Modified stem cell memory T cells, methods for making and using same

Abstract

To provide 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, wherein the disclosure provides a solution to the following long-felt but unmet need: 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.SOLUTION: Unlike traditional biological preparation 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.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application Nos. 62 / 402,707, filed September 30, 2016, 62 / 502,508, filed May 5, 2017, 62 / 553,058, filed August 31, 2017, and 62 / 556,309, filed September 8, 2017, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference The contents of the filed text file entitled "POTH-012_001WO_SeqList.txt", created on October 2, 2017, and measuring 110KB in size, are hereby incorporated by reference in their entirety.

[0003] FIELD OF THE INVENTION The present disclosure is directed to molecular biology, and more specifically, to methods of making and using engineered stem-cell memory T cells. [Background technology]

[0004] background There is a long-standing unmet need in the art for methods of generating engineered stem cell memory T cells for administration to a subject, e.g., as adoptive cell therapy. The present disclosure provides a solution to this long-standing unmet need. Summary of the Invention [Means for solving the problem]

[0005] Abstract Unlike conventional biologics and chemotherapeutic agents, the modified T cells of the present disclosure have the ability to rapidly regenerate upon antigen recognition, thereby potentially eliminating the need for repeated treatment. To achieve this, the modified T cells of the present disclosure not only initially drive tumor destruction but are also maintained in patients as a stable population of viable memory T cells to prevent potential cancer recurrence. Thus, the modified T cells of the present disclosure are capable of targeting antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as early memory cells, particularly stem cell memory (T SCM Intensive efforts have focused on the development of modified T cell products containing stem cell-like modified-T cells. The stem cell-like modified-T cells of the present disclosure have the greatest capacity for self-renewal as well as the ability to generate central memory T cells (T CM ), effector memory T cells (T EM ) and effector T cells (T E ), resulting in better tumor eradication and long-term engraftment of the engineered T cells. Engineered T cells of the present disclosure include, but are not limited to, cells expressing antigen receptors comprising the protein scaffolds of the present disclosure. Engineered T cells of the present disclosure include, but are not limited to, cells expressing chimeric antigen receptors (CARs) (i.e., CAR-T cells of the present disclosure). Chimeric antigen receptors (CARs) of the present disclosure can comprise one or more sequences that each specifically bind to an antigen, including, but not limited to, single-chain antibodies (e.g., scFvs), sequences comprising one or more fragments of antibodies (e.g., VHHs, referred to as VCARs for CARs), antibody mimetics, and centyrin (referred to as CARTyrin for CARs).

[0006] The modified cells of the present disclosure can be further subjected to genome editing.For example, a genome editing construct can be introduced into the modified cells of the present disclosure by transposon or other delivery means by electroporation or nucleofection, and then be integrated into the genome of the cell during the subsequent incubation step.The resulting cells are modified T cells with an edited genome that maintains a stem-like phenotype.This modified T cell with an edited genome that maintains a stem-like phenotype can be used as cell therapy.Alternatively, or in addition, the modified cells of the present disclosure can be subjected to first electroporation or nucleofection and then subsequent electroporation or nucleofection to introduce a genome editing construct.

[0007] Specifically, the present disclosure provides modified stem memory T cells (T SCM ), the method comprising introducing (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 into primary human T cells to generate modified T cells, wherein the modified T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of modified stem memory T cells (T SCM ) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCM ), the method comprising introducing (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 into a plurality of primary human T cells to generate 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 therebetween, of the plurality of modified T cells are stem memory T cells (T SCM), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells are stem memory T cells (T SCM), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. SCM The cell surface markers of CAR-T include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, the transposon is a plasmid DNA transposon, and the sequence encoding the antigen receptor or therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly embodiments in which the transposon is a piggyBac transposon, the transposase is a piggyBac™ or Super piggyBac™ (SPB) transposase.

[0008] In certain embodiments of the disclosed methods, the transposon is a plasmid DNA transposon, and the sequence encoding the antigen receptor or therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac™ or Super piggyBac™ (SPB) transposase. In certain embodiments, particularly in embodiments where the transposase is a Super piggyBac™ (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

[0009] In certain embodiments of the disclosed methods, the transposase enzyme is a piggyBac™ (PB) transposase enzyme. The piggyBac (PB) transposase enzyme is [ka] It may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to the amino acid sequence of the present invention.

[0010] In certain embodiments of the disclosed methods, a transposase enzyme is an array [ka] and a piggyBac™ (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of:

[0011] In certain embodiments, the transposase enzyme is a piggyBac™ (PB) transposase enzyme comprising or consisting 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 piggyBac™ (PB) transposase enzyme comprising or consisting 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 piggyBac™ (PB) transposase enzyme comprising or consisting 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 valine (V) with isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of serine (S) with glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of valine (V) with methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of lysine (K) with asparagine (N).

[0012] In certain embodiments of the disclosed methods, the transposase enzyme is a Super piggyBac™ (SPB) transposase enzyme. In certain embodiments, the Super piggyBac™ (SPB) transposase enzyme of the present disclosure may comprise or consist of the amino acid sequence of SEQ ID NO: 4, wherein the amino acid substitution at position 30 is a substitution of valine (V) with isoleucine (I), the amino acid substitution at position 165 is a substitution of serine (S) with glycine (G), the amino acid substitution at position 282 is a substitution of valine (V) with methionine (M), and the amino acid substitution at position 538 is a substitution of lysine (K) with asparagine (N). In certain embodiments, the Super piggyBac™ (SPB) transposase enzyme is [ka] It may comprise or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to the amino acid sequence of the present invention.

[0013] In certain embodiments of the disclosed methods, including those in which the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, piggyBac™ or Super The piggyBac™ 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 of the disclosed methods, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the piggyBac™ or Super piggyBac™ 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 asparagine (N) with 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 serine (S) with 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 threonine (T) with 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 tryptophan (W) with 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 proline (P) with 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 proline (P) with 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 alanine (A) with 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 leucine (L) with 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 lysine (K) with 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 histidine (H) with 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 leucine (L) with 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 phenylalanine (F) for isoleucine (I). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO:4 or SEQ ID NO:5 is a substitution of phenylalanine (F) for valine (V). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO:4 or SEQ ID NO:5 is a substitution of methionine (M) for leucine (L). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO:4 or SEQ ID NO:5 is a substitution of alanine (A) for glycine (G). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO:4 or SEQ ID NO:5 is a substitution of phenylalanine (F) for tryptophan (W). In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO:4 or SEQ ID NO:5 is a substitution of valine (V) for proline (P). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO:4 or SEQ ID NO:5 is a substitution of phenylalanine (F) for 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 phenylalanine (F) with 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 arginine (R) with 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 lysine (K) with 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 leucine (L) with 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 lysine (K) with 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 serine (S) with 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 tryptophan (W) with 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 tyrosine (Y) with 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 phenylalanine (F) with 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 leucine (L) with 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 alanine (A) with 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 valine (V) with 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 isoleucine (I) with 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 valine with 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 lysine (K) with 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 glycine (G) with 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 arginine (R) with 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 valine (V) with 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 glycine (G) with 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 leucine (L) with 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 histidine (H) with 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 isoleucine (I) with 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 tyrosine (Y) with 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 phenylalanine (F) with 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 lysine (K) with 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 leucine (L) with 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 isoleucine (I) with 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 lysine (K) with 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 threonine (T) with 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 proline (P) with 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 arginine (R) with glutamine (Q).

[0014] In certain embodiments of the disclosed methods, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the piggyBac™ transposase enzyme may comprise, and the Super piggyBac™ transposase enzyme may further comprise, amino acid substitutions 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 disclosed methods, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the piggyBac™ transposase enzyme may comprise, and the Super piggyBac™ transposase enzyme may further comprise, amino acid substitutions 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 of the disclosed methods, including those in which the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the piggyBac™ transposase enzyme may comprise, and the Super piggyBac™ transposase enzyme may further comprise, amino acid substitutions 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 proline (P) with 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 valine (V) with 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 alanine (A) with 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 alanine (A) with 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 asparagine (N) with 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 glycine (G) with serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO:4 or SEQ ID NO:5 is a substitution of serine (S) with asparagine (N). In certain embodiments, the piggyBac™ transposase enzyme may comprise a substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO:4. In certain embodiments, including embodiments in which the piggyBac™ transposase enzyme may comprise a substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO:4, the piggyBac™ transposase enzyme may further comprise amino acid substitutions at positions 372, 375, and 450 of the sequence of SEQ ID NO:4 or SEQ ID NO:5. In certain embodiments, the piggyBac™ transposase enzyme can comprise a substitution of valine (V) at position 194 of SEQ ID NO:4 with methionine (M), an alanine (A) at position 372 of SEQ ID NO:4 with arginine (R), and an alanine (A) at position 375 of SEQ ID NO:4 with lysine (K). In certain embodiments, the piggyBac™ transposase enzyme can comprise a substitution of valine (V) at position 194 of SEQ ID NO:4 with methionine (M), an alanine (A) at position 372 of SEQ ID NO:4 with arginine (R), an alanine (A) at position 375 of SEQ ID NO:4 with lysine (K), and an asparagine (N) at position 450 of SEQ ID NO:4.

[0015] The present disclosure provides modified stem memory T cells (T SCM ), the method comprising introducing (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 into primary human T cells to generate modified T cells, wherein the modified T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of modified stem memory T cells (T SCM) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCM ), the method comprising introducing (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 into a plurality of primary human T cells to generate 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 therebetween, of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCMIn certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. SCM The cell surface markers of CAR-T include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly those in which the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SB100X).

[0016] In certain embodiments of the disclosed methods, the Sleeping Beauty transposase enzyme is [ka] The amino acid sequence may be at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to the amino acid sequence of the present invention.

[0017] In certain embodiments of the disclosed methods, the hyperactive Sleeping Beauty (SB100X) transposase enzyme is [ka] The amino acid sequence may be at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween identical to the amino acid sequence of the present invention.

[0018] The present disclosure provides modified stem memory T cells (T SCM ), the method comprising introducing (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 into primary human T cells to generate modified T cells, wherein the modified T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of modified stem memory T cells (T SCM ) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCM), the method comprising introducing (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 into a plurality of primary human T cells to generate 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 therebetween, of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells are stem memory T cells (T SCM), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. SCM The cell surface markers of CAR-T include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, particularly those in which the transposon is a Helraiser transposon, the transposase is a Helitron transposase.

[0019] In certain embodiments of the methods of the present disclosure, the transposase is a Helitron transposase. The Helitron transposase mobilizes the Helraiser transposon, an ancient element from the bat genome that was active approximately 30-36 million years ago. Exemplary Helraiser transposons of the present disclosure include: [ka] [ka] [ka] [ka] Helibat1, which comprises a nucleic acid sequence comprising:

[0020] Unlike other transposases, Helitron transposase does not contain an RNase-H-like catalytic domain, but instead contains a RepHel motif, which is composed of a replication initiation domain (Rep) and a DNA helicase domain. The Rep domain is the nuclease domain of the HUH superfamily of nucleases.

[0021] Exemplary Helitron transposases of the present disclosure include: [ka] The amino acid sequence includes:

[0022] In Helitron transposition, a hairpin near the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of adjacent sequences. Furthermore, Helraiser transposition generates a covalently closed circular intermediate. Furthermore, Helitron transposition can lack target site overlap. In the Helraiser sequence, the transposase is flanked by left and right end sequences, designated LTS and RTS. These sequences terminate with a conserved 5'-TC / CTAG-3' motif. A 19-bp palindromic sequence, which potentially forms a hairpin termination structure, is located 11 nucleotides upstream of the RTS and consists of the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 29).

[0023] The present disclosure provides modified stem memory T cells (T SCM ), the method comprising introducing (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 into primary human T cells to generate modified T cells, wherein the modified T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of modified stem memory T cells (T SCM ) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCM ), the method comprising introducing (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 into a plurality of primary human T cells to generate 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 therebetween, of the plurality of modified T cells are stem memory T cells (T SCM), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells are stem memory T cells (T SCM), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells are stem memory T cells (T SCM ), thereby producing a plurality of modified stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. SCM The cell surface markers of CAR-T include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, including embodiments in which the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

[0024] In certain embodiments of the disclosed method, the transposase is a Tol2 transposase. The Tol2 transposon can be isolated or derived from the medaka genome and can be similar to the hAT family of transposons. An exemplary Tol2 transposon of the present disclosure contains a gene encoding a Tol2 transposase, which is encoded by a sequence comprising approximately 4.7 kilobases and contains four exons. An exemplary Tol2 transposase of the present disclosure is as follows: [ka] The amino acid sequence includes:

[0025] Exemplary Tol2 transposases of the present disclosure include an inverted repeat, a subterminal sequence, and a Tol2 transposase, as follows: [ka] [ka] [ka] It is encoded by the nucleic acid sequence of

[0026] The present disclosure provides modified central memory T cells (T CM The present invention provides a method for producing central memory T cells (T), the method comprising introducing (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 into primary human T cells to produce modified T cells, wherein the modified T cells are central memory T cells (T CM ), thereby expressing one or more cell surface markers of modified central memory T cells (T CM The present disclosure provides a method for producing multiple modified central memory T cells (T CM ), the method comprising introducing (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 into a plurality of primary human T cells to generate 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 therebetween, of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells are central memory T cells (T CM), thereby producing a plurality of modified central memory T cells (T CM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CM In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CMIn certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells are central memory T cells (T CM ), thereby producing a plurality of modified central memory T cells (T CM ) is generated. In certain embodiments, the cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments of this method, the transposon is a plasmid DNA transposon, and the sequence encoding the antigen receptor or therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly in embodiments where the transposon is a piggyBac transposon, the transposase is piggyBac™ or Super piggyBac™ (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly in embodiments where the transposon is a Sleeping Beauty transposon, the transposase is Sleeping Beauty transposase or hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, particularly in embodiments in which the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, including embodiments in which the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

[0027] The present disclosure provides a method for the production of multiple engineered stem memory T cells (T SCM ) and multiple modified central memory T cells (T CMand a method for making a composition comprising a plurality of modified T SCM and multiple modified T CM and (b) a transposase composition comprising a transposase or a sequence encoding the transposase into a plurality of primary human T cells to generate a composition comprising the plurality of modified T cells. SCM express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ, and express multiple modified T CM express one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby facilitating the development of multiple modified T SCM and multiple modified T CM In certain embodiments of this method, a composition comprising modified stem memory T cells (T SCM ) account for 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, modified central memory T cells (T CM ) 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 90% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM) comprise at least 10% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 80% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 20% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 70% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 30% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 60% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 60% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 40% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 50% of the total number of cells in the composition, and modified central memory T cells (T CM) account for at least 50% of the total number of cells in the composition. In certain embodiments of this method, the transposon is a plasmid DNA transposon, and the sequence encoding the antigen receptor or therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly in embodiments where the transposon is a piggyBac transposon, the transposase is piggyBac™ or Super piggyBac™ (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly in embodiments where the transposon is a Sleeping Beauty transposon, the transposase is Sleeping Beauty transposase or hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments of this method, the transposon is a Helraiser transposon. In certain embodiments, particularly in embodiments in which the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, including embodiments in which the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

[0028] In certain embodiments of the disclosed methods, the transposon may be derived from or recombinantly derived from any species. Alternatively, or additionally, the transposon may be a synthetic transposon.

[0029] In certain embodiments of the disclosed methods, 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, particularly embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to a wild-type T cell receptor. In certain embodiments, particularly embodiments where the T cell receptor is not naturally occurring, the T cell receptor is a recombinant T cell receptor. In certain embodiments of the 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 sequences. In certain embodiments, the CAR is a VCAR.

[0030] In certain embodiments of the methods of the present disclosure, including embodiments in which the method comprises introducing into primary human T cells (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase or a transposase composition comprising a sequence encoding the transposase, the method further comprises introducing into the primary human T cells (c) a second transposon composition comprising a transposon comprising a therapeutic protein, to generate modified T cells, wherein the modified T cells are capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein, and the method generates modified T cells capable of secreting the therapeutic protein. In certain embodiments, the transposase composition in (b) is transposed with the transposon in (a) and the transposon in (c). In certain embodiments, the method further comprises introducing into primary human T cells (d) a second transposase composition comprising a transposase or a sequence encoding the transposase. In certain embodiments, the second transposase composition is transposed with the transposon in (c). In certain embodiments, the transposase composition of (b) transposes with the transposon of (a), and the transposase composition of (d) transposes with the transposon of (c). In certain embodiments of this method, the transposon is a plasmid DNA transposon having a sequence encoding an antigen receptor or a therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly embodiments in which the transposon is a piggyBac transposon, the transposase is a piggyBac™ or Super piggyBac™ (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon.In certain embodiments, particularly in embodiments where 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, particularly in embodiments where the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, including embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

[0031] The present disclosure provides a method for the production of multiple engineered stem memory T cells (T SCM ), comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to generate a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with 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 complement to generate 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 are stem memory T cells (T SCM ), thereby generating a plurality of modified stem memory T cells (T SCM The present disclosure provides a method for producing a plurality of modified stem memory T cells (T SCM1. A method for producing stem memory T cells (T), comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to produce a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with 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 complement 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 therebetween, of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 50% of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells are stem memory T cells (TSCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modified T cells are stem memory T cells (T SCM ), thereby generating a plurality of activated modified stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated modified T SCM The cell surface markers of include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, the activated modified T SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

[0032] (a) producing modified T cells by introducing a composition comprising an antigen receptor into primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the modified T cells with a T cell activation 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 complement to produce activated modified T cells. SCM In certain embodiments of the disclosed method of generating stem memory T cells (T), the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method further comprises (c) contacting the activated modified T cells 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 generate a plurality of expanded modified T cells, wherein at least 2% of the plurality of expanded modified T cells are stem memory T cells (T SCM 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 therebetween of the plurality of expanded engineered T cells are stem memory T cells (T SCM In certain embodiments of this method, at least 60% of the plurality of expanded modified T cells express cell surface markers of stem memory T cells (T SCM In certain embodiments, the method further comprises (d) enriching the plurality of expanded modified T cells to produce stem memory T cells (T SCMIn certain embodiments, the method further comprises (d) enriching the plurality of expanded modified T cells to produce stem memory T cells (T). SCM In certain embodiments of this method, the enriching step further comprises producing a composition comprising at least 60% modified T cells that express cell surface markers for stem memory T cells (T). In certain embodiments of this method, the enriching step further comprises selecting stem memory T cells (T) from the plurality of enriched modified T cells. SCM In certain embodiments of this method, the enriching step comprises isolating a plurality of expanded, enriched modified T cells that express one or more cell surface markers of SCM To generate the isolated modified T SCMwith 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. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol. In certain embodiments of this method, the T cell expansion composition further comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and a sterol at a concentration of about 1 mg / kg. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive.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 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg.

[0033] The present disclosure provides modified central memory T cells (T CM (b) contacting the modified T cells with a T cell activation 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 complement to produce activated modified T cells; wherein the activated modified T cells are central memory T cells (T CM ), thereby expressing one or more cell surface markers of central memory T cells (T CM The present disclosure provides a method for producing multiple modified central memory T cells (T CM (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to generate a plurality of engineered T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of engineered T cells with a T cell activation 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 complement to generate a plurality of activated engineered 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 therebetween, of the plurality of activated engineered T cells are central memory T cells (T CM), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells are central memory T cells (T CM ), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 50% of the plurality of activated modified T cells are central memory T cells (T CM ), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells are central memory T cells (T CM ), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells are central memory T cells (T CM ), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells are central memory T cells (T CM ), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells are central memory T cells (T CM), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells are central memory T cells (T CM ), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modified T cells are central memory T cells (T CM ), thereby generating a plurality of activated modified central memory T cells (T CM In certain embodiments, an activated modified T CM The cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0034] and (b) contacting the modified T cells with a T cell activation 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 complement to produce activated modified T cells. CMIn certain embodiments of the disclosed methods of generating central memory T cells (T), the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method further comprises (c) contacting the activated modified T cells 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 generate a plurality of expanded modified T cells, wherein at least 2% of the plurality of expanded modified T cells are central memory T cells (T CM 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 therebetween of the plurality of expanded modified T cells are central memory T cells (T CM In certain embodiments of this method, at least 60% of the plurality of expanded modified T cells express cell surface markers of central memory T cells (T CM In certain embodiments, the method further comprises (d) enriching the plurality of expanded modified T cells to identify central memory T cells (T CM In certain embodiments, the method further comprises (d) enriching the plurality of expanded engineered 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 therebetween, engineered T cells expressing cell surface markers of central memory T cells (T). CM In certain embodiments of this method, the enriching step further comprises producing a composition comprising at least 60% modified T cells that express cell surface markers for central memory T cells (T). In certain embodiments of this method, the enriching step further comprises selecting central memory T cells (T) from the plurality of enriched modified T cells. CMIn certain embodiments of this method, the enriching step comprises isolating a plurality of expanded, enriched modified T cells that express one or more cell surface markers of CM To generate the isolated modified T CMwith 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. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol. In certain embodiments of this method, the T cell expansion composition further comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and a sterol at a concentration of about 1 mg / kg. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive.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 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg.

[0035] The present disclosure provides a method for the production of multiple engineered stem memory T cells (T SCM ) and multiple modified central memory T cells (T CM ), the method comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to produce a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with a T cell activation 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 complement to produce a plurality of activated modified stem memory T cells (T SCM ) and multiple activated modified central memory T cells (T CM and (iii) a plurality of activated modified T SCM express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ and express multiple activated modified T CM express one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby facilitating the development of multiple modified T SCM and multiple modified T CM In certain embodiments of this method, a composition comprising modified stem memory T cells (T SCM ) account for 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 90% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 10% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 80% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 20% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 70% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 30% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM) comprise at least 60% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 60% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 40% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 50% of the total number of cells in the composition, and modified central memory T cells (T CM ) constitute at least 50% of the total number of cells in the composition.

[0036] (a) producing a plurality of modified T cells by introducing a composition comprising an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with a T cell activation 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 complement to produce a plurality of activated modified stem memory T cells (T SCM ) and multiple activated modified central memory T cells (T CM ), comprising the step of generating a composition comprising a plurality of modified stem memory T cells (T SCM ) and multiple modified central memory T cells (T CM In certain embodiments of the disclosed method of producing a composition comprising (b), the T cell activation composition further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method further comprises (c) combining the composition, a plurality of activated modified stem memory T cells (T cells), to produce a plurality of expanded modified T cells. SCM ) and multiple activated modified central memory T cells (T CM ) 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, SCMexpress one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ, and express multiple expanded modified T CM express one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby resulting in multiple expanded modified T SCM and multiple increased modified T CM In certain embodiments of this method, the enriching step further comprises generating a composition comprising stem memory T cells (T SCM ), or isolating modified T cells that express one or more cell surface markers of central memory T cells (T CM In certain embodiments of this method, the enriching step includes isolating modified T cells that express one or more cell surface markers of stem memory T cells (T). In certain embodiments of this method, the enriching step includes isolating stem memory T cells (T) from the plurality of enriched modified T cells. SCM ), and from the plurality of enriched modified T cells, central memory T cells (T CM In certain embodiments of this method, the enriching step comprises isolating a plurality of expanded, enriched modified T cells that express one or more cell surface markers of SCM and multiple increased enriched modified T CM To make a composition comprising an isolated modified TSCM and an isolated modified T CMand contacting the composition comprising the compound 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. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol. In certain embodiments of this method, the T cell expansion composition further comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and a sterol at a concentration of about 1 mg / kg. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive.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 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and sterol at a concentration of about 2.5 μmol / kg. In certain embodiments of this method, the modified stem memory T cells (T. SCM ) account for 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, modified central memory T cells (T CM ) 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 90% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 10% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 80% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM) comprise at least 20% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 70% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 30% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 60% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 60% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 40% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 50% of the total number of cells in the composition, and modified central memory T cells (T CM ) constitute at least 50% of the total number of cells in the composition.

[0037] and (b) contacting the plurality of engineered T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complexes. SCM or T CMIn certain embodiments of the methods of generating a T cell, the introducing step comprises homologous recombination. In certain embodiments of the introducing step comprising homologous recombination, the genome editing composition is contacted with the genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introducing step comprising homologous recombination, the genome editing composition is contacted with the 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 therebetween of the total number of primary T cells in the plurality of T cells. In certain embodiments of the introducing step comprising homologous recombination, the genome editing composition is contacted with the genomic sequence of each primary T cell of the plurality of T cells. In certain embodiments of the introducing step involving homologous recombination, a single-strand break is induced by the genome editing composition. In certain embodiments of the introducing step involving homologous recombination, a double-strand break is induced by the genome editing composition. In certain embodiments of the introducing step involving homologous recombination, the introducing step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition comprises a sequence encoding an antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding an antigen receptor, a 5' genomic sequence, and a 3' genomic sequence, wherein the 5' genomic sequence is homologous or identical to the genomic sequence of the primary T cell 5' to the breakpoint induced by the genome editing composition, and the 3' genomic sequence is homologous or identical to the genomic sequence of the primary T cell 3' to the breakpoint induced by the genome editing composition. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition and the donor sequence composition are contacted with the genomic sequence simultaneously or sequentially. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition and the donor sequence composition are contacted with the genomic sequence sequentially, resulting in the genome editing composition first. In certain embodiments of the introducing step comprising homologous recombination, the genome editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain.In certain embodiments of the introducing step involving homologous recombination, the genome editing composition comprises a DNA-binding domain and a nuclease domain. In certain embodiments of the genome editing composition, the DNA-binding domain comprises a guide RNA (gRNA). In certain embodiments of the genome editing composition, the DNA-binding domain comprises a TALEN DNA-binding domain. In certain embodiments of the genome editing composition, the DNA-binding domain comprises a ZFN DNA-binding domain. In certain embodiments of the genome editing composition, the nuclease domain comprises a Cas9 nuclease or a sequence thereof. In certain embodiments of the genome editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising a substitution of alanine (A) at position 10 with aspartic acid (D) (D10A) and a substitution of alanine (A) at position 840 with histidine (H) (H840A)). In certain embodiments of the genome editing composition, the nuclease domain comprises a short, inactive Cas9 (SEQ ID NO: 32, comprising a substitution of alanine (A) at position 10 with aspartic acid (D) (D10A) and a substitution of alanine (A) at position 540 with asparagine (N) (N540A)). In certain embodiments of the genome editing composition, the nuclease domain comprises, or further comprises, a Type IIS endonuclease. In certain embodiments of the genome editing composition, the Type IIS endonuclease is selected from the group consisting of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, B pmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bp10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI, or Clo051. In certain embodiments, the Type IIS endonuclease comprises Clo051.In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises a TALEN or its nuclease domain. In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises a ZFN or its nuclease domain. In certain embodiments of the introduction step involving homologous recombination, the genome editing composition induces a break in the genome sequence, and the donor sequence composition is inserted using the endogenous DNA repair mechanism of the primary T cell. In certain embodiments of the introduction step involving homologous recombination, the insertion of the donor sequence composition eliminates the DNA binding site of the genome editing composition, thereby preventing further activity of the genome editing composition.

[0038] Activated modified T cells of the present disclosure include a method comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to generate a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with a T cell activation 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 complement. SCM or T CM In certain embodiments of the method for producing the viral vector, the viral vector comprises an antigen receptor. In certain embodiments, the viral vector comprises one or more sequences isolated from, derived from, or recombined with an RNA virus. In certain embodiments, the RNA virus is a single-stranded or double-stranded virus. In certain embodiments, the viral vector comprises one or more sequences isolated from, derived from, or recombined with a DNA virus. In certain embodiments, the DNA virus is a single-stranded or double-stranded virus. In certain embodiments, the virus is replication-deficient.

[0039] Activated modified T cells of the present disclosure include a method comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to generate a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with a T cell activation 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 complement. SCM or T CM In certain embodiments of the method of making a viral vector, the viral vector comprises an 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.

[0040] Activated modified T cells of the present disclosure include a method comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to generate a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with a T cell activation 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 complement. SCM or T CM In certain embodiments of the method of making a viral vector, the viral vector comprises an 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 gammaretrovirus.

[0041] Activated modified T cells of the present disclosure include a method comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to generate a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with a T cell activation 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 complement. SCM or T CM In certain embodiments of the method for producing a viral vector, the viral vector comprises an 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 of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV comprises a sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV comprises a sequence isolated from, derived from, or recombined with one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV comprises sequences isolated from, derived from, or recombined with AAV2. In certain embodiments, including those in which the vector crosses the blood-brain barrier (BBB), the AAV comprises sequences isolated from, derived from, or recombined with AAV9. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present 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 present disclosure include, but are not limited to, rAAV-LK03, rAAV-NP59, and rAAV-NP84.

[0042] The activated modified T SCM or T CM In certain embodiments of the method for producing an antigen receptor, the nucleic acid vector comprises an antigen receptor. In certain embodiments, the DNA vector comprises an antigen receptor. In certain embodiments, the mRNA vector comprises an antigen receptor. In certain embodiments, the nucleic acid vector is a plasmid or a minicircle vector.

[0043] The activated modified T SCM or T CM In certain embodiments of the method of making the nanoparticle vector, the nanoparticle vector comprises an antigen receptor. The nanoparticle may be composed of polymers disclosed in, for example, International Patent Publication No. WO2012 / 094679, International Patent Publication No. WO2016 / 022805, International Patent Publication No. WO / 2011 / 133635, International Patent Publication No. WO / 2016 / 090111, International Patent Publication No. WO / 2017 / 004498, International Patent Publication No. WO / 2017 / 004509, International Patent Application No. PCT / US2017 / 030271, U.S. Patent No. 6,835,394, U.S. Patent No. 7,217,427, and U.S. Patent No. 7,867,512.

[0044] The activated modified T SCM or T CM In certain embodiments of the method of making an antigen receptor, 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, particularly embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to a wild-type T cell receptor. In certain embodiments, particularly embodiments where the T cell receptor is not naturally occurring, the T cell receptor is a recombinant T cell receptor. In certain embodiments of the 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 sequences. In certain embodiments, the CAR is a VCAR.

[0045] Activated modified T cells of the present disclosure include a method comprising: (a) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to generate a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in a transposon; and (b) contacting the plurality of modified T cells with a T cell activation 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 complement. SCM or T CM In certain embodiments of the method for producing a T cell, the method further comprises introducing a composition comprising a therapeutic protein into primary human T cells to generate modified T cells capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein, and the method generates modified T cells capable of secreting the therapeutic protein. In certain embodiments, the introducing comprises homologous recombination, and the donor sequence comprises a sequence encoding a therapeutic protein. In certain embodiments, the donor sequence comprising an antigen receptor further comprises a therapeutic protein. In certain embodiments, the first donor sequence comprises an antigen receptor, and the second donor sequence comprises a therapeutic protein. In certain embodiments, the vector comprises a sequence encoding a therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle. In certain embodiments, the vector comprising an antigen receptor further comprises a therapeutic protein. In certain embodiments, the first vector comprises an antigen receptor, and the second vector template comprises a therapeutic protein.

[0046] The present disclosure provides modified stem memory T cells (T SCM(b) contacting the modified T cells with a T cell activation 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 complement to produce activated modified T cells, wherein the activated modified T cells are stem memory T cells (T SCM ), thereby identifying modified stem and memory T cells (T SCM ) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCM (b) contacting the plurality of modified T cells with a T cell activation 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 complement 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 are stem memory T cells (T SCM ), thereby identifying modified stem and memory T cells (T SCM In certain embodiments of this method, at least 60% of the plurality of activated modified-T cells are stem memory T cells (T SCM ) express one or more cell surface markers of the T cell activating composition of (b). In certain embodiments of this method, the T cell activating composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. The present disclosure provides modified stem memory T cells (T SCM(b) contacting the CAR-T cells with a T cell activation 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 complement to generate activated CAR-T cells, wherein the activated CAR-T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of CAR-expressing stem memory T cells (T SCM )(CAR-T SCM ) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCM (b) contacting the plurality of CAR-T cells with a T cell activation 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 complement to generate 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 therebetween of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCMIn certain embodiments, the method includes (c) contacting the activated modified T cells 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 generate a plurality of expanded modified T cells, wherein at least 2% of the plurality of expanded modified T cells are stem memory T cells (T SCM), expressing one or more cell surface markers of the following: In certain embodiments, the T cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg. 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 sterols at a concentration of about 1.01 mg / kg (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 about 2.13 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and a sterol at a concentration of 2.61 μmol / kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of expanded engineered T cells are stem memory T cells (T). SCM In certain embodiments, at least 60% of the plurality of expanded modified T cells express cell surface markers of stem memory T cells (T SCM In certain embodiments, the method further comprises (d) enriching the plurality of expanded modified T cells to obtain stem memory T cells (T SCMIn certain embodiments, the method further comprises (d) enriching the plurality of expanded modified T cells to produce stem memory T cells (T SCM In certain embodiments, the enriching step further comprises generating a composition comprising at least 60% modified T cells that express cell surface markers for stem memory T cells (T). In certain embodiments, the enriching step further comprises generating a composition comprising at least 60% modified T cells that express cell surface markers for stem memory T cells (T). SCM In certain embodiments, the enriching step further comprises isolating modified T cells that express one or more cell surface markers of the following: SCM To generate the isolated modified T SCMwith a T cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements. In certain embodiments, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg. 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 sterols at a concentration of about 1.01 mg / kg (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 sterols at a concentration of about 1.01 mg / kg. oleic acid at a concentration of 1.01 mg / kg (mg / kg = parts per million), and a sterol at a concentration of 1.01 mg / kg. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and sterols at a concentration of 2.61 μmol / kg.

[0047] The present disclosure provides modified central memory T cells (T CM (b) contacting the modified T cells with a T cell activation 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 complement to produce activated modified T cells; wherein the activated modified T cells are central memory T cells (TCM ), thereby expressing one or more cell surface markers of modified central memory T cells (T CM The present disclosure provides a method for producing multiple modified central memory T cells (T CM (b) contacting the plurality of modified T cells with a T cell activation 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 complement to generate 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 are central memory T cells (T CM ), thereby expressing one or more cell surface markers of modified central memory T cells (T CM In certain embodiments of this method, at least 60% of the plurality of activated modified-T cells are central memory T cells (T CM ). In certain embodiments of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method further comprises (c) contacting the activated modified T cells 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 generate a plurality of expanded modified T cells, wherein at least 2% of the plurality of expanded modified T cells are central memory T cells (T CM), expressing one or more cell surface markers of the following: In certain embodiments, the T cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg. 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 sterols at a concentration of about 1.01 mg / kg (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 about 2.13 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and a sterol at a concentration of 2.61 μmol / kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of expanded engineered T cells are central memory T cells (T). CM In certain embodiments, at least 60% of the plurality of expanded modified T cells express cell surface markers of central memory T cells (T CM In certain embodiments, the method (d) enriches the plurality of expanded modified T cells to produce central memory T cells (T CMIn certain embodiments, the method further comprises (d) enriching the plurality of expanded engineered 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 therebetween, engineered T cells expressing cell surface markers of central memory T cells (T). CM In certain embodiments, the enriching step further comprises generating a composition comprising at least 60% modified T cells that express cell surface markers for central memory T cells (T). In certain embodiments, the enriching step further comprises generating a composition comprising at least 60% modified T cells that express cell surface markers for central memory T cells (T). CM In certain embodiments, the enriching step further comprises isolating modified T cells that express one or more cell surface markers of the following: CM To generate the isolated modified T CMwith a T cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements. In certain embodiments, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg. 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 sterols at a concentration of about 1.01 mg / kg (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 (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and sterols at a concentration of 2.61 μmol / kg.

[0048] The present disclosure provides a method for the production of multiple engineered stem memory T cells (T SCM ) and multiple modified central memory T cells (T CM ), comprising: (a) a plurality of modified stem memory T cells (T SCM ) and multiple modified central memory T cells (T CM(b) introducing a composition comprising an antigen receptor into a plurality of primary human T cells to produce a composition comprising an antigen receptor, wherein the transposon comprises the antigen receptor; and (b) contacting the composition with a T cell activation 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 complement to produce a plurality of activated modified stem memory T cells (T SCM ) and multiple activated modified central memory T cells (T CM and (iii) a plurality of activated modified T SCM express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ, and CM express one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby providing multiple modified T SCM and multiple modified T CM In certain embodiments of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method further comprises (c) contacting the composition with a T cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T cells, wherein at least 2% of the composition comprising the plurality of expanded modified T cells are stem memory T cells (T SCM In certain embodiments, the method further comprises (c) contacting the composition 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 generate a plurality of expanded engineered T cells, wherein at least 2% of the composition comprising the plurality of expanded engineered T cells are central memory T cells (T CM), expressing one or more cell surface markers of the following: In certain embodiments, the T cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg. 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 sterols at a concentration of about 1.01 mg / kg (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 about 2.13 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and a sterol at a concentration of 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises a plurality of expanded modified T. SCM and multiple increased modified T CM 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 therebetween of the cells of the composition are stem memory T cells (T SCM ) cell surface markers. In certain embodiments, multiple expanded modified T SCM and multiple increased modified T CMAt least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the cells of the composition are central memory T cells (T CM In certain embodiments, the method further comprises (d) enriching the composition for stem and memory T cells (T SCM In certain embodiments, the method further comprises (d) enriching the composition for central memory T cells (T), producing 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 therebetween, modified T cells expressing cell surface markers of T. CM In certain embodiments, the enrichment step further comprises generating 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 therebetween, modified T cells that express cell surface markers of stem memory T cells (T). ... SCM ), or isolating modified T cells that express one or more cell surface markers of central memory T cells (T CM In certain embodiments, the enriching step further comprises isolating modified T cells that express one or more cell surface markers of stem memory T cells (T). SCM and isolating modified T cells that express one or more cell surface markers of central memory T cells (T CM In certain embodiments, the enriching step further comprises isolating modified T cells that express one or more cell surface markers of the following: SCM and / or T CM To generate a composition comprising the isolated modified T SCM and / or T CMwith a T cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements. In certain embodiments, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg. 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 sterols at a concentration of about 1.01 mg / kg (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 (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 64 μmol / kg, about 7 μmol / kg. The T cell expansion composition comprises one or more of palmitic acid at a concentration of about 7.5 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and sterols at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and sterols at a concentration of 2.61 μmol / kg. In certain embodiments of this method, modified stem memory T cells (T SCM) account for 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, modified central memory T cells (T CM ) 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 therebetween of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 90% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 10% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 80% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 20% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 70% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM) comprise at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 30% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 60% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 60% of the total number of cells in the composition, and modified central memory T cells (T CM ) comprise at least 40% of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T SCM ) comprise at least 50% of the total number of cells in the composition, and modified central memory T cells (T CM ) constitute at least 50% of the total number of cells in the composition.

[0049] In certain embodiments of the methods of the present disclosure, including embodiments in which the method comprises the steps of: (a) introducing into primary human T cells a composition comprising an antigen receptor, wherein the transposon comprises the antigen receptor, to generate modified T cells; and (b) contacting the modified T cells with a T cell activation 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 complement to generate activated modified T cells, the method further comprises the step of (c) introducing into the primary human T cells a second transposon composition comprising a transposon comprising a therapeutic protein, wherein the modified T cells are capable of expressing the therapeutic protein, to generate modified T cells. In certain embodiments, the therapeutic protein is a secreted protein, and the method generates modified T cells capable of secreting the therapeutic protein. In certain embodiments, the method further comprises the step of introducing a transposase composition. In certain embodiments, the transposase composition transposes the transposon of (a) and the second transposon. In certain embodiments, the method comprises introducing a first transposase composition and a second transposase composition. In certain embodiments, including embodiments in which the method comprises introducing a first transposase composition and a second transposase composition, the first transposase composition transposes with the transposon of (a), and the second transposase composition transposes with the second transposon. In certain embodiments of this method, the transposon is a plasmid DNA transposon having a sequence encoding an antigen receptor or a therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly embodiments in which the transposon is a piggyBac transposon, the transposase is a piggyBac™ or Super piggyBac™ (SPB) transposase.In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly in embodiments where 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, particularly in embodiments where the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, including embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

[0050] In certain embodiments of the methods of the present disclosure, including embodiments in which the method comprises the steps of: (a) introducing into primary human T cells a composition comprising an antigen receptor, wherein the transposon comprises the antigen receptor, to generate modified T cells; and (b) contacting the modified T cells with a T cell activation 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 complement to generate activated modified T cells, the method further comprises introducing into the primary human T cells a sequence encoding a therapeutic protein, wherein the modified T cells are capable of expressing the therapeutic protein, to generate modified T cells. In certain embodiments of the step of introducing a sequence encoding a therapeutic protein, the introducing step comprises homologous recombination. In certain embodiments of the step of introducing a sequence encoding a therapeutic protein, a vector comprises a sequence encoding a therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle.

[0051] In certain embodiments of the disclosed methods, the introducing step further comprises a composition comprising a genome editing construct. In certain embodiments, the genome 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 genome editing construct comprises a DNA-binding domain and a type IIS endonuclease. In certain embodiments, the genome editing construct encodes a fusion protein. In certain embodiments, the genome editing construct encodes a DNA-binding domain and a type IIS endonuclease, and the expressed DNA-binding domain and the expressed type IIS endonuclease are non-covalently linked. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the genome editing construct comprises a sequence derived from a Cas9 endonuclease. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the sequence derived from a Cas9 endonuclease is a DNA-binding domain. In certain embodiments, including those in which the sequence derived from a Cas9 endonuclease is a DNA-binding domain, the sequence derived from a Cas9 endonuclease encodes an inactive Cas9. In certain embodiments, including those in which the sequence derived from a Cas9 endonuclease is a 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) at position 10 with an aspartic acid (D) (D10A). In certain embodiments, the sequence derived from a Cas9 endonuclease comprises an amino acid substitution of an alanine (A) at position 840 with a histidine (H) (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) at position 580 with an asparagine (N) (N580A).In certain embodiments, the sequence derived from Cas9 endonuclease comprises dSaCas9 (SEQ ID NO: 32). In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the genome editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN). In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the sequence derived from a TALEN is a DNA-binding domain. In certain embodiments, the genome editing construct comprises a TALEN. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the genome editing construct comprises a sequence derived from a zinc finger nuclease (ZFN). In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the sequence derived from a ZFN is a DNA-binding domain. In certain embodiments, the genome editing construct comprises a zinc finger nuclease (ZFN).

[0052] In certain embodiments of the disclosed methods, the transposon is a plasmid DNA transposon having a sequence encoding an antigen receptor or a therapeutic protein flanked by two cis-regulatory insulator elements. In certain embodiments of the 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, particularly embodiments in which the transposon is a piggyBac transposon, the transposase is a Super piggyBac™ (SPB) transposase. In certain embodiments, particularly embodiments in which the transposase is a Super piggyBac™ (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, the hyperactive variant comprising 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 valine (V) with isoleucine (I) (I30V). In certain embodiments, the amino acid substitution at position 165 of SEQ ID NO:4 is a substitution of serine (S) with glycine (G) (G165S). In certain embodiments, the amino acid substitution at position 282 of SEQ ID NO:4 is a substitution of valine (V) with methionine (M) (M282V). In certain embodiments, the amino acid substitution at position 538 of SEQ ID NO:4 is a substitution of lysine (K) with asparagine (N) (N538K). In certain embodiments, the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO:5. In certain embodiments, the transposon is a Sleeping Beauty transposon.In certain embodiments, particularly in embodiments where 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, particularly in embodiments where the transposon is a Helraiser transposon, the transposase is a Helitron transposase. In certain embodiments, the transposon is a Tol2 transposon. In certain embodiments, particularly in embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase. In certain embodiments, the sequence encoding the transposase is an mRNA sequence. In certain embodiments, the transposon may be derived from or recombinantly derived from any species. Alternatively or additionally, the transposon may be a synthetic transposon.

[0053] In certain embodiments of the methods of the present disclosure, the transposon further comprises a selection gene. In certain embodiments, the T cell expansion composition further comprises a selection agent.

[0054] In certain embodiments of the disclosed methods, 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, particularly embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to a wild-type T cell receptor. In certain embodiments, particularly embodiments where the T cell receptor is not naturally occurring, the T cell receptor is a recombinant T cell receptor. In certain embodiments of the 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 sequences. In certain embodiments, the CAR is a VCAR.

[0055] In certain embodiments of the disclosed methods, modified T SCM Cell surface markers for the T include CD62L and CD45RA. SCM The cell surface markers of include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

[0056] In certain embodiments of the disclosed methods, the plurality of expanded engineered T cells is a subset of naive T cells (engineered T N ) including CAR-T N The cell surface markers of include one or more of CD45RA, CCR7, and CD62L. In certain embodiments, the plurality of expanded modified T cells are central memory T cells (modified T CM ) including CAR-T CM The cell surface markers of include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, the plurality of expanded modified T cells are effector memory T cells (modified T EM ) including CAR-T EM The cell surface markers of include one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, the plurality of expanded engineered T cells are designated as effector T cells (engineered T EFF ) including CAR-T EFF The cell surface markers include one or more of CD45RA, CD95, and IL-2Rβ.

[0057] In certain embodiments of the disclosed methods, the plurality of expanded modified T cells is a central memory T cell (modified T CM ) including CAR-T CMThe cell surface markers for include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, the most abundant T cells in the plurality of expanded modified T cells are central memory T cells (modified T CM ) and CAR-T CM The cell surface markers for the modified T cells include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. The most abundant T cells in the expanded modified T cells are central memory T cells (modified T CM In certain embodiments, the plurality of expanded engineered T cells is T SCM cells, including T SCM The cell surface markers of the cells include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ.

[0058] The present disclosure provides modified stem memory T cells (T SCM (b) contacting the CAR-T cells with a T cell activation 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 complement to generate activated CAR-T cells, wherein the activated CAR-T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of CAR-expressing stem memory T cells (T SCM )(CAR-T SCM ) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCMand (b) contacting the plurality of CAR-T cells with a T cell activation 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 complement to generate 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 therebetween of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCMIn certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated CAR-T SCM The cell surface markers of include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, activated CAR-TSCM The cell surface markers of include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. The present disclosure provides modified stem memory T cells (T SCM (b) contacting the CAR-T cells with a T cell activation 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 complement to generate activated CAR-T cells; wherein the activated CAR-T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of CAR-expressing stem memory T cells (T SCM )(CAR-T SCM ) is produced.

[0059] The present disclosure provides a method for the production of multiple engineered stem memory T cells (T SCM (b) contacting the plurality of CAR-T cells with a T cell activation 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 complement to generate 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 therebetween of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T SCM), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells are stem memory T cells (T SCM), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated CAR-T SCM The cell surface markers of include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, activated CAR-T SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

[0060] In certain embodiments, the method further comprises (c) contacting the activated CAR-T cells 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 generate a plurality of expanded CAR-T cells, wherein at least 2% of the plurality of expanded CAR-T cells are stem memory T cells (T SCM )(CAR-T SCM), expressing one or more cell surface markers of the following: In certain embodiments, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg. 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 sterols at a concentration of about 1.01 mg / kg (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 about 2.13 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and a sterol at a concentration of 2.61 μmol / kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of expanded CAR-T cells are stem memory T cells (T). SCM )(CAR-T SCM In certain embodiments, the plurality of expanded CAR-T cells expresses cell surface markers of stem memory T cells (T SCM ) cell surface markers. SCM ) such that after the enrichment step, the method further comprises enriching for stem memory T cells (T SCM )(CAR-T SCMIn certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the CAR-T cells may be enriched to 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 therebetween. ... SCM The cell surface markers of CAR-T include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. SCM The cell surface markers of include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells are derived from naive T cells (CAR-T N ) including CAR-T N The cell surface markers of include one or more of CD45RA, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells are referred to as central memory T cells (CAR-T CM ) including CAR-T CM The cell surface markers of include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells are referred to as effector memory T cells (CAR-T EM ) including CAR-T EM The cell surface markers of include one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, the plurality of expanded CAR-T cells are designated as effector T cells (CAR-T EFF ) including CAR-T EFF Cell surface markers for IL-2Rβ include one or more of CD45RA, CD95, and IL-2Rβ. Additional cell surface markers are described in Gattinoni et al. (Nat Med. 2011 Sep 18;17(10):1290-7; the contents of which are incorporated herein by reference in their entirety).

[0061] The present disclosure provides modified stem memory T cells (T SCM (b) contacting the CAR-T cells with a T cell activation 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 complement to generate activated CAR-T cells; wherein the activated CAR-T cells are stem memory T cells (T SCM ), thereby expressing one or more cell surface markers of CAR-expressing stem memory T cells (T SCM )(CAR-T SCM ) are generated. The present disclosure provides a method for producing multiple modified stem memory T cells (T SCM (b) contacting the plurality of CAR-T cells with a T cell activation 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 complement to generate 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 therebetween of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCMIn certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCMIn certain embodiments, the method generates a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ), thereby generating multiple activated CAR stem memory T cells (T SCM In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated CAR-T SCM The cell surface markers of include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, activated CAR-T SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

[0062] In certain embodiments of the disclosed methods, the plurality of expanded CAR-T cells are derived from naive T cells (CAR-T N ) including CAR-T N The cell surface markers of include one or more of CD45RA, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells are referred to as central memory T cells (CAR-T CM ) including CAR-T CM The cell surface markers of include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, the plurality of expanded CAR-T cells are referred to as effector memory T cells (CAR-T EM ) including CAR-T EM The cell surface markers of include one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, the plurality of expanded CAR-T cells are designated as effector T cells (CAR-T EFF ) including CAR-T EFF The cell surface markers include one or more of CD45RA, CD95, and IL-2Rβ.

[0063] In certain embodiments of the methods of the present disclosure, the transposon comprises a chimeric antigen receptor (CAR) of the present disclosure. The transposon may be a plasmid DNA transposon having 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, the step of introducing a composition comprising a chimeric antigen receptor (CAR) of the present disclosure may further comprise introducing a composition comprising an mRNA sequence encoding a transposase. In certain preferred embodiments, the transposase is a Super piggyBac™ (SPB) transposase.

[0064] In certain embodiments, the transposon of the present disclosure may further comprise a selection gene. When the transposon of the present disclosure comprises a selection gene, the T cell expansion composition of the method of the present disclosure may further comprise a selection agent to simultaneously select and expand the activated or modified T cells of the present disclosure.

[0065] In certain embodiments, the CAR of the present disclosure can be a CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

[0066] Modified T of the present disclosure SCMIn certain embodiments of the method of producing a T cell-expressing cell line, the introducing step may include electroporation or nucleofection. When the introducing step includes nucleofection, the nucleofection may include (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 electric pulses to the cuvette, and (c) incubating the composition comprising the plurality of primary human T cells at 37° C. 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. In certain embodiments, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and a sterol (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg (mg / kg = parts per million), inclusive. In certain embodiments, the T cell expansion composition comprises one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol (mg / kg = parts per million) at a concentration of about 1 mg / kg.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 sterols at a concentration of about 1.01 mg / kg (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 about 2.13 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and a sterol at a concentration of 2.61 μmol / kg.In certain embodiments of nucleofection, the transposon composition is a 0.5 μg / μl solution containing nuclease-free water, and the cuvette contains 2 μl of transposon composition, resulting in 1 μg of transposon. The transposon composition may include a piggyBac transposon. The transposon composition may include a Sleeping Beauty transposon. In certain embodiments of nucleofection, the transposase composition includes 5 μg of transposase. The transposase composition may include a hyperactive piggyBac™ or Super piggyBac™ (SPB) transposase. The transposase composition may include a hyperactive Sleeping Beauty (SB100X) transposase. In certain embodiments, the transposon may include a Helraiser transposon, and the transposase composition may include a Helitron transposase. In certain embodiments, the transposon may include a Tol2 transposon, and the transposase composition includes a Tol2 transposase.

[0067] In certain embodiments of the disclosed methods, including those in which the introducing step comprises nucleofection or electroporation, nucleofection comprises contacting a composition comprising a first transposon composition, a first transposase composition, and a plurality of primary human T cells in a cuvette. In certain embodiments of the disclosed methods, including those in which the introducing step comprises nucleofection or electroporation, nucleofection comprises contacting a composition comprising a first transposon composition, a second transposon composition, a first transposase composition, and a plurality of primary human T cells in a cuvette. In certain embodiments of the disclosed methods, including those in which the introducing step comprises nucleofection or electroporation, nucleofection comprises contacting a composition comprising a first transposon composition, a second transposon composition, a first transposase composition, a second transposase composition, and a plurality of primary human T cells in a cuvette. In certain embodiments, the first transposon comprises a sequence encoding an antigen receptor. In certain embodiments, the second transposon comprises a sequence encoding a therapeutic protein. In certain embodiments, the first transposon composition and the second transposon composition are identical. In certain embodiments, the first transposon composition and the second transposon composition are not identical. In certain embodiments, the first transposase mobilizes the first transposon composition and the second transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition but not the second transposon composition. In certain embodiments, the second transposase mobilizes the second transposon composition but not the first transposon composition. In certain embodiments, the first transposase mobilizes the first transposon composition and the second transposase mobilizes the second transposon composition. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding an antigen receptor.In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments, the first transposon composition comprises a sequence encoding an antigen receptor, and the second transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein is a secreted or secretable protein. In certain embodiments of the disclosed methods, including those in which the introducing step comprises nucleofection or electroporation, nucleofection comprises contacting the transposon composition, the first transposase composition, the 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 an antigen receptor. In certain embodiments, the transposon composition comprises a sequence encoding a therapeutic protein. In certain embodiments of the disclosed methods, including those in which the introducing step comprises nucleofection or electroporation, 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 an antigen receptor, and the transposon comprises a sequence encoding a therapeutic protein. In certain embodiments, the exogenous donor molecule comprises a sequence encoding a therapeutic protein, and the transposon comprises a sequence encoding an antigen receptor. In certain embodiments, a composition comprising a transposon, a composition comprising a transposase, and a composition capable of inducing homologous recombination at a specific site in the genome are simultaneously contacted with a composition comprising a plurality of primary human T cells. In certain embodiments, first, a composition comprising a transposon and a composition comprising a transposase are contacted with a composition comprising a plurality of primary human T cells, and second, a composition capable of inducing homologous recombination at a specific site in the genome is contacted with a composition comprising a plurality of primary human T cells.In certain embodiments, first, a composition capable of inducing homologous recombination at a specific site in the genome is contacted with a composition comprising a plurality of primary human T cells, and second, a composition comprising a transposon and a composition comprising a transposase are contacted with a composition comprising a plurality of primary human T cells. SCM In certain embodiments of the method of making a primary human T cell, the composition comprising the primary human T cells comprises a buffer that maintains or enhances the level of cell viability and / or stem-like phenotype of the primary human T cells. In certain embodiments, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of the primary human T cells prior to nucleofection. In certain embodiments, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of the primary human T cells during nucleofection. In certain embodiments, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of the primary human T cells after nucleofection. In certain embodiments, the buffer comprises P3 Primary Cell Solution (Lonza). In certain embodiments, the buffer comprises one or more of KCl, MgCl, ClNa, glucose, and Ca(NO) at any absolute or relative abundance or concentration; optionally, the buffer further comprises a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO). In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), and supplements comprising 20 mM HEPES and 75 mM Tris / HCl. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), and supplements comprising 40 mM NaHPO / NaHPO, pH 7.2. In certain embodiments, a composition comprising primary human T cells comprises 100 μl of buffer and 5×10 cells. 6 pieces~25×106 Includes pieces.

[0068] Modified T of the present disclosure SCM In certain embodiments of the method for making a composition comprising primary human T cells, the composition is depleted of cells expressing CD14, CD56, and / or CD19. In certain embodiments, the composition comprises primary human T cells, and the composition is prepared by mixing 100 μl of buffer and 5×10 cells. 6 pieces~25×10 6 Includes pieces.

[0069] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements at 37°C. Alternatively, or in addition, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of phosphorus, octanoic fatty acid, palmitic fatty acid, linoleic fatty acid, and oleic acid. In certain embodiments, the medium comprises 10-fold the amount of phosphorus found in, for example, Iscove's Modified Dulbecco's Medium (IMDM); available at ThermoFisher Scientific under catalog number 12440053).

[0070] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements at 37°C. Alternatively, or in addition, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of the following elements present at the corresponding average concentrations: boron, 3.7 mg / L, sodium, 3000 mg / L, magnesium, 18 mg / L, phosphorus, 29 mg / L, potassium, 15 mg / L, and calcium, 4 mg / L.

[0071] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements, at 37°C. Alternatively, or in addition, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be any of the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 112-80-1), methylparaben (MCP) (CAS No. 112-80-2), methylparaben (MCP) (CAS No. 112-80-3), methylparaben (MCP) (CAS No. 112-80-2), methylparaben (MCP) (CAS No. 112-80-3), methylparaben (MCP) (CAS No. 112-80-3), methylparaben (MCP) (CAS No. 112-80-4), methylparaben (MCP) (CAS No. 112-80-4), methylparaben (MCP) (CAS No. 112-80-5 ... The medium may be used interchangeably with media containing one or more of: benzophenone (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterols (e.g., cholesterol) (CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5).In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" refers to a composition that is formulated with the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 112-80-2), methylparaben (MCP) (CAS No. 112-80-3), methylparaben (MCP) (CAS No. 112-80-4), methylparaben (MCP) (CAS No. 112-80-5 ... The medium may be used interchangeably with media containing one or more of the following: benzophenone (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterols (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 term "supplemented T cell expansion composition" or "T cell expansion composition" refers to a composition that is formulated with the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 112-80-2), methylparaben (MCP) (CAS No. 112-80-3), methylparaben (MCP) (CAS No. 112-80-4), methylparaben (MCP) (CAS No. 112-80-5 ... The medium may be used interchangeably with media containing one or more of: benzophenone (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS No. 143-74-8), and lanolin alcohol.

[0072] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements, at 37° C. Alternatively, or in addition, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride ions, sulfate, and phosphate.

[0073] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements, at 37° C. Alternatively or additionally, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of the following free amino acids: histidine, asparagine, serine, glutamine, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine, and tryptophan. In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of the following free amino acids in the corresponding average molar 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 term "supplemented T cell expansion composition" or "T cell expansion composition" refers to a composition that is a T cell-enriched ... The medium may be used interchangeably with media containing one or more of the following: alanine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine ​​(about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine (about 3.14%), leucine (about 3.3%), phenylalanine (about 1.64%), and tryptophan (about 0.37%).

[0074] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium 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" can be used interchangeably to refer to a medium comprising one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, inclusive; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive; and sterols at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive (mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium 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 sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of: octanoic acid at a concentration of 9.19 mg / kg, palmitic acid at a concentration of 1.86 mg / kg, linoleic acid at a concentration of about 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of 9.19 mg / kg, palmitic acid at a concentration of 1.86 mg / kg, linoleic acid at a concentration of 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with medium comprising one or more of: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, inclusive; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, inclusive; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, inclusive; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg, inclusive. In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and sterols at a concentration of 2.61 μmol / kg.

[0075] As used herein, the term "P3 buffer" can be used interchangeably with a buffer containing one or more of KCl, MgCl, ClNa, glucose, and Ca(NO) at any absolute or relative abundance or concentration, optionally further containing a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. The term "P3 buffer" can be used interchangeably with a buffer containing 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), optionally further containing a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. The term "P3 buffer" can be used interchangeably with a buffer containing 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), and a supplement containing 20 mM HEPES and 75 mM Tris / HCl. The term "P3 buffer" can be used interchangeably with a buffer containing 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and supplements including 0.4 mM Ca(NO) and 40 mM NaHPO / NaHPO, pH 7.2.

[0076] As used herein, the term "supplemented RPMI-1640 medium" or "T cell conditioned medium (TCCM)" refers to a medium containing water, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium hydrogen phosphate (anhydrous), L-alanyl-L-glutamine, L-arginine, L-asparagine (anhydrous), L-aspartic acid, L-cysteine ​​2HCl, L-glutamic acid, glycine, L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine, L-lysine HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine 2Na. It can be used interchangeably with media containing one or more of the following in any absolute or relative abundance or concentration: 2H2O, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, p-aminobenzoic acid, D-pantothenic acid (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12, D-glucose, glutathione (reduced form), L-glutamine, and 2-mercaptoethanol.The term "supplemented RPMI-1640 medium" or "T cell conditioned medium (TCCM)" refers to a medium containing water, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium hydrogen phosphate (anhydrous), L-alanyl-L-glutamine, L-arginine, L-asparagine (anhydrous), L-aspartic acid, L-cysteine ​​2HCl, L-glutamic acid, glycine, L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine. Media containing niacin, L-lysine HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine 2Na2HO, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, p-aminobenzoic acid, D-pantothenic acid (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12, D-glucose, glutathione (reduced form), L-glutamine, and 2-mercaptoethanol at any absolute or relative abundance or concentration can be used interchangeably.

[0077] As used herein, the term "supplemented AIM-V" or "supplemented AIMV" medium refers to a medium containing water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium hydrogen phosphate (anhydrous), L-alanyl-L-glutamine, L-arginine, L-asparagine (anhydrous), L-aspartic acid, L-cysteine ​​2HCl, L-glutamic acid, glycine, L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine, L-lysine HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine 2Na. It can be used interchangeably with media containing one or more of the following in any absolute or relative abundance or concentration: 2H2O, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, p-aminobenzoic acid, D-pantothenic acid (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12, D-glucose, glutathione (reduced form), L-glutamine, and 2-mercaptoethanol.The terms "supplemented AIM-V" or "supplemented AIMV" medium refer to the following: water, human serum albumin, streptomycin sulfate, gentamicin, fetal bovine serum, HEPES, sodium pyruvate, one or more non-essential amino acids, phenol red indicator, calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium hydrogen phosphate (anhydrous), L-alanyl-L-glutamine, L-arginine, L-asparagine (anhydrous), L-aspartic acid, L-cysteine ​​2HCl, L-glutamic acid, glycine, L-histidine, hydroxy-L-proline, L- Media containing isoleucine, L-leucine, L-lysine HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine 2Na2HO, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, p-aminobenzoic acid, D-pantothenic acid (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12, D-glucose, glutathione (reduced form), L-glutamine, and 2-mercaptoethanol at any absolute or relative abundance or concentration can be used interchangeably.

[0078] As used herein, the term "ImmunoCult™ Medium" means a medium containing 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 ​​2HCl, L-glutamic acid, L-glutamine, L-histidine hydrochloride H2O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan. The present invention may be used interchangeably with media containing one or more of the following in any absolute or relative abundance or concentration: niacin, 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, monobasic sodium phosphate, sodium selenite, D-glucose, HEPES, and sodium pyruvate.The term "ImmunoCult™ Medium" refers to a mixture 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 ​​2HCl, L-glutamic acid, L-glutamine, L-histidine hydrochloride H2O, 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, monobasic sodium phosphate, sodium selenite, D-glucose, HEPES, and sodium pyruvate at any absolute or relative abundance or concentration can be used interchangeably.

[0079] Modified T of the present disclosure SCM and / or T CM The modified T cells of the present disclosure, including, can be incubated, cultured, grown, stored, or otherwise combined with growth medium containing one or more inhibitor components of the PI3K pathway at any step of the procedural method. Exemplary inhibitor components of the PI3K pathway include, but are not limited to, TWS119 (also known as GSK 3B inhibitor XII; CAS number 601514-19-6, chemical formula C 18 H 14 Exemplary inhibitor components of the PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO™).

[0080] As used herein, the terms "electroporation" and "nucleofection" are intended to describe alternative means for delivering a nucleic acid, transposon, vector, or composition of the disclosure to a cell by providing an electric pulse to induce the cell membrane (cell membrane, nuclear membrane, or both) to become permeable or more permeable to the nucleic acid, transposon, vector, or composition of the disclosure.

[0081] In certain nucleofection embodiments, the method is performed simultaneously in one or more cuvettes. In certain nucleofection embodiments, the method is performed simultaneously in two cuvettes. For processes performed on a larger scale for clinical or commercial applications, for example, nucleofection can be performed in large volume cassettes, with many steps occurring simultaneously. In certain nucleofection embodiments, the incubating step includes incubating a composition comprising a plurality of primary human T cells in a pre-warmed T cell expansion composition. The duration of the incubation step can be at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or any number of hours / portions therebetween. The duration of the incubation step can be at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or any number of days / parts therebetween. The duration of the incubation step can be at least 1 week. In certain embodiments of nucleofection, the duration of the incubation step is 2 days. In certain embodiments of nucleofection, the application step can include applying one or more of the following programs: EI-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, EO-151, EO-148, EO-156, EO-210, EO-213, and FI-156. In certain embodiments, the applying step may include applying one or more of the following programs: EI-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, EO-151, EO-148, EO-156, EO-210, EO-213, and FI-156, or a program that results in the same number of electrical pulses, each pulse having the same duration and intensity, and substantially similar intermediate pulse durations.In certain embodiments, the applying step can be performed using a known electroporation / nucleofection device, including, but not limited to, Lonza Amaxa, MaxCyte technology, BTX PulseAgile, and BioRad GenePulser. In certain embodiments of nucleofection, the applying step can include applying at least one electric pulse. In certain embodiments of nucleofection, the applying step can include applying at least one electric pulse sufficient to induce the cell membrane and / or nuclear membrane of the cell to become permeable to the composition of the present disclosure.

[0082] The amounts presented herein are exemplary and not limiting, but the relationships between these amounts (e.g., ratios or relative abundances) can be used to modify the methods exemplified herein for larger scale processes and manufacturing.

[0083] The modified T cells of the disclosure (e.g., T SCM and / or T CM In certain embodiments of the method for producing a medicament for the treatment of leukemia, the activation supplement comprises one or more cytokines. The one or more cytokines may include any cytokine, including but not limited to lymphokines. Exemplary lymphokines 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 (INFγ). The one or more cytokines may include IL-2.

[0084] The modified T cells of the disclosure (e.g., T SCM and / or T CMIn certain embodiments of the method for producing a medicament for the treatment of leukemia, the expansion supplement comprises one or more cytokines. The one or more cytokines may include any cytokine, including but not limited to lymphokines. Exemplary lymphokines 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 (INFγ). The one or more cytokines may include IL-2.

[0085] The modified T cells of the disclosure (e.g., T SCM and / or T CM In certain embodiments of the methods for producing a modified T cell of the present disclosure, the primary human T cells are naive T cells. Naive T cells may express CD45RA, CCR7, and CD62L. In certain embodiments, the methods are applied to a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween of naive T cells. In certain embodiments, the modified T cell of the present disclosure is SCM and / or T CM The efficiency of production of can be increased by increasing the rate or percentage of naive T cells within the cell population to which the methods of the present disclosure are applied.

[0086] Modified T of the present disclosure SCM and / or T CM In certain embodiments of the method of producing the primary human T cells, the primary human T cells are memory T cells.

[0087] Modified T of the present disclosure SCM and / or T CMIn certain embodiments of the method of producing the primary human T cells, the primary human T cells express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ.

[0088] Modified T of the present disclosure SCM and / or T CM In certain embodiments of the method of producing the modified T cells, the primary human T cells are naive T cells (modified T cells). N ) and modified T N The modified T of the present disclosure expresses one or more of CD45RA, CCR7, and CD62L. SCM and / or T CM In certain embodiments of the method of producing a modified T SCM T memory stem cells (modified T SCM ) and modified T SCM The modified T of the present disclosure express one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. SCM and / or T CM In certain embodiments of the method for generating the primary human T cells, the primary human T cells are central memory T cells (modified T CM ) and modified T CM The modified T of the present disclosure expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. SCM and / or T CM In certain embodiments of the method of generating the primary human T cells, the primary human T cells are effector memory T cells (modified T EM ) and modified T EM The modified T of the present disclosure expresses one or more of CD45RO, CD95, and IL-2Rβ. SCM and / or T CM In certain embodiments of the method of generating the primary human T cells, the primary human T cells are effector T cells (modified T EFF ) and modified T EFF express one or more of CD45RA, CD95, and IL-2Rβ.

[0089] Modified T of the present disclosure SCMand / or T CM In certain embodiments of the method for producing a T cell, 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 in various ratios. In certain embodiments, the primary human T cell may express non-naturally occurring CD4 and / or CD8 in various ratios. In certain embodiments, the primary human T cell expressing non-naturally occurring CD4 and / or CD8 in various ratios is a heterogeneous cell population.

[0090] Modified T of the present disclosure SCM and / or T CM In certain embodiments of the method for producing T cells, primary human T cells can be isolated, prepared, or drawn from, for example, whole blood, peripheral blood, umbilical cord blood, lymph, lymph node tissue, bone marrow, and cerebrospinal 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) obtained or prepared from the circulating pool of blood and not sequestered in the lymphatic system, spleen, liver, or bone marrow. Umbilical cord blood is distinct from peripheral blood and blood sequestered in the lymphatic system, spleen, liver, or bone marrow. The terms "umbilical cord blood," "umbilical blood," or "cord blood" can be used interchangeably and refer to blood that remains in the placenta and attached to the umbilical cord after birth. Umbilical cord blood often contains stem cells, including hematopoietic cells.

[0091] The primary human T cells of the present disclosure can include pan T cells. As used herein, pan T cells include all T lymphocytes isolated from a biological sample without sorting by subtype, activation state, maturation state, or expression of cell surface markers.

[0092] In certain embodiments of the methods of the present disclosure, the method comprises: SCM or T CMThe method further includes introducing a composition comprising a genome editing construct or composition into the cell. In certain embodiments, the genome 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 genome editing construct comprises a DNA-binding domain and a type IIS endonuclease. In certain embodiments, the genome editing construct encodes a fusion protein. In certain embodiments, the genome editing construct encodes a DNA-binding domain and a type IIS endonuclease, and the expressed DNA-binding domain and the expressed type IIS endonuclease are non-covalently linked. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the genome editing construct comprises a sequence derived from Cas9 endonuclease. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the sequence derived from the Cas9 endonuclease is a DNA-binding domain. In certain embodiments, including embodiments in which the sequence derived from the Cas9 endonuclease is a DNA-binding domain, the sequence derived from the Cas9 endonuclease encodes an inactive Cas9. In certain embodiments, including embodiments in which the sequence derived from the Cas9 endonuclease is a DNA-binding domain, the sequence derived from the Cas9 endonuclease encodes a truncated Cas9. In certain embodiments, the sequence derived from the Cas9 endonuclease comprises an amino acid substitution of aspartic acid (D) for alanine (A) at position 10 (D10A). In certain embodiments, the sequence derived from the Cas9 endonuclease comprises or further comprises an amino acid substitution of histidine (H) for alanine (A) at position 840 (H840A). In certain embodiments, the sequence derived from the Cas9 endonuclease comprises inactivated Cas9 (dCas9) (SEQ ID NO: 33).In certain embodiments, the sequence derived from Cas9 endonuclease comprises an amino acid substitution of asparagine (N) for alanine (A) at position 580 (N580A). In certain embodiments, the sequence derived from Cas9 endonuclease comprises a truncated and inactivated Cas9 (dSaCas9) (SEQ ID NO: 32). In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the genome editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN). In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the sequence derived from a TALEN is a DNA-binding domain. In certain embodiments, the genome editing construct comprises a TALEN. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and a type IIS endonuclease, the genome editing construct comprises a sequence derived from a zinc finger nuclease (ZFN). In certain embodiments, including embodiments in which the genome editing construct comprises a DNA binding domain and a type IIS endonuclease, the sequence derived from ZFN is a DNA binding domain. In certain embodiments, the genome editing construct comprises a zinc finger nuclease (ZFN).

[0093] Modified T of the present disclosure SCM and / or T CM The method of generating cells may result in a greater number or a greater proportion of modified T SCM and / or T CM For example, the population of cells subjected to the methods of the present disclosure can be enriched to contain an increased number or a greater proportion of naive T cells. As the number and / or proportion of naive T cells in the population of T cells subjected to the methods of the present disclosure increases, the number and / or proportion of modified T cells produced by the present disclosure increases. SCM and / or T CMAlternatively, or in addition, the length or duration of time required to perform the disclosed method is reduced, as is the number and / or proportion of cells produced by the disclosed modified T SCM and / or T CM The number and / or proportion of cells increases. The length or duration of time required to perform the disclosed methods, or "production period," can also be referred to as the "out-of-life period" of the T cells subjected to the disclosed methods.

[0094] In certain embodiments of the methods of generating modified T cells of the present disclosure, the primary human T cells express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, the primary human T cells are naive T cells (T N ) and T N In certain embodiments, the primary human T cells are T memory stem cells (T SCM ) and T SCM In certain embodiments, the primary human T cells express one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments, the primary human T cells are central memory T cells (T CM ) and T CM express one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, the primary human T cells are effector memory T cells (T EM ) and EM express one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, primary human T cells are effector T cells (T EFF ) and T EFF express one or more of CD45RA, CD95, and IL-2Rβ. In certain embodiments, the primary human T cells express CD4 and / or CD8.

[0095] The present disclosure provides modified T SCM The present disclosure provides a composition comprising a modified T produced by the method of the disclosure. CM The present disclosure provides a composition comprising a modified T produced by the method of the disclosure. SCM and modified T CM The modified T produced by the methods of the present disclosure is provided. SCM and modified T CM In certain embodiments of the composition comprising a plurality of T SCM 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% of the composition. SCM and modified T CM In certain embodiments of the composition comprising a plurality of T CM 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% of the composition.

[0096] The present disclosure also provides a method for the manufacture of a medicament for treating a subject in need thereof, comprising administering to a subject a modified T SCM and / or T CM In certain embodiments of the use, the modified T SCM and / or T CM is autologous. In certain embodiments of this use, the modified T SCM and / or T CMis allogeneic. In certain embodiments, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, particularly in embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to a wild-type T cell receptor. In certain embodiments, particularly in embodiments where 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 sequences. In certain embodiments, the CAR is a VCAR.

[0097] The present disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering to a subject a modified T SCM and / or T CM In certain embodiments of the method, the modified T SCM and / or T CM is autologous. In certain embodiments of this method, the modified T SCM and / or T CMis allogeneic. In certain embodiments, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, particularly embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to a wild-type T cell receptor. In certain embodiments, particularly embodiments where 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 sequences. 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 antigen, a bacterial antigen, a yeast antigen, or a microbial antigen. In certain embodiments, the disease or disorder is a disease or disorder caused by the absence of activity or insufficient amount of a secreted protein. In certain embodiments, the disease or disorder is a disease or disorder that is treated by replacing the activity of a therapeutic protein or by increasing the amount of a therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein. In certain embodiments, the activity of the secreted protein in a localized region of the body is absent or the amount is insufficient. In certain embodiments, the localized region of the body is accessible by native T cells or engineered T cells. In certain embodiments, the engineered T cells are generated in vivo, ex vivo, in vitro, or in situ. [Brief explanation of the drawings]

[0098] [Figure 1]Figure 1 is a series of plots showing the appearance 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.

[0099] [Figure 2] Figure 2 is a series of plots showing the purity of CAR-TSCM produced by the method of Example 1 at day 19. The population of CAR-TSCM cells produced by the method described in Example 1 at day 19 contained no B cells or lymphocytes. The majority of cells are CD3+ T cells. Only 1.1% are natural killer cells and 1.7% are natural killer T cells.

[0100] [Figure 3] FIG. 3 is a plot showing that at day 11 of the method described in Example 1, the majority of T cells generated express CARTyrin.

[0101] [Figure 4] Figure 4 is a series of plots showing CAR-TSCM phenotypic enrichment at day 19 of the method described in Example 1. Cells were nucleofected with a surrogate CARTyrin plasmid. CAR-TSCM cells express CD62L and CD45RA, as shown in the bottom two plots.

[0102] [Figure 5]Figure 5 is a series of plots showing the absence of T cell exhaustion on day 19 of the method described in Example 1. On day 19, the cell population generated by this method does not express PD1, a marker of T cell activation and exhaustion. These cells, which express CARTyrin, almost successfully reached a resting state after production. These cells do not show signs of antigen-independent (tonic) signaling, which would otherwise drive high levels of PD1 expression. It is hypothesized that tonic signaling is caused by some CAR molecules, which leads to early exhaustion and reduces the effectiveness of CAR T cell therapy.

[0103] [Figure 6A] Figure 6A is a series of plots showing T cells transfected with a plasmid containing a sequence encoding a transposon containing sequences encoding an inducible caspase polypeptide (safety switch, "iC9"), CARTyrin (anti-BCMA), and a selectable marker. The plot on the left shows live T cells exposed to the transposase in the absence of the plasmid. The plot on the right shows live T cells exposed to the transposase in the presence of the plasmid. Cells were exposed to either a hyperactive transposase ("Super piggyBac") or wild-type piggyBac transposase.

[0104] [Figure 6B] Figure 6B is a series of plots showing T cells transfected with a plasmid containing a sequence encoding green fluorescent protein (GFP). The plot on the left shows live T cells exposed to transposase in the absence of the plasmid. The plot on the right shows live T cells exposed to transposase in the presence of the plasmid. Cells were exposed to either a hyperactive transposase ("Super piggyBac") or wild-type piggyBac transposase.

[0105] [Figure 6C]Figure 6C is a table showing the percentage of transformed T cells resulting from transposition with WT versus superactive piggyBac transposase. T cells exposed to the superactive piggyBac transposase were transformed at a rate fourfold higher than that of the WT transposase.

[0106] [Figure 6D] Figure 6D is a table showing the percentage of transformed T cells resulting from transfection with WT versus superactive piggyBac transposase 5 days after nucleofection. T cells exposed to the superactive piggyBac transposase were transformed at a much higher rate than those exposed to the WT transposase.

[0107] [Figure 7] Figure 7 is a graph showing the phenotypic differences between piggyBac™-generated CAR+ T cells and lentiviral-generated CAR+ T cells. CAR+ T cells were generated using either piggyBac transduction or lentiviral transduction. Human pan-T cells were transduced with piggyBac encoding a CAR, stimulated with anti-CD3 / CD28 beads two days after transduction, expanded, and tested 19 days after transduction. For lentiviral generation, pan-T cells were stimulated with CD3 / CD28 beads, transduced with lentivirus encoding a CAR (MOI 5), expanded, and tested 18 days after stimulation. Each population of CAR+ T cells was then characterized based on the expression of standard memory markers CD62L, CD45RA, and CD95. The percentages of each CAR+ T cell subset were defined as naive (CD62L+CD45RA+), Tcm (CD62L+CD45RA-), Tem (CD62L-CD45RA-), and Teff (CD62L-CD45RA+). All CAR+ T cells were CD95+.

[0108] [Figure 8A] Figures 8A-B are a pair of graphs showing that piggyBac™ preferentially induces metastasis in naive T cells. Human pan-T cells were sorted into naive (CD62L+CD45RA+), Tcm (CD62L+CD45RA-), Tem (CD62L-CD45RA-), and Teff (CD62L-CD45RA+) subsets using a BD FACSAria II flow cytometer. The selected subsets were either transduced with piggyBac-GFP or transduced with lentivirus-GFP. For the former, each sorted subset was transduced with PiggyBac-GFP, stimulated and expanded with anti-CD3 / CD28 beads 2 days after transduction, and tested 19 days after transduction. For the latter, selected subsets were stimulated with CD3 / CD28 beads, transduced with lentivirus encoding GFP (MOI 5), expanded, and tested 19 days post-stimulation. n=3 donors. [Figure 8B] Figures 8A-B are a pair of graphs showing that piggyBac™ preferentially induces metastasis in naive T cells. Human pan-T cells were sorted into naive (CD62L+CD45RA+), Tcm (CD62L+CD45RA-), Tem (CD62L-CD45RA-), and Teff (CD62L-CD45RA+) subsets using a BD FACSAria II flow cytometer. The selected subsets were either transduced with piggyBac-GFP or transduced with lentivirus-GFP. For the former, each sorted subset was transduced with PiggyBac-GFP, stimulated and expanded with anti-CD3 / CD28 beads 2 days after transduction, and tested 19 days after transduction. For the latter, selected subsets were stimulated with CD3 / CD28 beads, transduced with lentivirus encoding GFP (MOI 5), expanded, and tested 19 days post-stimulation. n=3 donors.

[0109] [Figure 9] Figure 9 is a pair of graphs showing that the piggyBac™ 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 for MM patients and healthy donors, and plots are shown. T cells from MM patients generally have low frequencies of naive and TSCM cells but high frequencies of Teffs, which differs from healthy normal donors, where the reverse is known. Regardless of the input frequencies of naive and Tscm from different MM patients, production of P-BCMA-101 using the Poseida manufacturing process resulted in a product exhibiting high levels of CD8+ Tscm (E). This was also true for MM patients who were actively receiving treatment (red triangles).

[0110] [Figure 10-1]Figure 10 is a series of fluorescence-activated cell sorting (FACs) plots characterizing T cell and TSCM cell markers in human pan T cells transformed using the Sleeping Beauty (SB100x) transfection system and the disclosed method. Sleeping Beauty (SB100x) transfection using the Poseida manufacturing process results in a predominantly Tscm phenotype. Human pan T cells were transfected using 1 μg of either Sleeping Beauty transposon plasmid or piggyBac transposon plasmid, and SB100x or SPB mRNA, respectively, as indicated. After transfection, cells were expanded ex vivo, and all non-transfected cells were depleted using the Poseida manufacturing drug selection system. After 18 days of culture, cells were stained for phenotypic markers CD4, CD8, CD45RA, and CD62L. The stem cell memory phenotype (Tscm) was defined by CD45RA and CD62L double-positive cells, comprising >65% of cells in all samples. All panels within a column share common x- and y-axis parameters. Each row shows, from top to bottom, data from T cells transfected with 2.5 micrograms (µg) of the Sleeping Beauty transposon SB100x (top), data from T cells transfected with 5 µg of SB100x (second from top), data from T cells transfected with 10 µg of SB100x (third from top), data from T cells transfected with 5 µg of the piggyBac transposon P-BCMA-101 (second from bottom), and data from T cells transfected with an unstained control (bottom). The first and second columns of the x-axis, from left to right, show forward scatter (FSC) in units of 0 to 250 thousand (abbreviated as "k") in 50k increments. The third column of the x-axis, from left to right, shows CD8 expression, with reads marked from 0 to 105 in increments of 10. The rightmost column shows CD62L expression, with readings marked in powers of 10 increments from 0 to 105. The y-axis of the first column shows side scatter (SSC) in units from 0 to 250k in 50k increments.The y-axis of the second column, from left to right, shows the expression of the cell viability marker 7-aminoactinomycin D (7AAD), increasing in powers of 10 from 0 to 105. The y-axis of the third column, from left to right, shows the expression of the marker CD4, increasing in powers of 10 from 0 to 105. The y-axis of the right column shows the expression of the marker CD45RA, increasing in powers of 10 from 0 to 105. [Figure 10-2] Same as above

[0111] [Figure 11] Figure 11 is a schematic diagram showing the human coagulation pathways that lead to blood clotting. The intrinsic coagulation pathway is activated by contact activation, for example, due to endothelial injury. The extrinsic coagulation pathway is activated by tissue factor, for example, after trauma. Both pathways converge on the conversion of prothrombin to thrombin, which catalyzes the conversion of fibrinogen to fibrin. Platelets and polymerized fibrin form a clot. In the absence of factor IX (circled), clotting is defective. Factor VIII (FVIII) deficiency leads to the development of hemophilia A. Factor IX (FIX) deficiency leads to the development of hemophilia B. Hemophilia B is a rare disease occurring in approximately 1 in 25,000 to 30,000 cases. Sixty percent of hemophilia B cases are severe. Fewer than 1 percent of individuals with hemophilia B have normal FIX levels. Prior to the compositions and methods of the present disclosure, the standard treatment for hemophilia B involved infusion of recombinant FIX every 2-3 days, costing approximately $250,000 per year. In stark contrast to this standard treatment option, the TSCM cells of the present disclosure can be maintained in humans for decades.

[0112] [Figure 12]Figure 12 is a series of fluorescence-activated cell sorting (FACS) plots showing T cells secreting FIX. T cells encoding a human factor IX transgene exhibited a TSCM phenotype with approximately 80% of the cells displaying this phenotype. Six panels are depicted from left to right: (1) Forward scatter (FSC) on the x-axis versus side scatter (SSC) on the y-axis. The x-axis is 0 to 250 kJ (abbreviated as k) in 50 kJ increments, and the y-axis is 0 to 250 kJ in 50 kJ increments. (2) FSC on the x-axis versus the cell viability marker 7-aminoactinomycin D (7AAD). The x-axis is labeled from 0 to 250 kJ in 50 kJ increments. The y-axis readings, from top to bottom, are -103, 0, 103, 104, and 105. (3) On the x-axis, anti-CD56-APC conjugated to Cy7 dye (CDC56-APC-Cy7) is displayed in increments of 10 from 0 to 105. On the y-axis, anti-CD3 conjugated to phycoerythrin (PE) is displayed in increments of 10 from 0 to 105. (4) On the x-axis, anti-CD8 conjugated to fluorescein isothiocyanate (FITC) is displayed in increments of 10 from 0 to 105. On the y-axis, anti-CD4 conjugated to Brilliant Violet 650 dye (BV650) is displayed in increments of 10 from 0 to 105. (5) On the x-axis, anti-CD62L antibody conjugated to Brilliant Violet 421 dye (BV421) is displayed in increments of 10 from 0 to 105. The y-axis shows anti-CD45RA antibodies conjugated with PE and Cy7 in increments of 10 from 0 to 105. This panel is boxed. (6) The x-axis shows anti-CCR7 antibodies conjugated with Brilliant Violet 786 (BV786) in increments of 10 from 0 to 105. The y-axis shows anti-CD45RA antibodies conjugated with PE and Cy7 in increments of 10 from 0 to 105.

[0113] [Figure 13A]13A is a graph showing human Factor IX secretion during the generation of modified T cells of the present disclosure. The y-axis is Factor IX concentration in nanograms (ng) per milliliter (mL) from 0 to 80 in increments of 20. The x-axis shows T cells at days 9 and 12.

[0114] [Figure 13B] Figure 13B is a graph showing the clotting activity of secreted factor IX produced by T cells. The y-axis shows percent factor IX activity relative to human plasma, ranging from 0 to 8 in increments of 2. The x-axis shows T cells on days 9 and 12.

[0115] [Figure 14A] Figures 14A-E are a series of plasmid maps for site-specific integration into the AAVS1 site using either HR or MMEJ and the 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 piggyBac™ transposition. For the HR donor and MMEJ donor plasmids, the GFP-2A-DHFR gene expression cassette was flanked by CRISPR / Cas9 target sites and homology arms for AAVS1 site integration; for the piggyBac™ donor plasmid, the GFP-2A-DHFR gene expression cassette was flanked by piggyBac™ transposon elements. The homology arms for the HR and MMEJ plasmids are 500 bp and 25 bp, respectively. Panels D and E, and F show SEQ ID NOs: 41 and 42, respectively. [Figure 14B] Same as above [Figure 14C] Same as above [Figure 14D] Same as above [Figure 14E] Same as above

[0116] [Figure 15]Figure 15 is a graph showing transgene (GFP) expression in primary human pan T cells 3 days after nucleofection. HR or MMEJ donor plasmids were co-delivered to pan T cells by nucleofection with or without CRISPR ribonucleoprotein (RNP) targeting reagents. T cells receiving donor plasmid alone were included as controls. Pan T cells were also modified using the piggyBac™ transposon delivery system. T cells were activated by TCR stimulation on day 0, and the percentage of GFP+ T cells was assessed by flow cytometry 3 days after nucleofection; the data are summarized in a bar graph.

[0117] [Figure 16] Figure 16 is a graph showing transgene (GFP) expression in primary human pan T cells 11 days after nucleofection and selection. Activated T cells with stably integrated transgenes were selected by adding methotrexate using the DHFR selection gene encoded by a bicistronic GFP-2A-DHFR integration cassette. Eleven days after nucleofection, the percentage of GFP+ cells was assessed by flow cytometry, and the data are summarized in a bar graph. Upon selection, GFP+ cells were highly enriched in pan T cells that received transfer reagent, RNP plus HR, or MMEJ donor plasmid, but not in T cells that received donor plasmid alone.

[0118] [Figure 17A]Figure 17A-C are a series of graphs showing the phenotype of primary human pan T cells modified at the AAVS1 site by HR and MMEJ. The phenotype of GFP+CD8+ pan T cells was analyzed by flow cytometry 11 days after nucleofection. A) Cells were stained with 7AAD (cell viability), CD4, CD8, CD45RA, and CD62L, and the gating strategy is shown on the FACS plot. CD8+ T cell subsets were defined by expression: CD45RA+CD62L+ (stem cell memory T cells (Tscm)), CD45RA-CD62L+ (central memory T cells (Tcm)), CD45RA-CD62L- (effector memory T cells (Tem)), and CD45RA+CD62L- (T effector (Teff)). B) The percentage of total GFP+CD8+ T cells in each T cell subset is summarized in a bar graph. Using the piggyBac™ transposon system, or the combination of HR and Cas9 RNP, and the combination of MMEJ and Cas9 RNP, enriched populations of GFP+ Tscm were achieved in all cases. C) Total pan-T cell numbers were analyzed 13 days post-nucleofection, and the data are summarized in a bar graph. [Figure 17B] Same as above [Figure 17C] Same as above

[0119] [Figure 18]Figures 18A-B are a pair of photographs of gel electrophoresis results showing site-specific integration into the AAVS1 site. Selected cells from each group were harvested, genomic DNA extracted, and used as a template for PCR to confirm site-specific integration into the AAVS1 site for A) HR and B) MMEJ. Two primer pairs separately amplified the 5'-end junction of the AAVS1 target site (one primer primed the promoter region of the insert EF1a-2r CACCGGAGCCAATTCCCACT (SEQ ID NO: 36), and the other primer primed the AAVS1 region beyond the 500 bp homolog arm of the 5'-end AAVS-3r CTGCACCACGTGATGTCCTC (SEQ ID NO: 37), yielding a 0.73 kb DNA fragment for either HR or MMEJ) and the 3'-end junction (one primer primed the polyA signaling region SV40pA-1r GTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38), and the other primer primed the AAVS1 region beyond the 500 bp 5' homolog arm AAVS-2f CTGGGGACTCTTTAAGGAAAGAAG (SEQ ID NO: 39), yielding a 0.76 kb DNA fragment for either HR or MMEJ). PCR products were displayed on an agarose gel. Non-specific bands in the HR samples were likely the result of only a single round of PCR and would have been resolved by performing additional rounds. DETAILED DESCRIPTION OF THE INVENTION

[0120] Detailed Description The present disclosure provides a method for generating stem cell memory T cells (T cells) with potent CAR activity by using the piggyBac™ Transposon System to generate T cells expressing human chimeric antigen receptors (CARs). SCM ) (herein, CAR-T SCM The present disclosure provides methods for producing CAR-Ts under conditions in which the CAR-Ts are stored or induced. SCM The composition comprising SCMand display a distinct functional profile consistent with this T cell subset. Other T cell subsets found in the compositions of the present disclosure include, but are not limited to, central memory CAR-T cells (CAR-T CM ), effector memory CAR-T cells (CAR-T EM ), effector CAR-T cells (CAR-T E ), and well-differentiated effector CAR-T cells (CAR-T TE ) are listed. T N is the parent progenitor cell, and T SCM directly, which in turn produces T CM A linear pathway of differentiation, such as directly giving rise to naive T cells (T N )>T SCM >T CM >T EM >T E >T TE may be involved in the generation of these cells. SCM , CARTyrin-T SCM and / or VCAR-T SCM The composition comprising the CAR-T may comprise one or more of each parental CAR-T cell subset, SCM are the most abundant (e.g., T SCM >T CM >T EM >T E >T TE The absolute quantity / abundance and relative proportions of each parental T cell subset may vary between patient blood samples and naturally occurring cell populations, which may be T SCM and may be present in high abundance and / or proportions, and are not naturally occurring in the treatment of patients for disease and cancer. SCM The compositions of the present disclosure comprising are more potent and effective.

[0121] Immunotherapy using chimeric antigen receptor (CAR)-T cells is emerging as an exciting therapeutic approach for cancer therapy. Autologous CAR-modified T cells targeting tumor-associated antigens (Ag) can result in robust tumor killing, and in some cases, CD19 +This results in complete remission of hematological malignancies. Unlike traditional biologics and chemotherapy drugs, CAR-T cells have the ability to rapidly regenerate upon Ag recognition, potentially eliminating the need for repeated treatment. To achieve this, CAR-T cells must not only initially drive tumor destruction but also be sustained as a stable population of viable memory T cells in the patient to prevent potential cancer recurrence. Therefore, a CAR molecule that does not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as a CAR molecule that can stimulate early memory cells, particularly stem cell memory (T SCM Intensive efforts are focused on developing CAR-T products containing stem cell-like T cells (T cells). Stem cell-like CAR-T cells have the greatest capacity for self-renewal and central memory T cells (T cells). CM ), effector memory T cells (T EM ) and effector T cells (T E ), resulting in better tumor eradication and long-term CAR-T engraftment.

[0122] CAR-T of the present disclosure SCM The cells may contain a centrin-based CAR, termed CARTyrin (thus, the cells may be CARTyrin-T SCM Centirin is an alternative scaffold molecule based on the human consensus tenascin FN3 domain, which is smaller than scFv molecules and can be selected for its monomeric nature, which favors stability and reduces the potential for tonic signaling in CAR molecules. The CARTyrin of the present disclosure can be introduced into T cells using a plasmid DNA transposon encoding CARTyrin flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking inappropriate gene activation or silencing.

[0123] CAR-T of the present disclosure SCM The cells may contain a VHH-based CAR, referred to as a VCAR (thus, the cells may be referred to as VCAR-T SCMThe VCARs of the present disclosure can be introduced into T cells using a plasmid DNA transposon encoding a VHH flanked by two cis-regulatory insulator elements to help stabilize VHH expression by blocking inappropriate gene activation or silencing.

[0124] In certain embodiments of the disclosed methods, the piggyBac™ (PB) Transposon System, termed Super piggyBac™ (SPB), can be used to stably integrate antigen-specific CARTyrin or VCAR (including cancer antigen-specific CARTyrin or VCAR) into resting pan T cells, in which a transposon is co-delivered with an mRNA transposase enzyme in a single electroporation reaction (even though the transposon and transposase are contained in separate compositions until they are introduced into cells). Delivery of piggyBac™ transposons into intact resting primary human pan T cells resulted in 20-30% of cells with stable integration and expression of the PB-delivered gene. Unexpectedly, the majority of these modified CARTyrin-expressing T cells were stem memory T cells (T SCM The T cells were positive for expression of CD62L and CD45RA, markers commonly associated with CAR T cells. To confirm that this phenotype was retained during CAR T cell stimulation and expansion, modified CARTyrin-expressing T cells positive for CD62L and CD45RA were activated by stimulation with CD3 and CD28. As a result of stimulation with CD3 and CD28, >60% of CARTyrin+ T cells displayed a stem cell memory phenotype. Furthermore, these cells, which expressed CARTyrin specific for cancer antigens, were fully capable of expressing potent antitumor effector functions.

[0125] To determine whether the PB system directly contributes to enhanced expression of stem-like markers, we compared the phenotype of CAR-T cells generated by either PB transfer or lentiviral (LV) transduction. To do this, we constructed a new vector by subcloning the CARTyrin transgene into a common LV construct for viral production. After CARTyrin was introduced into intact resting T cells by PB transfer or LV transduction, CARTyrin was expressed in the CAR-T cells. + Cells were expanded and then returned to a quiescent state. Various phenotypic and functional characteristics were measured, including kinetic analysis of memory and exhaustion-related markers, secondary proliferation in response to homeostatic cytokines or tumor-associated Ags, cytokine production, and lytic ability in response to target tumor cells. + Unlike T cells, LV-transduced CARTyrin + T cells did not show an enhanced memory phenotype. Furthermore, PB-transferred cells showed equal or greater capacity for secondary proliferation and target tumor cell killing. Collectively, these data suggest that PB-transferred CAR-T cells are primarily T cells, a product phenotype that is highly desirable in the CAR-T field. SCM Furthermore, these CARTyrin + T cells exhibit potent antitumor activity and can give rise to longer-persistent cells in vivo due to the use of centilin-based CARs, which may be less prone to tonic signaling and functional exhaustion. Chimeric Antigen Receptor

[0126] The present disclosure provides chimeric antigen receptors (CARs) comprising: (a) an ectodomain comprising an antigen-recognition region, the antigen-recognition region comprising one or more sequences that specifically bind to 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 to an antigen, resulting in a bispecific or tandem CAR. In certain embodiments, the antigen-recognition region may comprise three sequences that each specifically bind to an antigen, resulting in a trispecific 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 to an antigen may include, but are not limited to, single-chain antibodies (e.g., scFvs), sequences comprising one or more fragments of antibodies (e.g., VHHs, referred to as VCARs for CARs), antibody mimetics, and centyrins (referred to as CARTyrin for CARs).

[0127] In certain embodiments of CARs of the present disclosure, the signal peptide can comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In certain embodiments of CARs of the present disclosure, the signal peptide can comprise a sequence encoding a human CD8α signal peptide. The human CD8α signal peptide can comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide can 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 an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide can be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgcacgcagctagacca (SEQ ID NO: 9).

[0128] In certain embodiments of the CARs of the present disclosure, the transmembrane domain can comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. In certain embodiments of the CARs of the present disclosure, the transmembrane domain can comprise a sequence encoding a human CD8α transmembrane domain. The CD8α transmembrane domain can comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8α transmembrane domain can be encoded by a nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 11).

[0129] In certain embodiments of a CAR of the present disclosure, the endodomain can comprise a human CD3ζ endodomain.

[0130] In certain embodiments of the CARs of the present disclosure, at least one costimulatory domain can comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CARs of the present disclosure, at least one costimulatory domain can comprise a CD28 and / or 4-1BB costimulatory domain. The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12). The CD28 costimulatory domain can be encoded by a nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (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 an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB costimulatory domain may be encoded by a nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

[0131] In certain embodiments of the CARs of the present disclosure, the hinge can comprise a sequence derived from a human CD8α, IgG4, and / or CD4 sequence. In certain embodiments of the CARs of the present disclosure, the hinge can comprise a sequence derived from a human CD8α sequence. The hinge can comprise a human CD8α amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16). The human CD8α hinge amino acid sequence can be encoded by a nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).

[0132] The present disclosure provides a composition comprising a CAR of the present disclosure and at least one pharmaceutically acceptable carrier.

[0133] The present disclosure provides a transposon that comprises the CAR of the present disclosure.The transposon of the present disclosure is maintained as an episome or is integrated into the genome of recombinant / modified cell.The transposon can be part of a two-component piggyBac system that utilizes a transposon and a transposase to enhance non-viral gene transfer.

[0134] The transposons of the present disclosure may contain a selection gene for identifying, enriching, and / or isolating cells that express the transposon. Exemplary selection genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for cell viability and survival. Exemplary selection genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for conferring resistance to attack by a drug to which the cell is sensitive (or which may 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., transcripts, proteins, enzymes) essential for viability and / or survival in a cell culture medium 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 (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), and NKX2.2 (encodes NK2 homeobox 2).

[0135] The transposon of the present disclosure may, for example, comprise at least one self-cleaving peptide located between one or more of the sequences that specifically bind to the antigen and selection gene of the present disclosure. The at least one self-cleaving peptide may comprise, for example, a T2A peptide, a 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. The 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 an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The GSG-E2A peptide can 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). The F2A peptide can 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).The GSG-F2A peptide can 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The P2A peptide can 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0136] The transposon of the present disclosure may comprise a first self-cleaving peptide and a second self-cleaving peptide, where the first self-cleaving peptide is located, for example, one or more upstream of a sequence that specifically binds to an antigen of the present disclosure, and the second self-cleaving peptide is located, for example, one or more downstream of a sequence that specifically binds to an antigen of the present disclosure. The first and / or second self-cleaving peptide may comprise, for example, a T2A peptide, a 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. The 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 an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).The 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 023). The 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0137] The present disclosure provides a composition comprising the transposon of the present disclosure.In certain embodiments, the method of introducing the composition can further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme.The sequence encoding a transposase enzyme can be an mRNA sequence.

[0138] The transposon of the present disclosure can include a piggyBac transposon. The transposase enzyme of the present disclosure can include a piggyBac transposase or a compatible enzyme.

[0139] The present disclosure provides a vector comprising the CAR of the present disclosure. In certain embodiments, the vector is a viral vector. The vector can be a recombinant vector.

[0140] The viral vectors of the present disclosure may contain sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vectors may contain sequences isolated or derived from adeno-associated viruses (AAV). The viral vectors may include recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure contain two or more inverted terminal repeat (ITR) sequences located in cis adjacent to one or more sequences that specifically bind to an antigen. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present 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 present disclosure include, but are not limited to, rAAV-LK03.

[0141] The viral vectors of the present disclosure may contain 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 with selective cell culture conditions. The selective cell culture conditions may include a compound that is detrimental to cell viability or survival, and the gene product confers resistance to the compound. Exemplary selection genes of the present disclosure can include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0142] The viral vector of the present disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein a first self-cleaving peptide is located upstream of the CAR and a second self-cleaving peptide is located downstream of the CAR. The self-cleaving peptide may comprise, for example, a T2A peptide, a 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. The 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 an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).The 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). The 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0143] The present disclosure provides a vector comprising the CAR of the present disclosure.In certain embodiments, the vector is an mRNA vector.The vector can be a recombinant mRNA vector.The T cells of the present disclosure can be expanded before contacting the T cells with the mRNA vector comprising the CAR of the present disclosure.Then, the T cells comprising the mRNA vector, modified T cells can be administered to a subject.

[0144] The present disclosure provides a vector comprising the CAR of the present disclosure.In certain embodiments, the vector is a nanoparticle.Exemplary nanoparticle vectors of the present disclosure include, but are not limited to, nucleic acids (for example, 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 (for example, polymersomes), micelles, lipids (for example, liposomes), organic molecules (for example, carbon atoms, sheets, fibers, tubes), inorganic molecules (for example, calcium phosphate or gold), or any combination thereof.Nanoparticle vectors can be passively or actively transported across cell membranes.

[0145] The nanoparticle vectors of the present disclosure may include 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. The selective cell culture conditions may include a compound that is detrimental to cell viability or survival, and the gene product confers resistance to the compound. Exemplary selection genes of the present disclosure can include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0146] The nanoparticle vector of the present disclosure may comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the nanoparticle. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located upstream of the CAR and the second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle and the second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle and the second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and the selection gene. The self-cleaving peptide may include, for example, a T2A peptide, a 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. The T2A peptide may include an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may include an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20).The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO:21). The 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:22). The 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:23). The GSG-F2A peptide can 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The P2A peptide can 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0147] The present disclosure provides compositions comprising the vectors of the present disclosure. CARTyrin

[0148] The present disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen-recognition region, the antigen-recognition region comprising at least one centirin; (b) a transmembrane domain; and (c) an endodomain comprising at least one costimulatory domain. As used throughout this disclosure, a CAR comprising centirin is referred to as a CARTyrin. In certain embodiments, the antigen-recognition region may comprise two centirins to generate a bispecific or tandem CAR. In certain embodiments, the antigen-recognition region may comprise three centirins to generate a trispecific CAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen-recognition region and the transmembrane domain.

[0149] The present disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen-recognition region, the ectodomain comprising at least one protein scaffold or antibody mimic; (b) a transmembrane domain; and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen-recognition region may comprise two protein scaffolds or antibody mimics to produce a bispecific or tandem CAR. In certain embodiments, the antigen-recognition region may comprise three protein scaffolds or antibody mimics to produce a trispecific 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.

[0150] In certain embodiments of CARs of the present disclosure, the signal peptide can comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In certain embodiments of CARs of the present disclosure, the signal peptide can comprise a sequence encoding a human CD8α signal peptide. The human CD8α signal peptide can comprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide can 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 an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide can be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgcacgcagctagacca (SEQ ID NO: 9).

[0151] In certain embodiments of the CARs of the present disclosure, the transmembrane domain can comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. In certain embodiments of the CARs of the present disclosure, the transmembrane domain can comprise a sequence encoding a human CD8α transmembrane domain. The CD8α transmembrane domain can comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8α transmembrane domain can be encoded by a nucleic acid sequence comprising atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 11).

[0152] In certain embodiments of a CAR of the present disclosure, the endodomain can comprise a human CD3ζ endodomain.

[0153] In certain embodiments of the CARs of the present disclosure, at least one costimulatory domain can comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CARs of the present disclosure, at least one costimulatory domain can comprise a CD28 and / or 4-1BB costimulatory domain. The CD28 costimulatory domain may comprise an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12). The CD28 costimulatory domain can be encoded by a nucleic acid sequence comprising cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (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 an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB costimulatory domain may be encoded by a nucleic acid sequence comprising aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

[0154] In certain embodiments of the CARs of the present disclosure, the hinge can comprise a sequence derived from a human CD8α, IgG4, and / or CD4 sequence. In certain embodiments of the CARs of the present disclosure, the hinge can comprise a sequence derived from a human CD8α sequence. The hinge can comprise a human CD8α amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16). The human CD8α hinge amino acid sequence can be encoded by a nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).

[0155] The centilin of the present disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding to an antigen. The centilin of the present 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 to 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 LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). The consensus sequence can be encoded by a nucleic acid sequence comprising atgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacgcagccttcgatagttttatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtcctgacagtgccagggtccgaacgctcttatgacctgacagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccgcaatcttcaccacca (SEQ ID NO: 3).The consensus sequence may be (a) an AB loop comprising or consisting of amino acid residues TEDS at positions 13 to 16 of the consensus sequence; (b) a BC loop comprising or consisting of amino acid residues TAPDAAF at positions 22 to 28 of the consensus sequence; (c) a CD loop comprising or consisting of amino acid residues SEKVGE at positions 38 to 43 of the consensus sequence; (d) a DE loop comprising or consisting of amino acid residues GSER at positions 51 to 54 of the consensus sequence; (e) an EF loop comprising or consisting of amino acid residues GLKPG at positions 60 to 64 of the consensus sequence; (f) an FG loop comprising or consisting of amino acid residues KGGHRSN at positions 75 to 81 of the consensus sequence; or (g) modified at one or more positions within any combination of (a) to (f). The centrin of the present 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. -9 Less than or equal to M, 10 -10 Less than or equal to M, 10 -11 Less than or equal to M, 10 -12 Less than or equal to M, 10 -13 Less than or equal to M, 10 -14 Less than or equal to M and 10 -15 K is smaller than or equal to M D can bind with at least one affinity selected from D can be determined by surface plasmon resonance.

[0156] The present disclosure provides a composition comprising a CAR of the present disclosure and at least one pharmaceutically acceptable carrier.

[0157] The present disclosure provides a transposon that comprises the CAR of the present disclosure.The transposon of the present disclosure is maintained as an episome or is integrated into the genome of recombinant / modified cell.The transposon can be part of a two-component piggyBac system that utilizes a transposon and a transposase to enhance non-viral gene transfer.

[0158] The transposons of the present disclosure may contain a selection gene for identifying, enriching, and / or isolating cells that express the transposon. Exemplary selection genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for cell viability and survival. Exemplary selection genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for conferring resistance to attack by a drug to which the cell is sensitive (or which may 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., transcripts, proteins, enzymes) essential for viability and / or survival in a cell culture medium 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 (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), and NKX2.2 (encodes NK2 homeobox 2).

[0159] The transposon of the present disclosure may include at least one self-cleaving peptide located between, for example, one or more of the protein scaffolds of the present disclosure, such as centilin or CARTyrin, and a selected gene of the present disclosure. The at least one self-cleaving peptide may include, for example, a T2A peptide, a 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. The T2A peptide may include an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The GSG-E2A peptide can 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). The F2A peptide can 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).The GSG-F2A peptide can 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The P2A peptide can 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0160] The transposon of the present disclosure may comprise a first self-cleaving peptide and a second self-cleaving peptide, where the first self-cleaving peptide is located upstream of, for example, one or more of the protein scaffolds of the present disclosure, such as centilin or CARTyrin, and the second self-cleaving peptide is located downstream of, for example, one or more of the protein scaffolds of the present disclosure, such as centilin or CARTyrin. The first and / or second self-cleaving peptides may comprise, for example, a T2A peptide, a 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. The 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 an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).The 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). The 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0161] The present disclosure provides a composition comprising the transposon of the present disclosure.In certain embodiments, the method of introducing the composition can further comprise a composition comprising a plasmid comprising a sequence encoding a transposase enzyme.The sequence encoding a transposase enzyme can be an mRNA sequence.

[0162] The transposon of the present disclosure can include a piggyBac transposon. The transposase enzyme of the present disclosure can include a piggyBac transposase or a compatible enzyme.

[0163] The present disclosure provides a vector comprising the CAR of the present disclosure. In certain embodiments, the vector is a viral vector. The vector can be a recombinant vector.

[0164] The viral vectors of the present disclosure may contain sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vectors may contain sequences isolated or derived from adeno-associated viruses (AAV). The viral vectors may include recombinant AAVs (rAAVs). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure contain two or more inverted terminal repeat (ITR) sequences located in cis next to the sequences encoding the protein scaffolds, centrin, or CARTyrin of the present disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present 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 present disclosure include, but are not limited to, rAAV-LK03.

[0165] The viral vectors of the present disclosure may contain 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 with selective cell culture conditions. The selective cell culture conditions may include a compound that is detrimental to cell viability or survival, and the gene product confers resistance to the compound. Exemplary selection genes of the present disclosure can include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0166] The viral vector of the present disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein a first self-cleaving peptide is located upstream of the CAR and a second self-cleaving peptide is located downstream of the CAR. The self-cleaving peptide may comprise, for example, a T2A peptide, a 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. The 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 an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).The 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). The 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0167] The present disclosure provides a vector comprising the CAR of the present disclosure.In certain embodiments, the vector is an mRNA vector.The vector can be a recombinant mRNA vector.The T cells of the present disclosure can be expanded before contacting the T cells with the mRNA vector comprising the CAR of the present disclosure.Then, the T cells comprising the mRNA vector, modified T cells can be administered to a subject.

[0168] The present disclosure provides a vector comprising the CAR of the present disclosure.In certain embodiments, the vector is a nanoparticle.Exemplary nanoparticle vectors of the present disclosure include, but are not limited to, nucleic acids (for example, 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 (for example, polymersomes), micelles, lipids (for example, liposomes), organic molecules (for example, carbon atoms, sheets, fibers, tubes), inorganic molecules (for example, calcium phosphate or gold), or any combination thereof.Nanoparticle vectors can be passively or actively transported across cell membranes.

[0169] The nanoparticle vectors of the present disclosure may include 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. The selective cell culture conditions may include a compound that is detrimental to cell viability or survival, and the gene product confers resistance to the compound. Exemplary selection genes of the present disclosure can include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0170] The nanoparticle vector of the present disclosure may comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the nanoparticle. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located upstream of the CAR and the second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle and the second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle and the second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and the selection gene. The self-cleaving peptide may include, for example, a T2A peptide, a 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. The T2A peptide may include an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). The GSG-T2A peptide may include an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20).The 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 an amino acid sequence comprising QCTNYALLKLAGDVESNPGP (SEQ ID NO:21). The 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 an amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:22). The 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 an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:23). The GSG-F2A peptide can 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 an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The P2A peptide can 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 an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The 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 an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0171] The present disclosure provides compositions comprising the vectors of the present disclosure. Scaffold proteins

[0172] Centilin is one example of a protein scaffold of the present disclosure. The antigen recognition region of a CAR of the present disclosure can comprise at least one protein scaffold.

[0173] The protein scaffolds of the present disclosure can be derived from fibronectin type III (FN3) repeat proteins, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods for making and using them. In a preferred embodiment, the protein scaffold is composed of a consensus sequence of multiple FN3 domains derived from human tenascin-C (hereinafter "tenascin"). In a more preferred embodiment, the protein scaffold of the present disclosure is a consensus sequence of 15 FN3 domains. The protein scaffolds of the present disclosure can be designed to bind to various molecules, for example, cellular target proteins. In a preferred embodiment, the protein scaffolds of the present disclosure can be designed to bind to epitopes of wild-type and / or variant forms of antigens.

[0174] The protein scaffolds of the present disclosure may include additional molecules or moieties, such as the Fc region of an antibody, an albumin binding domain, or other moieties that affect half-life. In further embodiments, the protein scaffolds of the present disclosure may be attached to a nucleic acid molecule that can encode the protein scaffold.

[0175] The present disclosure provides at least one method for expressing in a host cell at least one protein scaffold based on a consensus sequence of multiple FN3 domains, the method comprising culturing a host cell described herein under conditions wherein the at least one protein scaffold is expressed in detectable and / or recoverable amounts.

[0176] The present disclosure provides at least one composition comprising: (a) a protein scaffold and / or an encoding nucleic acid based on the consensus sequence of a number of FN3 domains described herein; and (b) a suitable and / or pharmaceutically acceptable carrier or diluent.

[0177] The present disclosure provides methods for generating libraries of protein scaffolds based on fibronectin type III (FN3) repeat proteins, preferably the consensus sequence of multiple FN3 domains, more preferably the consensus sequence of multiple FN3 domains derived from human tenascin. The libraries are formed by creating successive generations of scaffolds by altering (through mutation) specific portions of the scaffold, such as amino acids or the number of amino acids within the molecule in the loop region. Libraries can be generated by altering the amino acid composition of a single loop or by simultaneously altering multiple loops or additional portions of the scaffold molecule. Altered loops can be lengthened or shortened as appropriate. Such libraries can be generated to include all possible amino acids at each position, or a subset of amino acids can be designed. Library members can be used for screening in vitro or by display, such as CIS display (e.g., DNA, RNA, ribosome display), yeast display, bacterial display, and phage display.

[0178] The protein scaffolds of the present disclosure offer enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations, and can be expressed and folded in prokaryotic systems such as E. coli, eukaryotic systems such as yeast, and in vitro transcription / translation systems such as the rabbit reticulocyte lysate system.

[0179] The present disclosure provides isolated recombinant and / or synthetic protein scaffolds based on the consensus sequence of fibronectin type III (FN3) repeat proteins, including, but not limited to, mammalian-derived scaffolds, as well as compositions comprising and encoding nucleic acid molecules containing at least one polynucleotide encoding a protein scaffold based on the consensus FN3 sequence. The present disclosure further includes methods of making and using such nucleic acid and protein scaffolds, including, but not limited to, diagnostic and therapeutic compositions, methods, and devices.

[0180] The protein scaffolds of the present disclosure offer advantages over conventional therapeutics, such as the ability to be administered topically, orally, or across the blood-brain barrier; to be expressed in E. coli, thereby increasing resource-dependent protein expression relative to the expression capacity of mammalian cells; to be engineered into bispecific or tandem molecules that bind to multiple targets or multiple epitopes on the same target; to be conjugated with drugs, polymers, and probes; to be formulated at high concentrations; and to allow such molecules to effectively penetrate diseased tissues and tumors.

[0181] Furthermore, protein scaffolds have many of the properties of antibodies, with their folding mimicking the variable region of antibodies. This orientation allows the FN3 loops to be exposed in the same way as antibody complementarity determining regions (CDRs). Protein scaffolds should be able to bind to cellular targets, and loops can be modified, for example, affinity matured, to improve certain binding or related properties.

[0182] Three of the six loops in the protein scaffolds of the present disclosure topologically correspond to the complementarity determining regions (CDRs) (CDRs 1-3) of an antibody, i.e., the antigen-binding regions, while the remaining three loops are surface-exposed in a manner similar to antibody CDRs. These loops span residues 13-16 or about 13-16, residues 22-28 or about 22-28, residues 38-43 or about 38-43, residues 51-54 or about 51-54, residues 60-64 or about 60-64, and residues 75-81 or about 75-81 of SEQ ID NO: 1. The loop regions at residues 22-28 or about 22-28, residues 51-54 or about 51-54, and resi...

Claims

1. 1. A method of generating a plurality of modified T cells, the method comprising: (i) introducing into a plurality of primary human T cells (a) a transposon composition comprising a nucleic acid sequence encoding an antigen receptor or a therapeutic protein, and (b) a transposase composition comprising a transposase or a sequence encoding a transposase, to generate a plurality of modified T cells; At least 25% of the plurality of modified T cells comprise a sequence encoding the antigen receptor or therapeutic protein, and are stem memory T cells (T SCM ) or T SCM express one or more cell surface markers of a mitochondrial cell-like cell; and The method, wherein the one or more cell surface markers comprise CD45RA and CD62L.

2. At least 40% of the plurality of modified T cells are stem memory T cells (T SCM ) or T SCM The method of claim 1, wherein the cell expresses one or more cell surface markers of a cellular endothelial cell-like cell.

3. At least 50% of the plurality of modified T cells are stem memory T cells (T SCM ) or T SCM The method of claim 1, wherein the cell expresses one or more cell surface markers of a cellular endothelial cell-like cell.

4. At least 60% of the plurality of modified T cells are stem memory T cells (T SCM ) or T SCM The method of claim 1, wherein the cell expresses one or more cell surface markers of a cellular endothelial cell-like cell.

5. The method according to any one of claims 1 to 4, wherein the transposon is a piggyBac transposon, a Sleeping Beauty transposon, a Helraiser transposon, or a Tol2 transposon.

6. The method according to any one of claims 1 to 5, wherein the transposase is a piggyBac transposase, a super piggyBac transposase, a Sleeping Beauty transposase, a hyperactive Sleeping Beauty transposase (SB100X), a Helitron transposase, or a Tol2 transposase.

7. The method according to any one of claims 1 to 6, wherein the sequence encoding the transposase is an mRNA sequence.

8. The method according to any one of claims 1 to 7, wherein the antigen receptor is a T cell receptor (TCR).

9. The method of claim 8, wherein the TCR is non-naturally occurring.

10. The method according to any one of claims 1 to 7, wherein the antigen receptor is a chimeric antigen receptor (CAR).

11. 11. The method of claim 10, wherein the CAR comprises at least one of a single chain variable region (scFv), a single chain antibody, a VHH, an antibody mimetic, a protein scaffold, or a centrin.

12. The method of any one of claims 1 to 7, wherein the therapeutic protein is a secreted or secretable therapeutic protein.

13. 13. The method of any one of claims 1-12, wherein the method further comprises contacting the plurality of modified T cells with a T cell activation 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 complement to generate a plurality of activated modified T cells.

14. The method of claim 13, wherein the T cell activation composition further comprises an anti-human CD2 monospecific tetrameric antibody complex.

15. 15. The method of any one of claims 1-14, wherein at least 25% or at least 30% of the modified T cells of the population further express one or more of CD28, CCR7, CD127, CD45RO, CD95 and IL-2Rβ.

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