Modified stem cell memory T cells, method for producing them, and method for using them.

Abstract

This disclosure provides a method for producing modified stem cell memory T cells (e.g., CAR-T cells) for administration to a subject, for example, as adoptive cell therapy. There is a long-unaddressed need in the art for a method for producing modified stem cell memory T cells for administration to a subject, for example, as adoptive cell therapy. This disclosure provides a solution to this long-unaddressed need. [Solution] Unlike conventional biological agents and chemotherapy drugs, the modified T cells of this disclosure have the ability to rapidly regenerate once an antigen is recognized, thereby potentially eliminating the need for repeated treatments.

Description

[Technical Field]

[0001] Related applications This application claims the interests of U.S. Provisional Application No. 62 / 402,707 filed on 30 September 2016, No. 62 / 502,508 filed on 5 May 2017, No. 62 / 553,058 filed on 31 August 2017, and No. 62 / 556,309 filed on 8 September 2017, the contents of each of these applications, in whole, are incorporated herein by reference.

[0002] Use of sequence listings The contents of the filed text file named "POTH-012_001WO_SeqList.txt", created on October 2, 2017, with a size of 110KB, are incorporated herein by reference in their entirety.

[0003] Field of Invention This disclosure relates to molecular biology, and more specifically, to methods for creating and using modified stem cell memory T cells. [Background technology]

[0004] background For example, there is a long-unaddressed need in the art for a method of producing modified stem cell memory T cells for administration to subjects as adoptive cell therapy. This disclosure provides a solution to this long-unaddressed need. [Overview of the project] [Means for solving the problem]

[0005] Abstract Unlike conventional biological and chemotherapeutic agents, the modified T cells of this disclosure have the ability to rapidly regenerate upon antigen recognition, thereby potentially eliminating the need for repeated treatments. To achieve this, the modified T cells of this disclosure not only initially drive tumor destruction but also persist in the patient as a stable population of viable memory T cells to prevent potential cancer recurrence. Therefore, 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) cells, are important. SCM The focus of efforts has been on developing modified T cell products containing ). The stem cell-like modified T cells of this disclosure have the greatest capacity for self-renewal and central memory T cells (T CM ), effector memory T cells (T EM ) and effector T cells (T E They exhibit multipotency that elicits ) and thereby result in better tumor eradication and long-term engraftment of modified T cells. Modified T cells of the Disclosure include, but are not limited to, cells expressing antigen receptors including the protein scaffolds of the Disclosure. Modified T cells of the Disclosure include, but are not limited to, cells expressing chimeric antigen receptors (CARs) (i.e., CAR-T cells of the Disclosure). Chimeric antigen receptors (CARs) of the Disclosure may include, but are not limited to, one or more sequences that specifically bind to an antigen, including a single-chain antibody (e.g., scFv), a sequence containing one or more fragments of an antibody (e.g., VHH, referred to as VCAR in the case of CARs), an antibody mimetic, and centyrin (referred to as CARTyrin in the case of 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 a transposon or other delivery means such as electroporation or nucleofection, and can be integrated into the genome of the cells during a subsequent incubation step. The resulting cells are modified T cells having an edited genome that retains a stem-like phenotype. This modified T cell having an edited genome that retains a stem-like phenotype can be used as a cell therapy. Alternatively, or in addition thereto, the modified cells of the present disclosure can be subjected to a first electroporation or nucleofection and a subsequent electroporation or nucleofection to introduce a genome editing construct.

[0007] Specifically, the present disclosure provides a method for generating modified stem memory T cells (T SCM ), the method comprising, for generating modified T cells, the step of 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 a transposase into primary human T cells, wherein the modified T cells express one or more cell surface markers of stem memory T cells (T SCM ), thereby generating modified stem memory T cells (T SCM ). The present disclosure provides a method for generating a plurality of modified stem memory T cells (T SCM ), the method comprising, for generating a plurality of modified T cells, the step of 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 a transposase into a plurality of primary human T cells, wherein at least 2%, 5%, ɪ0%, 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) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 25% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 50% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 60% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 75% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 80% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 85% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 90% of the multiple modified T cells are stem memory T cells (T SCM) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 95% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, CAR-T SCM The cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, CAR-T 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 those where the transposon is a piggyBac transposon, the transposase is a piggyBac® or Super piggyBac® (SPB) transposase.

[0008] In certain embodiments of the methods of this disclosure, the transposon is a plasmid DNA transposon, and the sequence encoding an antigen receptor or therapeutic protein is adjacent to two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac® or Super piggyBac® (SPB) transposase. In certain embodiments, particularly those in which the transposase is a Super piggyBac® (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

[0009] In certain embodiments of the methods disclosed herein, the transposase enzyme is piggyBac(PB) transposase enzyme. piggyBac(PB) transposase enzyme is [ka] It may contain, or consist of, at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, identical amino acid sequences.

[0010] In certain embodiments of the methods disclosed herein, a transposase enzyme is an array [ka] The piggyBac(PB) transposase enzyme contains or consists of an amino acid sequence having one or more amino acid substitutions at the 30th, 165th, 282nd, or 538th positions.

[0011] In certain embodiments, the transposase enzyme is a piggyBac(PB) transposase enzyme comprising or comprising an amino acid sequence having two or more amino acid substitutions at positions 30, 165, 282, or 538 of the sequence of Sequence ID No. 4. In certain embodiments, the transposase enzyme is a piggyBac(PB) transposase enzyme comprising or comprising an amino acid sequence having three or more amino acid substitutions at positions 30, 165, 282, or 538 of the sequence of Sequence ID No. 4. In certain embodiments, the transposase enzyme is a piggyBac(PB) transposase enzyme comprising an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of Sequence ID No. 4. In certain embodiments, the amino acid substitution at position 30 of the sequence of Sequence 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 methods of the present disclosure, the transposase enzyme is Super piggyBac®(SPB) transposase enzyme. In certain embodiments, the Super piggyBac®(SPB) transposase enzyme of the present disclosure may include, or be derived from, the amino acid sequence of Sequence 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 contain, or consist of, at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between these, identical amino acid combinations.

[0013] In certain embodiments of the methods of this disclosure, including embodiments in which the transposase has the above mutations at positions 30, 165, 282 and / or 538, piggyBac® or Super The piggyBac® transposase enzyme may further include amino acid substitutions at one or more 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 methods of the present disclosure, including embodiments in which the transposase includes the above mutations at positions 30, 165, 282 and / or 538, the piggyBac® or Super piggyBac® transposase enzyme may further include amino acid substitutions at one or more of the following 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the substitution of isoleucine (I) with phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is the substitution of valine (V) with phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 4 or SEQ ID NO: 5 is the substitution of leucine (L) with methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is the substitution of glycine (G) with alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 4 or SEQ ID NO: 5 is the substitution of tryptophan (W) with phenylalanine (F). In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO: 4 or SEQ ID NO: 5 is the substitution of proline (P) with valine (V). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 4 or SEQ ID NO: 5 is the substitution of phenylalanine (F) with valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the substitution of valine (V) with proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 methods of the present disclosure, including embodiments in which the transposase includes the above mutations at positions 30, 165, 282 and / or 538, the piggyBac® transposase enzyme may include amino acid substitutions at one or more positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, and the Super piggyBac® transposase enzyme may further include this. In certain embodiments of the methods of the present disclosure, including embodiments in which the transposase includes the above mutations at positions 30, 165, 282 and / or 538, the piggyBac® transposase enzyme may include two, three, four, five, six or more 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, and the Super piggyBac® transposase enzyme may further include this. In certain embodiments of the methods of the present disclosure, including embodiments in which the transposase includes the above mutations at positions 30, 165, 282 and / or 538, the piggyBac® transposase enzyme may include 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, and the Super piggyBac® transposase enzyme may further include this. 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 the 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 the 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 include a substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO: 4. Including embodiments in which the piggyBac® transposase enzyme may include a substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO: 4, in certain embodiments, the piggyBac® transposase enzyme may further include 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 may include substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO: 4, substitution of alanine (A) with arginine (R) at position 372 of SEQ ID NO: 4, and substitution of alanine (A) with lysine (K) at position 375 of SEQ ID NO: 4. In certain embodiments, the piggyBac® transposase enzyme may include substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO: 4, substitution of alanine (A) with arginine (R) at position 372 of SEQ ID NO: 4, substitution of alanine (A) with lysine (K) at position 375 of SEQ ID NO: 4, and substitution of asparagine (N) with aspartic acid (D) at position 450 of SEQ ID NO: 4.

[0015] This disclosure relates to modified stem memory T cells (T SCM The present invention provides a method for producing modified T cells, the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor or therapeutic protein, and (b) a transposase composition comprising a transposase or a transposase encoding sequence into primary human T cells, wherein the modified T cells are stem memory T cells (T SCM ) express one or more cell surface markers, thereby modifying stem memory T cells (T SCM) is produced. This disclosure describes how multiple modified stem memory T cells (T) are created. SCM The present invention provides a method for producing multiple modified T cells, the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor or therapeutic protein, and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into multiple primary human 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 between these, of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 25% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 50% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 60% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 75% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 80% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 85% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 90% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 95% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, CAR-T SCM The cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, CAR-T 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 hyperactive Sleeping Beauty transposase (SB100X).

[0016] In certain embodiments of the method disclosed herein, the Sleeping Beauty transposase enzyme is [ka] It contains at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between these identical amino acid sequences.

[0017] In certain embodiments of the methods disclosed herein, the overactive Sleeping Beauty (SB100X) transposase enzyme is [ka] It contains at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between these identical amino acid sequences.

[0018] This disclosure relates to modified stem memory T cells (T SCM The present invention provides a method for producing modified T cells, the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor or therapeutic protein, and (b) a transposase composition comprising a transposase or a transposase encoding sequence into primary human T cells, wherein the modified T cells are stem memory T cells (T SCM ) express one or more cell surface markers, thereby modifying stem memory T cells (T SCM ) is produced. This disclosure describes how multiple modified stem memory T cells (T) are created. SCMThe present invention provides a method for producing multiple modified T cells, the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor, and (b) a transposase or a transposase composition comprising a sequence encoding a transposase into multiple primary human 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 between these, of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 25% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 50% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 60% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 75% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 80% of the multiple modified T cells are stem memory T cells (T SCM) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 85% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 90% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 95% of the multiple modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby creating multiple modified stem memory T cells (T SCM ) is produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, CAR-T SCM The cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, CAR-T 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 this disclosure, the transposase is a Helitron transposase. The Helitron transposase moves the Helraiser transposon, an ancient element from the bat genome that was active approximately 30 to 36 million years ago. An example of a Helraiser transposon of this disclosure is: [ka] [ka] [ka] [ka] Helibat1, which contains a nucleic acid sequence including the above, is an example.

[0020] Unlike other transposases, Helitron transposases do not contain an RNase-H-like catalytic domain, but instead contain a RepHel motif consisting 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] The exemplary Helitron transposases described herein are [ka] It contains an amino acid sequence that includes [specific amino acids].

[0022] In the Helitron transposition, a hairpin near the 3' end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, thereby potentially leading to transduction of an adjacent sequence. Furthermore, the Helraiser transposition results in a covalently closed cyclic intermediate. Additionally, the Helitron transposition may lack target site duplication. In the Helraiser sequence, the transposase is flanked by left- and right-terminal sequences, referred to as LTS and RTS. These sequences terminate with a conserved 5'-TC / CTAG-3' motif. A 19 bp palindrome sequence, potentially forming a hairpin termination structure, is located 11 nucleotides upstream of the RTS and consists of the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 29).

[0023] This disclosure relates to modified stem memory T cells (T SCM The present invention provides a method for producing modified T cells, the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor or therapeutic protein, and (b) a transposase composition comprising a transposase or a transposase encoding sequence into primary human T cells, wherein the modified T cells are stem memory T cells (T SCM ) express one or more cell surface markers, thereby modifying stem memory T cells (T SCM ) is produced. This disclosure describes how multiple modified stem memory T cells (T) are created. SCM The present invention provides a method for producing multiple modified T cells, the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor, and (b) a transposase or a transposase composition comprising a sequence encoding a transposase into multiple primary human 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 between these, of the multiple modified T cells are stem memory T cells (T SCM) express one or more cell surface markers, whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM ) whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM ) whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM ) whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM ) whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM ) whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM ) whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM) express one or more cell surface markers, whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the method produces a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells express one or more cell surface markers of stem memory T cells (T SCM ) whereby a plurality of modified stem memory T cells (T SCM ) are produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the cell surface markers of CAR-T SCM include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell surface markers of CAR-T SCM 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 where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

[0024] In certain embodiments of the methods of the present disclosure, the transposase is a Tol2 transposase. The Tol2 transposon can be isolated or derived from the genome of medaka and can be similar to transposons of the hAT family. Exemplary Tol2 transposons of the present disclosure contain a gene encoding a Tol2 transposase, which is encoded by a sequence containing approximately 4.7 kilobases and contains four exons. Exemplary Tol2 transposases of the present disclosure are as follows:

Chemical formula

[0025] Exemplary Tol2 transposases of the present disclosure include, among others, inverted repeats, sequences near the ends, and Tol2 transposases: [ka] [ka] [ka] It is encoded in the nucleic acid sequence.

[0026] This disclosure relates to modified central memory T cells (T CM The present invention provides a method for producing a central memory T cell (T), the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor or therapeutic protein, and (b) a transposase or transposase composition comprising a transposase-encoding sequence into primary human T cells, wherein the modified T cells are central memory T cells (T). CM ) expresses one or more cell surface markers, thereby modifying central memory T cells (T CM ) is produced. This disclosure describes how multiple modified central memory T cells (T) are created. CM The present invention provides a method for producing multiple modified T cells, the method comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor, and (b) a transposase or a transposase composition comprising a sequence encoding a transposase into multiple primary human 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 between these, of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 25% of the multiple modified T cells are central memory T cells (T CM) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 50% of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 60% of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 75% of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 80% of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 85% of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM ) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 90% of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM) is produced. In a particular embodiment, the method produces multiple modified T cells, where at least 95% of the multiple modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby creating multiple modified central memory T cells (T CM A transposon is produced. 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, in particular, the embodiment in which 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, in particular, the embodiment in which 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 those 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 those in which the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

[0027] This disclosure describes multiple modified stem memory T cells (T SCM ) and multiple modified central memory T cells (T CMThe present invention provides a method for preparing a composition containing ), and this method provides multiple modified T SCM and multiple modified T CM To prepare a composition comprising (a) a transposon composition comprising a transposon comprising an antigen receptor or therapeutic protein, and (b) a transposase composition comprising a transposase or a transposase-encoding sequence, the step of introducing these into a plurality of primary human T cells, wherein the plurality of modified T SCM It expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ, and multiple modified T CM It expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby producing multiple modified T SCM and multiple modified T CM A composition containing is prepared. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these. In certain embodiments of this method, modified central memory T cells (T) CM ) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T) SCM ) constitute at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 90% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM) account for at least 10% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 80% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 20% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 70% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 30% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 60% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 60% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 40% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute 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. 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, in particular, the embodiment in which 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, in particular, the embodiment in which 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 Hellaiser transposon. In certain embodiments, particularly those 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 those in which the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

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

[0029] In certain embodiments of the methods of this disclosure, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor. In certain embodiments, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor is a recombinant T cell receptor. In certain embodiments of this method, the antigen receptor is a chimeric antigen receptor (CAR). In certain embodiments, the CAR is 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 Method of the Disclosure, which includes embodiments comprising the step of introducing a transposon composition comprising (a) a transposon comprising an antigen receptor and (b) a transposase or a transposase composition comprising a transposase-encoding sequence into primary human T cells, the Method further comprises the step of introducing a second transposon composition comprising (c) a transposon comprising a therapeutic protein into primary human T cells in order to produce modified T cells, wherein the modified T cells are capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretory protein, and the Method produces modified T cells capable of secreting the therapeutic protein. In certain embodiments, the transposase composition of (b) undergoes transposition at the transposon of (a) and the transposon of (c). In certain embodiments, the Method further comprises the step of introducing a second transposase composition comprising (d) a transposase or a transposase-encoding sequence into primary human T cells. In certain embodiments, the second transposase composition undergoes transposition at the transposon of (c). In certain embodiments, the transposase composition of (b) undergoes transposition with the transposon of (a), and the transposase composition of (d) undergoes transposition with the transposon of (c). In certain embodiments of this method, the transposon is a plasmid DNA transposon having sequences encoding an antigen receptor or therapeutic protein adjacent to two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly those where 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 those in which the transposon is the Sleeping Beauty transposon, the transposase is Sleeping Beauty transposase or hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments of this method, the transposon is the Helraiser transposon. In certain embodiments, particularly those in which the transposon is the Helraiser transposon, the transposase is Helitron transposase. In certain embodiments of this method, the transposon is the Tol2 transposon. In certain embodiments, including those in which the transposon is the Tol2 transposon, the transposase is Tol2 transposase.

[0031] This disclosure describes multiple modified stem memory T cells (T SCM A method for producing stem memory T cells (T cells), comprising: (a) introducing a composition containing an antigen receptor into a plurality of primary human T cells in order to produce a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) contacting the plurality of modified T cells with a T cell activator composition containing one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances in order 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 cells). SCM ) express one or more cell surface markers, thereby enabling the expression of multiple modified stem memory T cells (T SCM This disclosure provides a method for producing multiple modified stem memory T cells (T). SCMA method for producing stem memory T cells (T cells), comprising: (a) introducing a composition containing an antigen receptor into a plurality of primary human T cells in order to produce a plurality of modified T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) contacting the plurality of modified T cells with a T cell activator composition containing one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances in order 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 between these, of the plurality of activated modified T cells are stem memory T cells (T cells). SCM ) express one or more cell surface markers, thereby enabling the expression of multiple modified stem memory T cells (T SCM The present invention provides a method for producing stem memory T cells (T). In a particular embodiment, the method produces 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 ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 50% of the plurality of activated modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 60% of the plurality of activated modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 75% of the plurality of activated modified T cells are stem memory T cells (TSCM ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated modified T cells, where at least 80% of the multiple activated modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated modified T cells, where at least 85% of the multiple activated modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 90% of the plurality of activated modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 95% of the plurality of activated modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated modified stem memory T cells (T SCM ) is produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated modified T SCM The cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, activated modified T SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

[0032] (a) A step of producing modified T cells by introducing a composition containing an antigen receptor into primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) A step of producing activated modified T cells by contacting the modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances. SCM In a particular embodiment of the method of the present disclosure for producing (b) T cell activation composition further comprises an anti-human CD2 monospecific tetrameric antibody complex. In a particular embodiment, the method is a step of (c) contacting activated modified T cells with a T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements to produce a plurality of augmented modified T cells, wherein at least 2% of the plurality of augmented modified T cells are stem memory T cells (T SCM The method further comprises the step of expressing one or more cell surface markers of a plurality of augmented modified T cells (T). 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 between these is expressed as stem memory T cells (T). SCM ) express cell surface markers. In a particular embodiment of this method, at least 60% of multiple enlarged modified T cells are stem memory T cells (T SCM ) express cell surface markers. In certain embodiments, this method enriches multiple augmented modified T cells with stem memory T cells (T SCM) The method further comprises the step of preparing 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 between these, of modified T cells expressing the cell surface marker of ). In a particular embodiment, the method comprises (d) enriching a plurality of enlarged modified T cells to make stem memory T cells (T SCM The method further comprises the step of preparing a composition containing at least 60% modified T cells expressing the cell surface marker of ). In a particular embodiment of this method, the enrichment step involves extracting stem memory T cells (T) from a plurality of enriched modified T cells. SCM This method includes isolating modified T cells expressing one or more cell surface markers of ). In certain embodiments of this method, the enrichment step involves multiple enhanced enriched modified T cells. SCM To produce it, isolated modified T SCMThe method further comprises the step of contacting the T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements. In certain embodiments of this method, the T cell augmentation composition further comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments of this method, the T cell augmentation composition further comprises octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols. In a particular embodiment of this method, the T cell enlargement composition further comprises one or more octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including the endpoint; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; and sterols at a concentration of about 0.1 mg / kg to 10 mg / kg including the endpoint. In a particular embodiment of this method, the T cell enlargement composition further comprises one or more 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. In a particular embodiment of this method, the T cell enlargement composition further comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoints; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; and one or more sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoints.In a particular embodiment of this method, the T cell enlargement composition further comprises one or more of the following: 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 sterols at a concentration of about 2.5 μmol / kg.

[0033] This disclosure relates to modified central memory T cells (T CM The present invention provides a method for producing central memory T cells (T), comprising the steps of (a) producing modified T cells by introducing a composition containing an antigen receptor into primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon, and (b) producing activated modified T cells by contacting the modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances, wherein the activated modified T cells are central memory T cells (T). CM ) express one or more cell surface markers, thereby central memory T cells (T CM ) is produced. This disclosure describes how multiple modified central memory T cells (T) are created. CM The present invention provides a method for producing central memory T cells (T), comprising the steps of (a) producing multiple modified T cells by introducing a composition containing an antigen receptor into multiple primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon, and (b) producing multiple activated modified T cells by contacting the multiple modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances, 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 between these is central memory T cells (T). CM) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 25% of the plurality of activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 50% of the plurality of activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 60% of the plurality of activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces multiple activated modified T cells, where at least 75% of the multiple activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces multiple activated modified T cells, where at least 80% of the multiple activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces multiple activated modified T cells, where at least 85% of the multiple activated modified T cells are central memory T cells (T CM) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 90% of the plurality of activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) are produced. In a particular embodiment, the method produces a plurality of activated modified T cells, where at least 95% of the plurality of activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby enabling multiple activated modified central memory T cells (T CM ) is produced. In a particular embodiment, an activated modified T CM The cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0034] (a) A step of producing modified T cells by introducing a composition containing an antigen receptor into primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) A step of producing activated modified T cells by contacting the modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances. CMIn a particular embodiment of the method of the present disclosure for producing (b) central memory T cells (T), the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In a particular embodiment, the method is a step of (c) contacting activated modified T cells with a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements to produce a plurality of augmented modified T cells, wherein at least 2% of the plurality of augmented modified T cells are central memory T cells (T). CM The method further comprises the step of expressing one or more cell surface markers of a plurality of augmented modified T cells (T). 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 between these is expressed as central memory T cells (T). CM ) express cell surface markers. In a particular embodiment of this method, at least 60% of multiple enlarged modified T cells express central memory T cells (T CM ) express cell surface markers. In certain embodiments, this method enriches a plurality of augmented modified T cells with central memory T cells (T CM ) The method further comprises the step of preparing 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 between these, of modified T cells expressing the cell surface marker of ). CM The method further comprises the step of preparing a composition comprising at least 60% modified T cells expressing the cell surface marker of (T). In a particular embodiment of this method, the enrichment step involves extracting central memory T cells (T) from a plurality of enriched modified T cells. CMThis method includes isolating modified T cells expressing one or more cell surface markers of ). In certain embodiments of this method, the enrichment step involves multiple enhanced enriched modified T cells. CM To produce it, isolated modified T CMThe method further comprises the step of contacting a T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements. In certain embodiments of this method, the T cell augmentation composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments of this method, the T cell augmentation composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols. In a particular embodiment of this method, the T cell enlargement composition further comprises one or more octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including the endpoint; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; and sterols at a concentration of about 0.1 mg / kg to 10 mg / kg including the endpoint. In a particular embodiment of this method, the T cell enlargement composition further comprises one or more 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. In a particular embodiment of this method, the T cell enlargement composition further comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoints; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; and one or more sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoints.In a particular embodiment of this method, the T cell enlargement composition further comprises one or more of the following: 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 sterols at a concentration of about 2.5 μmol / kg.

[0035] This disclosure describes multiple modified stem memory T cells (T SCM ) and multiple modified central memory T cells (T CM A method for producing a composition containing (a) a T cell, comprising the steps of (a) introducing a composition containing 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 the transposon, and (b) contacting the plurality of modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances to produce a plurality of activated modified stem memory T cells (T SCM ) and multiple activated modified central memory T cells (T CM The present invention provides a method comprising the step of preparing a composition containing ), and a plurality of activated modified T SCM It expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ, and multiple activated modified T CM It expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby producing multiple modified T SCM and multiple modified T CM A composition containing is prepared. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these. In certain embodiments of this method, modified central memory T cells (T)CM ) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T) SCM ) constitute at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 90% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 10% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 80% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 20% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 70% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 30% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM) occupies at least 60% of the total number of cells in the composition. In certain embodiments of this method, the modified stem memory T cells (T SCM ) occupy at least 60% of the total number of cells in the composition, and the modified central memory T cells (T CM ) occupy at least 40% of the total number of cells in the composition. In certain embodiments of this method, the modified stem memory T cells (T SCM ) occupy at least 50% of the total number of cells in the composition, and the modified central memory T cells (T CM ) occupy at least 50% of the total number of cells in the composition.

[0036] (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 antibody complex of a tetramer specific for anti-human CD3, an antibody complex of a tetramer specific for anti-human CD28, and an activation supplement to produce a composition comprising a plurality of activated modified stem memory T cells (T SCM ) and a plurality of activated modified central memory T cells (T CM ). In certain embodiments of the method of the present disclosure for producing a composition comprising a plurality of modified stem memory T cells (T SCM ) and a plurality of modified central memory T cells (T CM ), the T cell activation composition in (b) further comprises an antibody complex of a tetramer specific for anti-human CD2. In certain embodiments, the method comprises (c) contacting the composition, the plurality of activated modified stem memory T cells (T SCM ) and the plurality of 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 to produce a plurality of expanded modified T SCMIt expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ, and multiple augmented modified T CM It expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby expressing multiple augmented modified T SCM and multiple increased modified T CM The method further includes the step of preparing a composition containing the following. In a particular embodiment of this method, the enrichment step involves preparing stem memory T cells (T) from a plurality of enriched modified T cells. SCM Isolating modified T cells expressing one or more cell surface markers, or central memory T cells (T) from multiple enriched modified T cells. CM This method includes isolating modified T cells expressing one or more cell surface markers of the stem memory T cells (T). In certain embodiments of this method, the enrichment step involves isolating stem memory T cells (T) from a plurality of enriched modified T cells. SCM Modified T cells expressing one or more cell surface markers, and central memory T cells (T) derived from multiple enriched modified T cells. CM This method includes isolating modified T cells expressing one or more cell surface markers of ). In certain embodiments of this method, the enrichment step involves multiple enhanced enriched modified T cells. SCM and multiple enlarged enriched modified T CM To prepare a composition containing isolated modified TSCM and isolated modified T CMThe method further comprises the step of contacting a composition containing with a T cell augmentation composition containing one or more of the following: human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements. In certain embodiments of this method, the T cell augmentation composition further comprises one or more of the following: octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, hydrazide stearate, oleamide, sterols, and alkanes. In certain embodiments of this method, the T cell augmentation composition further comprises one or more of the following: octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols. In a particular embodiment of this method, the T cell enlargement composition further comprises one or more octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including the endpoint; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; and sterols at a concentration of about 0.1 mg / kg to 10 mg / kg including the endpoint. In a particular embodiment of this method, the T cell enlargement composition further comprises one or more 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. In a particular embodiment of this method, the T cell enlargement composition further comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoints; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; and one or more sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoints.In a particular embodiment of this method, the T cell enlargement composition further comprises one or more of the following: 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 sterols at a concentration of about 2.5 μmol / kg. In a particular embodiment of this method, modified stem memory T cells (T. SCM ) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these. In certain embodiments of this method, modified central memory T cells (T) CM ) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T) SCM ) constitute at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 90% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 10% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 80% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM) account for at least 20% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 70% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 30% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 60% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 60% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 40% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute 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] (a) a step of producing multiple modified T cells by introducing a composition containing an antigen receptor into multiple primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) a step of contacting the multiple modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, comprising the method of the activated modified T cells of the present disclosure. SCM or T CMIn certain embodiments of the method for producing the genome editing composition, the introduction step includes homologous recombination. In certain embodiments of the introduction step including homologous recombination, the genome editing composition is brought into contact with the genome sequence of at least one primary T cell in a plurality of T cells. In certain embodiments of the introduction step including homologous recombination, the genome editing composition is brought into contact with the genome sequence of a subset of primary T cells in a plurality of T cells. In certain embodiments, the subset of primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage in between. In certain embodiments of the introduction step including homologous recombination, the genome editing composition is brought into contact with the genome sequence of each primary T cell in a plurality of T cells. In certain embodiments of the introduction step including homologous recombination, a single-strand break is induced by the genome editing composition. In certain embodiments of the introduction step including homologous recombination, a double-strand break is induced by the genome editing composition. In certain embodiments of the introduction step including homologous recombination, the introduction step further includes a donor sequence composition. In certain embodiments, the donor sequence composition includes a sequence encoding an antigen receptor. In certain embodiments, the donor sequence composition includes a sequence encoding an antigen receptor, a 5' genome sequence, and a 3' genome sequence, where the 5' genome sequence is homologous or identical to the genome sequence of a primary T cell located 5' to the breakpoint induced by the genome editing composition, and the 3' genome sequence is homologous or identical to the genome sequence of a primary T cell located 3' to the breakpoint induced by the genome editing composition. In certain embodiments of the introduction step including homologous recombination, the genome editing composition and the donor sequence composition are brought into contact with the genome sequence simultaneously or sequentially. In certain embodiments of the introduction step including homologous recombination, the genome editing composition and the donor sequence composition are brought into contact with the genome sequence sequentially, first bringing the genome editing composition into contact. In certain embodiments that include an introduction step involving homologous recombination, the genome editing composition includes a sequence encoding a DNA-binding domain and a sequence encoding a nuclease domain.In certain embodiments of the genome editing composition, which include an introduction 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 its sequence. In certain embodiments of the genome editing composition, the nuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, including substitution of alanine (A) with aspartic acid (D) at position 10 (D10A) and substitution of alanine (A) with histidine (H) at position 840 (H840A)). In certain embodiments of the genome editing composition, the nuclease domain includes a short inactive Cas9 (SEQ ID NO: 32, including substitution of alanine (A) with aspartic acid (D) at position 10 (D10A) and substitution of alanine (A) with asparagine (N) at position 540 (N540A)). In certain embodiments of the genome editing composition, the nuclease domain includes or further includes an IIS-type endonuclease. In certain embodiments of the genome editing composition, the IIS-type endonuclease includes AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, B This includes pmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI, or Clo051. In certain embodiments, the IIS type endonuclease includes Clo051.In certain embodiments of the genome editing composition, the nuclease domain comprises, further comprises or consists of a TALEN or its nuclease domain. In certain embodiments of the genome editing composition, the nuclease domain comprises, further comprises or consists of a ZFN or its nuclease domain. In certain embodiments of the introduction step involving homologous recombination, the genome editing composition induces a cleavage within the genomic sequence and uses the endogenous DNA repair mechanism of primary T cells to insert a donor sequence composition. In certain embodiments of the introduction step involving homologous recombination, 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] (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 within 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 monomeric specific tetrameric antibody complex, an anti-human CD28 monomeric specific tetrameric antibody complex and an activation supplement. The activated modified T of the present disclosure comprises a method comprising SCM or T CM In certain embodiments of the method for generating, the viral vector comprises an antigen receptor. In certain embodiments, the viral vector comprises one or more sequences isolated from, derived from or recombinated from 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 recombinated from 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] (a) a step of producing multiple modified T cells by introducing a composition containing an antigen receptor into multiple primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) a step of contacting the multiple modified T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complex, anti-human CD28 monospecific tetrameric antibody complex, and activation replacement material, the activated modified T of the present disclosure. SCM or T CM In certain embodiments of the method for producing the vector, the viral vector includes an antigen receptor. In certain embodiments, the viral vector includes a sequence isolated from or derived from a retrovirus. In certain embodiments, the viral vector includes a sequence isolated from or derived from a lentivirus.

[0040] (a) a step of producing multiple modified T cells by introducing a composition containing an antigen receptor into multiple primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) a step of contacting the multiple modified T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complex, anti-human CD28 monospecific tetrameric antibody complex, and activation replacement material, the activated modified T of the present disclosure. SCM or T CM In certain embodiments of the method for producing the viral vector, the viral vector includes an antigen receptor. In certain embodiments, the viral vector includes a sequence isolated from or derived from a retrovirus. In certain embodiments, the viral vector includes a sequence isolated from or derived from a gamma retrovirus.

[0041] (a) a step of producing multiple modified T cells by introducing a composition containing an antigen receptor into multiple primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) a step of contacting the multiple modified T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complex, anti-human CD28 monospecific tetrameric antibody complex, and activation replacement material, the activated modified T of the present disclosure. SCM or T CM In certain embodiments of the method for producing the vector, the viral vector includes an antigen receptor. In certain embodiments, the viral vector includes a sequence isolated from or derived from adeno-associated virus (AAV). In certain embodiments, the AAV is serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV includes a sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In certain embodiments, the AAV includes a sequence isolated from, derived from, or recombinant 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 recombinant from AAV2. In certain embodiments, including embodiments in which the vector crosses the blood-brain barrier (BBB), the AAV comprises a sequence isolated from, derived from, or recombinant from AAV9. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include, but are not limited to, self-complementary AAVs (scAAVs) 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 this disclosure include, but are not limited to, rAAV-LK03, rAAV-NP59, and rAAV-NP84.

[0042] Activation Modification T of this Disclosure SCM or T CM In certain embodiments of the method for producing the antigen, the nucleic acid vector includes an antigen receptor. In certain embodiments, the DNA vector includes an antigen receptor. In certain embodiments, the mRNA vector includes an antigen receptor. In certain embodiments, the nucleic acid vector is a plasmid or a minicircle vector.

[0043] Activation Modification T of this Disclosure SCM or T CM In certain embodiments of the method for producing the nanoparticle vector, the nanoparticle vector includes an antigen receptor. The nanoparticles may consist of polymers disclosed, for example, in International Patent Publication WO2012 / 094679, International Patent Publication WO2016 / 022805, International Patent Publication WO / 2011 / 133635, International Patent Publication WO / 2016 / 090111, International Patent Publication WO / 2017 / 004498, International Patent Publication WO / 2017 / 004509, International Patent Application 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] Activated modified version of this disclosure T SCM or T CM In certain embodiments of the method for producing the 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, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor. In certain embodiments, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor is a recombinant T cell receptor. In certain embodiments of this method, the antigen receptor is a chimeric antigen receptor (CAR). In certain embodiments, the CAR is CARTyrin. In certain embodiments, the CAR comprises one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

[0045] (a) a step of producing multiple modified T cells by introducing a composition containing an antigen receptor into multiple primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (b) a step of contacting the multiple modified T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complex, anti-human CD28 monospecific tetrameric antibody complex, and activation replacement material, the activated modified T of the present disclosure. SCM or T CM In certain embodiments of the method for producing a therapeutic protein, the method further includes the step of introducing a composition containing a therapeutic protein into primary human T cells in order to produce modified T cells capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secretory protein, and the method produces modified T cells capable of secreting the therapeutic protein. In certain embodiments, the introduction step includes homologous recombination, and the donor sequence includes a sequence encoding the therapeutic protein. In certain embodiments, the donor sequence containing an antigen receptor further includes the therapeutic protein. In certain embodiments, the first donor sequence contains an antigen receptor, and the second donor sequence contains the therapeutic protein. In certain embodiments, the vector includes a sequence encoding the therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle. In certain embodiments, the vector containing an antigen receptor further includes the therapeutic protein. In certain embodiments, the first vector contains an antigen receptor, and the second vector template contains the therapeutic protein.

[0046] This disclosure relates to modified stem memory T cells (T SCMThe present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing an antigen receptor into primary human T cells in order to produce modified T cells, wherein the transposon contains the antigen receptor, and (b) contacting the modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacements to produce activated modified T cells, wherein the activated modified T cells are stem memory T cells (T SCM ) express one or more cell surface markers, thereby modifying stem memory T cells (T SCM ) is produced. This disclosure describes how multiple modified stem memory T cells (T) are created. SCM The present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing an antigen receptor into a plurality of primary human T cells in order to produce a plurality of modified T cells, wherein the transposons contain the antigen receptor, and (b) contacting the plurality of modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances 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 ) express one or more cell surface markers, thereby modifying stem memory T cells (T SCM ) is produced. In a particular embodiment of this method, at least 60% of the multiple activated modified T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers. In a particular embodiment of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. This disclosure relates to modified stem memory T cells (T SCMThe present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing a chimeric antigen receptor (CAR) into primary human T cells to produce CAR-T cells, and (b) contacting the CAR-T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, anti-human CD2 monospecific tetrameric antibody complexes, and activation replacement substances to produce activated CAR-T cells, wherein the activated CAR-T cells are stem memory T cells (T). SCM ) express one or more cell surface markers, thereby CAR-expressing stem memory T cells (T SCM )(CAR-T SCM ) is produced. This disclosure describes how multiple modified stem memory T cells (T) are created. SCM The present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing a chimeric antigen receptor (CAR) into a plurality of primary human T cells in order to produce a plurality of CAR-T cells, and (b) contacting the plurality of CAR-T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, anti-human CD2 monospecific tetrameric antibody complexes, and activation replacement substances to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage between these is a stem memory T cell (T) SCM ) expresses one or more cell surface markers, thereby enabling multiple activated CAR stem memory T cells (T SCM) are produced. In a particular embodiment, the method is (c) to produce a plurality of augmented modified T cells, an activated modified T cell and a T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements, wherein at least 2% of the plurality of augmented modified T cells are stem memory T cells (T SCMThe method further comprises the step of expressing one or more cell surface markers. In certain embodiments, the T cell augmentation composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell augmentation composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 a particular embodiment, the T cell enlargement 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 sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoint; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoint; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint. In certain embodiments, the T cell enlargement composition comprises one or more 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 sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell enlargement composition comprises one or more of the following: 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 enlargement 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, 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 between these is amplified modified T cells (T). SCM ) express cell surface markers. In certain embodiments, at least 60% of multiple enlarged modified T cells are stem memory T cells (T SCM ) express cell surface markers. In a particular embodiment, the method involves (d) enriching multiple enlarged modified T cells with stem memory T cells (T SCMThe method further comprises the step of preparing 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 between these, of modified T cells expressing the cell surface marker of (T). In a particular embodiment, the method comprises (d) enriching a plurality of enlarged modified T cells with stem memory T cells (T SCM The further step includes preparing a composition comprising at least 60% modified T cells expressing the cell surface marker of ). In a particular embodiment, the enrichment step involves extracting stem memory T cells (T) from a plurality of enriched modified T cells. SCM The method further includes isolating modified T cells expressing one or more cell surface markers of ). In certain embodiments, the enrichment step involves isolating multiple enhanced enriched modified T cells. SCM To produce it, isolated modified T SCMThe process further comprises contacting the T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements. In certain embodiments, the T cell augmentation composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell augmentation composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg. In certain embodiments, the T cell enlargement composition comprises one or more of the following: 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 enlargement 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, and about 2.13 mg / The composition contains oleic acid at a concentration of 1.01 mg / kg and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell enlargement composition contains one or more of the following, including endpoints: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 sterols at a concentration of about 2.5 μmol / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 a particular embodiment, the T cell enlargement 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] This disclosure relates to modified central memory T cells (T CM The present invention provides a method for producing a central memory T cell (T), comprising the steps of (a) introducing a composition containing an antigen receptor into primary human T cells in order to produce a modified T cell, wherein the transposon contains the antigen receptor, and (b) contacting the modified T cell with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances to produce an activated modified T cell, wherein the activated modified T cell is a central memory T cell (TCM ) expresses one or more cell surface markers, thereby modifying central memory T cells (T CM ) is produced. This disclosure describes how multiple modified central memory T cells (T) are created. CM The present invention provides a method for producing multiple modified T cells, comprising the steps of (a) introducing a composition containing an antigen receptor into multiple primary human T cells in order to produce multiple modified T cells, wherein the transposons contain the antigen receptor, and (b) contacting the multiple modified T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacements to produce multiple activated modified T cells, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the multiple activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers, thereby modifying central memory T cells (T CM ) is produced. In a particular embodiment of this method, at least 60% of the multiple activated modified T cells are central memory T cells (T CM ) expresses one or more cell surface markers. In a particular embodiment of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In a particular embodiment, the method is to (c) contact activated modified T cells with a T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements in order to produce a plurality of augmented modified T cells, wherein at least 2% of the plurality of augmented modified T cells are central memory T cells (T CMThe method further comprises the step of expressing one or more cell surface markers of ) in certain embodiments. In certain embodiments, the T cell enlargement composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell enlargement composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 a particular embodiment, the T cell enlargement 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 sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoint; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoint; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint. In certain embodiments, the T cell enlargement composition comprises one or more 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 sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell enlargement composition comprises one or more of the following: 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 enlargement 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, 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 between these is amplified modified T cells (CMS). CM ) express cell surface markers. In certain embodiments, at least 60% of multiple enlarged modified T cells are central memory T cells (T CM ) express cell surface markers. In certain embodiments, the method, (d) enriches multiple augmented modified T cells with central memory T cells (T CMThe method further comprises the step of preparing 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 between these, of modified T cells expressing the cell surface marker of (T). In a particular embodiment, the method comprises (d) enriching a plurality of enlarged modified T cells with central memory T cells (T CM The further step includes preparing a composition comprising at least 60% modified T cells expressing the cell surface marker of ). In a particular embodiment, the enrichment step involves selecting central memory T cells (T) from a plurality of enriched modified T cells. CM The method further includes isolating modified T cells expressing one or more cell surface markers of ). In certain embodiments, the enrichment step involves isolating multiple enhanced enriched modified T cells. CM To produce it, isolated modified T CMThe process further comprises contacting the T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements. In certain embodiments, the T cell augmentation composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell augmentation composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following at a concentration of 9.19 mg / kg: octanoic acid, 1.86 mg / kg: palmitic acid, about 2.12 mg / kg: linoleic acid, about 2.13 mg / kg: oleic acid, and about 1.01 mg / kg: sterols (mg / kg = parts per million).In a particular embodiment, the T cell enlargement 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 approximately 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million). In a particular embodiment, the T cell enlargement composition comprises one or more of the following at concentrations: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoints; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoints. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 sterols at a concentration of about 2.5 μmol / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 a particular embodiment, the T cell enlargement 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] This disclosure describes multiple modified stem memory T cells (T SCM ) and multiple modified central memory T cells (T CM A method for preparing a composition containing (a) a plurality of modified stem memory T cells (T SCM ) and multiple modified central memory T cells (T CM(b) To prepare a composition containing an antigen receptor, the step is to introduce a composition containing an antigen receptor into multiple primary human T cells, wherein the transposon contains an antigen receptor, and (b) to contact the composition with a T cell activating composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances to produce multiple activated modified stem memory T cells (T SCM ) and multiple activated modified central memory T cells (T CM The present invention provides a method comprising the step of preparing a composition containing ), and a plurality of activated modified T SCM It expresses one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ, and multiple activated modified T CM It expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, thereby expressing multiple modified T SCM and multiple modified T CM A composition comprising is prepared. In a particular embodiment of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In a particular embodiment, the method is to (c) contact the composition with a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements in order to produce a plurality of augmented modified T cells, wherein at least 2% of the composition comprising the plurality of augmented modified T cells is stem memory T cells (T SCM The method further comprises the step of expressing one or more cell surface markers of ) to produce a plurality of augmented modified T cells, wherein at least 2% of the composition containing the plurality of augmented modified T cells is a central memory T cell (T) CMThe method further comprises the step of expressing one or more cell surface markers of ) in certain embodiments. In certain embodiments, the T cell enlargement composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell enlargement composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 a particular embodiment, the T cell enlargement 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 sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoint; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoint; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint. In certain embodiments, the T cell enlargement composition comprises one or more 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 sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell enlargement composition comprises one or more of the following: 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 enlargement 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, multiple enlarged modified T cells. 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 between these, of the cells in the composition containing stem memory T cells (T SCM ) expresses the cell surface marker. In certain embodiments, multiple augmented 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 between these, of the cells in the composition containing the following: CM ) express cell surface markers. In a particular embodiment, the method involves (d) enriching the composition with stem memory T cells (T SCM The method further comprises the step of preparing 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 between these, of modified T cells expressing the cell surface marker of (T). In a particular embodiment, the method comprises (d) enriching the composition with central memory T cells (T CM The further step includes preparing 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 between these, of modified T cells expressing the cell surface marker of (T). In certain embodiments, the enrichment step involves enriching the composition with stem memory T cells (T) from the composition. SCM Isolating modified T cells expressing one or more cell surface markers of ) or central memory T cells (T) from a composition. CM The method further includes isolating modified T cells expressing one or more cell surface markers of ) from stem memory T cells (T). In certain embodiments, the enrichment step involves isolating modified T cells expressing one or more cell surface markers of ). SCM To isolate modified T cells expressing one or more cell surface markers of ) and to isolate central memory T cells (T) from the composition. CM The method further includes isolating modified T cells expressing one or more cell surface markers of ). In certain embodiments, the enrichment step involves isolating multiple enhanced enriched modified T cells. SCM and / or T CM To prepare a composition containing isolated modified T SCM and / or T CMThe process further comprises contacting the T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements. In certain embodiments, the T cell augmentation composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell augmentation composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following at a concentration of 9.19 mg / kg: octanoic acid, 1.86 mg / kg: palmitic acid, about 2.12 mg / kg: linoleic acid, about 2.13 mg / kg: oleic acid, and about 1.01 mg / kg: sterols (mg / kg = parts per million).In a particular embodiment, the T cell enlargement 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 approximately 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million). In a particular embodiment, the T cell enlargement composition comprises one or more of the following at concentrations: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoints; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoints. In a particular embodiment, the T cell enlargement composition contains octanoic acid at a concentration of about 64 μmol / kg and about 7 μmol / kg. The T cell enlargement composition comprises one or more of the following: palmitic acid at a concentration of approximately 7.5 μmol / kg, linoleic acid at a concentration of approximately 7.5 μmol / kg, oleic acid at a concentration of approximately 7.5 μmol / kg, and sterols at a concentration of approximately 2.5 μmol / kg. In certain embodiments, the T cell enlargement composition comprises one or more of the following: octanoic acid at a concentration of approximately 63.75 μmol / kg, palmitic acid at a concentration of approximately 7.27 μmol / kg, linoleic acid at a concentration of approximately 7.57 μmol / kg, oleic acid at a concentration of approximately 7.56 μmol / kg, and sterols at a concentration of approximately 2.61 μmol / kg. In certain embodiments, the T cell enlargement composition comprises octanoic acid at a concentration of approximately 63.75 μmol / kg, palmitic acid at a concentration of approximately 7.27 μmol / kg, linoleic acid at a concentration of approximately 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 a specific embodiment of this method, modified stem memory T cells (T SCM) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these. In certain embodiments of this method, modified central memory T cells (T) CM ) constitute at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of cells in between these of the total number of cells in the composition. In certain embodiments of this method, modified stem memory T cells (T) SCM ) constitute at least 10% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 90% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 90% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 10% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 20% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 80% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 80% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 20% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 30% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 70% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM) constitute at least 70% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 30% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 40% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 60% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute at least 60% of the total number of cells in the composition, and modified central memory T cells (T CM ) account for at least 40% of the total number of cells in the composition. In a particular embodiment of this method, modified stem memory T cells (T SCM ) constitute 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 Method of the Disclosure, the Method further comprises the step of introducing into primary human T cells a composition comprising an antigen receptor to produce modified T cells, wherein the transposon comprises an antigen receptor, and (b) contacting the modified T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacements to produce activated modified T cells. In certain embodiments, the method includes the step of introducing a first transposase composition and a second transposase composition. In certain embodiments, the method includes the step of introducing a first transposase composition and a second transposase composition, the first transposase composition is transposed by the transposon (a), and the second transposase composition is transposed by the second transposon. In certain embodiments of the method, the transposon is a plasmid DNA transposon having sequences encoding an antigen receptor or therapeutic protein adjacent to two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, in particular embodiments where 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 those in which 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 those in which the transposon is a Helraiser transposon, the transposase is Helitron transposase. In certain embodiments of this method, the transposon is a Tol2 transposon. In certain embodiments, including those in which the transposon is a Tol2 transposon, the transposase is Tol2 transposase.

[0050] In certain embodiments of the Method of the Disclosure, the Method further comprises the step of introducing a sequence encoding a therapeutic protein into primary human T cells, wherein the transposon comprises the antigen receptor, and the Method comprises the steps of introducing a sequence encoding a therapeutic protein into primary human T cells, wherein the transposon comprises the antigen receptor, and the Method comprises the steps of contacting the modified T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacements to produce activated modified T cells.

[0051] In certain embodiments of the methods of this disclosure, the introduction step further comprises a composition comprising a genome editing construct. In certain embodiments, the genome editing construct comprises a guide RNA and a clustered and regularly arranged short palindromic sequence repeat (CRISPR)-associated protein 9 (Cas9) DNA endonuclease. In certain embodiments, the genome editing construct comprises a DNA-binding domain and an IIS-type 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 an IIS-type endonuclease, and the expressed DNA-binding domain and the expressed IIS-type endonuclease are non-covalently linked. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and an IIS-type 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 an IIS-type endonuclease, the sequence derived from Cas9 endonuclease is the DNA-binding domain. In certain embodiments, including embodiments in which the sequence derived from Cas9 endonuclease is a DNA-binding domain, the sequence derived from Cas9 endonuclease encodes inactive Cas9. In certain embodiments, including embodiments in which the sequence derived from Cas9 endonuclease is a DNA-binding domain, the sequence derived from Cas9 endonuclease encodes a truncated Cas9. In certain embodiments, the sequence derived from Cas9 endonuclease includes an amino acid substitution of alanine (A) with aspartic acid (D) at position 10 (D10A). In certain embodiments, the sequence derived from Cas9 endonuclease includes an amino acid substitution of alanine (A) with histidine (H) at position 840 (H840A). In certain embodiments, the sequence derived from Cas9 endonuclease includes dCas9 (SEQ ID NO: 33). In certain embodiments, the sequence derived from Cas9 endonuclease includes an amino acid substitution of alanine (A) with asparagine (N) at position 580 (N580A).In certain embodiments, the sequence derived from Cas9 endonuclease includes dSaCas9 (SEQ ID NO: 32). In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the genome editing construct includes a sequence derived from a transcription activator-like effector nuclease (TALEN). In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the sequence derived from TALEN is the DNA-binding domain. In certain embodiments, the genome editing construct includes TALEN. In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the genome editing construct includes a sequence derived from a zinc finger nuclease (ZFN). In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the sequence derived from ZFN is the DNA-binding domain. In certain embodiments, the genome editing construct includes a zinc finger nuclease (ZFN).

[0052] In certain embodiments of the methods of the present disclosure, the transposon is a plasmid DNA transposon having sequences encoding an antigen receptor or therapeutic protein adjacent to two cis-regulatory insulator elements. In certain embodiments of the methods, the introduction step further comprises a composition comprising an mRNA sequence encoding a transposase. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, in particular, in embodiments where the transposon is a piggyBac transposon, the transposase is a Super piggyBac® (SPB) transposase. In certain embodiments, in particular, in embodiments where 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 an overactive variant, the overactive variant comprising amino acid substitutions at one or more 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 the substitution of valine (V) with isoleucine (I) (I30V). In certain embodiments, the amino acid substitution at position 165 of SEQ ID NO: 4 is the substitution of serine (S) with glycine (G) (G165S). In certain embodiments, the amino acid substitution at position 282 of SEQ ID NO: 4 is the substitution of valine (V) with methionine (M) (M282V). In certain embodiments, the amino acid substitution at position 538 of SEQ ID NO: 4 is the substitution of lysine (K) with asparagine (N) (N538K). In certain embodiments, the Super piggyBac (SPB) transposase contains the amino acid sequence including SEQ ID NO: 5. In certain embodiments, the transposon is the Sleeping Beauty transposon.In certain embodiments, particularly those in which the transposon is a Sleeping Beauty transposon, the transposase is Sleeping Beauty transposase or hyperactive Sleeping Beauty transposase (SB100X). In certain embodiments, the transposon is a Helraiser transposon. In certain embodiments, particularly those in which the transposon is a Helraiser transposon, the transposase is Helitron transposase. In certain embodiments, the transposon is a Tol2 transposon. In certain embodiments, particularly those in which the transposon is a Tol2 transposon, the transposase is Tol2 transposase. In certain embodiments, the sequence encoding the transposase is an mRNA sequence. In certain embodiments, the transposon may be derived from or recombinant from any species. Alternatively or additionally, the transposon may be a synthetic transposon.

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

[0054] In certain embodiments of the methods of this disclosure, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor. In certain embodiments, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor is a recombinant T cell receptor. In certain embodiments of this method, the antigen receptor is a chimeric antigen receptor (CAR). In certain embodiments, the CAR is 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 method disclosed herein, Modified T SCM The cell surface markers include CD62L and CD45RA. In certain embodiments, modified T SCM The cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, modified T SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

[0056] In certain embodiments of the method disclosed herein, a plurality of enlarged modified T cells are naive T cells (modified T cells). N ) including CAR-T N The cell surface markers include one or more of CD45RA, CCR7, and CD62L. In certain embodiments, multiple enlarged modified T cells are central memory T cells (modified T cells). CM ) including CAR-T CM The cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, multiple enlarged modified T cells are effector memory T cells (modified T cells). EM ) including CAR-T EM The cell surface markers include one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, multiple enlarged modified T cells are effector T cells (modified 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 method disclosed herein, a plurality of augmented modified T cells are central memory T cells (modified T cells). CM ) including CAR-T CMThe cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, the most abundant T cells in a plurality of expanded modified T cells are central memory T cells (modified T CM ), and the cell surface markers of CAR-T CM include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments where the most abundant T cells in a plurality of expanded modified T cells are central memory T cells (modified T CM ), the plurality of expanded modified T cells include T SCM cells, and the cell surface markers of T SCM cells include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ.

[0058] The present disclosure provides a method for generating modified stem memory T cells (T SCM ), comprising: (a) introducing a composition comprising a chimeric antigen receptor (CAR) into primary human T cells to generate CAR-T cells, and (b) contacting the CAR-T cells with a T cell activation composition comprising one or more of a tetrameric antibody complex specific for anti-human CD3, a tetrameric antibody complex specific for anti-human CD28, a tetrameric antibody complex specific for anti-human CD2, and an activation supplement to generate activated CAR-T cells, wherein the activated CAR-T cells express one or more cell surface markers of stem memory T cells (T SCM ), thereby generating CAR-expressing stem memory T cells (T SCM ) (CAR-T SCM ). The present disclosure provides a plurality of modified stem memory T cells (T SCMThe present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing a chimeric antigen receptor (CAR) into a plurality of primary human T cells in order to produce a plurality of CAR-T cells, and (b) contacting the plurality of CAR-T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, anti-human CD2 monospecific tetramer antibody complexes, and activation replacement substances to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage between these is stem memory T cells (T). SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 80% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 85% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 90% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 95% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) is produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated CAR-T SCM The cell surface markers 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 include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. This disclosure relates to modified stem memory T cells (T SCM The present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing a chimeric antigen receptor (CAR) into primary human T cells to produce CAR-T cells, and (b) contacting the CAR-T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances to produce activated CAR-T cells, wherein the activated CAR-T cells are stem memory T cells (T SCM ) express one or more cell surface markers, thereby CAR-expressing stem memory T cells (T SCM )(CAR-T SCM ) is produced.

[0059] This disclosure describes multiple modified stem memory T cells (T SCM The present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing a chimeric antigen receptor (CAR) into a plurality of primary human T cells in order to produce a plurality of CAR-T cells, and (b) contacting the plurality of CAR-T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances in order to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage between these is stem memory T cells (T). SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T SCM) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 80% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 85% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 90% of the multiple activated CAR-T cells are stem memory T cells (T SCM) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 95% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) is produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated CAR-T SCM The cell surface markers 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 a particular embodiment, the method is a step of (c) contacting activated CAR-T cells with a T cell augmentation composition comprising human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements in order to produce a plurality of augmented CAR-T cells, wherein at least 2% of the plurality of augmented CAR-T cells are stem memory T cells (T SCM )(CAR-T SCMThe step may further include expressing one or more cell surface markers of ). In certain embodiments, the T cell augmentation composition further includes one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell augmentation composition includes one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg. In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 a particular embodiment, the T cell enlargement 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 sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoint; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoint; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint. In certain embodiments, the T cell enlargement composition comprises one or more 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 sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell enlargement composition comprises one or more of the following: 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 enlargement 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 a particular embodiment, 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 between these, of the multiple enlarged CAR-T cells are stem memory T cells (T. SCM )(CAR-T SCM ) express cell surface markers. In certain embodiments, multiple enlarged CAR-T cells express stem memory T cells (T SCM CAR-T cells expressing cell surface markers (CAR-T SCM ) can be enriched, and by doing so, after the enrichment step, the method is to enrich stem memory T cells (T SCM )(CAR-T SCMEnriched compositions can be prepared containing at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, of CAR-T cells expressing the cell surface markers. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, CAR-T SCM The cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, CAR-T SCM The cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments, multiple enlarged CAR-T cells are naive T cells (CAR-T). N ) including CAR-T N The cell surface markers include one or more of CD45RA, CCR7, and CD62L. In certain embodiments, multiple enlarged CAR-T cells are central memory T cells (CAR-T). CM ) including CAR-T CM The cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, multiple enlarged CAR-T cells are effector memory T cells (CAR-T EM ) including CAR-T EM The cell surface markers include one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, multiple enlarged CAR-T cells are effector T cells (CAR-T EFF ) including CAR-T EFF The cell surface markers include one or more of CD45RA, CD95, and IL-2Rβ. Additional cell surface markers are described by Gattinoni et al. (Nat Med. 2011 Sep 18;17(10):1290-7; their contents are incorporated herein by reference in their entirety).

[0061] This disclosure relates to modified stem memory T cells (T SCM The present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing a chimeric antigen receptor (CAR) into primary human T cells to produce CAR-T cells, and (b) contacting the CAR-T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances to produce activated CAR-T cells, wherein the activated CAR-T cells are stem memory T cells (T SCM ) express one or more cell surface markers, thereby CAR-expressing stem memory T cells (T SCM )(CAR-T SCM ) is produced. This disclosure describes how multiple modified stem memory T cells (T) are created. SCM The present invention provides a method for producing stem memory T cells (T), comprising the steps of (a) introducing a composition containing a chimeric antigen receptor (CAR) into a plurality of primary human T cells in order to produce a plurality of CAR-T cells, and (b) contacting the plurality of CAR-T cells with a T cell activation composition containing one or more anti-human CD3 monospecific tetrameric antibody complexes, anti-human CD28 monospecific tetrameric antibody complexes, and activation replacement substances to produce a plurality of activated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage between these is a stem memory T cell (T) SCM ) expresses one or more cell surface markers, thereby enabling multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces a plurality of activated CAR-T cells, where at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 80% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 85% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 90% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM) are produced. In a particular embodiment, the method produces multiple activated CAR-T cells, where at least 95% of the multiple activated CAR-T cells are stem memory T cells (T SCM ) expresses one or more cell surface markers, thereby activating multiple activated CAR stem memory T cells (T SCM ) is produced. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, activated CAR-T SCM The cell surface markers 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 method of the present disclosure, a plurality of enlarged CAR-T cells are naive T cells (CAR-T N ) including CAR-T N The cell surface markers include one or more of CD45RA, CCR7, and CD62L. In certain embodiments, multiple enlarged CAR-T cells are central memory T cells (CAR-T). CM ) including CAR-T CM The cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, multiple enlarged CAR-T cells are effector memory T cells (CAR-T EM ) including CAR-T EM The cell surface markers include one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, multiple enlarged CAR-T cells are 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 the 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 the chimeric antigen receptor (CAR) of the present disclosure may further include introducing a composition comprising an mRNA sequence encoding a transposase. In certain preferred embodiments, the transposase is Super piggyBac® (SPB) transposase.

[0064] In certain embodiments, the transposons of the Disclosure may further comprise a selection gene. If the transposons of the Disclosure comprise a selection gene, the T cell augmentation composition of the Method of the Disclosure may further comprise a selector for simultaneously selecting and augmenting the activated or modified T cells of the Disclosure.

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

[0066] Modification of this disclosure SCMIn certain embodiments of the method for producing the T cells, the introduction step may include electroporation or nucleofection. If the introduction step includes nucleofection, the nucleofection may include (a) contacting a composition comprising a transposon composition, a transposase composition, and a plurality of primary human T cells in a cuvette; (b) applying one or more electrical pulses to the cuvette; and (c) incubating the composition comprising the plurality of primary human T cells at 37°C in a composition comprising a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements. In certain embodiments, the T cell enlargement composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell enlargement composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoints; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoints; and sterols (mg / kg = parts per million) at concentrations of about 0.1 mg / kg to 10 mg / kg including the endpoints. In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at concentrations of about 9 mg / kg, palmitic acid at concentrations of about 2 mg / kg, linoleic acid at concentrations of about 2 mg / kg, oleic acid at concentrations of about 2 mg / kg, and sterols (mg / kg = parts per million) at concentrations of about 1 mg / kg.In a particular embodiment, the T cell enlargement composition comprises one or more of the following: 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 a particular embodiment, the T cell enlargement 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 sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell enlargement composition comprises one or more octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoint; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoint; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint. In certain embodiments, the T cell enlargement composition comprises one or more 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 sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell enlargement composition comprises one or more of the following: 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 enlargement 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 nucleofection, the transposon composition is a 0.5 μg / μl solution containing nuclease-free water, and the cuvette contains 2 μl of the transposon composition, producing 1 μg of transposon. The transposon composition may contain piggyBac transposons. The transposon composition may contain Sleeping Beauty transposons. In certain embodiments of nucleofection, the transposase composition contains 5 μg of transposase. The transposase composition may contain overactive piggyBac® or Super piggyBac® (SPB) transposase. The transposase composition may contain overactive Sleeping Beauty (SB100X) transposase. In certain embodiments, the transposon may contain Helraiser transposase, and the transposase composition may contain Helitron transposase. In certain embodiments, the transposon may contain Tol2 transposase, and the transposase composition contains Tol2 transposase.

[0067] In certain embodiments of the methods of the present disclosure, including embodiments in which the introduction step includes nucleofection or electroporation, the nucleofection includes 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 methods of the present disclosure, including embodiments in which the introduction step includes nucleofection or electroporation, the nucleofection includes 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, the first transposon includes a sequence encoding an antigen receptor. In certain embodiments, the second transposon includes 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 transposon composition and the second transposon composition are moved by the first transposase. In certain embodiments, the first transposon composition is moved by the first transposase, but the second transposon composition is not. In certain embodiments, the second transposon composition is moved by the second transposase, but the first transposon composition is not. In certain embodiments, the first transposon composition is moved by the first transposase, and the second transposon composition is moved by the second transposase. In certain embodiments, the first transposon composition or the second transposon composition includes a sequence encoding an antigen receptor.In certain embodiments, the first transposon composition or the second transposon composition includes a sequence encoding a therapeutic protein. In certain embodiments, the first transposon composition includes a sequence encoding an antigen receptor, and the second transposon composition includes a sequence encoding a therapeutic protein. In certain embodiments, the therapeutic protein is a secretory or secretible protein. In certain embodiments of the methods of the present disclosure, including embodiments in which the introduction step includes nucleofection or electroporation, the nucleofection includes contacting a composition comprising a 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 transposon composition includes a sequence encoding an antigen receptor. In certain embodiments, the transposon composition includes a sequence encoding a therapeutic protein. In certain embodiments of the methods of the present disclosure, including embodiments in which the introduction step includes nucleofection or electroporation, the nucleofection further includes 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 includes an exogenous donor molecule. In certain embodiments, the exogenous donor molecule includes a sequence encoding an antigen receptor, and the transposon includes a sequence encoding a therapeutic protein. In certain embodiments, the composition containing a transposon, the composition containing a transposase, and the composition capable of inducing homologous recombination at a specific site in the genome are brought into contact with a composition containing a plurality of primary human T cells. In certain embodiments, firstly, the composition containing a transposon and the composition containing a transposase are brought into contact with a composition containing a plurality of primary human T cells, and secondly, the composition capable of inducing homologous recombination at a specific site in the genome is brought into contact with a composition containing a plurality of primary human T cells.In certain embodiments, firstly, a composition capable of inducing homologous recombination at a specific site in the genome is brought into contact with a composition containing a plurality of primary human T cells; secondly, a composition containing a transposon and a composition containing a transposase are brought into contact with a composition containing a plurality of primary human T cells. Modified T cells of the present disclosure. SCM In certain embodiments of the method for producing primary human T cells, the composition comprising primary human T cells comprises a buffer that maintains or enhances the level of viability and / or stem-like phenotype of the primary human T cells. In certain embodiments, the buffer maintains or enhances the level of viability and / or stem-like phenotype of the primary human T cells before nucleofection. In certain embodiments, the buffer maintains or enhances the level of 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 viability and / or stem-like phenotype of the primary human T cells after nucleofection. In certain embodiments, the buffer comprises a P3 primary cell solution (Lonza). In certain embodiments, the buffer comprises one or more of KCl, MgCl2, ClNa, glucose, and Ca(NO3)2 in any absolute or relative abundance or concentration, and optionally, the buffer further comprises a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. In certain embodiments, the buffer contains 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO3)2. In certain embodiments, the buffer contains a supplement comprising 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO3)2, as well as 20 mM HEPES and 75 mM Tris / HCl. In certain embodiments, the buffer contains a supplement comprising 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO3)2, as well as 40 mM Na2HPO4 / NaH2PO4, pH 7.2. In certain embodiments, the composition containing primary human T cells comprises 100 μl of buffer and 5 × 10 cells. 6 pieces~25×106 Includes one.

[0068] Modification of this disclosure SCM In a particular embodiment of the method for producing the cells, the composition containing primary human T cells is depleted of cells expressing CD14, CD56, and / or CD19. In a particular embodiment, the composition containing primary human T cells is 100 μl of buffer and 5 × 10 cells. 6 pieces~25×10 6 Includes one.

[0069] As used herein, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a medium containing one or more of the following: human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove MDM, and enlargement supplements, at 37°C. Alternatively, or in addition, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a medium containing one or more of the following: phosphorus, octane fatty acids, palmitic fatty acids, linoleic fatty acids, and oleic acid. In certain embodiments, the medium contains, for example, ten times the amount of phosphorus found in Iscove's Modified Dulbecco's Medium (IMDM); available from ThermoFisher Scientific under catalog number 12440053.

[0070] As used herein, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a medium containing one or more of the following elements at 37°C: human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and enlargement supplements. Alternatively, or in addition to, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a medium containing one or more of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. In certain embodiments, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a medium containing one or more of the following elements present at 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 enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following: human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and enlargement supplements, at 37°C. Instead, or in addition to the above, the term “supplemented T cell enlargement composition” or “T cell enlargement composition” refers to the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butylbenzenesulfonamide (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), hydrazide stearate (CAS It can be used interchangeably with media containing one or more of the following: 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 enlargement composition” or “T cell enlargement composition” refers to the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butylbenzenesulfonamide (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), hydrazide stearate (CAS It can be used interchangeably with media containing one or more of the following: 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 enlargement composition” or “T cell enlargement composition” refers to the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butylbenzenesulfonamide (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), hydrazide stearate (CAS It can be used interchangeably with media containing one or more of the following: (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 terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following ions: human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and enlargement supplements, at 37°C. Alternatively, or in addition to, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride ions, sulfate, and phosphoric acid.

[0073] As used herein, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a medium containing one or more of the following free amino acids: human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and enlargement supplements, at 37°C. Alternatively or additionally, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a medium containing 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 enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing, in corresponding average molar percentages, one or more of the following free amino acids: 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 enlargement composition” or “T cell enlargement composition” refers to the following free amino acids in their corresponding average molar percentages: histidine (approx. 0.78%), asparagine (approx. 0.4%), serine (approx. 1.6%), glutamine (approx. 67.01%), arginine (approx. 1.67%), glycine (approx. 1.72%), aspartic acid (approx. 1.00%), glutamic acid (approx. 1.93%), threonine (approx. It can be used interchangeably with media containing one or more of the following: 2.38%, alanine (approx. 1.11%), proline (approx. 1.49%), cysteine ​​(approx. 1.65%), lysine (approx. 2.84%), tyrosine (approx. 1.62%), methionine (approx. 0.85%), valine (approx. 3.45%), isoleucine (approx. 3.14%), leucine (approx. 3.3%), phenylalanine (approx. 1.64%), and tryptophan (approx. 0.37%).

[0074] As used herein, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with media containing one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with media containing one or more of the following: octanoic acid at concentrations of 0.9 mg / kg to 90 mg / kg including the endpoint; palmitic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoint; linoleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoint; oleic acid at concentrations of 0.2 mg / kg to 20 mg / kg including the endpoint; and sterols (mg / kg = parts per million) at concentrations of approximately 0.1 mg / kg to 10 mg / kg including the endpoint. In certain embodiments, the term “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following: 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 term “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following: 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 enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following: 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 approximately 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 enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium comprising one or more sterols and octanoic acid at concentrations of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at concentrations of 0.7 μmol / kg to 70 μmol / kg including endpoints; linoleic acid at concentrations of 0.75 μmol / kg to 75 μmol / kg including endpoints; oleic acid at concentrations of 0.75 μmol / kg to 75 μmol / kg including endpoints; and one or more sterols at concentrations of 0.25 μmol / kg to 25 μmol / kg including endpoints. In certain embodiments, the term “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following: 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 sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the term “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following: 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 terms “supplemented T cell enlargement composition” or “T cell enlargement composition” can be used interchangeably with a culture medium containing one or more of the following: 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, MgCl2, ClNa, glucose, and Ca(NO3)2 in any absolute or relative abundance or concentration, and 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 MgCl2, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO3)2, and 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 a supplement containing 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO3)2, and 20 mM HEPES and 75 mM Tris / HCl. The term "P3 buffer" can be used interchangeably with a buffer containing a supplementary substance that includes 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM glucose, 0.4 mM Ca(NO3)2, and 40 mM Na2HPO4 / NaH2PO4, pH 7.2.

[0076] As used herein, the terms “replenished RPMI-1640 medium” or “T-cell conditioned medium (TCCM)” refer to 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 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 in any absolute or relative amount or concentration.The terms "Replenished RPMI-1640 Medium" or "T-cell conditioned medium (TCCM)" refer to 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 It can be used interchangeably with media containing any absolute or relative amount or concentration of lysine, L-lysine HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine 2Na2H2O, 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.

[0077] As used herein, the terms “Replenished AIM-V” or “Replenished AIMV” medium refer to 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 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 in any absolute or relative amount or concentration.The term "Replenished AIM-V" or "Replenished AIMV" medium refers to 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- It can be used interchangeably with media containing isoleucine, L-leucine, L-lysine HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine 2Na2H2O, 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 in any absolute or relative amount or concentration.

[0078] As used herein, the term “ImmunoCult® medium” refers to 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 It can be used interchangeably with culture media containing one or more of the following in any absolute or relative amount or concentration: L-tyrosine disodium salt, L-valine, biotin, choline chloride, calcium D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol, anhydrous calcium chloride, anhydrous magnesium sulfate, 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 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, and L-tryptophan. It can be used interchangeably with culture media containing L-tyrosine disodium salt, L-valine, biotin, choline chloride, calcium D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol, anhydrous calcium chloride, anhydrous magnesium sulfate, potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, monobasic sodium phosphate, sodium selenite, D-glucose, HEPES, and sodium pyruvate in any absolute or relative abundance or concentration.

[0079] Modification of this disclosure SCM and / or T CM Modified T cells of this disclosure, including those mentioned above, can be incubated, cultured, grown, stored, or otherwise combined with a growth medium containing one or more inhibitor components of the PI3K pathway at any step of the procedure. Examples of 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 C3K). 18 H 14 Examples of GSK3β inhibitors include those containing N4O2. Examples of exemplary inhibitor components of the PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO®).

[0080] As used herein, the terms “electropermeation” and “nucleofection” are intended to describe alternative means for delivering the nucleic acids, transposons, vectors, or compositions of the Disclosure to cells by supplying electrical pulses to induce the cell membrane (cell membrane, nuclear membrane, or both) to become permeable or more permeable to the nucleic acids, transposons, vectors, or compositions of the Disclosure.

[0081] In certain embodiments of nucleofection, the method is carried out simultaneously in one or more cuvettes. In certain embodiments of nucleofection, the method is carried out simultaneously in two cuvettes. With respect to processes carried out on a larger scale for clinical or commercial application, for example, nucleofection can be carried out by performing many steps simultaneously in a large volume cassette. In certain embodiments of nucleofection, the incubation step includes incubating a composition containing a plurality of primary human T cells in a pre-warmed T cell augmentation composition. The duration of the incubation step may 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 / parts in between. The incubation step may be at least one day, two days, three days, four days, five days, six days, or seven days, or any number of days / parts in between. The incubation step may be at least one week. In certain embodiments of nucleofection, the incubation step is two days. In certain embodiments of nucleofection, the application 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. In certain embodiments, the application 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 produces the same number of electrical pulses, each having the same duration and intensity, as well as substantially similar intermediate pulse durations.In certain embodiments, the application step can be carried out using known electroporation / nucleofection devices, including, but not limited to, Lonza Amaxa, MaxCyte technology, BTX PulseAgile, and BioRad GenePulser. In certain embodiments of nucleofection, the application step may include applying at least one electrical pulse. In certain embodiments of nucleofection, the application step may include applying at least one electrical pulse sufficient to induce the cell membrane and / or nuclear membrane of a cell to become permeable to the composition of the Disclosure.

[0082] The quantities presented herein are illustrative and not limiting; however, the relationships between these quantities (e.g., ratios or relative abundances) can be used to modify the methods illustrated herein for larger-scale processes and manufacturing.

[0083] Modified T cells (e.g., T) as described herein SCM and / or T CM In certain embodiments of the method for producing the ) the activation replacement substance comprises one or more cytokines. The one or more cytokines may include, but are not limited to, any cytokines, including, 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] Modified T cells (e.g., T) as described herein SCM and / or T CMIn a particular embodiment of the method for producing ), the enhancement supplement comprises one or more cytokines. The one or more cytokines may include, but are not limited to, any cytokines, including, 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] Modified T cells (e.g., T) as described herein SCM and / or T CM In certain embodiments of the method for producing ) primary human T cells are naive T cells. Naive T cells may express CD45RA, CCR7 and CD62L. In certain embodiments, the method is applied to a cell population containing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between these naive T cells. In certain embodiments, modified T cells of the present disclosure SCM and / or T CM The efficiency of production can be increased by increasing the proportion or percentage of naive T cells in the cell population to which the method of this disclosure is applied.

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

[0087] Modification of this disclosure SCM and / or T CMIn a particular embodiment of the method for producing these cells, primary human T cells express one or more of the following: CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ.

[0088] Modification of this disclosure SCM and / or T CM In a specific embodiment of the method for producing primary human T cells, naive T cells (modified T cells) are used. N ) and modified T N It expresses one or more of CD45RA, CCR7, and CD62L. Modified T of the present disclosure SCM and / or T CM In a specific embodiment of the method for producing modified T cells, primary human T cells are used. SCM T memory stem cells (modified T) SCM ) and modified T SCM It expresses one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. Modified T of the present disclosure SCM and / or T CM In a specific embodiment of the method for producing primary human T cells, central memory T cells (modified T cells) are used. CM ) and modified T CM It expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. Modified T of the present disclosure SCM and / or T CM In a specific embodiment of the method for producing them, primary human T cells are effector memory T cells (modified T cells). EM ) and modified T EM It expresses one or more of CD45RO, CD95, and IL-2Rβ. Modified T of the present disclosure SCM and / or T CM In a specific embodiment of the method for producing them, primary human T cells are effector T cells (modified T cells). EFF ) and modified T EFF It expresses one or more of the following: CD45RA, CD95, and IL-2Rβ.

[0089] Modification of this disclosure SCMand / or T CM In certain embodiments of the method for producing primary human T cells, primary human T cells may express CD4 and / or CD8. In certain embodiments, primary human T cells may express CD4 and / or CD8 in various ratios. In certain embodiments, primary human T cells may express CD4 and / or CD8, which are not naturally occurring, in various ratios. In certain embodiments, primary human T cells expressing CD4 and / or CD8, which may not be naturally occurring, in various ratios constitute a heterogeneous cell population.

[0090] Modification of this disclosure SCM and / or T CM In certain embodiments of the method for producing primary human 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 the cellular components of blood (e.g., red blood cells, white blood cells, and platelets) obtained from or prepared from the circulating pool of blood and not segregated within the lymphatic system, spleen, liver, or bone marrow. Umbilical cord blood is distinct from peripheral blood and blood segregated within 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 the blood that remains in the placenta after birth and is attached to the umbilical cord. Cord blood often contains stem cells, including hematopoietic cells.

[0091] The primary human T cells in this disclosure may 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 disclosed herein, the method is modified T SCM or T CMThe method further includes the step of introducing a composition comprising a genome editing construct or composition into cells. In certain embodiments, the genome editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR)-related protein 9 (Cas9) DNA endonuclease. In certain embodiments, the genome editing construct comprises a DNA-binding domain and an IIS-type 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 an IIS-type endonuclease, and the expressed DNA-binding domain and the expressed IIS-type endonuclease are non-covalently linked. In certain embodiments, including embodiments in which the genome editing construct comprises a DNA-binding domain and an IIS-type endonuclease, the genome editing construct comprises a sequence derived from Cas9 endonuclease. In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the sequence derived from Cas9 endonuclease is the DNA-binding domain. In certain embodiments, including embodiments in which the sequence derived from Cas9 endonuclease is the DNA-binding domain, the sequence derived from Cas9 endonuclease encodes inactive Cas9. In certain embodiments, including embodiments in which the sequence derived from Cas9 endonuclease is the DNA-binding domain, the sequence derived from Cas9 endonuclease encodes a truncated Cas9. In certain embodiments, the sequence derived from Cas9 endonuclease includes an amino acid substitution of alanine (A) with aspartic acid (D) at position 10 (D10A). In certain embodiments, the sequence derived from Cas9 endonuclease includes or further includes an amino acid substitution of alanine (A) with histidine (H) at position 840 (H840A). In certain embodiments, the sequence derived from Cas9 endonuclease includes inactivated Cas9 (dCas9) (SEQ ID NO: 33).In certain embodiments, the sequence derived from Cas9 endonuclease includes an amino acid substitution of alanine (A) with asparagine (N) at position 580 (N580A). In certain embodiments, the sequence derived from Cas9 endonuclease includes a truncated and inactivated Cas9 (dSaCas9) (SEQ ID NO: 32). In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the genome editing construct includes a sequence derived from a transcription activator-like effector nuclease (TALEN). In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the sequence derived from TALEN is the DNA-binding domain. In certain embodiments, the genome editing construct includes TALEN. In certain embodiments, including embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease, the genome editing construct includes a sequence derived from a zinc finger nuclease (ZFN). Embodiments in which the genome editing construct includes a DNA-binding domain and an IIS-type endonuclease are included, and in certain embodiments, the sequence derived from the ZFN is the DNA-binding domain. In certain embodiments, the genome editing construct includes a zinc finger nuclease (ZFN).

[0093] Modification of this disclosure SCM and / or T CM The method for creating cells involves a larger number or a larger proportion of modified T cells. SCM and / or T CM The cells can be optimized to produce more cells. For example, the population of cells subjected to the method of the disclosure can be enriched to contain an increased number or a larger proportion of naive T cells. As the number and / or proportion of naive T cells in the population of T cells subjected to the method of the disclosure increases, the modified T cells produced will be more efficient. SCM and / or T CMThe number and / or proportion of cells also increases. Instead, or in addition, as the length or duration of time required to perform the disclosed method decreases, the modified T of the disclosed method produced by said method SCM and / or T CM The number and / or proportion of cells increases. The length or duration of time required to precede the method of disclosure, or the “manufacturing period,” may also be referred to as the “out-of-life period” of the T cells subjected to the method of disclosure.

[0094] In certain embodiments of the method for producing modified T cells according to the Disclosure, primary human T cells express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, primary human T cells express naive T cells (T N ) and T N It expresses one or more of CD45RA, CCR7, and CD62L. In certain embodiments, primary human T cells are T memory stem cells (T SCM ) and T SCM It expresses one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments, primary human T cells are central memory T cells (T CM ) and T CM These cells express one or more of the following: CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, primary human T cells are effector memory T cells (T EM ) and EM These cells 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 These cells express one or more of CD45RA, CD95, and IL-2Rβ. In certain embodiments, primary human T cells express CD4 and / or CD8.

[0095] This disclosure is a modified version of T created in the manner of this disclosure. SCM The present disclosure provides a composition containing [a specific compound]. This disclosure provides a modified T prepared by the method of this disclosure. CM The present disclosure provides a composition containing [a specific compound]. This disclosure provides a modified T prepared by the method of this disclosure. SCM and modified T CM A composition containing is provided. Modified T prepared by the method of this disclosure SCM and modified T CM In certain embodiments of a composition containing multiple T SCM This may constitute 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. Modified T prepared by the method of this disclosure SCM and modified T CM In certain embodiments of a composition containing multiple T CM It may constitute 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] This disclosure relates to a modified T made by the method of this disclosure for manufacturing a pharmaceutical product for treating subjects that require it. SCM and / or T CM The use of a composition containing the above is provided. In certain embodiments of this use, modified T SCM and / or T CM It is self-derived. In certain embodiments of this use, modified T SCM and / or T CMThey are 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, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor includes one or more mutations compared to the wild-type T cell receptor. In certain embodiments, in particular 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 CARTyrin. In certain embodiments, the CAR includes one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

[0097] This disclosure relates to a method for treating a disease or disorder in a subject that requires it, and a modified T made by the method of this disclosure. SCM and / or T CM The present invention provides a method comprising the step of administering a therapeutically effective amount of a composition containing to a target. In certain embodiments of this method, modified T SCM and / or T CM It is self-derived. In a particular embodiment of this method, modified T SCM and / or T CMThey are 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, in particular embodiments where the T cell receptor is not naturally occurring, the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor. In certain embodiments, in particular 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 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, bacterial antigen, yeast antigen, or microbial antigen. In certain embodiments, the disease or disorder is caused by a lack of activity or insufficient quantity of a secreted protein. In certain embodiments, the disease or disorder is treated by replacing the activity of a therapeutic protein or by increasing the quantity of a therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein. In certain embodiments, the activity of a secreted protein is lacking or insufficient in a local area of ​​the body. In certain embodiments, the local area of ​​the body is accessible by native T cells or modified T cells. In certain embodiments, the modified T cells are produced in vivo, ex vivo, in vitro, or in situ. [Brief explanation of the drawing]

[0098] [Figure 1]Figure 1 is a series of plots showing the emergence of the CAR-TSCM phenotype on 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 two plots below.

[0099] [Figure 2] Figure 2 shows a series of plots indicating the purity of CAR-TSCM cells prepared by the method of Example 1 on day 19. The population of CAR-TSCM cells prepared by the method described in Example 1 on day 19 contained neither B cells nor lymphocytes. The majority of cells were CD3+ T cells. Only 1.1% were natural killer cells, and 1.7% were natural killer T cells.

[0100] [Figure 3] Figure 3 is a plot showing that the majority of T cells produced by the method described in Example 1 express CARTyrin on day 11.

[0101] [Figure 4] Figure 4 is a series of plots showing the enrichment of the CAR-TSCM phenotype at day 19 using 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 two plots below.

[0102] [Figure 5]Figure 5 shows a series of plots demonstrating the absence of T cell exhaustion at day 19 in the method described in Example 1. At day 19, the cell population produced by this method does not express PD1, a marker of T cell activation and exhaustion. These cells expressing CARTyrin reached a quiescent state almost successfully after production. These cells do not show signs of antigen-independent (tonic) signaling, which otherwise drives high levels of PD1 expression. It is hypothesized that tonic signaling is triggered by certain CAR molecules that lead to early exhaustion and reduce the efficacy of CAR T cell therapy.

[0103] [Figure 6A] Figure 6A shows a series of plots showing T cells transposed using a plasmid containing an inducible caspase polypeptide (safety switch, "iC9"), CARTyrin (anti-BCMA), and a sequence encoding a transposon containing a sequence encoding a selection 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 hyperactive transposase ("Super piggyBac") or wild-type piggyBac transposase.

[0104] [Figure 6B] Figure 6B shows a series of plots illustrating T cells transposed using 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 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 transposase using WT piggyBac transposase and transposase using hyperactive piggyBac transposase. T cells exposed to hyperactive piggyBac transposase (Super piggyBac transposase) were transformed at a rate four times higher than those exposed to WT transposase.

[0106] [Figure 6D] Figure 6D is a table showing the percentage of transformed T cells 5 days after nucleofection, resulting from transposition using WT piggyBac transposase and, in comparison, transposition using hyperactive piggyBac transposase. T cells exposed to hyperactive piggyBac transposase (Super piggyBac transposase) were transformed at a much higher rate than those exposed to WT transposase.

[0107] [Figure 7] Figure 7 is a graph showing the phenotypic differences between CAR+ T cells generated by piggyBac® and CAR+ T cells generated by lentivirus. CAR+ T cells were generated using either piggyBac-mediated translocation or lentivirus-mediated transduction. Human pan-T cells were translocated using piggyBac encoding CAR, stimulated with anti-CD3 / CD28 beads two days after translocation to increase cell volume, and tested 19 days after translocation. For lentivirus-mediated generation, pan-T cells were stimulated with CD3 / CD28 beads, transduced with a lentivirus encoding CAR (MOI 5), increased cell volume, 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 and 8B are pairs of graphs showing preferential metastasis in naive T cells using piggyBac®. Human pan-T cells were sorted into naive (CD62L+CD45RA+) subsets, Tcm (CD62L+CD45RA-) subsets, Tem (CD62L-CD45RA-) subsets, and Teff (CD62L-CD45RA+) subsets (using a BD FACSAria II flow cytometer). Each sorted subset was either metastasized using piggyBac-GFP or transduced using lentivirus-GFP. For the former, each sorted subset was metastasized using PiggyBac-GFP, stimulated with anti-CD3 / CD28 beads 2 days after metastasis to increase cell volume, and tested 19 days after metastasis. For the latter, selected subsets were stimulated with CD3 / CD28 beads, transduced and amplified using a lentivirus encoding GFP (MOI 5), and tested 19 days after stimulation. n=3 donors. [Figure 8B] Figures 8A and 8B are pairs of graphs showing preferential metastasis in naive T cells using piggyBac®. Human pan-T cells were sorted into naive (CD62L+CD45RA+) subsets, Tcm (CD62L+CD45RA-) subsets, Tem (CD62L-CD45RA-) subsets, and Teff (CD62L-CD45RA+) subsets (using a BD FACSAria II flow cytometer). Each sorted subset was either metastasized using piggyBac-GFP or transduced using lentivirus-GFP. For the former, each sorted subset was metastasized using PiggyBac-GFP, stimulated with anti-CD3 / CD28 beads 2 days after metastasis to increase cell volume, and tested 19 days after metastasis. For the latter, selected subsets were stimulated with CD3 / CD28 beads, transduced and amplified using a lentivirus encoding GFP (MOI 5), and tested 19 days after stimulation. n=3 donors.

[0109] [Figure 9] Figure 9 is a pair of graphs demonstrating that the piggyBac® manufacturing process yields high levels of TSCM in samples derived from multiple myeloma (MM) patients, even when naive T cells are scarce. T cells from MM patients (triangles) and T cells from healthy donors (circles) were characterized for memory marker expression by flow cytometry before (left) and after (right) the Poseida manufacturing process. CD45RA and CD62L expression were assessed by FACS for MM patients and healthy donors and are plotted. MM patient-derived T cells generally have a lower frequency of naive and TSCM cells but a higher frequency of Teff, which differs from healthy, normal donors where the opposite is known. Regardless of the input frequency of naive and Tscm from different MM patients, the production of P-BCMA-101 using the Poseida manufacturing process resulted in a product exhibiting high levels of CD8+ Tscm (E). This also applies to MM patients who received active treatment (red triangles).

[0110] [Figure 10-1]Figure 10 shows a series of fluorescence-activated cell selection (FAC) plots characterizing T cell and TSCM cell markers in human pan-T cells transformed using the Sleeping Beauty (SB100x) transposition system and the method of this disclosure. Sleeping Beauty (SB100x) transposition using the Poseida manufacturing process primarily yields the Tscm phenotype. As shown, transposition was induced in human pan-T cells using either 1 μg of the Sleeping Beauty transposon plasmid or the piggyBac transposon plasmid, and either SB100x or SPB mRNA, respectively. After transposition, cells were grown ex vivo, and all non-transposition cells were depleted using the Poseida manufactured drug selection system. After 18 days of culture, cells were stained with phenotypic markers CD4, CD8, CD45RA, and CD62L. The stem cell memory phenotype (Tscm) is defined by CD45RA and CD62L double-positive cells, which constitute >65% of the cells in the total sample. All panels in the column share common x and y axis parameters. Each row, from top to bottom, shows data from T cells that induced transposition using 2.5 micrograms (μg) of the Sleeping Beauty transposon SB100x (top), data from T cells that induced transposition using 5 μg of SB100x (second from the top), data from T cells that induced transposition using 10 μg of SB100x (third from the top), data from T cells that induced transposition using 5 μg of the piggyBac transposon P-BCMA-101 (second from the bottom), and at the bottom, data from T cells that induced transposition using an unstained control. 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 increments of 50k. The x-axis of the third column, from left to right, shows CD8 expression, with readings marked from 0 to 105, increasing in powers of 10. The rightmost column shows CD62L expression, with readings marked from 0 to 105, increasing in powers of 10. The y-axis of the first column shows side scattering (SSC) in units of 0 to 250k, in increments of 50k.The y-axis of the second column, from left to right, shows the expression of 7-aminoactinomycin D (7AAD), a marker of cell viability, 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 rightmost 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 pathway that leads to blood clotting. For example, the endogenous coagulation pathway is activated by contact activation due to endothelial damage. For example, the exogenous coagulation pathway is activated by tissue factor after trauma. Both pathways converge on the conversion of prothrombin to thrombin, which catalyzes the conversion of fibrinogen to fibrin. Fibrin polymerizes with platelets to form a blood clot. In the absence of factor IX (circled), coagulation is incomplete. 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 at a frequency of approximately 1 in 25,000 to 30,000 people. 60 percent of hemophilia B cases are severe. Less than 1 percent of individuals with hemophilia B have normal FIX levels. Prior to the compositions and methods of this disclosure, the standard treatment for hemophilia B involved infusions 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 this disclosure can be maintained in humans for several decades.

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

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

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

[0115] [Figure 14A] Figures 14A–E are a series of plasmid maps for site-specific integration to the AAVS1 site using either HR or MMEJ and the corresponding sequences. A) Site-specific (AAVS1) homologous recombination (HR), B) Site-specific (AAVS1) microhomology-mediated end-joining (MMEJ) recombination, and C) Donor plasmids for testing stable integration into the genome of human pan-T cells by TTAA-specific piggyBac® transposation. For the HR and MMEJ donor plasmids, the GFP-2A-DHFR gene expression cassette was flanked by homology arms for CRISPR / Cas9 target site and 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, as well as F, show sequence numbers 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 the donor plasmid alone were included as a control. 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 evaluated by flow cytometry 3 days after nucleofection. The data are summarized in the 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 methotrexate addition using the DHFR selection gene encoded in the Bicistron GFP-2A-DHFR integration cassette. On day 11 after nucleofection, the percentage of GFP+ cells was evaluated by flow cytometry, and the data are summarized in a bar graph. GFP+ cells were highly enriched by selection in pan-T cells that received transfer reagents, RNP plus HR, or MMEJ donor plasmids, but not in T cells that received the donor plasmid alone.

[0118] [Figure 17A]Figures 17A-C are a series of graphs showing the phenotypes 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 in 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 the bar graph. Using the piggyBac™ transposon system, or combinations of HR and Cas9 RNP, and MMEJ and Cas9 RNP, GFP+Tscm-enriched populations were achieved in all cases. C) The total number of pan-T cells was analyzed 13 days after nucleofection, and the data are summarized in bar graphs. [Figure 17B] Same as above [Figure 17C] Same as above

[0119] [Figure 18]Figures 18A and 18B are pairs of photographs of gel electrophoresis results showing site-specific integration into the AAVS1 site. Cells selected from each group were collected, genomic DNA was 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 were used to individually amplify the 5' 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 500bp homologous arm of the 5' AAVS-3r CTGCACCACGTGATGTCCTC (SEQ ID NO: 37), thereby obtaining a 0.73kb DNA fragment for either HR or MMEJ) and the 3' junction (one primer primed the poly(A) signaling region SV40pA-1r GTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38), and the other primer primed the AAVS1 region beyond the 500bp 5' homologous arm AAVS-2f CTGGGGACTCTTTAAGGAAAGAAG (SEQ ID NO: 39), thereby obtaining a 0.76kb DNA fragment for either HR or MMEJ). PCR products were presented on an agarose gel. Nonspecific bands in the HR sample are the result of only one round of PCR and are likely to be resolved by performing additional rounds. [Modes for carrying out the invention]

[0120] Detailed explanation This disclosure describes how to create stem cell memory T cells (T) expressing human chimeric antigen receptors (CARs) using the piggyBac® Transposon System, thereby enabling the creation of T cells with potent CAR activity. SCM )(In this specification, CAR-T SCM The present disclosure provides a method for producing CAR-T (referred to as) under conditions in which it is stored or induced. SCM Compositions containing ≥60% CAR-T SCMThis includes and exhibits a distinct functional profile consistent with this T cell subset. Other T cell subsets found in the compositions of this 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 highly differentiated effector CAR-T cells (CAR-T TE ) are mentioned. N These are the parent progenitor cells, and T SCM It directly causes T CM A linear differentiation pathway that directly generates T cells: naive T cells (T N )>T SCM >T CM >T EM >T E >T TE These cells may be involved in the generation of these cells. CAR-T SCM CARTyrin-T SCM and / or VCAR-T SCM A composition containing may contain one or more of each parental CAR-T cell subset, and CAR-T SCM The most abundant (for example, T SCM >T CM >T EM >T E >T TE The absolute amount / abundance and relative proportion of each parental T cell subset may vary between patient blood samples and naturally occurring cell populations, and naturally occurring cell populations are T SCM CAR-T, which may be present in high concentrations and / or proportions, is not naturally present in patient treatment for disease and cancer. SCM The compositions of this disclosure, including [the specified element], are more potent and effective.

[0121] Immunotherapy using chimeric antigen receptor (CAR) T cells is emerging as an exciting treatment approach for cancer. Autologous CAR-modified T cells that target tumor-associated antigens (Ag) can lead to robust tumor death, and in some cases, CD19 +Complete remission of hematological malignancies is achieved. Unlike conventional biological agents and chemotherapy drugs, CAR-T cells have the ability to rapidly regenerate upon recognition of Ag, thereby potentially eliminating the need for repeated treatments. To achieve this, CAR-T cells must not only initially drive tumor destruction but also be sustained in the patient as a stable population of viable memory T cells to prevent potential cancer recurrence. Therefore, CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as early memory cells, especially stem cell memory (T) cells, are needed. SCM The focus of efforts is on developing CAR-T products containing ) stem cells. Stem cell-like CAR-T cells have the greatest capacity for self-renewal and central memory T cells (T CM ), effector memory T cells (T EM ) and effector T cells (T E It exhibits multipotency that elicits ) results in better tumor eradication and long-term CAR-T engraftment.

[0122] The CAR-T of this disclosure SCM It may contain a centintin-based CAR called CARTyrin (therefore, the cell is CARTyrin-T SCM (It can be referred to as [this]). Centilin is an alternative scaffold molecule based on the human consensus tenasin FN3 domain, smaller than scFv molecules, can be selectively sized in terms of monomericity which is advantageous for stability, and also reduces the potential for tonic signaling in CAR molecules. The CARTyrin of this 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] The CAR-T of this disclosure SCM This may include VCARs, which are VHH-based CARs (therefore, these cells are VCAR-T SCM(This may be referred to as) The VCAR of this disclosure can be introduced into T cells using a plasmid DNA transposon encoding 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 methods of this disclosure, the piggyBac®(PB) Transposon System, referred to as Super piggyBac®(SPB), can be used to stably incorporate antigen-specific CARTyrin or VCAR (including cancer antigen-specific CARTyrin or VCAR) into quiescent pan-T cells, in which the transposon is co-delivered with the mRNA transposase enzyme in a single electroporation reaction (even though the transposon and transposase are contained in separate compositions until they are introduced into the cells). As a result of delivery of piggyBac® transposons to as-is quiescent primary human pan-T cells, 20–30% of the cells exhibited stable incorporation and expression of the gene delivered by PB. Unexpectedly, the majority of these modified CARTyrin-expressing T cells were identified as stem memory T cells (T SCM The cells were positive for the 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 growth, modified CARTyrin-expressing T cells positive for CD62L and CD45RA were activated by stimulation with CD3 and CD28. As a result of CD3 and CD28 stimulation, >60% of the CARTyrin+ T cells exhibited the stem cell memory phenotype. Furthermore, these cells expressing CARTyrin specific to cancer antigens were well capable of exhibiting potent antitumor effector function.

[0125] To determine whether the PB system directly contributes to the enhancement of stem-like marker expression, we compared the phenotypes of CAR-T cells generated by either PB-mediated translocation or lentiviral (LV) transduction. To do this, we constructed a novel vector by subcloning the CARTyrin transgene into a common LV construct for viral production. After introducing CARTyrin into original quiescent T cells via PB-mediated translocation or LV transduction, the phenotypes of CARTyrin + Cells were enlarged and then returned to a resting state. Various phenotypic and functional characteristics were measured, including dynamic analysis of memory and exhaustion-related markers, secondary proliferation in response to homeostatic cytokines or tumor-associated Ag, cytokine production, and lytic capacity in response to target tumor cells. CARTyrin cells metastasized by PB were also examined. + Unlike T cells, CARTyrin underwent transduction via LV. + In T cells, no enhancement of the memory phenotype was observed. Furthermore, cells metastasized by PB showed comparable or greater ability in secondary proliferation and target tumor cell death. In summary, these data suggest that CAR-T cells generated by PB metastasis primarily exhibit the highly desirable product phenotype in the CAR-T field. SCM It is proven that these are cells. Furthermore, these CARTyrin + T cells exhibit potent antitumor activity and may be less susceptible to tonic signaling and functional exhaustion. The use of centiriin-based CARs can also lead to the generation of longer-lasting cells in vivo. Chimeric antigen receptor

[0126] This disclosure provides a chimeric antigen receptor (CAR) comprising (a) an external domain comprising an antigen recognition region, wherein the antigen recognition region comprises 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, such that a bispecific or tandem CAR is produced. In certain embodiments, the antigen recognition region may comprise three sequences that each specifically bind to an antigen, such that a triplicate CAR is produced. In certain embodiments, the external domain may further comprise a signal peptide. Alternatively, or in addition thereto, in certain embodiments, the external domain may further comprise a hinge between the antigen recognition region and the transmembrane domain. Sequences that each specifically bind to an antigen include, but are not limited to, single-chain antibodies (e.g., scFv), sequences comprising one or more fragments of an antibody (e.g., VHH, referred to as VCAR in the case of CAR), antibody mimes, and centintin (referred to as CARTyrin in the case of CAR).

[0127] In certain embodiments of the CARs of this disclosure, the signal peptide may include 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 the CARs of this disclosure, the signal peptide may include a sequence encoding a human CD8α signal peptide. The human CD8α signal peptide may include an amino acid sequence containing MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide may include an amino acid sequence containing MALPVTALLLPLALLLHAARP (SEQ ID NO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide can be encoded by a nucleic acid sequence containing atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 9).

[0128] In certain embodiments of the CARs of this disclosure, the transmembrane domain may include 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 this disclosure, the transmembrane domain may include a sequence encoding a human CD8α transmembrane domain. The CD8α transmembrane domain may include an amino acid sequence containing IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8α transmembrane domain may be encoded by a nucleic acid sequence containing atctacatttgggcaccactggccgggacctgtggagctgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 11).

[0129] In certain embodiments of the CAR of this disclosure, the end domain may include a human CD3ζ end domain.

[0130] In certain embodiments of the CARs of this disclosure, at least one co-stimulatory domain may include human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segments, or any combination thereof. In certain embodiments of the CARs of this disclosure, at least one co-stimulatory domain may include CD28 and / or a 4-1BB co-stimulatory domain. The CD28 co-stimulatory domain may contain an amino acid sequence containing RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12). The CD28 co-stimulatory domain can be encoded by a nucleic acid sequence containing cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccccggagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 13).The 4-1BB co-stimulatory domain may contain an amino acid sequence including KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB co-stimulatory domain may be encoded by a nucleic acid sequence including aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BB co-stimulatory domain may be located between the transmembrane domain and the CD28 co-stimulatory domain.

[0131] In certain embodiments of the CARs of this disclosure, the hinge may include sequences derived from human CD8α, IgG4, and / or CD4 sequences. In certain embodiments of the CARs of this disclosure, the hinge may include sequences derived from human CD8α sequences. The hinge may include sequences that are at least 70%, 80%, 90%, 95%, or 99% identical to a human CD8α amino acid sequence containing TTTPARPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or an amino acid sequence containing TTTPARPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16). The human CD8α hinge amino acid sequence can be encoded by a nucleic acid sequence containing actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).

[0132] This disclosure provides compositions comprising the CAR of this disclosure and at least one pharmaceutically acceptable carrier.

[0133] This disclosure provides transposons containing the CARs of this disclosure. The transposons of this disclosure are maintained as episomes or incorporated into the genome of recombinant / modified cells. The transposons may be part of a two-component piggyBac system that utilizes transposons and transposases to enhance nonviral gene transfer.

[0134] The transposons of this disclosure may include select genes for identifying, enriching, and / or isolating cells that express the transposons. Exemplary select genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for cell viability and survival. Exemplary select genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for conferring resistance to drug attack to which the cell is susceptible (or may be lethal) in the absence of the gene product encoded by the select gene. Exemplary select genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for cell viability and / or survival in cell media lacking one or more nutrients essential for survival in the absence of the select gene. Examples of select genes, though not limited to these, include neo (which confers resistance to neomycin), DHFR (which encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (which encodes thymidylate synthetase), MGMT (which encodes O(6)-methylguanine-DNA methyltransferase), the multidrug resistance gene (MDR1), ALDH1 (which encodes member A1 of the aldehyde dehydrogenase 1 family), FRANCF, RAD51C (which encodes RAD51 paralog C), GCS (which encodes glucosylceramide synthase), and NKX2.2 (which encodes NK2 homeobox 2).

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

[0136] The transposons of this disclosure may include a first self-cleaving peptide and a second self-cleaving peptide, the first self-cleaving peptide being located, for example, one or more upstream of a sequence that specifically binds to the antigen of this disclosure, and the second self-cleaving peptide being located, for example, one or more downstream of a sequence that specifically binds to the antigen of this disclosure. The first and / or second self-cleaving peptides 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 containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). GSG-T2A peptides may contain an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). GSG-T2A peptides may contain a nucleic acid sequence including ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). E2A peptides may contain an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The GSG-E2A peptide may contain an amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).F2A peptides may contain an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity with an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 023). GSG-F2A peptides may contain an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity with an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). P2A peptides may contain an amino acid sequence including ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). GSG-P2A peptides may contain an amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0137] This disclosure provides compositions comprising the transposons of this disclosure. In certain embodiments, a method for introducing a composition may further comprise a composition comprising a plasmid containing a sequence encoding a transposase enzyme. The sequence encoding the transposase enzyme may be an mRNA sequence.

[0138] The transposons of this disclosure may include the piggyBac transposon. The transposase enzymes of this disclosure may include the piggyBac transposase or a compatible enzyme.

[0139] This disclosure provides vectors comprising CARs of this disclosure. In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.

[0140] The viral vectors of this disclosure may include sequences isolated from or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vectors may include sequences isolated from or derived from adeno-associated viruses (AAVs). The viral vectors may include recombinant AAVs (rAAVs). The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include two or more terminal inversion (ITR) sequences located in cis adjacent to one or more sequences that specifically bind to an antigen. The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include, but are not limited to, self-complementary AAVs (scAAVs) and AAV hybrids (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8) containing the genome of one serotype and the capsid of another serotype. The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include, but are not limited to, rAAV-LK03.

[0141] The viral vectors of this disclosure may include a select gene. The select gene may encode a gene product essential for cell viability and survival. The select gene may encode a gene product essential for cell viability and survival when attacked by selective cell culture conditions. The selective cell culture conditions may include compounds that are harmful to cell viability or survival, and the gene product confers resistance to such compounds. Examples of select genes in this disclosure include, but are not limited to, neo (which confers resistance to neomycin), DHFR (which encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (which encodes thymidylate synthetase), MGMT (which encodes O(6)-methylguanine-DNA methyltransferase), the multidrug resistance gene (MDR1), ALDH1 (which encodes member A1 of the aldehyde dehydrogenase 1 family), FRANCF, RAD51C (which encodes RAD51 paralog C), GCS (which encodes glucosylceramide synthase), NKX2.2 (which encodes NK2 homeobox 2), or any combination thereof.

[0142] The viral vectors of this disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide, where the self-cleaving peptide is located between the CAR and the selected gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, where 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 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 containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). GSG-T2A peptides may contain an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). GSG-T2A peptides may contain a nucleic acid sequence including ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). E2A peptides may contain an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The GSG-E2A peptide may contain an amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).F2A peptides may contain an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). GSG-F2A peptides may contain an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). P2A peptides may contain an amino acid sequence containing ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The GSG-P2A peptide may contain an amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0143] This disclosure provides a vector containing the CAR of this disclosure. In certain embodiments, the vector is an mRNA vector. The vector may be a recombinant mRNA vector. The T cells of this disclosure can be enlarged before contacting the T cells with the CAR-containing mRNA vector of this disclosure. The T cells containing the mRNA vector, the modified T cells, can then be administered to the target.

[0144] This disclosure provides vectors comprising CARs of the Disclosure. In certain embodiments, the vector is a nanoparticle. Exemplary nanoparticle vectors of the Disclosure include, but are not limited to, nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides, or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymersomes), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold), or any combination thereof. Nanoparticle vectors can be transported passively or actively across cell membranes.

[0145] The nanoparticle vectors of this disclosure may include a select gene. The select gene may encode a gene product essential for cell viability and survival. The select gene may encode a gene product essential for cell viability and survival when attacked by selective cell culture conditions. The selective cell culture conditions may include compounds that are harmful to cell viability or survival, and the gene product confers resistance to such compounds. Examples of select genes in this disclosure include, but are not limited to, neo (which confers resistance to neomycin), DHFR (which encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (which encodes thymidylate synthetase), MGMT (which encodes O(6)-methylguanine-DNA methyltransferase), the multidrug resistance gene (MDR1), ALDH1 (which encodes member A1 of the aldehyde dehydrogenase 1 family), FRANCF, RAD51C (which encodes RAD51 paralog C), GCS (which encodes glucosylceramide synthase), NKX2.2 (which encodes NK2 homeobox 2), or any combination thereof.

[0146] The nanoparticle vectors of this disclosure may comprise at least one self-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, where 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, where a first self-cleaving peptide is located upstream of the CAR and a second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, where a first self-cleaving peptide is located between the CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, where a first self-cleaving peptide is located between the CAR and the nanoparticle and a second self-cleaving peptide is located downstream of the CAR, for example, between the CAR and the selection gene. Self-cleaving peptides may include, for example, T2A peptides, GSG-T2A peptides, E2A peptides, GSG-E2A peptides, F2A peptides, GSG-F2A peptides, P2A peptides, or GSG-P2A peptides. T2A peptides may include an amino acid sequence containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). GSG-T2A peptides may include an amino acid sequence containing GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). The GSG-T2A peptide may contain a nucleic acid sequence including ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20).E2A peptides may contain an amino acid sequence containing QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). GSG-E2A peptides may contain an amino acid sequence containing GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). F2A peptides may contain an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). The GSG-F2A peptide may contain an amino acid sequence including GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The P2A peptide may contain an amino acid sequence including ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The GSG-P2A peptide may contain an amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0147] This disclosure provides compositions comprising the vectors of this disclosure. CARTyrin

[0148] This disclosure provides a chimeric antigen receptor (CAR) comprising (a) an external domain comprising an antigen-recognition region, wherein the antigen-recognition region comprises at least one centinlin; (b) a transmembrane domain; and (c) an endodomain comprising at least one costimulatory domain. As used throughout this disclosure, a centinlin-containing CAR is referred to as CARTyrin. In certain embodiments, the antigen-recognition region may comprise two centinlins so as to result in a bispecific or tandem CAR. In certain embodiments, the antigen-recognition region may comprise three centinlins so as to result in a triplicate CAR. In certain embodiments, the external domain may further comprise a signal peptide. Alternatively, or in addition thereto, in certain embodiments, the external domain may further comprise a hinge between the antigen-recognition region and the transmembrane domain.

[0149] This disclosure provides a chimeric antigen receptor (CAR) comprising (a) an external domain including an antigen recognition region, wherein the antigen recognition region includes at least one protein scaffold or antibody mimetic; (b) a transmembrane domain; and (c) an endodomain including at least one costimulatory domain. In certain embodiments, the antigen recognition region may include two protein scaffolds or antibody mimetic so as to result in a bispecific or tandem CAR. In certain embodiments, the antigen recognition region may include three protein scaffolds or antibody mimetic so as to result in a triplicate CAR. In certain embodiments, the external domain may further include a signal peptide. Alternatively, or in addition thereto, in certain embodiments, the external domain may further include a hinge between the antigen recognition region and the transmembrane domain.

[0150] In certain embodiments of the CARs of this disclosure, the signal peptide may include 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 the CARs of this disclosure, the signal peptide may include a sequence encoding a human CD8α signal peptide. The human CD8α signal peptide may include an amino acid sequence containing MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide may include an amino acid sequence containing MALPVTALLLPLALLLHAARP (SEQ ID NO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing MALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide can be encoded by a nucleic acid sequence containing atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 9).

[0151] In certain embodiments of the CARs of this disclosure, the transmembrane domain may include 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 this disclosure, the transmembrane domain may include a sequence encoding a human CD8α transmembrane domain. The CD8α transmembrane domain may include an amino acid sequence containing IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10). The CD8α transmembrane domain may be encoded by a nucleic acid sequence containing atctacatttgggcaccactggccgggacctgtggagctgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 11).

[0152] In certain embodiments of the CAR of this disclosure, the end domain may include a human CD3ζ end domain.

[0153] In certain embodiments of the CARs of this disclosure, at least one co-stimulatory domain may include human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segments, or any combination thereof. In certain embodiments of the CARs of this disclosure, at least one co-stimulatory domain may include CD28 and / or a 4-1BB co-stimulatory domain. The CD28 co-stimulatory domain may contain an amino acid sequence containing RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12). The CD28 co-stimulatory domain can be encoded by a nucleic acid sequence containing cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccccggagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 13).The 4-1BB co-stimulatory domain may contain an amino acid sequence including KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BB co-stimulatory domain may be encoded by a nucleic acid sequence including aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BB co-stimulatory domain may be located between the transmembrane domain and the CD28 co-stimulatory domain.

[0154] In certain embodiments of the CARs of this disclosure, the hinge may include sequences derived from human CD8α, IgG4, and / or CD4 sequences. In certain embodiments of the CARs of this disclosure, the hinge may include sequences derived from human CD8α sequences. The hinge may include sequences that are at least 70%, 80%, 90%, 95%, or 99% identical to a human CD8α amino acid sequence containing TTTPARPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or an amino acid sequence containing TTTPARPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16). The human CD8α hinge amino acid sequence can be encoded by a nucleic acid sequence containing actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).

[0155] The centinlin of this disclosure may include a protein scaffold capable of specifically binding to an antigen. The centinlin of this disclosure may include a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, 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 include LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). The consensus sequence can be encoded by a nucleic acid sequence containing atgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacgcagccttcgatagttttatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtccgacagtgccagggtccgaacgctcttatgacctgacagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccgcaatcttcaccaca (SEQ ID NO: 3).The consensus sequence may be modified at one or more positions within any combination of (a) to (f): (a) an AB loop containing or consisting of the amino acid residue TEDS at positions 13 to 16 of the consensus sequence; (b) a BC loop containing or consisting of the amino acid residue TAPDAAF at positions 22 to 28 of the consensus sequence; (c) a CD loop containing or consisting of the amino acid residue SEKVGE at positions 38 to 43 of the consensus sequence; (d) a DE loop containing or consisting of the amino acid residue GSER at positions 51 to 54 of the consensus sequence; (e) an EF loop containing or consisting of the amino acid residue GLKPG at positions 60 to 64 of the consensus sequence; (f) an FG loop containing or consisting of the amino acid residue KGGHRSN at positions 75 to 81 of the consensus sequence; or (g) any combination of (a) to (f). The centinlin of this disclosure may comprise at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains, or a consensus sequence of at least 15 fibronectin type III (FN3) domains. The scaffold is 10 on the antigen. -9 Smaller than or equivalent to M, 10 -10 Smaller than or equivalent to M, 10 -11 Smaller than or equivalent to M, 10 -12 Smaller than or equivalent to M, 10 -13 Smaller than or equivalent to M, 10 -14 Smaller than or equivalent to M, and 10 -15 K smaller than or equivalent to M D It can bind with at least one affinity selected from K. D This can be determined by surface plasmon resonance.

[0156] This disclosure provides compositions comprising the CAR of this disclosure and at least one pharmaceutically acceptable carrier.

[0157] This disclosure provides transposons containing the CARs of this disclosure. The transposons of this disclosure are maintained as episomes or incorporated into the genome of recombinant / modified cells. The transposons may be part of a two-component piggyBac system that utilizes transposons and transposases to enhance nonviral gene transfer.

[0158] The transposons of this disclosure may include select genes for identifying, enriching, and / or isolating cells that express the transposons. Exemplary select genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for cell viability and survival. Exemplary select genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for conferring resistance to drug attack to which the cell is susceptible (or may be lethal) in the absence of the gene product encoded by the select gene. Exemplary select genes encode any gene product (e.g., transcripts, proteins, enzymes) essential for cell viability and / or survival in cell media lacking one or more nutrients essential for survival in the absence of the select gene. Examples of select genes, though not limited to these, include neo (which confers resistance to neomycin), DHFR (which encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (which encodes thymidylate synthetase), MGMT (which encodes O(6)-methylguanine-DNA methyltransferase), the multidrug resistance gene (MDR1), ALDH1 (which encodes member A1 of the aldehyde dehydrogenase 1 family), FRANCF, RAD51C (which encodes RAD51 paralog C), GCS (which encodes glucosylceramide synthase), and NKX2.2 (which encodes NK2 homeobox 2).

[0159] The transposons of the Disclosure may include, for example, one or more of the protein scaffolds of the Disclosure, at least one self-cleaving peptide located between centintin or CARTyrin and a selected gene of the 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 containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). GSG-T2A peptides may contain an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). GSG-T2A peptides may contain a nucleic acid sequence including ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). E2A peptides may contain an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The GSG-E2A peptide may contain an amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity with the amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22). The F2A peptide may contain an amino acid sequence including VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity with the amino acid sequence including VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23).The GSG-F2A peptide may contain an amino acid sequence including GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). The P2A peptide may contain an amino acid sequence including ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The GSG-P2A peptide may contain an amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0160] The transposons of this disclosure may include a first self-cleaving peptide and a second self-cleaving peptide, the first self-cleaving peptide being located, for example, upstream of one or more of the protein scaffolds, centinlin, or CARTyrin of this disclosure, and the second self-cleaving peptide being located, for example, downstream of one or more of the protein scaffolds, centinlin, or CARTyrin of this disclosure. The first and / or second self-cleaving peptides 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 containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). GSG-T2A peptides may contain an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). GSG-T2A peptides may contain a nucleic acid sequence including ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). E2A peptides may contain an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The GSG-E2A peptide may contain an amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).F2A peptides may contain an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). GSG-F2A peptides may contain an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). P2A peptides may contain an amino acid sequence containing ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The GSG-P2A peptide may contain an amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0161] This disclosure provides compositions comprising the transposons of this disclosure. In certain embodiments, a method for introducing a composition may further comprise a composition comprising a plasmid containing a sequence encoding a transposase enzyme. The sequence encoding the transposase enzyme may be an mRNA sequence.

[0162] The transposons of this disclosure may include the piggyBac transposon. The transposase enzymes of this disclosure may include the piggyBac transposase or a compatible enzyme.

[0163] This disclosure provides vectors comprising CARs of this disclosure. In certain embodiments, the vector is a viral vector. The vector may be a recombinant vector.

[0164] The viral vectors of this disclosure may include sequences isolated from or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vectors may include sequences isolated from or derived from adeno-associated viruses (AAVs). The viral vectors may include recombinant AAVs (rAAVs). The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include two or more terminal inversion (ITR) sequences located in cis adjacent to sequences encoding the protein scaffolds, centinrin, or CARTyrin of this disclosure. The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include, but are not limited to, self-complementary AAVs (scAAVs) and AAV hybrids (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8) containing the genome of one serotype and the capsid of another serotype. The exemplary adeno-associated viruses and recombinant adeno-associated viruses of this disclosure include, but are not limited to, rAAV-LK03.

[0165] The viral vectors of this disclosure may include a select gene. The select gene may encode a gene product essential for cell viability and survival. The select gene may encode a gene product essential for cell viability and survival when attacked by selective cell culture conditions. The selective cell culture conditions may include compounds that are harmful to cell viability or survival, and the gene product confers resistance to such compounds. Examples of select genes in this disclosure include, but are not limited to, neo (which confers resistance to neomycin), DHFR (which encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (which encodes thymidylate synthetase), MGMT (which encodes O(6)-methylguanine-DNA methyltransferase), the multidrug resistance gene (MDR1), ALDH1 (which encodes member A1 of the aldehyde dehydrogenase 1 family), FRANCF, RAD51C (which encodes RAD51 paralog C), GCS (which encodes glucosylceramide synthase), NKX2.2 (which encodes NK2 homeobox 2), or any combination thereof.

[0166] The viral vectors of this disclosure may comprise at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide, where the self-cleaving peptide is located between the CAR and the selected gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, where 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 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 containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence containing EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). GSG-T2A peptides may contain an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). GSG-T2A peptides may contain a nucleic acid sequence including ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ ID NO: 20). E2A peptides may contain an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence including QCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). The GSG-E2A peptide may contain an amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).F2A peptides may contain an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). GSG-F2A peptides may contain an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24). P2A peptides may contain an amino acid sequence containing ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence containing ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). The GSG-P2A peptide may contain an amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence including GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

[0167] This disclosure provides a vector containing the CAR of this disclosure. In certain embodiments, the vector is an mRNA vector. The vector may be a recombinant mRNA vector. The T cells ...

Claims

1. Modified stem memory T cells (T ＳＣＭ A method for producing ) To produce modified T cells, the process involves introducing into primary human T cells (a) a transposon composition comprising a transposon containing an antigen receptor, a therapeutic protein, or a sequence encoding the same, and (b) a transposase or a transposase composition comprising a sequence encoding a transposase. The modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, This results in modified stem memory T cells (T ＳＣＭ A method for producing ).

2. Multiple modified stem memory T cells (T ＳＣＭ A method for producing ) To produce multiple modified T cells, the process includes the step of introducing into multiple primary human T cells (a) a transposon composition comprising a transposon containing an antigen receptor, a therapeutic protein, or a sequence encoding the same, and (b) a transposase or a transposase composition comprising a sequence encoding a transposase. At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the aforementioned modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, This results in multiple modified stem memory T cells (T ＳＣＭ A method for producing ).

3. At least 60% of the aforementioned modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 2, wherein one or more cell surface markers of ) are expressed.

4. Modified central memory T cells (T ＣＭ A method for producing ) To produce modified T cells, the process involves introducing into primary human T cells (a) a transposon composition comprising a transposon containing an antigen receptor, a therapeutic protein, or a sequence encoding the same, and (b) a transposase or a transposase composition comprising a sequence encoding a transposase. The modified T cells express one or more cell surface markers of central memory T cells (T ＣＭ ) and This results in modified central memory T cells (T ＣＭ A method for producing ).

5. Multiple modified central memory T cells (T ＣＭ A method for producing ) To produce multiple modified T cells, the process includes the step of introducing into multiple primary human T cells (a) a transposon composition comprising a transposon containing an antigen receptor, a therapeutic protein, or a sequence encoding the same, and (b) a transposase or a transposase composition comprising a sequence encoding a transposase. At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the aforementioned modified T cells are central memory T cells (T ＣＭ ) expressing one or more cell surface markers, This results in multiple modified central memory T cells (T ＣＭ A method for producing ).

6. At least 60% of the aforementioned modified T cells are central memory T cells (T ＣＭ The method according to claim 5, wherein one or more cell surface markers of ) are expressed.

7. The method according to any one of claims 1 to 6, wherein the transposon is a plasmid DNA transposon having a sequence encoding the antigen receptor or the therapeutic protein sandwiched between two cis-regulatory insulator elements.

8. The method according to any one of claims 1 to 7, wherein the transposon is a piggyBac transposon.

9. The method according to any one of claims 1 to 8, wherein the transposase is piggyBac transposase.

10. The method according to claim 9, wherein the piggyBac transposase comprises an amino acid sequence including SEQ ID NO:

4.

11. The method according to claim 9 or 10, wherein the piggyBac transposase is an overactive variant, and the overactive variant comprises an amino acid substitution at one or more positions 30, 165, 282, and 538 of SEQ ID NO:

4.

12. The method according to claim 11, wherein the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of valine (V) with isoleucine (I) (I30V).

13. The method according to claim 11, wherein the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of serine (S) with glycine (G) (G165S).

14. The method according to claim 11, wherein the amino acid substitution at position 282 of sequence number 4 is a substitution of valine (V) with methionine (M) (M282V).

15. The method according to claim 11, wherein the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of lysine (K) with asparagine (N) (N538K).

16. The method according to any one of claims 1 to 15, wherein the transposase is Super piggyBac (SPB) transposase.

17. The method according to claim 16, wherein the Super piggyBac (SPB) transposase comprises the amino acid sequence comprising SEQ ID NO:

5.

18. The method according to any one of claims 1 to 17, wherein the sequence encoding the transposase is an mRNA sequence.

19. The method according to any one of claims 1 to 6, wherein the transposon is a Sleeping Beauty transposon.

20. The method according to any one of claims 1 to 6 or 19, wherein the transposase is Sleeping Beauty transposase or hyperactive Sleeping Beauty transposase (SB100X).

21. The method according to any one of claims 1 to 6, wherein the transposon is a Hellraiser transposon.

22. The method according to any one of claims 1 to 6 or 21, wherein the transposase is Helitron transposase.

23. The method according to any one of claims 1 to 6, wherein the transposon is a Tol2 transposon.

24. The method according to any one of claims 1 to 6 or 23, wherein the transposase is Tol2 transposase.

25. The method according to any one of claims 1 to 6, wherein the transposon is derived from or recombinant from any species.

26. The method according to any one of claims 1 to 6 or 25, wherein the transposon is a synthetic transposon.

27. The method according to any one of claims 1 to 26, wherein the antigen receptor is a T cell receptor.

28. The method according to claim 27, wherein the T cell receptor is naturally present.

29. The method according to claim 27, wherein the T cell receptor does not exist in nature.

30. The method according to claim 29, wherein the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor.

31. The method according to claim 29 or 30, wherein the T cell receptor is a recombinant T cell receptor.

32. The method according to any one of claims 1 to 31, wherein the antigen receptor is a chimeric antigen receptor (CAR).

33. The method according to claim 32, wherein the CAR is CARTyrin.

34. The method according to claim 32, wherein the CAR comprises a 1-tertiary VHH sequence.

35. The method according to claim 34, wherein the CAR is a VCAR.

36. The method according to any one of claims 1 to 32, further comprising the step of introducing a composition comprising (c) a second transposon containing a sequence encoding the therapeutic protein into primary human T cells in order to produce modified T cells capable of expressing the therapeutic protein.

37. The method according to claim 33, wherein the therapeutic protein is a secretory protein or a secretory-type protein.

38. The method according to claim 33 or 34, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.

39. The method according to claim 35, wherein the sequence encoding the therapeutic protein is a DNA sequence.

40. The method according to any one of claims 33 to 36, wherein the transposase composition of (b) causes the transposon of (a) and the second transposon of (c) to move.

41. The method further includes the step of introducing (d) a transposase or a second transposase composition comprising a sequence encoding the transposase into the primary human T cells, The second transposase in (d) above can transpose the transposon in (c), The second transposase composition in (d) and the transposase composition in (b) are not the same. The method according to any one of claims 1 to 37.

42. The method according to claim 38, wherein the transposon (a) moves by the transposase composition (b), and the transposon (c) moves by the transposase composition (d).

43. Modified stem memory T cells (T ＳＣＭ A method for producing ) (a) A step of introducing a composition comprising an antigen receptor, a therapeutic protein, or a sequence encoding the same into primary human T cells in order to produce modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in the transposon. (b) To produce activated modified T cells, the step of contacting the modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances, The activated modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, This results in modified stem memory T cells (T ＳＣＭ A method for producing ).

44. Multiple modified stem memory T cells (T ＳＣＭ A method for producing ) (a) A step of introducing a composition comprising an antigen receptor, a therapeutic protein, or a sequence encoding the same into a plurality of primary human T cells in order to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in the transposon. (b) A step of contacting the plurality of modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances in order to produce a plurality of activated modified T cells, At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the aforementioned multiple activated modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, This results in multiple modified stem memory T cells (T ＳＣＭ A method for producing ).

45. At least 60% of the aforementioned multiple activated modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 41, wherein one or more cell surface markers of ) are expressed.

46. Modified central memory T cells (T ＣＭ A method for producing ) (a) A step of introducing a composition comprising an antigen receptor, a therapeutic protein, or a sequence encoding the same into primary human T cells in order to produce modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in the transposon. (b) To produce activated modified T cells, the step of contacting the modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances, The activated modified T cells are central memory T cells (T ＣＭ ) expressing one or more cell surface markers, This results in modified central memory T cells (T ＣＭ A method for producing ).

47. Multiple modified central memory T cells (T ＣＭ A method for producing ) (a) A step of introducing a composition comprising an antigen receptor, a therapeutic protein, or a sequence encoding the same into a plurality of primary human T cells in order to produce a plurality of modified T cells, wherein the antigen receptor or the therapeutic protein is not contained in the transposon. (b) A step of contacting the plurality of modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances in order to produce a plurality of activated modified T cells, At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the aforementioned multiple activated modified T cells are central memory T cells (T ＣＭ ) expressing one or more cell surface markers, This results in multiple modified central memory T cells (T ＣＭ A method for producing ).

48. At least 60% of the aforementioned multiple activated modified T cells are central memory T cells (T ＣＭ The method according to claim 44, wherein one or more cell surface markers of ) are expressed.

49. The method according to any one of claims 40 to 45, wherein the viral vector comprises the antigen receptor or therapeutic protein.

50. The method according to claim 46, wherein the viral vector comprises a sequence isolated from or derived from a lentivirus.

51. The method according to claim 46, wherein the viral vector comprises a sequence isolated from or derived from a retrovirus.

52. The method according to claim 48, wherein the retrovirus is a gamma retrovirus.

53. The method according to any one of claims 40 to 46, wherein the viral vector comprises a sequence isolated from or derived from adeno-associated virus (AAV).

54. The method according to any one of claims 40 to 45, wherein the nucleic acid vector comprises the antigen receptor or therapeutic protein.

55. The method according to claim 51, wherein the mRNA vector comprises the antigen receptor or therapeutic protein.

56. The method according to any one of claims 40 to 45, wherein the nanoparticle vector comprises the antigen receptor or therapeutic protein.

57. The method according to any one of claims 40 to 45, wherein the introducing step includes homologous recombination.

58. The method according to claim 54, wherein the homologous recombination comprises contacting the composition comprising the antigen receptor or the therapeutic protein, the genome editing construct, and the genome sequence of at least one of the plurality of primary human T cells.

59. The method according to claim 55, wherein the vector comprises the antigen receptor or therapeutic protein.

60. The method according to claim 56, wherein the vector is an adeno-associated vector (AAV).

61. The method according to any one of claims 54 to 57, wherein the genome editing construct comprises a guide RNA and a clustered and regularly arranged short palindromic sequence repeat (CRISPR)-associated protein 9 (Cas9) DNA endonuclease.

62. The method according to claim 58, wherein the genome editing construct comprises a DNA-binding domain and an IIS-type endonuclease.

63. The method according to claim 59, wherein the genome editing construct encodes a fusion protein.

64. The method according to claim 59, wherein the genome editing construct encodes the DNA-binding domain and the IIS-type endonuclease, and the expressed DNA-binding domain and the expressed IIS-type endonuclease are linked by a non-covalent bond.

65. The method according to any one of claims 58 to 62, wherein the genome editing construct comprises a sequence derived from Cas9 endonuclease.

66. The method according to claim 62, wherein the sequence derived from Cas9 endonuclease is a DNA-binding domain.

67. The method according to claim 62 or 63, wherein the sequence derived from the Cas9 endonuclease encodes inactive Cas9.

68. The method according to claim 64, wherein the sequence derived from the Cas9 endonuclease includes an amino acid substitution (H840A) of histidine (H) with alanine (A) at position 840.

69. The method according to any one of claims 62 to 65, wherein the sequence derived from the Cas9 endonuclease encodes a shortened form of Cas9.

70. The method according to claim 66, wherein the sequence derived from the Cas9 endonuclease includes an amino acid substitution (N580A) of asparagine (N) with alanine (A) at position 580.

71. The method according to any one of claims 62 to 67, wherein the sequence derived from the Cas9 endonuclease includes an amino acid substitution (D10A) of aspartic acid (D) with alanine (A) at position 10.

72. The method according to any one of claims 58 to 61, wherein the genome editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).

73. The method according to claim 69, wherein the sequence derived from TALEN is a DNA-binding domain.

74. The method according to claim 58, wherein the genome editing construct comprises TALEN.

75. The method according to any one of claims 58 to 61, wherein the genome editing construct comprises a sequence derived from a zinc finger nuclease (ZFN).

76. The method according to claim 72, wherein the sequence derived from the ZFN is a DNA-binding domain.

77. The method according to claim 58, wherein the genome editing construct comprises a zinc finger nuclease (ZFN).

78. The method according to any one of claims 58 to 74, wherein the genome editing construct targets a safe harbor site on a mammalian chromosome.

79. The method according to any one of claims 58 to 74, wherein the genome editing construct targets a safe harbor site on a human chromosome.

80. The method according to claim 75 or 76, wherein the chromosome is in vivo, in situ, ex vivo, or in vitro.

81. The method according to any one of claims 58 to 77, wherein the genome editing construct targets a sequence encoding a component of an endogenous T cell receptor or a sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a mammalian chromosome.

82. The method according to any one of claims 58 to 77, wherein the genome editing construct targets a sequence encoding a component of an endogenous T cell receptor or a sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a human chromosome.

83. The method according to any one of claims 40 to 79, wherein the antigen receptor is a T cell receptor.

84. The method according to claim 80, wherein the T cell receptor is naturally present.

85. The method according to claim 80, wherein the T cell receptor does not exist in nature.

86. The method according to claim 82, wherein the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor.

87. The method according to claim 82 or 83, wherein the T cell receptor is a recombinant T cell receptor.

88. The method according to any one of claims 40 to 79, wherein the antigen receptor is a chimeric antigen receptor (CAR).

89. The method according to claim 85, wherein the CAR comprises one or more centinline sequences.

90. The method according to claim 86, wherein the CAR is CARTyrin.

91. The method according to claim 85, wherein the CAR comprises one or more VHH sequences.

92. The method according to claim 88, wherein the CAR is a VCAR.

93. The method according to any one of claims 40 to 89, further comprising the step of introducing a composition containing a sequence encoding the therapeutic protein into primary human T cells in order to produce modified T cells capable of expressing the therapeutic protein.

94. The method according to any one of claims 40 to 90, wherein the therapeutic protein is a secretory protein or a secretory-type protein.

95. The method according to claim 90 or 91, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.

96. The method according to claim 92, wherein the sequence encoding the therapeutic protein is a DNA sequence.

97. The method according to any one of claims 90 to 93, wherein the introducing step includes homologous recombination.

98. The method according to any one of claims 40 to 94, wherein the T cell activator composition of (b) further comprises a tetrameric antibody complex that is monospecific to anti-human CD2.

99. (c) To produce a plurality of enlarged modified T cells, the step of contacting the activated modified T cells with a T cell enlargement composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and an enlargement supplement, At least 2% of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, The method according to any one of claims 40 to 42 or 46 to 95.

100. 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 between these, of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 96, wherein the cell surface marker of ) is expressed.

101. At least 60% of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 97, wherein the cell surface marker of ) is expressed.

102. (c) To produce a plurality of enlarged modified T cells, the step of contacting the activated modified T cells with a T cell enlargement composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and an enlargement supplement, At least 2% of the aforementioned multiple enlarged modified T cells are central memory T cells (T ＣＭ ) expressing one or more cell surface markers, The method according to any one of claims 43 to 95.

103. 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 between these, of the aforementioned multiple enlarged modified T cells are central memory T cells (T ＣＭ The method according to claim 99, wherein the cell surface marker of ) is expressed.

104. At least 60% of the aforementioned multiple enlarged modified T cells are central memory T cells (T ＣＭ The method according to claim 99, wherein the cell surface marker of ) is expressed.

105. (d) Stem memory T cells (T ＳＣＭ The method according to any one of claims 40 to 42 or 46 to 101, further comprising the step of enriching the plurality of enlarged modified T cells in order to prepare a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between.

106. (d) Stem memory T cells (T ＳＣＭ The method according to any one of claims 40 to 42 or 46 to 101, further comprising the step of enriching the plurality of augmented modified T cells in order to produce a composition comprising at least 60% modified T cells expressing the cell surface marker of ).

107. (d) Central memory T cells (T ＣＭ The method according to any one of claims 43 to 101, further comprising the step of enriching the plurality of enlarged modified T cells in order to prepare 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 between these.

108. (d) Central memory T cells (T ＣＭ The method according to any one of claims 43 to 101, further comprising the step of enriching the plurality of augmented modified T cells in order to prepare a composition comprising at least 60% modified T cells expressing the cell surface marker of ).

109. The enrichment step involves extracting stem memory T cells (T) from the plurality of enriched modified T cells. ＳＣＭ The method according to claim 102 or 103, comprising isolating modified T cells expressing one or more cell surface markers of the following:

110. The enrichment step involves a plurality of increased enriched modified T ＳＣＭ To produce the isolated modified T ＳＣＭ The method according to claim 106, further comprising contacting with a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements.

111. The enrichment step involves extracting central memory T cells from the plurality of enriched modified T cells. ＳＣＭ The method according to claim 104 or 105, comprising isolating modified T cells expressing one or more cell surface markers of the following:

112. The enrichment step involves a plurality of increased enriched modified T ＣＭ To produce it, isolated modified T ＣＭ The method according to claim 108, further comprising contacting with a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements.

113. The method according to any one of claims 40 to 109, wherein the T cell enlargement composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearate hydrazide, oleamide, sterols, and alkanes.

114. The method according to any one of claims 40 to 109, wherein the T cell enlargement composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols.

115. The method according to claim 111, wherein the T cell enlargement composition further comprises one or more sterols in a concentration of 0.9 mg / kg to 90 mg / kg including the endpoints: octanoic acid; palmitic acid in a concentration of 0.2 mg / kg to 20 mg / kg including the endpoints: linoleic acid in a concentration of 0.2 mg / kg to 20 mg / kg including the endpoints: oleic acid in a concentration of 0.2 mg / kg to 20 mg / kg including the endpoints: and sterols in a concentration of about 0.1 mg / kg to 10 mg / kg including the endpoints.

116. The method according to claim 111, wherein the T cell enlargement 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 sterols at a concentration of about 1 mg / kg.

117. The method according to claim 111, wherein the T cell enlargement composition further comprises one or more sterols and octanoic acid in a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoints; palmitic acid in a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoints; linoleic acid in a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; oleic acid in a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; and one or more sterols in a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoints.

118. The method according to claim 111, wherein the T cell enlargement 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 sterols at a concentration of about 2.5 μmol / kg.

119. Modified stem memory T cells (T ＳＣＭ A method for producing ) (a) A step of introducing a composition containing an antigen receptor or a therapeutic protein into primary human T cells in order to produce modified T cells, wherein the transposon contains the antigen receptor, (b) To produce activated modified T cells, the step of contacting the modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances, The activated modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, This results in modified stem memory T cells (T ＳＣＭ A method for producing ).

120. Multiple modified stem memory T cells (T ＳＣＭ A method for producing ) (a) A step of introducing a composition containing an antigen receptor or a therapeutic protein into multiple primary human T cells in order to produce multiple modified T cells, wherein the transposon contains the antigen receptor, (b) A step of contacting the plurality of modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances in order to produce a plurality of activated modified T cells, At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the aforementioned multiple activated modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, This results in multiple modified stem memory T cells (T ＳＣＭ A method for producing ).

121. At least 60% of the aforementioned multiple activated modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 117, wherein one or more cell surface markers of ) are expressed.

122. Modified central memory T cells (T ＣＭ A method for producing ) (a) A step of introducing a composition containing an antigen receptor or a therapeutic protein into primary human T cells in order to produce modified T cells, wherein the transposon contains the antigen receptor, (b) To produce activated modified T cells, the step of contacting the modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances, The activated modified T cells are central memory T cells (T ＣＭ ) expressing one or more cell surface markers, This results in modified central memory T cells (T ＣＭ A method for producing ).

123. Multiple modified central memory T cells (T ＣＭ A method for producing ) (a) A step of introducing a composition containing an antigen receptor or a therapeutic protein into multiple primary human T cells in order to produce multiple modified T cells, wherein the transposon contains the antigen receptor, (b) A step of contacting the plurality of modified T cells with a T cell activator composition comprising one or more anti-human CD3 monospecific tetramer antibody complexes, anti-human CD28 monospecific tetramer antibody complexes, and activation replacement substances in order to produce a plurality of activated modified T cells, At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the aforementioned multiple activated modified T cells are central memory T cells (T ＣＭ ) expressing one or more cell surface markers, This results in modified central memory T cells (T ＣＭ A method for producing ).

124. At least 60% of the aforementioned multiple activated modified T cells are central memory T cells (T ＣＭ The method according to claim 120, wherein one or more cell surface markers of ) are expressed.

125. The method according to any one of claims 116 to 121, wherein the T cell activator composition of (b) further comprises a tetrameric antibody complex that is monospecific to anti-human CD2.

126. (c) To produce a plurality of enlarged modified T cells, the step of contacting the activated modified T cells with a T cell enlargement composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and an enlargement supplement, At least 2% of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, The method according to any one of claims 116 to 118 or 122.

127. 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 between these, of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 123, wherein the cell surface marker of ) is expressed.

128. At least 60% of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 123, wherein the cell surface marker of ) is expressed.

129. (c) To produce a plurality of enlarged modified T cells, the step of contacting the activated modified T cells with a T cell enlargement composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and an enlargement supplement, At least 2% of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ ) expressing one or more cell surface markers, The method according to any one of claims 119 to 122.

130. 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 between these, of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 126, wherein the cell surface marker of ) is expressed.

131. At least 60% of the aforementioned multiple enlarged modified T cells are stem memory T cells (T ＳＣＭ The method according to claim 126, wherein the cell surface marker of ) is expressed.

132. (d) Stem memory T cells (T ＳＣＭ The method according to any one of claims 116 to 118 or 122 to 128, further comprising the step of enriching the plurality of enlarged modified T cells in order to prepare a composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between.

133. (d) Stem memory T cells (T ＳＣＭ The method according to any one of claims 116 to 118 or 122 to 128, further comprising the step of enriching the plurality of augmented modified T cells in order to produce a composition comprising at least 60% modified T cells expressing the cell surface marker of ).

134. (d) Stem memory T cells (T ＳＣＭ The method according to any one of claims 119 to 128, further comprising the step of enriching the plurality of enlarged modified T cells in order to prepare 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 between these.

135. (d) Stem memory T cells (T ＳＣＭ The method according to any one of claims 119 to 128, further comprising the step of enriching the plurality of augmented modified T cells in order to produce a composition comprising at least 60% modified T cells expressing the cell surface marker of ).

136. The enrichment step involves extracting stem memory T cells (T) from the plurality of enriched modified T cells. ＳＣＭ The method according to claim 129 or 130, comprising isolating modified T cells expressing one or more cell surface markers of the following:

137. The enrichment step involves a plurality of increased enriched modified T ＳＣＭ To produce the isolated modified T ＳＣＭ The method according to claim 133, further comprising contacting with a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements.

138. The enrichment step involves extracting stem memory T cells (T) from the plurality of enriched modified T cells. ＳＣＭ The method according to claim 131 or 132, comprising isolating modified T cells expressing one or more cell surface markers of the following:

139. The enrichment step involves a plurality of increased enriched modified T ＳＣＭ To produce it, isolated modified T ＳＣＭ The method according to claim 135, further comprising contacting with a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplements.

140. The method according to any one of claims 116 to 136, wherein the T cell enlargement composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decine-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butylbenzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, hydrazide stearate, oleamide, sterols, and alkanes.

141. The method according to any one of claims 116 to 137, wherein the T cell enlargement composition further comprises one or more octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols.

142. The method according to claim 138, wherein the T cell enlargement composition further comprises one or more sterols in a concentration of 0.9 mg / kg to 90 mg / kg including the endpoints: octanoic acid; palmitic acid in a concentration of 0.2 mg / kg to 20 mg / kg including the endpoints: linoleic acid in a concentration of 0.2 mg / kg to 20 mg / kg including the endpoints: oleic acid in a concentration of 0.2 mg / kg to 20 mg / kg including the endpoints: and sterols in a concentration of about 0.1 mg / kg to 10 mg / kg including the endpoints.

143. The method according to claim 138, wherein the T cell enlargement 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 sterols at a concentration of about 1 mg / kg.

144. The method according to claim 138, wherein the T cell enlargement composition further comprises one or more sterols and octanoic acid in a concentration of 6.4 μmol / kg to 640 μmol / kg including the endpoints; palmitic acid in a concentration of 0.7 μmol / kg to 70 μmol / kg including the endpoints; linoleic acid in a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; oleic acid in a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoints; and one or more sterols in a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoints.

145. The method according to claim 138, wherein the T cell enlargement 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 sterols at a concentration of about 2.5 μmol / kg.

146. The method according to any one of claims 116 to 142, further comprising the step of introducing a composition comprising (c) a second transposon containing a sequence encoding the therapeutic protein into primary human T cells in order to produce modified T cells capable of expressing the therapeutic protein.

147. The method according to claim 143, wherein the therapeutic protein is a secretory protein or a secretory-type protein.

148. The method according to claim 143 or 144, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.

149. The method according to claim 143 or 144, wherein the sequence encoding the therapeutic protein is a DNA sequence.

150. The method according to any one of claims 143 to 146, wherein the transposase composition of (b) causes the transposon of (a) and the second transposon of (c) to move.

151. The method further includes the step of introducing (d) a transposase or a second transposase composition comprising a sequence encoding the transposase into the primary human T cells, The second transposase in (d) above can transpose the transposon in (c), The second transposase composition in (d) and the transposase composition in (b) are not the same. The method according to any one of claims 143 to 147.

152. The method according to any one of claims 1 to 148, wherein the introduction step further comprises a composition comprising a genome editing construct.

153. The method according to claim 149, wherein the genome editing construct comprises a guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-related protein 9 (Cas9) DNA endonuclease.

154. The method according to claim 150, wherein the genome editing construct comprises a DNA-binding domain and an IIS-type endonuclease.

155. The method according to claim 151, wherein the genome editing construct encodes a fusion protein.

156. The method according to claim 151, wherein the genome editing construct encodes the DNA-binding domain and the IIS-type endonuclease, and the expressed DNA-binding domain and the expressed IIS-type endonuclease are linked by a non-covalent bond.

157. The method according to any one of claims 149 to 153, wherein the genome editing construct comprises a sequence derived from Cas9 endonuclease.

158. The method according to claim 154, wherein the sequence derived from Cas9 endonuclease is a DNA-binding domain.

159. The method according to claim 154 or 155, wherein the sequence derived from Cas9 endonuclease encodes inactive Cas9.

160. The method according to claim 156, wherein the sequence derived from Cas9 endonuclease includes an amino acid substitution (H840A) of alanine (A) by histidine (H) at position 840.

161. The method according to any one of claims 154 to 157, wherein the sequence derived from Cas9 endonuclease encodes a shortened form of Cas9.

162. The method according to claim 158, wherein the sequence derived from Cas9 endonuclease includes an amino acid substitution (N580A) of alanine (A) by asparagine (N) at position 580.

163. The method according to any one of claims 154 to 159, wherein the sequence derived from Cas9 endonuclease includes an amino acid substitution (D10A) of alanine (A) by aspartic acid (D) at position 10.

164. The method according to any one of claims 149 to 153, wherein the genome editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).

165. The method according to claim 161, wherein the sequence derived from TALEN is a DNA-binding domain.

166. The method according to claim 149, wherein the genome editing construct comprises TALEN.

167. The method according to any one of claims 149 to 153, wherein the genome editing construct comprises a sequence derived from a zinc finger nuclease (ZFN).

168. The method according to claim 164, wherein the sequence derived from the ZFN is a DNA-binding domain.

169. The method according to claim 149, wherein the genome editing construct comprises a zinc finger nuclease (ZFN).

170. The method according to any one of claims 149 to 153, further comprising the step of introducing a composition containing a sequence encoding a therapeutic protein into primary human T cells.

171. The method according to claim 167, wherein the therapeutic protein is a secretory protein or a secretory-type protein.

172. The method according to claim 168, wherein the therapeutic protein is an intracellular protein.

173. The method according to claim 168, wherein the therapeutic protein is a cytoplasmic protein.

174. The method according to claim 168, wherein the therapeutic protein is a membrane-bound protein.

175. The method according to claim 168, wherein the therapeutic protein is a transmembrane protein.

176. The aforementioned modified T ＳＣＭ The method according to any one of claims 1 to 172, wherein the cell surface markers include CD62L and CD45RA.

177. The aforementioned modified T ＳＣＭ The method according to any one of claims 1 to 172, wherein the cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ.

178. The aforementioned modified T ＳＣＭ The method according to any one of claims 1 to 172, wherein the cell surface marker comprises one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

179. The aforementioned modified T ＣＭ The method according to any one of claims 1 to 172, wherein the cell surface marker comprises one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

180. The aforementioned multiple enlarged modified T cells are naive T cells (modified T Ｎ ) includes CAR-T Ｎ The method according to any one of claims 96 to 176, wherein the cell surface marker comprises one or more of CD45RA, CCR7, and CD62L.

181. The aforementioned multiple enlarged modified T cells are central memory T cells (modified T ＣＭ ) includes CAR-T ＣＭ The method according to any one of claims 96 to 176, wherein the cell surface marker comprises one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

182. The aforementioned multiple enlarged modified T cells are effector memory T cells (modified T ＥＭ ) includes CAR-T ＥＭ The method according to any one of claims 96 to 176, wherein the cell surface marker comprises one or more of CD45RO, CD95, and IL-2Rβ.

183. The aforementioned multiple enlarged modified T cells are effector T cells (modified T ＥＦＦ ) includes CAR-T ＥＦＦ The method according to any one of claims 96 to 176, wherein the cell surface marker comprises one or more of CD45RA, CD95, and IL-2Rβ.

184. The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is a plasmid DNA transposon having sequences encoding the antigen receptor or therapeutic protein adjacent to two cis-regulatory insulator elements.

185. The method according to claim 181, wherein the introduction step further comprises a composition containing an mRNA sequence encoding a transposase.

186. The method according to claim 181 or 182, wherein the transposon is a piggyBac transposon.

187. The method according to any one of claims 181 to 183, wherein the transposase is piggyBac transposase.

188. The method according to claim 184, wherein the piggyBac transposase comprises an amino acid sequence including SEQ ID NO:

4.

189. The method according to claim 184 or 185, wherein the piggyBac transposase is an overactive variant, and the overactive variant comprises amino acid substitutions at one or more positions 30, 165, 282, and 538 of SEQ ID NO:

4.

190. The method according to claim 186, wherein the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of valine (V) with isoleucine (I) (I30V).

191. The method according to claim 186, wherein the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of serine (S) with glycine (G) (G165S).

192. The method according to claim 186, wherein the amino acid substitution at position 282 of sequence number 4 is a substitution of valine (V) with methionine (M) (M282V).

193. The method according to claim 186, wherein the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of lysine (K) with asparagine (N) (N538K).

194. The method according to any one of claims 183 to 190, wherein the transposase is Super piggyBac (SPB) transposase.

195. The method according to claim 191, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence including SEQ ID NO:

5.

196. The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is a Sleeping Beauty transposon.

197. The method according to claim 193, wherein the transposase is Sleeping Beauty transposase or hyperactive Sleeping Beauty transposase (SB100X).

198. The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is a Hellraiser transposon.

199. The method according to claim 195, wherein the transposase is Helitron transposase.

200. The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is a Tol2 transposon.

201. The method according to claim 197, wherein the transposase is Tol2 transposase.

202. The method according to any one of claims 1 to 39 or 116 to 198, wherein the sequence encoding the transposase is an mRNA sequence.

203. The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is derived from or recombinant from any species.

204. The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is a synthetic transposon.

205. The method according to any one of claims 1 to 39 or 116-180, wherein the transposon further comprises a selection gene.

206. The method according to claim 202, wherein the T cell enlargement composition further comprises a selector.

207. The method according to any one of claims 1 to 203, wherein the antigen receptor is a T cell receptor.

208. The method according to claim 204, wherein the T cell receptor is naturally present.

209. The method according to claim 204, wherein the T cell receptor does not exist in nature.

210. The method according to claim 206, wherein the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor.

211. The method according to claim 206 or 207, wherein the T cell receptor is a recombinant T cell receptor.

212. The method according to any one of claims 1 to 203, wherein the antigen receptor is a chimeric antigen receptor (CAR).

213. The method according to claim 209, wherein the CAR comprises one or more centinline sequences.

214. The method according to claim 210, wherein the CAR is CARTyrin.

215. The method according to claim 209, wherein the CAR comprises one or more VHH sequences.

216. The method according to claim 212, wherein the CAR is a VCAR.

217. The method according to any one of claims 1 to 39 and 116 to 213, wherein the introducing step includes electroporation or nucleofection.

218. The aforementioned introduction step includes nucleofection, and the nucleofection is (a) A step of bringing a transposon composition, a transposase composition, and a composition comprising a plurality of primary human T cells into contact in a cuvette, (b) The step of applying one or more electrical pulses to the cuvette, (c) The step of incubating the composition containing the plurality of primary human T cells at 37°C in a composition containing a T cell augmentation composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, ISCOFF MDM, and augmentation supplements. The method according to any one of claims 1 to 39 and 116 to 213, including the method described in any one of claims 1 to 39.

219. The method according to claim 215, wherein the transposon is a first transposon or a second transposon.

220. The method according to claim 215 or 216, wherein the transposase composition is the first transposase composition or the second transposase composition.

221. The method according to any one of claims 214 to 217, wherein, in order to obtain 1 μg of transposon, the transposon composition is a 0.5 μg / μl solution containing water that does not contain a nuclease, and the cuvette contains 2 μl of the transposon composition.

222. The method according to claim 218, wherein the transposon composition comprises a piggyBac transposon.

223. The method according to claim 219, wherein the transposon composition comprises a Sleeping Beauty transposon.

224. The method according to claim 219 or 220, wherein the transposase composition comprises 5 μg of transposase.

225. The method according to claim 221, wherein the transposase composition comprises Super piggyBac (SPB) transposase.

226. The method according to claim 221, wherein the transposase composition comprises an overactive Sleeping Beauty (SB100X) transposase.

227. The method according to claim 219, wherein the transposon includes a Hellraiser transposon.

228. The method according to claim 224, wherein the transposase composition comprises Helitron transposase.

229. The method according to claim 219, wherein the transposon includes a Tol2 transposon.

230. The method according to claim 226, wherein the transposase composition comprises Tol2 transposase.

231. The method according to any one of claims 215 to 227, wherein 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.

232. The method according to claim 228, wherein prior to nucleofection, the buffer is used to maintain or enhance the level of cell viability and / or stem-like phenotype of the primary human T cells.

233. The method according to claim 228, wherein the buffer solution maintains or enhances the level of cell viability and / or stem-like phenotype of the primary human T cells during the nucleofection.

234. The method according to claim 228, wherein, after nucleofection, the buffer is used to maintain or enhance the level of cell viability and / or stem-like phenotype of the primary human T cells.

235. The method according to any one of claims 228 to 231, wherein the buffer solution comprises a P3 primary cell solution.

236. The buffer solution contains KCl, MgCl ２ , ClNa, glucose and Ca(NO ３ ) ２ The method according to any one of claims 228 to 231, comprising one or more of in any absolute or relative abundance or concentration.

237. The method according to claim 228, wherein the buffer further comprises a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer.

238. The buffer solution is 5 mM KCl, 15 mM MgCl ２ , 90 mM ClNa, 10 mM glucose and 0.4 mM Ca(NO ３ ) ２ The method according to claim 228 or 229, comprising

239. The method according to claim 235, wherein the buffer further comprises a supplement containing 20 mM HEPES and 75 mM Tris / HCl.

240. The buffer solution is 40 mM Na ２ HPO ４ / NaH ２ PO ４ The method according to claim 236, further comprising a supplement containing a pH of 7.

2.

241. The method according to claim 215 or 228 to 237, wherein the composition comprising the primary human T cells is depleted of cells expressing CD14, CD56, and / or CD19.

242. The composition containing the primary human T cells contains 100 μl of the buffer and 5×10 ６ to 25×10 ６ cells, and the method according to any one of claims 215 to 238.

243. The method according to any one of claims 215 to 239, which is carried out simultaneously in one or more cuvettes.

244. The method according to any one of claims 215 to 240, wherein the incubation step comprises incubating the composition comprising the plurality of primary human T cells in a preheated T cell augmentation composition.

245. The method according to any one of claims 215 to 241, wherein the incubation step is performed for two days.

246. The method according to any one of claims 40 to 242, wherein the activating replacement substance comprises one or more cytokines.

247. The method according to claim 243, wherein the one or more cytokines include IL-2.

248. The method according to any one of claims 96 to 244, wherein the augmentation supplement comprises one or more cytokines.

249. The method according to claim 245, wherein the one or more cytokines include IL-2.

250. Modified T ＳＣＭ Cells or modified T cells ＣＭ The method according to any one of claims 1 to 246, further comprising the step of introducing a composition comprising a genome editing construct into cells.

251. The method according to claim 247, wherein the genome editing construct comprises a guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-related protein 9 (Cas9) DNA endonuclease.

252. The method according to claim 248, wherein the genome editing construct comprises a DNA-binding domain and an IIS-type endonuclease.

253. The method according to claim 249, wherein the genome editing construct encodes a fusion protein.

254. The method according to claim 249, wherein the genome editing construct encodes the DNA-binding domain and the IIS-type endonuclease, and the expressed DNA-binding domain and the expressed IIS-type endonuclease are linked by a non-covalent bond.

255. The method according to any one of claims 247 to 251, wherein the genome editing construct comprises a sequence derived from Cas9 endonuclease.

256. The method according to claim 252, wherein the sequence derived from Cas9 endonuclease is a DNA-binding domain.

257. The method according to claim 253, wherein the sequence derived from Cas9 endonuclease includes an amino acid substitution (H840A) of alanine (A) by histidine (H) at position 840.

258. The method according to any one of claims 252 to 254, wherein the sequence derived from the Cas9 endonuclease encodes a shortened form of Cas9.

259. The method according to claim 255, wherein the sequence derived from Cas9 endonuclease includes an amino acid substitution (N580A) of alanine (A) by asparagine (N) at position 580.

260. The method according to any one of claims 252 to 256, wherein the sequence derived from Cas9 endonuclease includes an amino acid substitution (D10A) of alanine (A) at position 10 by aspartic acid (D).

261. The method according to any one of claims 247 to 251, wherein the genome editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN).

262. The method according to claim 258, wherein the sequence derived from TALEN is a DNA-binding domain.

263. The method according to claim 247, wherein the genome editing construct comprises TALEN.

264. The method according to any one of claims 247 to 251, wherein the genome editing construct comprises a sequence derived from a zinc finger nuclease (ZFN).

265. The method according to claim 261, wherein the sequence derived from the ZFN is a DNA-binding domain.

266. The method according to claim 247, wherein the genome editing construct comprises a zinc finger nuclease (ZFN).

267. The method according to any one of claims 1 to 263, wherein the primary human T cells express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ.

268. The aforementioned primary human T cells are naive T cells (T Ｎ ) and the above T Ｎ The method according to any one of claims 1 to 263, wherein one or more of CD45RA, CCR7, and CD62L are expressed.

269. The aforementioned primary human T cells are T memory stem cells (T ＳＣＭ ) and the above T ＳＣＭ The method according to any one of claims 1 to 263, wherein the organism expresses one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

270. The aforementioned primary human T cells become central memory T cells (T ＣＭ ) and the above T ＣＭ The method according to any one of claims 1 to 263, wherein the present agent expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

271. The aforementioned primary human T cells become effector memory T cells (T ＥＭ ) and the above T ＥＭ The method according to any one of claims 1 to 263, wherein the present agent expresses one or more of CD45RO, CD95, and IL-2Rβ.

272. The aforementioned primary human T cells become effector T cells (T ＥＦＦ ) and the above T ＥＦＦ The method according to any one of claims 1 to 263, wherein the present agent expresses one or more of CD45RA, CD95, and IL-2Rβ.

273. The method according to any one of claims 1 to 269, wherein the primary human T cells express CD4 and / or CD8.

274. Modified T prepared by the method according to any one of claims 1 to 270 ＳＣＭ A composition containing the following:

275. Modified T prepared by the method according to any one of claims 1 to 270 ＣＭ A composition containing the following:

276. Use of the composition according to claim 271 or 272 for manufacturing a pharmaceutical for treating a subject that requires it.

277. The aforementioned modified T ＳＣＭ or modified T ＣＭ However, the use described in claim 273 is of self-origin.

278. The aforementioned modified T ＳＣＭ or modified T ＣＭ However, the use described in claim 274 is of the same type but different in nature.

279. The use according to any one of claims 273 to 275, wherein the antigen receptor is a T cell receptor.

280. The use according to claim 276, wherein the T cell receptor is naturally occurring.

281. The use according to claim 276, wherein the T cell receptor does not exist in nature.

282. The use according to claim 278, wherein the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor.

283. The use according to claim 278 or 279, wherein the T cell receptor is a recombinant T cell receptor.

284. The use according to any one of claims 273 to 275, wherein the antigen receptor is a chimeric antigen receptor (CAR).

285. The use according to claim 281, wherein the CAR comprises one or more centinline sequences.

286. The use according to claim 282, wherein the CAR is CARTyrin.

287. The method according to claim 283, wherein the CAR comprises one or more VHH sequences.

288. The method according to claim 284, wherein the CAR is a VCAR.

289. A method for treating a disease or disorder in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of the composition according to claim 271 or 272.

290. The aforementioned modified T ＳＣＭ or modified T ＣＭ The method according to claim 286, wherein the is of its own origin.

291. The aforementioned modified T ＳＣＭ or modified T ＣＭ The method according to claim 286, wherein the two are of the same type but different in origin.

292. The method according to any one of claims 286 to 288, wherein the antigen receptor is a T cell receptor.

293. The method according to claim 289, wherein the T cell receptor is naturally present.

294. The method according to claim 289, wherein the T cell receptor does not exist in nature.

295. The method according to claim 291, wherein the T cell receptor comprises one or more mutations compared to the wild-type T cell receptor.

296. The method according to claim 291 or 292, wherein the T cell receptor is a recombinant T cell receptor.

297. The method according to any one of claims 286 to 288, wherein the antigen receptor is a chimeric antigen receptor (CAR).

298. The method according to claim 294, wherein the CAR comprises one or more centinline sequences.

299. The method according to claim 295, wherein the CAR is CARTyrin.

300. The method according to claim 294, wherein the CAR comprises one or more VHH sequences.

301. The method according to claim 297, wherein the CAR is a VCAR.

302. The method according to any one of claims 286 to 298, wherein the disease or disorder is cancer, and the antigen receptor specifically targets a cancer antigen.

303. The method according to any one of claims 286 to 298, wherein 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.

304. The method according to any one of claims 286 to 298, wherein the disease or disorder is characterized by a lack of activity or low abundance of secretory proteins, or the disease or disorder is treated by increasing the activity or abundance of secretory proteins.

305. The method according to claim 301, wherein the secreted protein comprises coagulation factor VIII protein or coagulation factor IX protein.

306. The method according to claim 301 or 302, wherein the amount of the secreted protein is determined at a local site.

307. The aforementioned local area is modified T ＳＣＭ Cells or modified T cells ＣＭ The method according to claim 303, wherein the cells are accessible.

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