgctgcctcgtcttgcagttcattcagggcaccggacaggtcggtatgacaannagaaccvsgcgcccct gcgctga cagccggaacacggeggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagectctecacccaageg gccggag aacctgcgtgcaatccatcttgttcaatcataatattattgaagcatttatcagggttcgtctcgtcccgg tctcctcccaatgcatgicaata ttggccattagccatattattcattggttatatagcataaatcaatattggctattggccattgcatacgttgtatcta tatcataata (SEQ
ID NO: 35).
[02651 The poly-nucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

Date Recue/Date Received 2021-01-15 [0266] The DNA insert should be operatively linked to an appropriate promoter.
The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.
[0267] Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464;
5,770,359;
5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB
gene), G418/Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E.
coli and other bacteria or prokaryotics. Appropriate culture mediums and conditions for the above-described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
[0268] Expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure. Such cell surface markers include, e.g., but are not limited to, "cluster of designation" or "classification determinant"
proteins (often abbreviated as "CD") such as a truncated or full length form of CD19, CD271, CD34, CD22, Date Recue/Date Received 2021-01-15 CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8):1277-87).
[0269] Expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NI0(2.2, or any combination thereof.
[0270] At least one protein scaffold of the disclosure can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a protein scaffold to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a protein scaffold of the disclosure to facilitate purification. Such regions can be removed prior to final preparation of a protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74;
Ausubel, supra, Chapters 16, 17 and 18.
[0271] Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the disclosure.
Alternatively, nucleic acids of the disclosure can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding a protein scaffold of the disclosure. Such methods are well known in the art, e.g., as described in U.S.
Pat. Nos.
5,580,734, 5,641,670, 5,733,746, and 5,733,761.
[0272] Illustrative of cell cultures useful for the production of the protein scaffolds, specified portions or variants thereof, are bacterial, yeast, and mammalian cells as known in the art. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEI(293, BHI(21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC
CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, 5P2/0-Ag14, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of Date Recue/Date Received 2021-01-15 lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Ag14 cells (ATCC

Accession Number CRL-1851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8.653 or an SP2/0-Ag14 cell.
[0273] Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Pat. Nos.
5,168,062;
5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an 5V40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells useful for production of nucleic acids or proteins of the present invention are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.
[0274] When eukaryotic host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene.
Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from 5V40 (Sprague, et al., J. Virol. 45:773-781 (1983)).
Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.
Purification of a Protein Scaffold [0275] A protein scaffold can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10.

Date Recue/Date Received 2021-01-15 [0276] Protein scaffolds of the disclosure include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, E. coli, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the protein scaffold of the disclosure can be glycosylated or can be non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14.
Variants [0277] The amino acids that make up protein scaffolds of the disclosure are often abbreviated. The amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). A protein scaffold of the disclosure can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein. Amino acids in a protein scaffold of the disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15;
Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites that are critical for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).
[0278] As used throughout the disclosure, the term "substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.
[0279] As used throughout the disclosure, the term "substantially identical"
refers to a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or Date Recue/Date Received 2021-01-15 990/0 identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
[0280] As used throughout the disclosure, the term "variant" when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
[0281] As used throughout the disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid.
[02821 As used throughout the disclosure, the term "variant" when used to describe a peptide or poly:peptide, refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.
102831 A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge.
Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a Date Recue/Date Received 2021-01-15 useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference.
[0284] Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example inununogenicity.
Substitutions can be performed with amino acids having hydrophilicity values within 2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
[0285] As used herein, "conservative" amino acid substitutions may be defined as set out in Tables A, B, or C below. In some embodiments, fusion polypeptides and/or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention.
Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.
[0286] Table A ¨ Conservative Substitutions I
Side chain characteristics Amino Acid Aliphatic Non-polar GAPILVF
Polar uncharged CSTMNQ
Polar - charged DEKR
Aromatic HFWV
Other NQDE
[0287] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table B.
[0288] Table B -- Conservative Substitutions II
Side Chain Characteristic Amino Acid Date Recue/Date Received 2021-01-15 Non-polar (hydrophobic) Aliphatic: ALIVP
Aromatic: F W Y
Sulfur-containing: Ni Borderline: G Y
¨Uncharged-polar Hydroxyl: STY
Amides: NQ
Sulfhydryl:
Borderline: G Y
Positively Charged (Basic): K R H
Negatively Charged (Acidic): D E
[02891 Alternately, exemplary conservative substitutions are set out in Table C.
[0290] Table C -- Conservative Substitutions III
Original Residue Exemplary Substitution ¨Ala (A) Val Leu Ile Met ¨Arg (R) Lys His Asn (N) Gin Asp( D) Glu Cys (C) Ser Thr Gin (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His (H) Lys Arg Ile (I) Leu Val Met Ala Phe Leu (L) Ile Val Met Ala Phe Lys (K) Arg His Met (M) Leu Tie Val Ala Phe (F) Trp Tyr Ile Pro (P) Gly Ala Val Leu Ile Ser (S) Thr Thr (I) Ser Date Recue/Date Received 2021-01-15 Tyr (Y) Trp Phe Thr Ser Val (V) Ile Leu Met Ala [0291] It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.
[0292] As used throughout the disclosure, the term "more than one" of the aforementioned amino acid substitutions refers to 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, or 20 or more of the recited amino acid substitutions. The term "more than one"
may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.
[0293] Polypeptides and proteins of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring.
Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring.
[0294] As used throughout the disclosure, "sequence identity" may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (b12seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250. The terms "identical" or "identity" when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces Date Recue/Date Received 2021-01-15 one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (1) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2Ø
EXAMPLES
Example 1: Production of stem-like modified T-cells [0295] The following is an illustrative but nonlimiting example of one protocol for modifying T cells to express a chimeric antigen receptor (CAR) under conditions that induce or preserve desirable stein-like properties of the T cells.
[0296] Day 0: Nucleofection of T cells [0297] Pre-wann ImmunoCultTm-XF T cell expansion medium (Stemcell Technologies, Cat #: 10981) in 37 C, 5% CO2, high humidity incubator. For 5x106T cells/reaction (100 AL
cuvette size) warm 3 mL of media/reaction in a single well of a 6-well plate.
For 25x106 T
cells/reaction (100 1.tL cuvette size) warm 20 mL of media/reaction in a G-Rex 10 (Wilson Wolf, Cat #: 80040S).
[0298] Warm P3 primary cell solution (Lonza, Cat #: PBP3-02250) up to room temperature and add supplement if necessary.
[02991 Turn on the core unit (Lonza. Cat 14: AAF-1002B) of the 4D-NucleofectorTm System, which controls the X-unit (Lonza, Cat #: AAF-1002X). Program the number of nucleofections required to use P3 buffer. Program E0-210.
[0300] Label cuvettes, pre-open transfer pipettes (supplied with the Lonza P3 kit), and prepare proper dilutions of nucleic acids prior to working with the cells.
[0301] For a transposon plasmid, make a 0.5 fig/ 1, solution in nuclease free H20.
[03021 Count CD14, CD56, and CD19 depleted cells collected using the CliniMACs Prodigy and calculate the volume needed for the required cell number.
[03031 Centrifuge T cells at 90 g for 10 minutes with brake at 7 on a Heraeus Multifuge X3R benchtop centrifuge (Thennofisher Scientific). If performing multiple reactions using the same number of cells/reaction, centrifuge all the necessary cells in a single centrifuge tube. Either a 15 mL (Fisher, Cat #: 14-959-49B) or 50 mL (Fisher, Cat #: 14-959-49A) conical tube can be used depending on volume. During centrifugation add nucleic acids Date Recue/Date Received 2021-01-15 directly to the bottom of cuvettes that come with the P3 primary cell solution box (Lonza, Cat #: PBP3-02250). Add 2 j.iL of the 0.5 pg/pL transposon plasmid solution made in step 4 for a total of 1 lig transposon to one of the bottom comers of the cuvette. Add 5 pg of Super piggyBacTm (SPB) transposase mRNA to the other corner of the cuvette.
[03041 Because mRNA can be rapidly degraded, it is optimal to minimize the time it is in contact with other nucleic acid solutions and with cells prior to electroporation due to the potential presence of RNases. This is why, for example, the transposon and transposase are delivered to opposite corners of the cuvette to prevent mixing. In addition, it is optimal to keep the total volume of nucleic acids under 10 L (10%) of the total reaction volume.
[03051 The amount of both transposon (1 jig) and transposase (5 g) stays the same regardless of the number of cells/reaction. Transposition efficiencies remain unchanged between 5x106 cells/100 tiLL reaction and 25x106 cells/100 pL reaction.
[03061 Following centrifugation, completely aspirate off the media without disturbing the cell pellet.
[03071 Suspend the cell pellet in 100 pL of room temperature P3 buffer containing the supplement/reaction.
[03081 Transfer 100 pL of cells in P3 buffer to a cuvette containing the appropriate nucleic acids, optimally, taking care not to introduce any air bubbles into the solution. It is recommended that only up to 2 cuvettes should loaded with cells at a time.
After the addition of cells to the cuvette, it is optimal to work quickly and efficiently to reduce contact time of mRNA with cells prior to nucleofection. While no decrease in transposition efficiency has observed for cells resting in P3 buffer for up to 10 minutes, it is recommended to minimize the amount of time cells remain in P3.
[03091 Mix the contents of the cuvette by flicking several times and load up to two cuvettes into the 4DNucleofectorTM X-unit.
[03101 Pulse the cells with program EO-210 and ensure there was no effor recorded by the machine.
[03111 Immediately transfer the nucleofected cells into either the 6-well plate or G-Rex10 using the transfer pipettes provided with the Lonza P3 kit. To transfer the cells, first draw up a small amount of pre-wanncd media into the transfer pipette from either the 6-well plate or the G-Rex flask. Then pipette the media into the cuvette and transfer the entire contents of the cuvette using the pipette into the final culture dish. It is recommended not to pipette the cells up and down in either the cuvette or the final culture dish.

Date Recue/Date Received 2021-01-15 [03121 Repeat protocol from the transfer of cells in P3 buffer to a cuvette containing the appropriate nucleic acids through the mixing, pulsing, and transfer of the nucleofected cells into either the 6-well plate or G-Rexl 0 for any remaining reactions.
[03131 Place cells in incubator at 37 C, 5% CO2, high humidity.
[03141 Day 2: T cell Activation [03151 Add 25 pL/mL of lmmunoCultTM Human CD3/CD28/CD2 T cell Activator (Stemcell Technologies, Cat #: 10970) to the nucleofected cells.
[03161 Mix cells gently by pipetting.
[03171 Place cells back into the incubator at 370 C, 5% CO2, high humidity.
[03181 For cells being grown in G-Rex flask: It is essential not to disturb the cultures until visible cell clumping is observed. Thus, it is recommended to separate the media additions and changes from the disruption/mixing/pipetting of the cells.
[03191 Culture media notes: For growing cells in the G-Rex flask, media addition and/or changes should be done based off of glucose and other metabolite levels. If the glucose level (or another indicating metabolite) falls to a critical level (-100 mg/dL of glucose, for example) media volume should be doubled and/or replenished by a half-media change using pre-warmed ImmunoCultTm-XF T cell expansion medium. Media addition should be performed slowly and care taken to disrupt the cells as little as possible.
Half media changes should be performed at least 12 hours post mechanical disruption of the cell culture to allow the cells to fully settle to the bottom of the culture flask.
[03201 Cell Sampling and disruption: Cells should be left undisturbed during much of the culture period.
[03211 The first disruption of the cell culture following activation reagent addition should occur once large visible aggregates of cells have formed (aggregates will measure 3-4 squares by 3-4 squares of the grid that can be seen on the G-Rex membrane).
[03221 Once cell aggregates have reached the required size, they can be mechanically disrupted using a 10 mL serological pipette. This time point may occur between 11-14 days depending on donor and transposition efficiency. In certain circumstances, this time point may occur closer to day 14 than day 11, for example, when using a manual cassette, a large volume and/or a large cell number for nucleofection. A sampling of cells should be collected at this point for cell counts, viability, and flow analysis. Ideally the volume of culture medium at this point will have no more than doubled from the initial volume used (200mL

Date Recue/Date Received 2021-01-15 for a G-Rex100). It is recommended to collect all of the cells needed at once so that the cells do not need to be disturbed again.
[03231 Once the cells have been disrupted they should be left undisturbed for 12 hours in the same volume of media they started in. Cells should re-aggregate at this point; however, the aggregates will be smaller and more numerous. These aggregates should measure 1-2 squares by 1-2 squares on the G-Rex membrane grid.
[03241 Three days following the first disruption (day 14-17 depending on the culture) of the cells they can be pipetted a second time. Samples should be taken again for cell counts, viability, and flow cytometry. Once again the cells should be left undisturbed for at least 12 hours post sampling. It is recommended to collect all of the cells needed at once so that the cells do not need to be disturbed again.
[0325] Following this second disruption, the cells will likely not form any clumps and the rate of cell growth will slow considerably.
[03261 Cell harvest should be performed 3 days after the second disruption of cells between day 17 and day 20 of the culture.
[03271 Flow Cytometry [0328] Flow should be run on Day 5, D-Day, D-Day +3. and D-Day +6.
[03291 For Day 5, D-Day, and D-Day +3 use the CD45, CD4, CD8, and CARTyrin flow panel [03301 For D-Day +6, there are 3 target panels:
a. Panel 1: CD3, CD8, CD4, CARTyrin, CD45RA, CD45RO, CD62L
b. Panel 2: CD3, CD8, CD4, CARTyrin, CD25, CXCR4, PD-1 c. Panel 3: CD45, CD14, CD20, CD56, CD8, CD4, CD3 Example 2: Functional characterization of CARTyrin+ stem memory T cells [03311 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.
[03321 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 into resting pan T cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme, called Super piggyBacTM
(SPB), in a single electroporation reaction. Delivery of piggyBacTM transposon into untouched, resting Date Recue/Date Received 2021-01-15 primary human pan T cells resulted in 20-30% of cells with stable integration and expression of PB-delivered genes. Unexpectedly, a majority of these modified CARTyrin-expressing T
cells were positive for expression of CD62L and CD45RA, markers commonly associated with stem memory T-cells (Ism 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.
[0333] 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 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 CARTyrie T cells, the LV-transduced CARTyrin+ T cells did not exhibit an augmented memory phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of target tumor cells.
Together, these data demonstrate that CAR-T cells produced by PB transposition are predominantly Tscm cells, a highly desirable product phenotype in the CAR-T field. Furthermore, these CARTyrie 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.
Example 3: Sleeping Beauty Transposition Yields Predominantly Tscm phenotype [0334j Sleeping Beauty (SB100x) Transposition yielded a predominately Tscm phenotype using the methods of the disclosure. Human pan T cells were transposed using Ing of either a Sleeping Beau , or piggyBac transposon plasmid and SB100x or SPB mRNA, respectively as shown in Figure 10. Following transposition, cells were expanded ex vivo and all non-Date Recue/Date Received 2021-01-15 transposed cells were depleted using a 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.
Example 4: Expression of Factor IX in modified T-cells [0335] Genetic deficiencies in Factor IX (Figure 11) lead to a life threatening disease called Hemophila B. Hemophila B is a rare disease that affects 1 in 25,000 to 1 in 30,000 people.
Current Hemophilia B treatments involve an infusion of recombinant Factor IX
protein every 2-3 days, at a cost of around $250,000 per year.
[0336] Stem memory T cells (Tscm cells) are maintained in humans for several decades, and are therefore an ideal vehicle to secrete Factor IX, supplying the Factor IX
missing in Hemophilia B patients without the need for frequent transfusions. T cells were transformed with PiggyBac to secrete Factor IX. When transgenic T cells encoding a human Factor IX
transgene were examined for T and Tscm cell markers using FACS, approximately 80% of all cells showed a Tscm phenotype (Figure 12). These modified T cells were able to secrete human Factor IX (Figure 13A), and this secreted Factor IX provided clotting activity (Figure 13B).
[0337] The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a cited document, the meaning or definition assigned to that term in this document shall govern.
OTHER EMBODIMENTS
[0338] While particular embodiments of the disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and Date Recue/Date Received 2021-01-15 scope of the disclosure. The scope of the appended claims includes all such changes and modifications that are within the scope of this disclosure, Date Recue/Date Received 2021-01-15

Claims

What is claimed is:

1. A composition comprising a population of modified T-cells, wherein a plurality of modified T-cells of the population comprise a non-naturally occurring antigen receptor or a sequence encoding the same, wherein at least 25% of the population of modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm) or a Tscm-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L.

2. The composition of claim 1, wherein at least 25% of the modified T cells of the population express one or more of CD127, CD45RO, CD95 and IL-2RP.

3. The composition of claim 1, wherein the non-naturally occurring antigen receptor is a non-naturally occurring T-Cell Receptor (TCR).

4. The composition of claim 3, wherein the non-naturally occurring T-cell receptor comprises one or more mutation(s) compared to a wild-type TCR.

5. The composition of claim 3, wherein the non-naturally occurring T-cell receptor is a recombinant TCR.

6. The composition of claim 1, wherein the non-naturally occurring antigen receptor is a Chimeric Antigen Receptor (CAR).

7. The composition of claim 6, wherein the CAR comprises (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.

Date Recue/Date Received 2021-01-15

8. The composition of claim 7, wherein the antigen recognition region comprises at least one of a single chain variable fragment (scFv), a single domain antibody, a VHH, an antibody mimetic, a protein scaffold or a Centyrin.

9. The composition of claim 7, wherein the antigen recognition region comprises at least one single chain variable fragment (scFv).

10. The composition of claim 7, wherein the antigen recognition region comprises at least one VHH.

11. The composition of claim 7, wherein the antigen recognition region comprises at least one Centyrin.

12. The composition of claim 1, wherein the plurality of modified T-cells of the population further comprise a genomic editing composition.

13. The composition of claim 12, wherein the genomic editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain.

14. The composition of claim 12, wherein the plurality of modified T-cells of the population further comprise a donor sequence composition.

15. The composition of claim 14, wherein the donor sequence composition comprises a sequence encoding the non-naturally occurring antigen receptor.

16. The composition of claim 15, wherein the sequence encoding the non-naturally occurring antigen receptor comprises a single-stranded or double-stranded DNA
molecule.

Date Recue/Date Received 2021-01-15

17. The composition of claim 14, wherein the donor sequence composition is inserted into a specific site in the genome contacted with the composition by homologous recombination or by endogenous DNA repair mechanisms.

18. The composition of claim 1, wherein the plurality of modified T-cells of the population further comprise a transposon comprising a sequence encoding the non-naturally occurring antigen receptor.

19. The composition of claim 18, wherein the plurality of modified T-cells of the population further comprise a transposase composition comprising a transposase or a sequence encoding the transposase.

20. The composition of claim 19, wherein the transposase composition comprises a piggyBac transposase or a sequence encoding a piggyBac or piggyBac-like transposase.

21. The composition of claim 1, wherein a genomic safe harbor site comprises a sequence encoding the non-naturally occurring antigen receptor.

22. The composition of claim 1, wherein the composition further comprises a T-cell expansion composition.

23. The composition of claim 22, wherein the composition or the T-cell expansion composition 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 or an alkane.

24. The composition of claim 1, wherein at least 30% of the modified T-cells of the population express CD62L and CD45RA.

Date Recue/Date Received 2021-01-15

25. The composition of claim 1, wherein at least 30% of the modified T-cells of the population express one or more of CD127, CD45RO, CD95 and IL-2RP.

26. The composition of claim 1, wherein one or more of a stem cell-like T
cell, a stem cell memory T cell (Tscm) and a central memory T cell (Tcm) comprise at least 75%
of the population of modified T-cells.

27. The composition of claim 1, wherein central memory T cells (Tcms) comprise at least 15% of the population of modified T-cells.

28. The composition of claim 1, further comprising 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 anti-human CD2 monospecific tetrameric antibody complex.

29. The composition of claim 28, comprising a plurality of activated and modified T-cells.

30. The composition of claim 1, wherein at least 40% of the modified T-cells of the population express CD62L and CD45RA.

Date Recue/Date Received 2021-01-15