JP2019528760A - Modified stem cell memory T cell, method for producing the same and method for using the same - Google Patents

Abstract

The present disclosure provides a method of making modified stem memory T cells (eg, CAR-T cells) for administration to a subject, eg, as adoptive cell therapy. There is a need in the art that has not been addressed for many years of a method of making modified stem cell memory T cells for administration to a subject, eg, as adoptive cell therapy. The present disclosure provides a solution to this need that has not been addressed for many years. Unlike conventional biologics and chemotherapeutic agents, the modified T cells of the present disclosure have the ability to rapidly regenerate once the antigen is recognized, thereby potentially eliminating the need for repeated treatments. obtain.

Description

Related Applications This application is a provisional application of US Provisional Application No. 62 / 402,707 filed September 30, 2016, 62 / 502,508, filed May 5, 2017, August 2017. No. 62 / 553,058 filed on May 31, and 62 / 556,309 filed on September 8, 2017, the contents of each of which are as follows: , All of which are incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING The contents of the filed textle created on October 2, 2017 and having the name “POTH-012_001WO_SeqList.txt” having a size of 110 KB are hereby incorporated by reference in their entirety. .

FIELD OF THE DISCLOSURE The present disclosure is directed to molecular biology, and more specifically to methods of making and using modified stem-cell memory T cells.

Background There is a need in the art that has not been addressed for many years of a method of making modified stem cell memory T cells for administration to a subject, for example, as adoptive cell therapy. The present disclosure provides a solution to this need that has not been addressed for many years.

Summary Unlike conventional biologics and chemotherapeutic agents, the modified T cells of the present disclosure have the ability to rapidly regenerate once the antigen is recognized, thereby potentially necessitating repeated treatments. Disappear. To accomplish this, the modified T cells of the present disclosure not only drive tumor destruction initially, but also as a stable population of memory T cells that can survive in patients to prevent potential cancer recurrence. Persisted. Thus, antigen-independent (tonic) does not cause T cell exhaustion through signaling antigen receptor molecules, as well as the initial memory cell, in particular, the modified T cells products containing stem cells memory (stem cell _{memory) (T SCM)} The focus of intensive efforts on the development of The stem cell-like modified-T cells of the present disclosure are capable of maximum self-renewal and central memory T cells (T _CM ), effector memory T cells (T _EM ) and effector T cells (T _E). ), Which results in better tumor eradication and long-term altered T cell engraftment. Modified T cells of the present disclosure include, but are not limited to, cells that express an antigen receptor comprising the protein scaffold of the present disclosure. Modified T cells of the present disclosure include, but are not limited to, cells that express a chimeric antigen receptor (CAR) (ie, a CAR-T cell of the present disclosure). A chimeric antigen receptor (CAR) of the present disclosure is referred to as, but not limited to, a single chain antibody (eg, scFv), a sequence comprising one or more fragments of an antibody (eg, VHH, referred to as VCAR for CAR). ), Antibody mimetics, and Centyrin (referred to as CARTYrin for CAR), each of which may contain one or more sequences that specifically bind an antigen.

  The modified cells of the present disclosure can be further subjected to genome editing. For example, a genome editing construct can be introduced into a modified cell of the present disclosure by transposon or other delivery means by electroporation or nucleofection and incorporated into the cell's genome during the subsequent incubation step. The resulting cells are modified T cells with an edited genome that retains a stem-like phenotype. This modified T cell with an edited genome that retains a stem-like phenotype can be used as a cell therapy. Alternatively or in addition, the modified cells of the present disclosure can be subjected to a first electroporation or nucleofection and subsequent electroporation or nucleofection to introduce a genome editing construct.

Specifically, the present disclosure provides a method for producing modified stem memory T cells ( _TSCM ), which comprises (a) an antigen receptor or therapeutic protein for producing modified T cells. A transposon composition comprising a transposon comprising, and (b) introducing a transposase or transposase composition comprising a transposase-encoding sequence into a primary human T cell, wherein the modified T cell is a stem memory T cell (T Expresses one or more cell surface markers of _SCM ), thereby creating modified stem memory T cells (T _SCM ). The present disclosure provides a method of generating a plurality of modified stem memory T cells (T _SCM ), the method comprising (a) an antigen receptor or a therapeutic protein to generate a plurality of modified T cells. Transposon composition comprising a transposon, and (b) introducing a transposase or transposase composition comprising a transposase-encoding sequence into a plurality of primary human T cells, wherein at least 2% of the plurality of modified T cells, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 99%, or any percentage in between, expresses one or more cell surface markers of stem memory T cells (T _SCM ), thereby producing a plurality of modified stems Memory T cells (T _SCM ) are produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the cell surface marker for CAR-T _SCM comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell surface marker for CAR-T _SCM comprises 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 in which the transposon is a piggyBac transposon, the transposase is a piggyBac ™ or Super piggyBac ™ (SPB) transposase.

  In certain embodiments of the disclosed 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 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.

In certain embodiments of the disclosed method, the transposase enzyme is a piggyBac ™ (PB) transposase enzyme. The piggyBac (PB) transposase enzyme is
May comprise or consist of at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage amino acid sequence therebetween.

In certain embodiments of the disclosed methods, the transposase enzyme is a sequence.
A piggyBac ™ (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538.

  In certain embodiments, the transposase enzyme comprises or consists of an amino acid sequence having amino acid substitutions at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4 piggyBac ™ (PB) transposase enzyme. In certain embodiments, the transposase enzyme comprises or consists of an amino acid sequence having amino acid substitutions at 3 or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4 piggyBac ™ (PB) transposase enzyme. In certain embodiments, the transposase enzyme comprises a piggyBac (comprising or consisting of an amino acid sequence having an amino acid substitution at each of positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 4 below. (Trademark) (PB) transposase enzyme. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 4 is a substitution of valine (V) with isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of serine (S) with glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of valine (V) with methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of lysine (K) with asparagine (N).

In certain embodiments of the disclosed method, the transposase enzyme is a Super piggyBac ™ (SPB) transposase enzyme. In certain embodiments, the Super piggyBac ™ (SPB) transposase enzyme of the present disclosure may comprise or consist of the amino acid sequence of SEQ ID NO: 4, wherein the amino acid substitution at position 30 is , The substitution of valine (V) with isoleucine (I), the amino acid substitution at position 165 is the substitution of serine (S) with glycine (G), and the amino acid substitution at position 282 is a methionine of valine (V) ( 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
May comprise or consist of at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage of the same amino acid sequence therebetween.

  In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ or Super piggyBac ™ transposase enzyme is 3rd, 46th, 82th, 103th, 119th, 125th, 177th, 180th, 185th, 187th, 200th, 207th, 209th of the sequence of SEQ ID NO: 4 or 5 226th, 235th, 240th, 241st, 243th, 258th, 296th, 298th, 311th, 315th, 319th, 327th, 328th, 340th, 421st, 436th, 456th It may further comprise amino acid substitutions at one or more of positions 470, 486, 503, 552, 570 and 591In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ or Super piggyBac ™ transposase enzyme is 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 241, 243, 296, 298 Amino acid at one or more of positions 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570 Substitution may further be included. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of asparagine (N) with serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of serine (S) with alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of threonine (T) with alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tryptophan (W) with isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of proline (P) with serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of proline (P) with arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alanine (A) with cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of histidine (H) by tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of 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 a 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 a 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 a 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 a 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 a 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 a 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 a substitution of phenylalanine (F) with methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of arginine (R) with leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of serine (S) with asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tryptophan (W) with leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tyrosine (Y) with leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of phenylalanine (F) with leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alanine (A) with methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of valine (V) with methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of isoleucine (I) with proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of valine with proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with arginine (R). In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of glycine (G) with threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of arginine (R) with tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of valine (V) by tyrosine (Y). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of glycine (G) with cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of histidine (H) with aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of isoleucine (I) with valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tyrosine (Y) with methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of phenylalanine (F) with leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of isoleucine (I) with methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of threonine (T) with alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of proline (P) with glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of arginine (R) with glutamine (Q).

  In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ transposase enzyme is SEQ ID NO: 4 or May include amino acid substitutions in one or more of positions 103, 194, 372, 375, 450, 509, and 570 of the sequence of number 5, and the Super piggyBac ™ transposase enzyme further May be included. In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ transposase enzyme is SEQ ID NO: 4 or Amino acid substitutions at 2, 3, 4, 5, 6 or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of number 5. The Super piggyBac ™ transposase enzyme may further comprise this. In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ transposase enzyme is SEQ ID NO: 4 or The amino acid substitutions at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of number 5 may be included, 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 a substitution of alanine (A) with lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of asparagine (N) with aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of glycine (G) with serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of serine (S) with asparagine (N). In certain embodiments, the piggyBac ™ transposase enzyme may comprise a substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO: 4. In certain embodiments, including the embodiment where the piggyBac ™ transposase enzyme may include a substitution of valine (V) by methionine (M) at position 194 of SEQ ID NO: 4, the piggyBac ™ transposase enzyme comprises SEQ ID NO: It may further comprise amino acid substitutions at positions 372, 375 and 450 of the sequence of 4 or SEQ ID NO: 5. In certain embodiments, the piggyBac ™ transposase enzyme replaces valine (V) with methionine (M) at position 194 of SEQ ID NO: 4, arginine (R) of alanine (A) at position 372 of SEQ ID NO: 4. And a substitution of alanine (A) at position 375 of SEQ ID NO: 4 with lysine (K). In certain embodiments, the piggyBac ™ transposase enzyme replaces valine (V) with methionine (M) at position 194 of SEQ ID NO: 4, arginine (R) of alanine (A) at position 372 of SEQ ID NO: 4. Substitution of alanine (A) at position 375 of SEQ ID NO: 4 with lysine (K) and substitution of asparagine (N) at position 450 of SEQ ID NO: 4 with aspartic acid (D).

The present disclosure provides a method of making a modified stem memory T cell ( _TSCM ) comprising: (a) a transposon comprising a transposon comprising an antigen receptor or a therapeutic protein to produce a modified T cell. Introducing a composition and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into a primary human T cell, wherein the modified T cell is one of stem memory T cells ( _TSCM ) Alternatively, a plurality of cell surface markers are expressed, thereby producing modified stem memory T cells (T _SCM ). The present disclosure provides a method of generating a plurality of modified stem memory T cells (T _SCM ), the method comprising (a) an antigen receptor or a therapeutic protein to generate a plurality of modified T cells. Transposon composition comprising a transposon, and (b) introducing a transposase or transposase composition comprising a transposase-encoding sequence into a plurality of primary human T cells, wherein at least 2% of the plurality of modified T cells, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 99%, or any percentage in between, expresses one or more cell surface markers of stem memory T cells (T _SCM ), thereby producing a plurality of modified stems Memory T cells (T _SCM ) are produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the cell surface marker for CAR-T _SCM comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell surface marker for CAR-T _SCM comprises 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 an overactive Sleeping Beauty transposase (SB100X).

In certain embodiments of the disclosed methods, the Sleeping Beauty transposase enzyme is
At least 75%, 80%, 85%, 90%, 95%, 99% or any percentage amino acid sequence between them.

In certain embodiments of the disclosed methods, the overactive Sleeping Beauty (SB100X) transposase enzyme comprises:
At least 75%, 80%, 85%, 90%, 95%, 99% or any percentage amino acid sequence between them.

The present disclosure provides a method of making a modified stem memory T cell ( _TSCM ) comprising: (a) a transposon comprising a transposon comprising an antigen receptor or a therapeutic protein to produce a modified T cell. Introducing a composition and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into a primary human T cell, wherein the modified T cell is one of stem memory T cells ( _TSCM ) Alternatively, a plurality of cell surface markers are expressed, thereby producing modified stem memory T cells (T _SCM ). The present disclosure provides a method of generating a plurality of modified stem memory T cells (T _SCM ), the method comprising: (a) a transposon comprising a transposon comprising an antigen receptor to produce a plurality of modified T cells. Introducing a composition and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into a plurality of primary human T cells, wherein at least 2%, 5%, 10% of the plurality of modified T cells. %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, any percentage between 99% and of which express one or more cell surface markers of stem memory T cells (T _SCM), whereby a plurality of modified stem memory T cells (T _{S M)} is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the cell surface marker for CAR-T _SCM comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell surface marker for CAR-T _SCM comprises 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.

In certain embodiments of the disclosed methods, the transposase is a Helitron transposase. The Helitron transposase moves the Helraiser transposon, an ancient element from the bat genome that was active about 3000-36 million years ago. Exemplary Helraiser transposons of the present disclosure include:
Helibat1 containing a nucleic acid sequence containing

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

Exemplary Helitron transposases of the present disclosure are:
An amino acid sequence comprising

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

The present disclosure provides a method of making a modified stem memory T cell ( _TSCM ) comprising: (a) a transposon comprising a transposon comprising an antigen receptor or a therapeutic protein to produce a modified T cell. Introducing a composition and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into a primary human T cell, wherein the modified T cell is one of stem memory T cells ( _TSCM ) Alternatively, a plurality of cell surface markers are expressed, thereby producing modified stem memory T cells (T _SCM ). The present disclosure provides a method of generating a plurality of modified stem memory T cells (T _SCM ), the method comprising: (a) a transposon comprising a transposon comprising an antigen receptor to produce a plurality of modified T cells. Introducing a composition and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into a plurality of primary human T cells, wherein at least 2%, 5%, 10% of the plurality of modified T cells. %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, any percentage between 99% and of which express one or more cell surface markers of stem memory T cells (T _SCM), whereby a plurality of modified stem memory T cells (T _{S M)} is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells have one or more cell surface markers of stem memory T cells (T _SCM ). Expressed thereby creating a plurality of modified stem memory T cells ( _TSCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the cell surface marker for CAR-T _SCM comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell surface marker for CAR-T _SCM comprises 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.

In certain embodiments of the disclosed method, the transposase is a Tol2 transposase. Tol2 transposons can be isolated or derived from the medaka genome and can be similar to hAT family transposons. An exemplary Tol2 transposon of the present disclosure contains a gene encoding a Tol2 transposase that is encoded by a sequence comprising about 4.7 kilobases and contains four exons. An exemplary Tol2 transposase of the present disclosure is:
An amino acid sequence comprising

Exemplary Tol2 transposases of the present disclosure include inverted repeats, near-terminal sequences and Tol2 transposases, including:
Of the nucleic acid sequence.

The present disclosure provides a method for producing modified central memory T cells (T _CM ), which comprises (a) a transposon comprising a transposon comprising an antigen receptor or a therapeutic protein to produce modified T cells. And (b) introducing a transposase or a transposase composition comprising a transposase-encoding sequence into a primary human T cell, wherein the modified T cell is one of central memory T cells (T _CM ). Alternatively, a plurality of cell surface markers are expressed, thereby producing modified central memory T cells (T _CM ). The present disclosure provides a method of generating a plurality of modified central memory T cells (T _CM ), the method comprising: (a) a transposon comprising a transposon comprising an antigen receptor to produce a plurality of modified T cells. Introducing a composition and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into a plurality of primary human T cells, wherein at least 2%, 5%, 10% of the plurality of modified T cells. %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between expresses one or more cell surface markers of central memory T cells (T _CM ), whereby multiple modified central media Molly T cells (T _CM ) are generated. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 25% of the plurality of modified T cells have one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 50% of the plurality of modified T cells contain one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 60% of the plurality of modified T cells have one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 75% of the plurality of modified T cells have one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 80% of the plurality of modified T cells have one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 85% of the plurality of modified T cells have one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 90% of the plurality of modified T cells have one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the method generates a plurality of modified T cells, wherein at least 95% of the plurality of modified T cells have one or more cell surface markers of central memory T cells (T _CM ). It expressed, whereby a plurality of modified central memory T cells (T _CM) is produced. In certain embodiments, the cell surface marker comprises one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments of this method, the transposon is a plasmid DNA transposon and the sequence encoding the antigen receptor or therapeutic protein is flanked by two cis-regulatory insulator elements. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly those in which the transposon is a piggyBac transposon, the transposase is a piggyBac ™ or Super piggyBac ™ (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly those in which the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or an overactive 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 embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

The present disclosure provides a method of making 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 method comprising a plurality of modified T _SCMs and a plurality of modified T _SCMs in order to produce a composition comprising a modified T _CM, the transposase composition comprising a sequence encoding (a) a transposon composition comprising a transposon containing the antigen receptor or therapeutic protein, and (b) a transposase or transposase wherein the step of introducing a plurality of primary human T cells, wherein the plurality of modified _{T SCM,} expressed CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and one or more of IL-2R beta , the plurality of modified _{T CM, CD45RO, CD95, IL} -2R beta, CCR7, and express one or more of CD62L, whereby a composition comprising a plurality of modified _{T SCM} and more modifications _{T CM} is produced. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified central memory T cells (T _CM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 10% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 90% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 90% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 10% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 20% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 80% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 80% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 20% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 30% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 70% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 70% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 30% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 40% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 60% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 60% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 40% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 50% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 50% of the total number. 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 in which the transposon is a piggyBac transposon, the transposase is a piggyBac ™ or Super piggyBac ™ (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly those in which the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or an overactive 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 embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

  In certain embodiments of the disclosed methods, the transposon may be derived from or recombined from any species. Alternatively or in addition, a transposon. It can be a synthetic transposon.

  In certain embodiments of the disclosed method, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, particularly those in which 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, particularly those in which the T cell receptor does not naturally occur, 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 includes one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

  The present disclosure includes embodiments comprising the steps of introducing (a) a transposon composition comprising a transposon comprising an antigen receptor and (b) a transposase composition comprising a transposase or a transposase-encoding sequence into primary human T cells. In certain embodiments of the method, the method is the step of introducing (c) a second transposon composition comprising a transposon comprising a therapeutic protein into primary human T cells to generate modified T cells. The modified T cell further comprises a step capable of expressing the therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein, and modified T cells capable of secreting the therapeutic protein are produced by the method. In certain embodiments, the transposase composition of (b) undergoes transposition with the transposon of (a) and the transposon of (c). In certain embodiments, the method further comprises (d) introducing a second transposase composition comprising a transposase or a transposase-encoding sequence into primary human T cells. In certain embodiments, the second transposase composition undergoes metastasis with 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 a sequence 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 in which the transposon is a piggyBac transposon, the transposase is a piggyBac ™ or Super piggyBac ™ (SPB) transposase. In certain embodiments of this method, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly those in which the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or an overactive 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 embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

The present disclosure is a method for producing a plurality of modified stem memory T cells ( _TSCM ), wherein (a) a plurality of primary human T cells includes an antigen receptor in order to produce a plurality of modified T cells. Introducing a composition, wherein no antigen receptor or therapeutic protein is contained in the transposon; and (b) a plurality of modified T cells to generate a plurality of activated modified T cells. T cell activation comprising a cell and one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement Contacting the factor composition, wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T cells are stems Memory T cells ( _TSCM ) Wherein one or more cell surface markers are expressed, thereby generating a plurality of modified stem memory T cells (T _SCM ). The present disclosure is a method for producing a plurality of modified stem memory T cells ( _TSCM ), wherein (a) a plurality of primary human T cells includes an antigen receptor in order to produce a plurality of modified T cells. Introducing a composition, wherein no antigen receptor or therapeutic protein is contained in the transposon; and (b) a plurality of modified T cells to generate a plurality of activated modified T cells. T cell activation comprising a cell and one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement Contacting the factor composition with at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of the plurality of activated modified T cells. %, 50%, 60%, 65%, 70 , 75%, 80%, 85%, 90%, 95%, any percentage between 99% or they express one or more cell surface markers of stem memory T cells _{(T SCM),} whereby A method is provided wherein a plurality of modified stem memory T cells ( _TSCM ) are generated. In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the method creates a plurality of activated modified T cells, wherein at least 50% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the method creates a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the method creates a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ). Expresses one or more cell surface markers, thereby creating a plurality of activated modified stem memory T cells (T _SCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, cell surface markers of the modified _{T SCM} activated include CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, one or more of CD95 and IL-2R beta. In certain embodiments, cell surface markers of the modified _{T SCM} activated include CD45RA, CD95, IL-2Rβ, CR7, and one or more of CD62L.

(A) introducing a composition containing an antigen receptor into primary human T cells to produce a modified T cell, wherein the antigen receptor or therapeutic protein is not contained in the transposon; (B) one or more of a modified T cell and an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement. In certain embodiments of the methods of the present disclosure for producing modified stem memory T cells (T _SCM ) comprising contacting activated T cell activating composition to produce activated modified T cells, The T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method comprises (c) activated modified T cells and human serum albumin, recombinant human insulin, human transferrin, 2-to produce a plurality of expanded modified T cells. Contacting a T cell expansion composition comprising mercaptoethanol, Iskov MDM, and one or more augmentation supplements, wherein at least 2% of the plurality of altered T cells are stem memory T cells ( _TSCM A) expressing one or more cell surface markers. In certain embodiments of the method, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the plurality of increased modified T cells. %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between is a cell surface marker of stem memory T cells ( _TSCM ) Is expressed. In certain embodiments of this method, at least 60% of the plurality of augmented modified T cells express a cell surface marker of stem memory T cells (T _SCM ). In certain embodiments, the method comprises (d) enriching a plurality of augmented modified T cells to express at least 2% modified T cells expressing a cell surface marker of stem memory T cells (T _SCM ), 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 99%, or any percentage in between, is further included. In certain embodiments, the method comprises (d) at least 60% modified T cells enriched with a plurality of increased modified T cells and expressing a cell surface marker of stem memory T cells (T _SCM ). The method further includes the step of making a composition. In certain embodiments of this method, the step of enriching comprises modifying T cells expressing one or more cell surface markers of stem memory T cells (T _SCM ) from a plurality of enriched modified T cells. Including isolation. In certain embodiments of this method, the step of enriching is to produce modified T _SCM which is more increased enrichment, and isolated modified T _SCM, human serum albumin, recombinant human insulin , Further comprising contacting the T cell expansion composition comprising human transferrin, 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate. (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic hydrazide, one of oleamide, sterol and alkane Or further includes a plurality. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and sterol. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including endpoints; at a concentration of 0.2 mg / kg to 20 mg / kg including endpoints. Palmitic acid; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoint; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoint; and about 0.1 mg / kg including endpoint Further comprising one or more of sterols at a concentration of 10 mg / kg. In certain embodiments of this method, the T cell expansion composition comprises 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, about 2 mg / kg. It further comprises one or more of oleic acid at a concentration of kg and sterol at a concentration of about 1 mg / kg. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints. Palmitic acid; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including endpoint; and 0.25 μmol / kg to 25 μmol including the endpoint It further comprises one or more of sterols at a concentration of / kg. In certain embodiments of this method, the T cell expansion composition comprises 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, It further comprises one or more of oleic acid at a concentration of 7.5 μmol / kg and sterol at a concentration of about 2.5 μmol / kg.

The present disclosure is a method for producing a modified central memory T cell (T _CM ), and (a) a step of producing a modified T cell by introducing a composition containing an antigen receptor into a primary human T cell. Wherein no antigen receptor or therapeutic protein is contained in the transposon, and (b) a modified T cell and anti-human CD3 monospecific tetrameric antibody complex, anti-human CD28 monospecific Providing a modified T cell activated by contacting a T cell activation composition comprising a typical tetrameric antibody complex and one or more of the activation supplements; here, the modified T cells activated express one or more cell surface markers of central memory T cells (T _CM), which central memory T cells (T _CM) is prepared by . The present disclosure is a method for producing a plurality of modified central memory T cells (T _CM ), and (a) producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells. Wherein no antigen receptor or therapeutic protein is contained in the transposon, and (b) a plurality of modified T cells and an anti-human CD3 monospecific tetrameric antibody complex A plurality of activated modified T cells by contacting the body, a T cell activation composition comprising one or more of an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement. Providing a method comprising generating at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of a plurality of activated modified T cells 45%, 50%, 60%, 5%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or one or more of cell surface markers of any percentages central memory T cells between them _{(T CM)} Expressed thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method creates a plurality of activated modified T cells, wherein at least 25% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 50% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method creates a plurality of activated modified T cells, wherein at least 60% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 75% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 80% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method creates a plurality of activated modified T cells, wherein at least 85% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 90% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, the method generates a plurality of activated modified T cells, wherein at least 95% of the plurality of activated modified T cells is one of central memory T cells (T _CM ). One or more cell surface markers are expressed, thereby creating a plurality of activated modified central memory T cells (T _CM ). In certain embodiments, cell surface markers of activated modified _{T CM} includes CD45RO, CD95, IL-2Rβ, CCR7, and one or more of CD62L.

(A) introducing a composition comprising an antigen receptor into primary human T cells to produce a modified T cell, wherein the antigen receptor or therapeutic protein is not contained in the transposon; and (B) one or more of a modified T cell and an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement. In certain embodiments of the disclosed method of producing modified central memory T cells (T _CM ) comprising the step of producing activated modified T cells by contacting with a T cell activation composition comprising: The T cell activation composition of b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method comprises (c) activated modified T cells and human serum albumin, recombinant human insulin, human transferrin, 2-to produce a plurality of expanded modified T cells. mercaptoethanol, a step of contacting a T cell expansion composition comprising one or more of Iscove's MDM, and increased supplements, at least 2% more increased modified T cells, central memory T cells (T _CM A) expressing one or more cell surface markers. In certain embodiments of this method, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the plurality of increased modified T cells. %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between is a cell surface marker of central memory T cells (T _CM ) Is expressed. In certain embodiments of this method, at least 60% of the plurality of augmented modified T cells express a cell surface marker of central memory T cells (T _CM ). In certain embodiments, the method comprises (d) enriching a plurality of increased modified T cells to express at least 2% modified T cells expressing a cell surface marker of central memory T cells (T _CM ), 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 99%, or any percentage in between, is further included. In certain embodiments, the method comprises (d) at least 60% modified T cells enriched with a plurality of increased modified T cells and expressing a cell surface marker of central memory T cells (T _CM ). The method further includes the step of making a composition. In certain embodiments of this method, the step of enriching comprises modifying T cells that express one or more cell surface markers of central memory T cells (T _CM ) from the plurality of enriched modified T cells. Including isolation. In certain embodiments of this method, the step of enriching is to produce a plurality of increased enriched modified T _CM, a modified T _CM isolated, human serum albumin, recombinant human insulin Contacting with a T cell expansion composition comprising one or more of: human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplement. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate. (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic hydrazide, one of oleamide, sterol and alkane Or further includes a plurality. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and sterol. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including endpoints; at a concentration of 0.2 mg / kg to 20 mg / kg including endpoints. Palmitic acid; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoint; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoint; and about 0.1 mg / kg including endpoint Further comprising one or more of sterols at a concentration of 10 mg / kg. In certain embodiments of this method, the T cell expansion composition comprises 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, about 2 mg / kg. It further comprises one or more of oleic acid at a concentration of kg and sterol at a concentration of about 1 mg / kg. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints. Palmitic acid; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including endpoint; and 0.25 μmol / kg to 25 μmol including the endpoint It further comprises one or more of sterols at a concentration of / kg. In certain embodiments of this method, the T cell expansion composition comprises 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, It further comprises one or more of oleic acid at a concentration of 7.5 μmol / kg and sterol at a concentration of about 2.5 μmol / kg.

The present disclosure is a method of making a composition comprising a plurality of modified stem memory T cells (T _SCM ) and a plurality of modified central memory T cells (T _CM ), and (a) accepts a plurality of primary human T cells for antigen reception Introducing a composition comprising a body to generate a plurality of modified T cells, wherein no antigen receptor or therapeutic protein is contained in the transposon; and (b) a plurality of modified T cells. And a T cell activation composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement including the step of contacting the object 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) Provides a method, the plurality of activated modified _{T SCM, CD62L, CD45RA, CD28} , CCR7, CD127, CD45RO, CD95, CD95 and express one or more of IL-2R beta, was more activated modified _T CM is, CD45RO, CD95, IL-2Rβ , CCR7, and express one or more of CD62L, whereby a composition comprising a plurality of modified _{T SCM} and more modifications _{T CM} is produced. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified central memory T cells (T _CM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 10% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 90% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 90% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 10% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 20% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 80% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 80% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 20% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 30% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 70% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 70% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 30% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 40% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 60% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 60% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 40% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 50% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 50% of the total number.

(A) a step of producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon. , Steps, and (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement A plurality of activated modified stem memory T cells (T _SCM ) and a plurality of activated modified central memory T cells (T _CM ) in contact with a T cell activation composition comprising one or more of JP with a plurality of modified stem memory T cells (T _SCM) and methods of the present disclosure for producing a composition comprising a plurality of modified central memory T cells (T _CM) comprising the step of making a composition comprising In embodiments, T cell activation composition (b) further comprises an antibody conjugate of anti-human CD2 monospecific tetramer. In certain embodiments, the method comprises (c) a composition, a plurality of activated modified stem memory T cells (T _SCM ) and a plurality of activations to generate a plurality of augmented modified T cells. It has been modified central memory T cells (T _CM) of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, contact with Iscove's MDM, and T cell expansion composition comprising one or more of increasing supplements a step of, the plurality of increased modified _{T SCM, CD62L, CD45RA, CD28} , CCR7, CD127, CD45RO, CD95, CD95 and express one or more of IL-2R beta, modified _{T CM} in which a plurality of increased , CD45RO, CD95, IL-2Rβ, CCR7, and CD62L or Further comprising the step of expressing a plurality, thereby creating a composition comprising a plurality of increased modified T _SCMs and a plurality of increased modified T _CMs . In certain embodiments of this method, the step of enriching comprises modifying T cells expressing one or more cell surface markers of stem memory T cells (T _SCM ) from a plurality of enriched modified T cells. including isolating it, or a plurality of isolating the modified T cells that express one or more cell surface markers of the enriched modified T cells from central memory T cells (T _CM). In certain embodiments of this method, the enriching step includes isolating from a plurality of enriched modified T cells and expressing one or more cell surface markers of stem memory T cells (T _SCM ). And isolating modified T cells that express one or more cell surface markers of central memory T cells (T _CM ) from the plurality of enriched modified T cells. In certain embodiments of this method, step, to produce a composition comprising a plurality of increased enriched modified T _SCM and more increased enriched modified T _CM enriching single T a composition comprising isolated altered TSCM and isolated modified T _CM containing human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and one or more of increasing supplements It further comprises contacting with the cell augmentation composition. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate. (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic hydrazide, one of oleamide, sterol and alkane Or further includes a plurality. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and sterol. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including endpoints; at a concentration of 0.2 mg / kg to 20 mg / kg including endpoints. Palmitic acid; linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoint; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoint; and about 0.1 mg / kg including endpoint Further comprising one or more of sterols at a concentration of 10 mg / kg. In certain embodiments of this method, the T cell expansion composition comprises 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, about 2 mg / kg. It further comprises one or more of oleic acid at a concentration of kg and sterol at a concentration of about 1 mg / kg. In certain embodiments of this method, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints. Palmitic acid; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including endpoint; and 0.25 μmol / kg to 25 μmol including the endpoint It further comprises one or more of sterols at a concentration of / kg. In certain embodiments of this method, the T cell expansion composition comprises 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, It further comprises one or more of oleic acid at a concentration of 7.5 μmol / kg and sterol at a concentration of about 2.5 μmol / kg. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified central memory T cells (T _CM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 10% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 90% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 90% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 10% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 20% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 80% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 80% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 20% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 30% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 70% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 70% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 30% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 40% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 60% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 60% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 40% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 50% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 50% of the total number.

(A) A step of producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon. And b) contacting a plurality of modified T cells with a T cell activation composition comprising one or more anti-human CD3 monospecific tetrameric antibody complexes. in certain embodiments of the method of making an activated modified T _SCM or T _CM of the present disclosure including the step of introducing includes homologous recombination. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition is contacted with the genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition is contacted with the genomic sequence of a portion of the primary T cell of the plurality of T cells. In certain embodiments, the portion of primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25 of the total number of primary T cells in the plurality of T cells. %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or Any percentage between these. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition is contacted with the genomic sequence of each primary T cell of the plurality of T cells. In certain embodiments of the introducing step involving homologous recombination, single-strand breaks are induced by the genome editing composition. In certain embodiments of the introducing step involving homologous recombination, double-strand breaks are induced by the genome editing composition. In certain embodiments of the introducing step involving homologous recombination, the introducing step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition includes a sequence encoding an antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding an antigen receptor, a 5 ′ genomic sequence and a 3 ′ genomic sequence, wherein the 5 ′ genomic sequence is derived by a genomic editing composition. Homologous or identical to the genomic sequence of the primary T cell, 5 ′ to the breakpoint, and the 3 ′ genome sequence is 3 ′ to the breakpoint induced by the genome editing composition. Homologous or identical to the T cell genomic sequence. In certain embodiments of the introduction step involving homologous recombination, the genome editing composition and the donor sequence composition are contacted with the genome sequence simultaneously or sequentially. In certain embodiments of the introduction step involving homologous recombination, the genome editing composition and donor sequence composition are sequentially contacted with the genome sequence, resulting in the genome editing composition first. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition comprises a DNA binding domain and a nuclease domain. In certain embodiments of the genome editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genome editing composition, the DNA binding domain comprises a TALEN DNA binding domain. In certain embodiments of the genome editing composition, the DNA binding domain comprises a ZFN DNA binding domain. In certain embodiments of the genome editing composition, the nuclease domain comprises a Cas9 nuclease or sequence thereof. In certain embodiments of the genome editing composition, the nuclease domain comprises inactive Cas9 (SEQ ID NO: 33, substitution of alanine (A) at position 10 with aspartic acid (D) (D10A), and alanine at position 840 (A ) With histidine (H) (including H840A). In certain embodiments of the genome editing composition, the nuclease domain comprises short inactive Cas9 (SEQ ID NO: 32, substitution of alanine (A) at position 10 with aspartic acid (D) (D10A) and alanine at position 540 (A ) With asparagine (N) (including N540A). In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises a type IIS endonuclease. In certain embodiments of the genome editing composition, the type IIS endonuclease is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, MboII, MboII, Mbo1I PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsGE Contains BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments, the Type IIS endonuclease comprises Clo051. In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises TALEN or a nuclease domain thereof. In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises ZFN or a nuclease domain thereof. In certain embodiments of the introduction step involving homologous recombination, the genomic editing composition induces breaks in the genomic sequence and the primary T cell's endogenous DNA repair machinery is used to insert the 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.

(A) A step of producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon. , Steps, and (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement one or in certain embodiments of the methods of making the activated modified T _SCM or T _CM of the present disclosure includes a method comprising contacting a T cell activating composition comprising a plurality of viral vectors Includes antigen receptors. In certain embodiments, the viral vector comprises one or more sequences isolated from, derived from, or recombined 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 recombined 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 defective.

(A) A step of producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon. , Steps, and (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement one or in certain embodiments of the methods of making the activated modified T _SCM or T _CM of the present disclosure includes a method comprising contacting a T cell activating composition comprising a plurality of viral vectors Includes antigen receptors. In certain embodiments, the viral vector comprises a sequence isolated from or derived from a retrovirus. In certain embodiments, the viral vector comprises a sequence isolated from or derived from a lentivirus.

(A) A step of producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon. , Steps, and (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement one or in certain embodiments of the methods of making the activated modified T _SCM or T _CM of the present disclosure includes a method comprising contacting a T cell activating composition comprising a plurality of viral vectors Includes antigen receptors. In certain embodiments, the viral vector comprises a sequence isolated from or derived from a retrovirus. In certain embodiments, the viral vector comprises a sequence isolated from or derived from a gamma retrovirus.

(A) A step of producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon. , Steps, and (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement one or in certain embodiments of the methods of making the activated modified T _SCM or T _CM of the present disclosure includes a method comprising contacting a T cell activating composition comprising a plurality of viral vectors Includes antigen receptors. In certain embodiments, the viral vector comprises sequences isolated from or derived from an 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 comprises a sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11. In certain embodiments, the AAV is isolated from, derived from or recombined from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 Contains an array. In certain embodiments, the AAV comprises a sequence isolated from, derived from, or recombined from AAV2. In certain embodiments, including embodiments where the vector crosses the blood brain barrier (BBB), the AAV comprises sequences isolated, derived or recombinant from AAV9. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing one serotype genome and another serotype capsid. (For example, AAV2 / 5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, rAAV-LK03, rAAV-NP59 and rAAV-NP84.

In certain embodiments of the methods of making activated modified T _SCM or T _CM of the present disclosure, the nucleic acid vector comprises an antigen receptor. In certain embodiments, the DNA vector includes an antigen receptor. In certain embodiments, the mRNA vector comprises an antigen receptor. In certain embodiments, the nucleic acid vector is a plasmid or a minicircle vector.

In certain embodiments of the methods of making activated modified T _SCM or T _CM of the present disclosure, the nanoparticles vector comprises an antigen receptor. Nanoparticles are, for example, International Patent Publication No. WO2012 / 094679, International Patent Publication No. WO2016 / 022805, International Patent Publication No. WO / 2011/133635, International Patent Publication No. WO / 2016/090111, International Patent Publication No. WO / 2017/004498, WO / 2017/004509, International Patent Application No. PCT / US2017 / 030271, US Pat. No. 6,835,394, US Pat. No. 7,217,427, and US Patent It may be composed of a polymer disclosed in No. 7,867,512.

In certain embodiments of the method of making an activated modified T _SCM or T _CM of the present 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, particularly those in which 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, particularly those in which the T cell receptor does not naturally occur, 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 includes one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

(A) A step of producing a plurality of modified T cells by introducing a composition containing an antigen receptor into a plurality of primary human T cells, wherein the antigen receptor or therapeutic protein is not contained in the transposon. , Steps, and (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement a method of making an activated modified T _SCM or T _CM of the present disclosure includes a method comprising contacting a T cell activating composition comprising one or more in certain embodiments, the method In order to generate a modified T cell capable of expressing a therapeutic protein, the method further includes the step of introducing a composition containing the therapeutic protein into primary human T cells. In certain embodiments, the therapeutic protein is a secreted protein and the method produces modified T cells capable of secreting the therapeutic protein. In certain embodiments, the introducing step comprises homologous recombination and the donor sequence comprises a sequence encoding a therapeutic protein. In certain embodiments, the donor sequence comprising the antigen receptor further comprises a therapeutic protein. In certain embodiments, the first donor sequence includes an antigen receptor and the second donor sequence includes a therapeutic protein. In certain embodiments, the vector includes a sequence encoding a therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle. In certain embodiments, the vector comprising the antigen receptor further comprises a therapeutic protein. In certain embodiments, the first vector includes an antigen receptor and the second vector template includes a therapeutic protein.

The present disclosure is a method of producing modified stem memory T cells ( _TSCM ), and (a) introducing a composition containing an antigen receptor into primary human T cells in order to produce modified T cells. A transposon comprises an antigen receptor, (b) a modified T cell, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex Contacting the body with a T cell activation composition comprising one or more of the activation supplements to produce activated modified T cells, wherein the activated modification -T cells express one or more cell surface markers of stem memory T cells _{(T SCM),} whereby the modified stem memory T cells _{(T SCM)} is produced. The present disclosure is a method of producing a plurality of modified stem memory T cells ( _TSCM ), and (a) a composition comprising an antigen receptor in a plurality of primary human T cells in order to produce a plurality of modified T cells. A transposon contains an antigen receptor, (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific Contacting a T cell activation composition comprising a typical tetrameric antibody complex and one or more of the activation supplements to produce a plurality of activated modified T cells. And 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 ( _TSCM ) Emit one or more cell surface markers Thus, modified stem memory T cells ( _TSCM ) are produced thereby. In certain embodiments of this method, at least 60% of the plurality of activated modified-T cells express one or more cell surface markers of stem memory T cells (T _SCM ). In certain embodiments of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. The present disclosure is a method for producing a modified stem memory T cell ( _TSCM ), and (a) a composition comprising a chimeric antigen receptor (CAR) in a primary human T cell to produce a CAR-T cell. And (b) CAR-T cells and anti-human CD3 monospecific tetrameric antibody complex, anti-human CD28 monospecific tetrameric antibody complex, anti-human CD2 single Contacting the T-cell activation composition comprising a monospecific tetrameric antibody complex and one or more of the activation supplements to produce activated CAR-T cells. provided, where, CAR-T cells activated express one or more cell surface markers of stem memory T cells _{(T SCM),} whereby CAR expression stem memory T cells _{(T SCM)} ( _{CAR-T SCM)} is created It is. The present disclosure is a method for producing a plurality of modified stem memory T cells (T _SCM ). (A) In order to produce a plurality of CAR-T cells, a plurality of primary human T cells are treated with a chimeric antigen receptor (CAR). And b) a plurality of CAR-T cells and an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific tetramer of Contacting a T cell activation composition comprising one or more of an antibody conjugate, an anti-human CD2 monospecific tetrameric antibody conjugate and an activation supplement, a plurality of activated CAR- A method comprising the steps of generating T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35 of a plurality of activated CAR-T cells is provided. %, 40%, 45%, 50%, 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, any percentage between 99% or they express one or more cell surface markers of stem memory T cells _{(T SCM),} whereby A plurality of activated CAR stem memory T cells (T _SCM ) are generated. In certain embodiments, the method comprises (c) activated modified T cells and human serum albumin, recombinant human insulin, human transferrin, 2-mercapto to generate a plurality of expanded modified T cells. Contacting a T cell expansion composition comprising ethanol, Iskov MDM, and one or more augmentation supplements, wherein at least 2% of the plurality of altered T cells are stem memory T cells ( Expressing one or more cell surface markers of T _SCM ). In certain embodiments, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. Including or further including. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. Contains oleic acid at a concentration of 13 mg / kg and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60 of a plurality of increased modified T cells. %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between expresses cell surface markers of stem memory T cells (T _SCM ) . In certain embodiments, at least 60% of the plurality of augmented modified T cells express a cell surface marker of stem memory T cells (T _SCM ). In certain embodiments, the method comprises (d) at least 2%, 5% modified T cells enriched with a plurality of augmented modified T cells and expressing a cell surface marker of stem memory T cells (T _SCM ). 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or any percentage in between, is further included. In certain embodiments, the method comprises (d) a composition comprising at least 60% of modified T cells enriched with a plurality of increased modified T cells and expressing a cell surface marker of stem memory T cells (T _SCM ). The method further includes the step of making the object. In certain embodiments, the enriching step isolates modified T cells that express one or more cell surface markers of stem memory T cells (T _SCM ) from the plurality of enriched modified T cells. In addition. In certain embodiments, the step of enrichment is to produce modified T _SCM which is more increased enrichment, and isolated modified T _SCM, human serum albumin, recombinant human insulin, human transferrin , Further comprising contacting with a T cell expansion composition comprising one or more of: 2-mercaptoethanol, Iskov MDM, and augmentation supplement. In certain embodiments, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. In addition. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. 13mg /
Contains oleic acid at a concentration of kg, and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg.

The present disclosure is a method for producing modified central memory T cells (T _CM ), and (a) introducing a composition containing an antigen receptor into primary human T cells in order to produce modified T cells. A transposon comprises an antigen receptor, (b) a modified T cell, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex Contacting the body with a T cell activation composition comprising one or more of the activation supplements to produce activated modified T cells, wherein the activated modification -T cells express one or more cell surface markers of central memory T cells _{(T CM),} whereby the modified central memory T cells _{(T CM)} is produced. The present disclosure is a method for producing a plurality of modified central memory T cells (T _CM ), and (a) a composition comprising an antigen receptor in a plurality of primary human T cells to produce a plurality of modified T cells. A transposon contains an antigen receptor, (b) a plurality of modified T cells, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific Contacting a T cell activation composition comprising a typical tetrameric antibody complex and one or more of the activation supplements to produce a plurality of activated modified T cells. Where at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T cells are central memory T cells (T _CM ) one or more of Express cell surface markers, thereby creating modified central memory T cells (T _CM ). In certain embodiments of this method, at least 60% of the plurality of activated modified-T cells express one or more cell surface markers of central memory T cells (T _CM ). In certain embodiments of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method comprises (c) activated modified T cells and human serum albumin, recombinant human insulin, human transferrin, 2-mercapto to produce a plurality of expanded modified T cells. Contacting a T cell expansion composition comprising ethanol, Iskov MDM, and one or more augmentation supplements, wherein at least 2% of the plurality of altered T cells are central memory T cells (T _CM ) Expressing one or more cell surface markers. In certain embodiments, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. Including or further including. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. Contains oleic acid at a concentration of 13 mg / kg and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60 of a plurality of increased modified T cells. %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween express the cell surface marker of central memory T cells (T _CM ) . In certain embodiments, at least 60% of the plurality of expanded modified T cells express a cell surface marker of central memory T cells (T _CM ). In certain embodiments, the method, (d) enriched with a plurality of increased modified T cells to express at least 2%, 5% modified T cells expressing a cell surface marker of central memory T cells (T _CM ). 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or any percentage in between, is further included. In certain embodiments, the method comprises (d) a composition comprising at least 60% modified T cells enriched with a plurality of increased modified T cells and expressing a cell surface marker of central memory T cells (T _CM ). The method further includes the step of making the object. In certain embodiments, the enriching step isolates modified T cells that express one or more cell surface markers of central memory T cells (T _CM ) from the plurality of enriched modified T cells. Further includes. In certain embodiments, the step of enrichment is to produce a plurality of increased enriched modified T _CM, a modified T _CM isolated, human serum albumin, recombinant human insulin, human transferrin , Further comprising contacting a T cell expansion composition comprising one or more of: 2-mercaptoethanol, Iskov MDM, and augmentation supplement. In certain embodiments, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. In addition. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. Contains oleic acid at a concentration of 13 mg / kg and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg.

The present disclosure is a method of making a composition comprising a plurality of modified stem memory T cells (T _SCM ) and a plurality of modified central memory T cells (T _CM ), and (a) a plurality of modified stem memory T cells (T _SCM ) and a plurality of modified central memory T cells (T _CM ) to produce a composition comprising an antigen receptor in a plurality of primary human T cells, wherein the transposon is an antigen receptor. (B) a composition, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, and an activation supplement. one or T cell activating composition and contacting a plurality of activated modified stem memory T cells (T _SCM) and a plurality of activated modified central memory T cells, including a plurality (T _C ) Provides a method comprising making a composition comprising a plurality of activated modified _{T SCM} is CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and one or more of IL-2R beta expressing a plurality of activated modified _{T CM} is CD45RO, CD95, IL-2Rβ, CCR7, and express one or more of CD62L, thereby a plurality of modified _{T SCM} and a plurality of modified _{T CM} A composition comprising is made. In certain embodiments of this method, the T cell activation composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. In certain embodiments, the method comprises (c) a composition and human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, to generate a plurality of augmented modified T cells, And a T cell expansion composition comprising one or more of the augmented supplements, wherein at least 2% of the composition comprising a plurality of augmented modified T cells comprises stem memory T cells (T _SCM ) Further comprising expressing one or more cell surface markers. In certain embodiments, the method comprises (c) a composition and human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, to generate a plurality of augmented modified T cells, And a T cell expansion composition comprising one or more of the augmented supplements, wherein at least 2% of the composition comprising a plurality of augmented modified T cells comprises central memory T cells (T _CM ) Further comprising expressing one or more cell surface markers. In certain embodiments, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. Including or further including. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. Contains oleic acid at a concentration of 13 mg / kg and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg. In certain embodiments, at least 2% of the cells in a composition comprising a plurality of increased modified _{T SCM} and more increased modified _{T CM, 5%, 10%} , 15%, 20%, 25%, 30% , 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between Expresses cell surface markers of memory T cells (T _SCM ). In certain embodiments, at least 2% of the cells in a composition comprising a plurality of increased modified _{T SCM} and more increased modified _{T CM, 5%, 10%} , 15%, 20%, 25%, 30% , 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between is central expressing the cell surface marker of memory T cells _{(T CM).} In certain embodiments, the method comprises (d) at least 2%, 5%, 10% modified T cells enriched with the composition and expressing a cell surface marker of stem memory T cells (T _SCM ), 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% Or further comprising making a composition comprising any percentage between them. In certain embodiments, the method comprises (d) at least 2%, 5%, 10% modified T cells enriched with the composition and expressing a cell surface marker of central memory T cells (T _CM ), 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% Or further comprising making a composition comprising any percentage between them. In certain embodiments, the step of enriching comprises isolating modified T cells expressing one or more cell surface markers of stem memory T cells (T _SCM ) from the composition, or centralizing from the composition. Further comprising isolating a modified T cell that expresses one or more cell surface markers of memory T cells (T _CM ). In certain embodiments, the step of enriching comprises isolating modified T cells that express one or more cell surface markers of stem memory T cells (T _SCM ) and central memory T from the composition. modified T cells that express one or more cell surface markers of cells (T _CM) further comprises isolating. In certain embodiments, the enriching step comprises isolating the modified modified T _SCM and / or T to produce a composition comprising a plurality of increased enriched modified T _SCM and / or T _CM. Further comprising contacting the _CM with a T cell expansion 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 expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. In addition. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. Contains oleic acid at a concentration of 13 mg / kg and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified central memory T cells (T _CM ) are at least 1%, 2%, 5%, 7%, 10%, 15%, 20% of the total number of cells of the composition. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99 Occupy% or any percentage of cells between them. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 10% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 90% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 90% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 10% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 20% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 80% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 80% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 20% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 30% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 70% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 70% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 30% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 40% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 60% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 60% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 40% of the total number. In certain embodiments of this method, the modified stem memory T cells (T _SCM ) comprise at least 50% of the total number of cells of the composition, and the modified central memory T cells (T _CM ) It accounts for at least 50% of the total number.

  A method comprising introducing into a primary human T cell; (a) introducing a composition comprising an antigen receptor into the primary human T cell to produce a modified T cell, wherein the transposon comprises an antigen receptor; And (b) one of a modified T cell, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement. In certain embodiments of the disclosed methods, including embodiments comprising contacting a T cell activation composition comprising one or more to produce activated modified T cells, the method comprises modifying T (C) introducing a second transposon composition comprising a transposon comprising a therapeutic protein into primary human T cells, wherein the modified T cells express the therapeutic protein. Further comprising the step that can. In certain embodiments, the therapeutic protein is a secreted protein and the method produces modified T cells capable of secreting the therapeutic protein. In certain embodiments, the method further comprises introducing a transposase composition. In certain embodiments, the transposase composition is transferred with the transposon of (a) and the second transposon. In certain embodiments, the method includes introducing a first transposase composition and a second transposase composition. In certain embodiments, including embodiments where the method comprises introducing a first transposase composition and a second transposase composition, the first transposase composition is transferred with the transposon of (a), The second transposase composition is transferred with the second transposon. In certain embodiments of this method, the transposon is a plasmid DNA transposon having a sequence encoding an antigen receptor or therapeutic protein 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 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 an overactive 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 embodiments where the transposon is a Tol2 transposon, the transposase is a Tol2 transposase.

  A method comprising introducing into a primary human T cell; (a) introducing a composition comprising an antigen receptor into the primary human T cell to produce a modified T cell, wherein the transposon comprises an antigen receptor; And (b) one of a modified T cell, an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement. In certain embodiments of the disclosed methods, including embodiments comprising contacting a T cell activation composition comprising one or more to produce activated modified T cells, the method comprises modifying T Introducing a sequence encoding a therapeutic protein into primary human T cells to generate a cell, wherein the modified T cell further comprises a step capable of expressing the therapeutic protein. In certain embodiments of introducing a sequence encoding a therapeutic protein, the introducing step comprises homologous recombination. In certain embodiments of introducing a sequence encoding a therapeutic protein, the vector comprises a sequence encoding a therapeutic protein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a nanoparticle.

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

  In certain embodiments of the disclosed method, the transposon is a plasmid DNA transposon having a sequence encoding an antigen receptor or therapeutic protein adjacent to two cis-regulatory insulator elements. In certain embodiments of the method, the introducing step further comprises a composition comprising an mRNA sequence encoding a transposase. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly those in which the transposon is a piggyBac transposon, the transposase is a 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. 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, and the overactive variant comprises amino acid substitutions at one or more of positions 30, 165, 282, and 538 of SEQ ID NO: 4. In certain embodiments, the amino acid substitution at position 30 of SEQ ID NO: 4 is a substitution of valine (V) with isoleucine (I) (I30V). In certain embodiments, the amino acid substitution at position 165 of SEQ ID NO: 4 is a substitution of serine (S) with glycine (G) (G165S). In certain embodiments, the amino acid substitution at position 282 of SEQ ID NO: 4 is a substitution of valine (V) with methionine (M) (M282V). In certain embodiments, the amino acid substitution at position 538 of SEQ ID NO: 4 is a substitution of lysine (K) with asparagine (N) (N538K). In certain embodiments, the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5. In certain embodiments, the transposon is a Sleeping Beauty transposon. In certain embodiments, particularly those in which the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or an overactive 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 a 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 a Tol2 transposase. In certain embodiments, the transposase-encoding sequence is an mRNA sequence. In certain embodiments, the transposon may be derived from or recombined from any species. Alternatively or additionally, the transposon can be a synthetic transposon.

  In certain embodiments of the disclosed methods, the transposon further comprises a selection gene. In certain embodiments, the T cell expansion composition further comprises a selective agent.

  In certain embodiments of the disclosed method, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, particularly those in which 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, particularly those in which the T cell receptor does not naturally occur, 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 includes one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

In certain embodiments of the methods of the present disclosure, cell surface markers of the modified _{T SCM} include CD62L and CD45RA. In certain embodiments, cell surface markers of the modified _{T SCM} include CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, one or more of CD95 and IL-2R beta. In certain embodiments, cell surface markers of the modified _{T SCM} include CD45RA, CD95, IL-2Rβ, CR7, and one or more of CD62L.

In certain embodiments of the disclosed methods, the plurality of augmented modified T cells comprises naïve T cells (modified T _N ), and the CAR- _TN cell surface marker is one of CD45RA, CCR7 and CD62L. Or include multiple. In certain embodiments, the plurality of augmented modified T cells comprises a central memory T cell (modified T _CM ), and the cell surface markers for CAR-T _CM are CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. One or more of. In certain embodiments, the plurality of increased modified T cells, including effector memory T cells (modified _{T EM),} the cell surface markers of the _{CAR-T EM,} CD45RO, CD95, and one of the IL-2R beta or Includes multiple. In certain embodiments, the plurality of augmented modified T cells comprises effector T cells (modified T _EFF ), and the CAR-T _EFF cell surface marker is one or more of CD45RA, CD95, and IL-2Rβ. including.

In certain embodiments of the methods of the present disclosure, the plurality of increased modified T cells, including the central memory T cells (modified _{T CM),} the cell surface markers of the _{CAR-T CM, CD45RO, CD95} , IL-2Rβ , CCR7, and CD62L. In certain embodiments, the most abundant T cells in multiple increased modified T cells, a central memory T cells (modified _{T CM),} the cell surface markers of the _{CAR-T CM, CD45RO, CD95} , IL- Includes one or more of 2Rβ, CCR7, and CD62L. The most abundant T cell is located in central memory T cells (modified T _CM) specific embodiments of the plurality of increased modified T cells, a plurality of increased modified T cells include T _SCM cells, T _SCM cell The cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ.

The present disclosure is a method for producing a modified stem memory T cell ( _TSCM ), and (a) a composition comprising a chimeric antigen receptor (CAR) in a primary human T cell to produce a CAR-T cell. And (b) CAR-T cells and anti-human CD3 monospecific tetrameric antibody complex, anti-human CD28 monospecific tetrameric antibody complex, anti-human CD2 single Contacting the T-cell activation composition comprising a monospecific tetrameric antibody complex and one or more of the activation supplements to produce activated CAR-T cells. provided, where, CAR-T cells activated express one or more cell surface markers of stem memory T cells _{(T SCM),} whereby CAR expression stem memory T cells _{(T SCM)} ( _{CAR-T SCM)} is created It is. The present disclosure is a method for producing a plurality of modified stem memory T cells (T _SCM ). (A) In order to produce a plurality of CAR-T cells, a chimera antigen receptor (CAR) And b) a plurality of CAR-T cells and an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific tetramer of Contacting a T cell activation composition comprising one or more of an antibody conjugate, an anti-human CD2 monospecific tetrameric antibody conjugate and an activation supplement, a plurality of activated CAR- Providing a method comprising generating T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of a plurality of activated CAR-T cells %, 45%, 50%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 99%, or one or more of cell surface markers expressed, whereby a plurality of activation of any percentage stem memory T cells _{(T SCM)} between them CAR stem memory T cells ( _TSCM ) are produced. In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method creates a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the activated CAR-T _SCM cell surface marker comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the activated CAR-T _SCM cell surface marker comprises one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. The present disclosure is a method for producing a modified stem memory T cell ( _TSCM ), and (a) a composition comprising a chimeric antigen receptor (CAR) in a primary human T cell to produce CAR-T cells And (b) CAR-T cells, anti-human CD3 monospecific tetramer antibody complex, anti-human CD28 monospecific tetramer antibody complex and activation supplement Contacting with a T cell activation composition comprising one or more of the following to produce an activated CAR-T cell, wherein the activated CAR-T cell comprises: express one or more cell surface markers of stem memory T cells _{(T SCM),} whereby CAR expression stem memory T cells _{(T SCM) (CAR-T} SCM) is produced.

The present disclosure is a method for producing a plurality of modified stem memory T cells (T _SCM ), and (a) a chimeric antigen receptor (CAR) for a plurality of primary human T cells to produce a plurality of CAR-T cells. And b) a plurality of CAR-T cells and an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific tetramer of Contacting the antibody complex and a T cell activation composition comprising one or more of the activation supplements 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% of the plurality of activated CAR-T cells 70%, 75%, 80%, 85%, 90%, 95%, 9 % Or any percentage express one or more cell surface markers of stem memory T cells (T _SCM), whereby a plurality of activated CAR stem memory T cells (T _SCM) is produced between these Is done. In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method creates a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the activated CAR-T _SCM cell surface marker comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the activated CAR-T _SCM cell surface marker comprises one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

In certain embodiments, the method comprises (c) activated CAR-T cells and human serum albumin, recombinant human insulin, human transferrin, to generate a plurality of augmented CAR-T cells, Contacting a T cell expansion composition comprising 2-mercaptoethanol, Iskov MDM, and one or more augmentation supplements, wherein at least 2% of the plurality of augmented CAR-T cells are stem memory T cells ( Expressing one or more cell surface markers of T _SCM ) (CAR-T _SCM ) may further be included. In certain embodiments, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. In addition. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. Contains oleic acid at a concentration of 13 mg / kg and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the plurality of increased CAR-T cells, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between are stem memory T cells (T _SCM ) (CAR-T _SCM ) Expresses a cell surface marker. In certain embodiments, the plurality of _expanded CAR-T cells can be enriched for CAR-T cells (CAR-T _SCM ) that express a cell surface marker of stem memory T cells (T _SCM ), After the step of enriching in doing so, the method comprises at least 2%, 5%, 10% CAR-T cells expressing a cell surface marker of stem memory T cells (T _SCM ) (CAR-T _SCM ), 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% Or an enriched composition can be made that includes any percentage between them. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the cell surface marker for CAR-T _SCM comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell surface marker for CAR-T _SCM comprises one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments, the plurality of augmented CAR-T cells comprises naïve T cells (CAR-T _N ), and the CAR-T _N cell surface marker comprises one or more of CD45RA, CCR7 and CD62L. Including. In certain embodiments, the plurality of augmented CAR-T cells comprises a central memory T cell (CAR-T _CM ), and the cell surface markers for CAR-T _CM are CD45RO, CD95, IL-2Rβ, CCR7, And one or more of CD62L. In certain embodiments, the plurality of augmented CAR-T cells comprises effector memory T cells (CAR-T _EM ), and the CAR-T _EM cell surface markers are CD45RO, CD95, and IL-2Rβ 1 Contains one or more. In certain embodiments, the plurality of increased CAR-T cells, including effector T cells _{(CAR-T EFF),} the cell surface markers of the _{CAR-T EFF,} CD45RA, CD95, and one of the IL-2R beta Or include multiple. Additional cell surface markers are described in Gattinoni et al. (Nat Med. 2011 Sep 18; 17 (10): 1290-7; the contents of which are hereby incorporated by reference in their entirety).

The present disclosure is a method for producing a modified stem memory T cell ( _TSCM ), and (a) a composition comprising a chimeric antigen receptor (CAR) in a primary human T cell to produce CAR-T cells And (b) CAR-T cells, anti-human CD3 monospecific tetramer antibody complex, anti-human CD28 monospecific tetramer antibody complex and activation supplement Contacting with a T cell activation composition comprising one or more of the following to produce an activated CAR-T cell, wherein the activated CAR-T cell comprises: express one or more cell surface markers of stem memory T cells _{(T SCM),} whereby CAR expression stem memory T cells _{(T SCM) (CAR-T} SCM) is produced. The present disclosure is a method for producing a plurality of modified stem memory T cells (T _SCM ), and (a) a chimeric antigen receptor (CAR) for a plurality of primary human T cells to produce a plurality of CAR-T cells. And b) a plurality of CAR-T cells and an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific tetramer of Contacting the antibody complex and a T cell activation composition comprising one or more of the activation supplements 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% of the plurality of activated CAR-T cells 70%, 75%, 80%, 85%, 90%, 95%, 9 %, Or any percentage between these express one or more cell surface markers of stem memory T cells (T _SCM), whereby the plurality of activated CAR stem memory T cells (T _SCM) Is produced. In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 25% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 50% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 60% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 75% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method creates a plurality of activated CAR-T cells, wherein at least 80% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method creates a plurality of activated CAR-T cells, wherein at least 85% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 90% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the method produces a plurality of activated CAR-T cells, wherein at least 95% of the plurality of activated CAR-T cells are stem memory T cells (T _SCM ). Expressing one or more cell surface markers, thereby generating a plurality of activated CAR stem memory T cells (T _SCM ). In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the activated CAR-T _SCM cell surface marker comprises one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the activated CAR-T _SCM cell surface marker comprises one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

In certain embodiments of the disclosed methods, the plurality of augmented CAR-T cells comprises naive T cells (CAR- _TN ), and the CAR- _TN cell surface markers are CD45RA, CCR7 and CD62L. Includes one or more. In certain embodiments, the plurality of augmented CAR-T cells comprises a central memory T cell (CAR-T _CM ), and the cell surface markers for CAR-T _CM are CD45RO, CD95, IL-2Rβ, CCR7, And one or more of CD62L. In certain embodiments, the plurality of augmented CAR-T cells comprises effector memory T cells (CAR-T _EM ), and the CAR-T _EM cell surface markers are CD45RO, CD95, and IL-2Rβ 1 Contains one or more. In certain embodiments, the plurality of increased CAR-T cells, including effector T cells _{(CAR-T EFF),} the cell surface markers of the _{CAR-T EFF,} CD45RA, CD95, and one of the IL-2R beta Or include multiple.

  In certain embodiments of the disclosed methods, the transposon comprises a chimeric antigen receptor (CAR) of the present disclosure. The transposon may be a plasmid DNA transposon having a sequence encoding CAR sandwiched between two cis-regulatory insulator elements. In certain preferred embodiments, the transposon is a piggyBac transposon. In certain embodiments, introducing a composition comprising a chimeric antigen receptor (CAR) of the present disclosure may further comprise introducing a composition comprising an mRNA sequence encoding a transposase. In certain preferred embodiments, the transposase is a Super piggyBac ™ (SPB) transposase.

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

  In certain embodiments, the CAR of the present disclosure may be CARTYrin. In certain embodiments, the CAR includes one or more VHH sequences. In certain embodiments, the CAR is a VCAR.

In certain embodiments of the methods of making a modified _{TSCM of the} present disclosure, the introducing step can include electroporation or nucleofection. Where the introducing step comprises nucleofection, the nucleofection comprises: (a) contacting a transposon composition, a transposase composition, and a composition comprising a plurality of primary human T cells in a cuvette; A) applying one or more electrical pulses to the cuvette; and (c) a composition comprising a plurality of primary human T cells, human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM. And incubating at 37 ° C. in a composition comprising a T cell expansion composition comprising one or more of the augmentation supplements. In certain embodiments, the T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA). , N-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterol and alkane. In addition. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and sterols (eg, cholesterol). In certain embodiments, the T cell expansion composition comprises 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 about 0.1 mg / kg to 10 mg / kg including the endpoint Of one or more of a concentration of sterols (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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, a concentration of about 2 mg / kg. Of oleic acid and one or more of sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of about 2.12 mg / kg linoleic acid, about 2 Oleic acid at a concentration of .13 mg / kg and one or more of sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises a concentration of 9.19 mg / kg octanoic acid, a concentration of 1.86 mg / kg palmitic acid, a concentration of 2.12 mg / kg linoleic acid, about 2. Contains oleic acid at a concentration of 13 mg / kg and sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 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 0.25 μmol / kg to 25 μmol / kg including the endpoint Contains one or more of a concentration of sterols. In certain embodiments, the T cell expansion composition has a concentration of about 64 μmol / kg octanoic acid, a concentration of about 7 μmol / kg palmitic acid, a concentration of about 7.5 μmol / kg linoleic acid, about 7.5 μmol. Oleic acid at a concentration of / kg and one or more sterols at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It includes one or more of oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, It contains oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg. In certain embodiments of nucleofection, the transposon composition is a 0.5 μg / μl solution containing nuclease-free water, and the cuvette contains 2 μl of the transposon composition, yielding 1 μg of transposon. The transposon composition can include a piggyBac transposon. The transposon composition may comprise a Sleeping Beauty transposon. In certain embodiments of nucleofection, the transposase composition comprises 5 μg transposase. The transposase composition may include overactive piggyBac ™ or Super piggyBac ™ (SPB) transposase. The transposase composition may comprise an overactive Sleeping Beauty (SB100X) transposase. In certain embodiments, the transposon may comprise a Helraiser transposon and the transposase composition may comprise a Helitron transposase. In certain embodiments, the transposon may comprise a Tol2 transposon and the transposase composition comprises a Tol2 transposase.

In certain embodiments of the disclosed methods, including embodiments where the introducing step comprises nucleofection or electroporation, the nucleofection comprises a first transposon composition and a first transposase composition and a plurality of Contacting the composition comprising primary human T cells in a cuvette. In certain embodiments of the disclosed methods, including embodiments where the introducing step comprises nucleofection or electroporation, the nucleofection comprises a first transposon composition, a second transposon composition, a first Contacting a transposase composition and a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments of the disclosed methods, including embodiments where the introducing step comprises nucleofection or electroporation, the nucleofection comprises a first transposon composition, a second transposon composition, a first Contacting a transposase composition, a second transposase composition and a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments, the first transposon includes a sequence encoding an antigen receptor. In certain embodiments, the second transposon includes a sequence that encodes a therapeutic protein. In certain embodiments, the first transposon composition and the second transposon composition are the same. In certain embodiments, the first transposon composition and the second transposon composition are not the same. In certain embodiments, the first transposase moves the first transposon composition and the second transposon composition. In certain embodiments, the first transposase moves the first transposon composition, but does not move the second transposon composition. In certain embodiments, the second transposase moves the second transposon composition, but does not move the first transposon composition. In certain embodiments, a first transposase moves a first transposon composition and a second transposase moves a second transposon composition. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence encoding an antigen receptor. In certain embodiments, the first transposon composition or the second transposon composition comprises a sequence that encodes a therapeutic protein. In certain embodiments, the first transposon composition includes a sequence that encodes an antigen receptor and the second transposon composition includes a sequence that encodes a therapeutic protein. In certain embodiments, the therapeutic protein is a secreted or secretable protein. In certain embodiments of the disclosed methods, including embodiments where the introducing step comprises nucleofection or electroporation, the nucleofection comprises a transposon composition, a first transposase composition, a second transposase composition. Contacting the composition and a composition comprising 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 that encodes a therapeutic protein. In certain embodiments of the disclosed methods, including embodiments where the introducing step includes nucleofection or electroporation, the nucleofection can induce homologous recombination at a particular site in the genome. Further comprising contacting the composition with a composition comprising a plurality of primary human T cells in a cuvette. In certain embodiments, a composition capable of inducing homologous recombination comprises an exogenous donor molecule. In certain embodiments, the exogenous donor molecule includes a sequence that encodes an antigen receptor, and the transposon includes a sequence that encodes a therapeutic protein. In certain embodiments, the exogenous donor molecule includes a sequence that encodes a therapeutic protein and the transposon includes a sequence that encodes an antigen receptor. In certain embodiments, a composition comprising a plurality of primary human T cells comprises a composition comprising a transposon, a composition comprising a transposase, and a composition capable of inducing homologous recombination at a particular site in the genome. Contact with the object at the same time. In certain embodiments, first, a composition comprising a transposon and a composition comprising a transposase are contacted with a composition comprising a plurality of primary human T cells, and second, a homologous set at a particular site in the genome. A composition capable of inducing replacement is contacted with a composition comprising a plurality of primary human T cells. In certain embodiments, first, a composition capable of inducing homologous recombination at a specific site in the genome is contacted with a composition comprising a plurality of primary human T cells, and second, a transposon. And a composition comprising a transposase are contacted with a composition comprising a plurality of primary human T cells. In certain embodiments of the methods of making the modified _{TSCM of the} present disclosure, a composition comprising primary human T cells maintains or enhances the level of cell viability and / or stem-like phenotype of primary human T cells. Contains buffer. In certain embodiments, prior to nucleofection, the buffer maintains or enhances the level of primary human T cell cell viability and / or the stem-like phenotype. In certain embodiments, during nucleofection, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of primary human T cells. In certain embodiments, following nucleofection, the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of primary human T cells. In certain embodiments, the buffer comprises P3 primary cell solution (Lonza). In certain embodiments, the buffer comprises one or more of KCl, MgCl ₂ , ClNa, glucose and Ca (NO ₃ ) ₂ in any absolute or relative abundance or concentration, optionally, The buffer further comprises a replenisher selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl ₂ , 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca (NO ₃ ) ₂ . In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl ₂ , 90 mM ClNa, 10 mM glucose and 0.4 mM Ca (NO ₃ ) ₂ , and 20 mM HEPES and 75 mM Tris / HCl. Contains supplements to contain. In certain embodiments, the buffer is 5 mM KCl, 15 mM MgCl ₂ , 90 mM ClNa, 10 mM glucose and 0.4 mM Ca (NO ₃ ) ₂ , and 40 mM Na ₂ HPO ₄ / NaH ₂ PO. _4. Contains supplements including pH 7.2. In certain embodiments, a composition comprising primary human T cells comprises 100 μl of buffer and 5 × 10 ⁶ to 25 × 10 ⁶ cells.

In certain embodiments of the methods of making a modified _{TSCM of the} present disclosure, a composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and / or CD19. In certain embodiments, a composition comprising primary human T cells comprises 100 μl of buffer and 5 × 10 ⁶ to 25 × 10 ⁶ cells.

  As used herein, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and A medium containing one or more augmentation supplements, 37 ° C. can be used interchangeably. Alternatively or additionally, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to one or more of phosphorus, octane fatty acid, palmitic fatty acid, linoleic fatty acid and oleic acid. The medium can be used interchangeably. In certain embodiments, the medium can be found in, for example, 10 times the amount of Iscove's Modified Dulbecco's Medium ((IMDM); available in the Catalog number 12440053 at ThermoFisher Scientific). Contains an amount of phosphorus.

  As used herein, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and A medium containing one or more augmentation supplements, 37 ° C. can be used interchangeably. Alternatively or additionally, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to one of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. Or it can use interchangeably with the culture medium containing two or more. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to the following elements present at corresponding average concentrations: boron, 3.7 mg / L, sodium, It can be used interchangeably with media containing one or more of 3000 mg / L, magnesium, 18 mg / L, phosphorus, 29 mg / L, potassium, 15 mg / L and calcium, 4 mg / L.

  As used herein, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and A medium containing one or more augmentation supplements, 37 ° C. can be used interchangeably. Alternatively or in addition, the term “supplemented T cell expansion composition” or “T cell expansion 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-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) ) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (CAS No. 6). 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS N) 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterol (eg, cholesterol) (CAS No. 57-88-5) , And medium containing one or more of alkanes (eg, nonadecane) (CAS No. 629-92-5) can be used interchangeably. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion 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-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) ( CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (CAS No. 84-) 69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112) 80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterol (for example, cholesterol) (CAS No. 57-88-5), alkane ( For example, it can be used interchangeably with media containing one or more of nonadecane (CAS No. 629-92-5) and phenol red (CAS No. 143-74-8). In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) ( CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (CAS No. 84-) 69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112) 80-1) one of stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS No. 143-74-8) and lanolin alcohol or It can be used interchangeably with media containing multiple.

  As used herein, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and A medium containing one or more augmentation supplements, 37 ° C. can be used interchangeably. Alternatively or in addition, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride ion, sulfuric acid And a medium containing one or more of phosphoric acid.

  As used herein, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and A medium containing one or more augmentation supplements, 37 ° C. can be used interchangeably. Alternatively or additionally, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to 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 can be used interchangeably. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to the following average 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%). %) Is used interchangeably with media containing one or more of It is possible. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to the following average free amino acids: histidine (about .78%), asparagine ( About 0.4%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00) %), Glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine ( About 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine (about 3.14%), leucine (about 3.3%) ), Phenylalanine (about 1.64%) and tri It can be the medium used interchangeably with including one or more Tofan (about 0.37%).

  The term “supplemented T cell expansion composition” or “T cell expansion composition” as used herein is one of octanoic acid, palmitic acid, linoleic acid, oleic acid and sterols (eg, cholesterol). Or it can use interchangeably with the culture medium containing two or more. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” includes octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg, including endpoints; Palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg; 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 medium containing one or more of sterols (mg / kg = parts per million) at a concentration of about 0.1 mg / kg to 10 mg / kg including endpoints. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” comprises octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, Compatible with medium containing one or more of linoleic acid at a concentration of 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterol at a concentration of about 1 mg / kg (mg / kg = parts per million) Can be used. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to 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 sterol at a concentration of about 1.01 mg / kg (mg / kg = parts per million) or It can be used interchangeably with media containing multiple. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to 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 sterol at a concentration of 1.01 mg / kg (mg / kg = parts per million). The medium can be used interchangeably. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” includes octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, including endpoints; 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 including the endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint Can be used interchangeably with media containing a concentration of 0.25 μmol / kg to 25 μmol / kg, including endpoints, and containing one or more of sterols. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” comprises octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, Can be used interchangeably with media containing one or more of linoleic acid at a concentration of 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg and sterol at a concentration of about 2.5 μmol / kg . In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to octanoic acid at a concentration of about 63.75 μmol / kg, at a concentration of about 7.27 μmol / kg. Use interchangeably with medium containing one or more of palmitic acid, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg and sterol at a concentration of about 2.61 μmol / kg can do. In certain embodiments, the term “supplemented T cell expansion composition” or “T cell expansion composition” refers to octanoic acid at a concentration of about 63.75 μmol / kg, at a concentration of about 7.27 μmol / kg. Use interchangeably with media containing one or more of palmitic acid, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg and sterol at a concentration of 2.61 μmol / kg. Can do.

As used herein, the term “P3 buffer” includes one or more of KCl, MgCl ₂ , ClNa, glucose and Ca (NO ₃ ) ₂ in any absolute or relative abundance or concentration. Optionally, it can be used interchangeably with a buffer further comprising a replenisher selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. The term “P3 buffer” includes 5 mM KCl, 15 mM MgCl ₂ , 90 mM ClNa, 10 mM glucose and 0.4 mM Ca (NO ₃ ) ₂ , optionally HEPES, Tris / HCl, and It can be used interchangeably with a buffer further comprising a replenisher selected from the group consisting of a phosphate buffer. The term “P3 buffer” is a supplement containing 5 mM KCl, 15 mM MgCl ₂ , 90 mM ClNa, 10 mM glucose and 0.4 mM Ca (NO ₃ ) ₂ and 20 mM HEPES and 75 mM Tris / HCl. Can be used interchangeably with a buffer solution containing The term “P3 buffer” refers to 5 mM KCl, 15 mM MgCl ₂ , 90 mM ClNa, 10 mM glucose and 0.4 mM Ca (NO ₃ ) ₂ and 40 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , pH 7 Can be used interchangeably with buffers containing supplements containing.

The term “supplemented RPMI-1640 medium” or “T cell conditioned medium (TCCM)” as used herein refers 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 2H ₂ O, L-valine, D- biotin, choline chloride, folic acid, myo-inositol, niacinamide, p- aminobenzoic acid, D- pantothenate (hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12 , D-glucose, glutathione (reduced), L-glutamine, and 2-mercaptoethanol can be used interchangeably with media containing any absolute or relative abundance or concentration. The terms “supplemented 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 2Na2H ₂ O, 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- A medium containing glucose, glutathione (reduced), L-glutamine and 2-mercaptoethanol in any absolute or relative abundance or concentration can be used interchangeably.

As used herein, the term "supplemented AIM-V" or "supplemented AIMV" medium includes water, human serum albumin, streptomycin sulfate, gentamicin, fetal calf 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- Phosphorus, L- threonine, L- tryptophan, L- tyrosine 2Na 2H ₂ O, L- valine, D- biotin, choline chloride, folic acid, myo-inositol, niacinamide, p- aminobenzoic acid, D- pantothenate (hemicalcium ), Pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12, D-glucose, glutathione (reduced), L-glutamine and 2-mercaptoethanol in any absolute or relative abundance or concentration It can be used interchangeably with the culture medium. The term “supplemented AIM-V” or “supplemented AIMV” medium refers to water, human serum albumin, streptomycin sulfate, gentamicin, fetal calf 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-threoni , L- tryptophan, L- tyrosine 2Na2H ₂ O, L- valine, D- biotin, choline chloride, folic acid, myo-inositol, niacinamide, p- aminobenzoic acid, D- pantothenate (hemicalcium), pyridoxine HCl, riboflavin , Thiamine HCl, vitamin B12, D-glucose, glutathione (reduced form), L-glutamine and 2-mercaptoethanol can be used interchangeably with any absolute or relative abundance or concentration.

  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 H20, L-isoleucine, L-leucine, L-lysine hydrochloride, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt, L-valine, biotin, choline chloride, calcium D-pantothenate, folic acid, niacinamide, Pyridoxal hydrochloride, riboflavin, Amine hydrochloride, vitamin B12, i-inositol, calcium chloride (anhydrous), magnesium sulfate (anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, monobasic sodium phosphate, sodium selenite, D-glucose, A medium containing one or more of HEPES and sodium pyruvate in any absolute or relative abundance or concentration can be used interchangeably. 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 H20, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt, L-valine, biotin, choline chloride, calcium D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride Salt, bita B12, i-inositol, calcium chloride (anhydrous), magnesium sulfate (anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, monobasic sodium phosphate, sodium selenite, D-glucose, HEPES and pyruvic acid It can be used interchangeably with media containing sodium in any absolute or relative abundance or concentration.

Modified T cells of the present disclosure, including the modified T _SCM and / or T _CM of the present disclosure, in any step of the method steps, incubated with growth medium containing one or more inhibitors components of the PI3K pathway, cultured Can be grown, stored, stored, or otherwise combined. Exemplary inhibitor components of the PI3K pathway include, but are not limited to, GSK3β such as TWS119 (also known as GSK 3B inhibitor XII; CAS number 601514-19-6, having the chemical formula C ₁₈ H ₁₄ N ₄ O ₂ ). Inhibitors are mentioned. Exemplary inhibitor components of the PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO ™).

  As used herein, the terms “electroporation” and “nucleofection” refer to a nucleic acid, transposon, vector or composition of the present disclosure supplied with an electrical pulse to the cell membrane (cell membrane, nuclear membrane, or both). Is intended to describe alternative means for delivering to cells by inducing them to become permeable or more permeable to nucleic acids, transposons, vectors or compositions of the disclosure .

  In certain embodiments of nucleofection, the method is performed simultaneously in one or more cuvettes. In certain embodiments of nucleofection, the method is performed in two cuvettes simultaneously. For processes that are performed on a larger scale for clinical or commercial applications, for example, nucleofection can be performed while performing many procedures simultaneously in a large volume cassette. In certain embodiments of nucleofection, the step of incubating comprises incubating a composition comprising a plurality of primary human T cells in a pre-warmed T cell expansion composition. The duration of the incubation step is 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. It can be hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or any number / part of time in between. The duration of the incubation step can be at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days or any number of days / parts therebetween. The duration of the incubation step can be at least one week. In certain embodiments of nucleofection, the duration of the incubation step is 2 days. In certain embodiments of nucleofection, the application step comprises the following program: EI-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-151, ES-115. , ES-151, EO-151, EO-148, EO-156, EO-210, EO-213, and FI-156. In certain embodiments, the applying step comprises the following program: 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 substantially the same number of electrical pulses, each pulse having the same duration and intensity, and substantially Applying a program that provides similar intermediate pulse durations may be included. In certain embodiments, the application step can be performed 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 applying step can include applying at least one electrical pulse. In certain embodiments of nucleofection, the applying step comprises at least one electrical pulse sufficient to induce the cell membrane and / or nuclear membrane of the cell to become permeable to the composition of the present disclosure. May include applying.

  The amounts presented herein are exemplary and include, but are not limited to, the relationships between these amounts (e.g., ratios or relative abundances) that are exemplified herein. The method being described can be modified for larger scale processes and manufacturing.

In certain embodiments of the methods of making the modified T cells of the present disclosure (eg, T _SCM and / or T _CM ), the activation supplement comprises one or more cytokines. The one or more cytokines can include any cytokine, including but not limited to lymphokines. Exemplary lymphokines include 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- Examples include, but are not limited to, gamma (INFγ). The one or more cytokines can include IL-2.

In certain embodiments of the methods of generating the disclosed modified T cells (eg, T _SCM and / or T _CM ), the augmentation supplement comprises one or more cytokines. The one or more cytokines can include any cytokine, including but not limited to lymphokines. Exemplary lymphokines include 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- Examples include, but are not limited to, gamma (INFγ). The one or more cytokines can include IL-2.

In certain embodiments of the methods of generating a modified T cell of the present disclosure (eg, T _SCM and / or T _CM ), the primary human T cell is a naive T cell. Naive T cells can express CD45RA, CCR7 and CD62L. In certain embodiments, the method comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, Applies to cell populations containing 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage of naive T cells therebetween. In certain embodiments, the efficiency of production of the modified T _SCM and / or T _CM of the present disclosure, be increased by increasing the proportion or percentage of naive T cells in the cell population to apply the method of the present disclosure Can do.

In certain embodiments of the method of producing a modified T _SCM and / or T _CM of the present disclosure, primary human T cells are memory T cells.

In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, primary human T cells, CD62L, CD45RA, CD28, CCR7 , CD127, CD45RO, CD95, CD95 and 1 of IL-2R beta Express one or more.

In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, the primary human T cells are naive T cells (modified _{T N),} the modified _{T N} is, CD45RA, CCR7 and CD62L of Express one or more. In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, primary human T cells are modified _{T SCM} T memory stem cells (modified _{T SCM),} the modified _{T SCM} is, CD45RA, CD95 Expresses one or more of IL-2Rβ, CR7, and CD62L. In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, the primary human T cells is a central memory T cells (modified _{T CM),} modified _T CM is, CD45RO, CD95, IL -Expresses one or more of -2Rβ, CCR7, and CD62L. In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, the primary human T cells are effector memory T cells (modified _{T EM),} modified _{T EM} is, CD45RO, CD95, and Express one or more of IL-2Rβ. In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, the primary human T cells and effector T cells (modified _{T EFF),} modified _{T EFF} is, CD45RA, CD95, and IL -Expresses one or more of -2Rβ.

In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, primary human T cells can express CD4 and / or CD8. In certain embodiments, primary human T cells can express CD4 and / or CD8 in various ratios. In certain embodiments, primary human T cells can express non-naturally occurring CD4 and / or CD8 in various ratios. In certain embodiments, the primary human T cells that express CD4 and / or CD8 in different ratios that may not occur in nature are heterogeneous cell populations.

In certain embodiments of the method of producing a modified _{T SCM} and / or _{T CM} of the present disclosure, primary human T cells, e.g., whole blood, peripheral blood, umbilical cord blood (umbilical cord blood), lymph, lymph node tissue , Bone marrow, and cerebrospinal fluid (CSF). The term “peripheral blood” as used herein refers to cellular components of blood obtained or prepared from a circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow (eg, Red blood cells, white blood cells and platelets). Umbilical cord blood is separate from peripheral blood and blood that is sequestered in the lymphatic system, spleen, liver or bone marrow. The terms “umbilical cord blood”, “umbilical blood” or “cord blood” can be used interchangeably and remain in the placenta after childbirth and adhere to the umbilical cord Refers to blood. Cord blood often contains stem cells, including hematopoietic cells.

  Primary human T cells of the present disclosure can include pan T cells. As used herein, pan-T cells are all T lymphocytes isolated from a biological sample without sorting by subtype, activation state, maturation state, or expression of cell surface markers. including.

In certain embodiments of the methods of the present disclosure, the method further comprises the step of introducing a composition comprising a modified T _SCM or T _CM cells genome editing construct or composition. In certain embodiments, the genome editing construct comprises a guide RNA and a clustered regularly interspersed short palindromic repeats (CRISPR) -related protein 9 (Cas9) DNA endonuclease . In certain embodiments, the genome editing construct comprises a DNA binding domain and a type IIS endonuclease. In certain embodiments, the genome editing construct encodes a fusion protein. In certain embodiments, the genome editing construct encodes a DNA binding domain and a type IIS endonuclease, wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked. In certain embodiments, including embodiments where the genome editing construct includes a DNA binding domain and a type IIS endonuclease, the genome editing construct includes a sequence derived from a Cas9 endonuclease. In certain embodiments, including embodiments in which the genome editing construct includes a DNA binding domain and a type IIS endonuclease, the sequence derived from the Cas9 endonuclease is a DNA binding domain. In certain embodiments, including those 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 where 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 the Cas9 endonuclease comprises an amino acid substitution (D10A) of alanine (A) with aspartic acid (D) at position 10. In certain embodiments, the sequence derived from the Cas9 endonuclease comprises or further comprises an amino acid substitution (H840A) of alanine (A) with histidine (H) at position 840. In certain embodiments, the sequence derived from Cas9 endonuclease comprises inactivated Cas9 (dCas9) (SEQ ID NO: 33). In certain embodiments, the sequence derived from Cas9 endonuclease comprises an amino acid substitution (N580A) at position 580 with an asparagine (N) of alanine (A). In certain embodiments, the sequence derived from Cas9 endonuclease comprises a truncated and inactivated Cas9 (dSaCas9) (SEQ ID NO: 32). In certain embodiments, including embodiments where the genome editing construct includes a DNA binding domain and a type IIS endonuclease, the genome editing construct includes a sequence derived from a transcriptional activator-like effector nuclease (TALEN). In certain embodiments, including embodiments in which the genome editing construct includes a DNA binding domain and a Type IIS endonuclease, the sequence derived from TALEN is a DNA binding domain. In certain embodiments, the genome editing construct comprises TALEN. In certain embodiments, including embodiments in which the genome editing construct includes a DNA binding domain and a type IIS endonuclease, the genome editing construct comprises a sequence derived from a zinc finger nuclease (ZFN). In certain embodiments, including embodiments in which the genome editing construct includes a DNA binding domain and a type IIS endonuclease, the sequence derived from ZFN is a DNA binding domain. In certain embodiments, the genome editing construct comprises a zinc finger nuclease (ZFN).

The methods of generating modified T _SCM and / or T _CM cells of the present disclosure can be optimized such that a greater number or a greater proportion of modified T _SCM and / or T _CM cells are generated. For example, the population of cells subjected to the disclosed method can be enriched to contain an increased number or a greater proportion of naïve T cells. According to the number and / or percentage of naive T cells within a population of T cells that are subjected to the method of the present disclosure is increased, the number and / or percentage of modified T _SCM and / or T _CM cells of the present disclosure to be prepared Will also increase. Alternatively, or in addition, with decreasing the length or duration of time required to perform the disclosed method above, the modified T _SCM and / or T _CM of the disclosure made by the method The number and / or percentage of cells increases. The length or duration of time required to perform the disclosed method first, or the “manufacturing period”, is the “out-of-life period” of the T cells subjected to the disclosed method. It can also be called.

In certain embodiments of the methods of generating the disclosed modified T cells, the primary human T cells express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. To do. In certain embodiments, primary human T cells are naive T cells _{(T N), T N} expresses one or more of CD45RA, CCR7 and CD62L. In certain embodiments, the primary human T cell is a T memory stem cell (T _SCM ) and the T _SCM expresses one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments, the primary human T cell is a central memory T cell (T _CM ), and the T _CM expresses one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, the primary human T cell is an effector memory T cell (T _EM ), and the _EM expresses one or more of CD45RO, CD95, and IL-2Rβ. In certain embodiments, the primary human T cell is an effector T cell (T _EFF ), and T _EFF expresses one or more of CD45RA, CD95, and IL-2Rβ. In certain embodiments, primary human T cells express CD4 and / or CD8.

The present disclosure provides a composition comprising a modified _TSCM made by the methods of the present disclosure. The present disclosure provides a composition comprising a modified T _CM produced by the method of the present disclosure. The present disclosure provides a composition comprising a fabricated modified T _SCM and modified T _CM in the methods of the present disclosure. In certain embodiments of modified T _SCMs and compositions comprising modified T _CMs made by the methods of the present disclosure, the plurality of T _SCMs are at least 1%, 2%, 5%, 10%, 15% of the composition %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, It can account for 97%, 98% or 99%. In certain embodiments of modified T _SCMs and compositions comprising modified T _CMs made by the disclosed methods, the plurality of T _CMs are at least 1%, 2%, 5%, 10%, 15% of the composition %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, It can account for 97%, 98% or 99%.

The present disclosure provides the use of a composition comprising for the manufacture of a medicament for treating a subject in need thereof, a modified T _SCM and / or T _CM produced by the method of the present disclosure. In certain embodiments of this use, the modified _{T SCM} and / or _{T CM} are autologous. In certain embodiments of this use, the modified T _SCM and / or T _CM is allogeneic. In certain embodiments, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, particularly those in which 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, particularly those in which the T cell receptor does not naturally occur, 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.

The present disclosure provides a method for treating a disease or disorder in a subject in need thereof, a subject a therapeutically effective amount of a composition comprising a modified T _SCM and / or T _CM produced by the method of the present disclosure A method comprising the steps of: In certain embodiments of this method, the modified _{T SCM} and / or _{T CM} are autologous. In certain embodiments of this method, the modified T _SCM and / or T _CM is allogeneic. In certain embodiments, the antigen receptor is a T cell receptor. In certain embodiments, the T cell receptor is naturally occurring. In certain embodiments, the T cell receptor is not naturally occurring. In certain embodiments, particularly those in which 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, particularly those in which the T cell receptor does not naturally occur, 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. In certain embodiments of this method, the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen. In certain embodiments of this method, the disease or disorder is an infectious disease or disorder and the antigen receptor specifically targets a viral, bacterial, yeast or microbial antigen. In certain embodiments, the disease or disorder is a disease or disorder caused by a lack of activity or an insufficient amount of secreted protein. In certain embodiments, the disease or disorder is a disease or disorder that is treated by replacing the activity of the therapeutic protein or by increasing the amount of the therapeutic protein. In certain embodiments, the therapeutic protein is a secreted protein. In certain embodiments, the activity of the secreted protein in a local region of the body is lacking or insufficient. In certain embodiments, a local region of the body is accessible by native or modified T cells. In certain embodiments, the modified T cells are generated in vivo, ex vivo, in vitro or in situ.

FIG. 1 is a series of plots showing the appearance of the CAR-T _SCM phenotype on day 11 of the method of Example 1. The cells were nucleofected with the surrogate CARTYrin plasmid. CAR-T _SCM cells express CD62L and CD45RA as shown in the lower two plots.

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

FIG. 3 is a plot showing that the majority of T cells generated express CARTYrin on day 11 of the method described in Example 1.

FIG. 4 is a series of plots showing the enrichment of the CAR-T _SCM phenotype on day 19 of the method described in Example 1. The cells were nucleofected with the surrogate CARTYrin plasmid. CAR-T _SCM cells express CD62L and CD45RA as shown in the lower two plots.

FIG. 5 is a series of plots showing the absence of T cell exhaustion on day 19 of the method described in Example 1. On day 19, the cell population generated by this method does not express PD1, a marker of T cell activation and exhaustion. Those cells expressing CARTYrin successfully reached dormancy after manufacture. These cells show no signs of antigen-independent (tonic) signaling that would otherwise drive high levels of PD1 expression. It is hypothesized that tonic signaling is caused by some CAR molecules that lead to early exhaustion and reduce the effectiveness of CAR T cell therapy.

FIG. 6A produced transfer using a plasmid containing a transposon-encoding sequence including an inducible caspase polypeptide (safety switch, “iC9”), CARTYrin (anti-BCMA), and a sequence encoding a selectable marker. Figure 2 is a series of plots showing T cells. The left plot shows live T cells exposed to transposase in the absence of plasmid. The right plot shows live T cells exposed to transposase in the presence of plasmid. Cells were exposed to either an overactive transposase (“Super piggyBac”) or a wild type piggyBac transposase.

FIG. 6B is a series of plots showing T cells that have undergone metastasis using a plasmid containing a sequence encoding green fluorescent protein (GFP). The left plot shows live T cells exposed to transposase in the absence of plasmid. The right plot shows live T cells exposed to transposase in the presence of plasmid. Cells were exposed to either an overactive transposase (“Super piggyBac”) or a wild type piggyBac transposase.

FIG. 6C is a table showing the percent of transformed T cells that resulted from metastasis using WT piggyBac transposase and metastasis using overactive piggyBac transposase. T cells contacted with overactive piggyBac transposase (Super piggyBac transposase) were transformed at a 4-fold higher rate than WT transposase.

FIG. 6D is a table showing the percent of transformed T cells that resulted from transfer with WT piggyBac transposase and transfer with overactive piggyBac transposase 5 days after nucleofection. T cells contacted with overactive piggyBac transposase (Super piggyBac transposase) were transformed at a much higher rate than WT transposase.

FIG. 7 is a graph showing the phenotypic differences between CAR + T cells produced by piggyBac ™ and CAR + T cells produced by lentivirus. CAR + T cells were generated using either piggyBac transfer or lentiviral transduction. Human pan-T cells were metastasized using piggyBac encoding CAR, stimulated with anti-CD3 / CD28 beads 2 days after metastasis, expanded and tested 19 days after metastasis. For production using lentivirus, pan-T cells are stimulated with CD3 / CD28 beads, transduced with lentivirus encoding CAR (MOI 5), expanded and 18 days after stimulation. Tested. Each population of CAR + T cells was then characterized based on the expression of standard memory markers CD62L, CD45RA and CD95. The percentage of each CAR + T cell subset was defined as naive (CD62L + CD45RA +), Tcm (CD62L + CD45RA−), Tem (CD62L-CD45RA−) and Teff (CD62L-CD45RA +). All CAR + T cells were CD95 +.

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

FIG. 9 is a graph showing that the piggyBac ™ manufacturing process yields high levels of _{TSCM in} samples from multiple myeloma (MM) patients even when naïve T cells are rare. It is a pair. T cells from MM patients (triangles) and healthy donor-derived T cells (circles) were characterized for memory marker expression by flow cytometry before (left) and after (right) the Posida manufacturing process. CD45RA and CD62L expression was assessed by FACS for MM patients and healthy donors, and plots are shown. T cells from MM patients will generally be less frequent naive cells and T _SCM cell high frequency of Teff, which is different from the healthy normal donor known converse. Regardless of the naive and Tscm input frequency from different MM patients, the production of P-BCMA-101 using the Posida manufacturing process resulted in a product exhibiting high levels of CD8 + Tscm (E). This is also true for MM patients who were actively treated (red triangles).

FIG. 10 is a series of fluorescence activated cell sorting (FACs) plots characterizing T cell and T _SCM cell markers in human pan T cells transformed using the Sleeping Beauty (SB100x) transfer system and the disclosed method. The Sleeping Beauty (SB100x) transition using the Poseida manufacturing process mainly yields the Tscm phenotype. As indicated, human pan-T cells were metastasized using either 1 μg of the Sleeping Beauty transposon plasmid or the piggyBac transposon plasmid, and SB100x or SPB mRNA, respectively. After metastasis, cells were expanded ex vivo and all non-metastatic cells were depleted using the Posida manufactured drug selection system. After culturing for 18 days, the cells were stained with the phenotypic markers CD4, CD8, CD45RA, and CD62L. The stem cell memory phenotype (Tscm) is defined by CD45RA and CD62L double positive cells and constitutes> 65% of the cells in all samples. All panels in the column share common x-axis and y-axis parameters. In each row, from top to bottom (top), data from T cells metastasized using 2.5 micrograms (μg) of Sleeping Beauty transposon SB100x, (second from top) 5 μg of SB100x. Data from T cells used to cause metastasis, (third from top) data from T cells produced using 10 μg SB100x, (second from bottom) 5 μg piggyBac transposon P-BCMA- Data from T cells that produced metastases using 101, and at the bottom, data from T cells that produced metastases using unstained controls are shown. The first and second columns of the x-axis show forward scatter (FSC) from left to right in units of 0 to 250,000 (abbreviated “k”) in 50k increments. The x-axis of the third column, from the left, shows CD8 expression and is marked with readings increasing from 0 to 10 ^{5 in} increments of 10. The rightmost column shows CD62L expression and is marked with readings increasing from 0 to 10 ^{5 in} increments of 10. The y-axis of the first column shows side scatter (SSC) in units from 0 to 250k, in increments of 50k. The y-axis in the second column, from the left, shows the expression of the cell viability marker 7 aminoactinomycin D (7AAD), increasing from 0 to 10 ^{5 in} increments of 10. The y-axis of the third column shows the expression of the marker CD4 from the left, increasing from 0 to 10 ⁵ by 10 steps. The y-axis in the right column shows the expression of the marker CD45RA, increasing from 0 to 10 ⁵ by 10 steps. Same as above

FIG. 11 is a schematic diagram showing the human coagulation pathway leading to blood coagulation. For example, contact activation due to endothelial damage activates the intrinsic coagulation pathway. For example, after trauma, the exogenous coagulation pathway is activated by tissue factor. Both pathways converge to the conversion of prothrombin to thrombin, which catalyzes the conversion of fibrinogen to fibrin. Fibrin polymerized with platelets forms a clot. In the absence of Factor IX (circled), clotting 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 that occurs at a frequency of about 1 out of 25,000-30,000. Sixty 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 the present disclosure, standard treatment for hemophilia B involves injecting recombinant FIX every 2-3 days, and the cost per year is approximately 250,000. It was a dollar. In contrast to this standard treatment option, the _TSCM cells of the present disclosure are maintained in humans for decades.

FIG. 12 is a series of fluorescence activated cell sorting (FACS plot) showing T cells secreting FIX. T cells encoding the human factor IX transgene exhibited approximately 80% of the cells with a _TSCM phenotype. Six panels are listed from left to right. (1) Forward scattering (FSC) on the x axis and side scattering (SSC) on the y axis. The x-axis is 0 to 250,000 (abbreviated as k) in 50k increments, and the y-axis is from 0 to 250k in 50k increments. (2) FSC on the x-axis against 7 aminoactinomycin D (7AAD), a cell viability marker. The x-axis is labeled from 0 to 250k in 50k increments. The y-axis readings are −10 ³ , 0, 10 ³ , 10 ⁴ , 10 ⁵ from top to bottom. (3) Cy7 dye on the x-axis-conjugated anti-CD56-APC (CDC56-APC- Cy7) is shown in units increments power of 10 0 to 10 ^5. The y-axis, anti-CD3 conjugated with phycoerythrin (PE) is shown in units increments power of 10 0 to 10 ^5. (4) the x-axis, anti-CD8 conjugated with fluorescein isothiocyanate (FITC) is shown in units increments power of 10 0 to 10 ^5. The y-axis, Brilliant Violet 650 dye (BV650) conjugated anti-CD4 is shown in units increments power of 10 0 to 10 ^5. (5) to the x-axis, the anti-CD62L antibody conjugated with Brilliant Violet 421 dye (BV421) is shown in units increments power of 10 0 to 10 ^5. The y-axis, anti-CD45RA antibody PE and Cy7 conjugated is shown in units increments power of 10 0 to 10 ^5. This panel is framed. The (6) x-axis, Brilliant Violet 786 (BV786) and anti-CCR7 antibody conjugated is shown in units increments power of 10 0 to 10 ^5. The y-axis, PE and Cy7-conjugated anti-CD45RA is shown in units increments power of 10 0 to 10 ^5.

FIG. 13A is a graph showing human factor IX secretion during production of the modified T cells of the present disclosure. The y-axis is the factor IX concentration in nanograms (ng) per milliliter (mL) in 20 steps from 0 to 80. On the x-axis, day 9 and day 12 T cells are shown.

FIG. 13B is a graph showing the clotting activity of secreted factor IX produced by T cells. On the y-axis, percent factor IX activity on human plasma is shown in two increments from 0 to 8. The x-axis is day 9 and day 12 T cells.

FIGS. 14A-E are a series of plasmid maps for site-specific integration into the AAVS1 site using either HR or MMEJ and corresponding sequences. A) site-specific (AAVS1) homologous recombination (HR), B) site-specific (AAVS1) microhomology-mediated end joining (MMEJ) recombination and C) human pan T cells with TTAA-specific piggyBac ™ transfer Donor plasmids for testing stable integration into the genome. For the HR and MMEJ donor plasmids, the GFP-2A-DHFR gene expression cassette was sandwiched by homology arms for CRISPR / Cas9 target site and AAVS1 site integration; for the piggyBac ™ donor plasmid, GFP-2A -The DHFR gene expression cassette was sandwiched between piggyBac ™ transposon elements. The homology arms for the HR and MMEJ plasmids are 500 bp and 25 bp, respectively. Panels D and E, and F show SEQ ID NOs 41 and 42, respectively. Same as above Same as above Same as above Same as above

FIG. 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 controls. Pan-T cells were also modified using the piggyBac ™ transposon delivery system. T cells were activated by TCR stimulation on day 0, the percentage of GFP + T cells assessed by flow cytometry on day 3 after nucleofection, and the data summarized in a bar graph.

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

FIGS. 17A-C are a series of graphs showing the phenotype of primary human pan-T cells modified at the AAVS1 site by HR and MMEJ. The GFP + CD8 + pan T cell phenotype was analyzed by flow cytometry 11 days after nucleofection. A) Cells are 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 a bar graph. Using the piggyBac ™ transposon system, or a combination of HR and Cas9 RNP and a combination of MMEJ and Cas9 RNP, an enriched population of GFP + Tscm was achieved in all cases. C) The total number of pan-T cells was analyzed 13 days after nucleofection and the data are summarized in a bar graph. Same as above Same as above

18A-B are a pair of photographs of gel electrophoresis results showing site-specific incorporation into the AAVS1 site. Selected cells from each group were harvested, 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 prime the 5 'end junction of the AAVS1 target site (inserted EF1a-2r CACCGGAGCCCAATTCCCACT (SEQ ID NO: 36) with one primer and the other primer with the 5' end AAVS-3r CTGCACCACGTGATGTCTCTC ( Priming the AAVS1 region beyond the 500 bp homologous arm of SEQ ID NO: 37, thereby obtaining a 0.73 kb DNA fragment for either HR or MMEJ) and the 3 ′ end junction (with one primer, polyA Signaling region SV40pA-1r GTAACCCATTATAAGCTGCCAATAAACAAG (SEQ ID NO: 38) was primed and 500 bp of 5 ′ homozygous with the other primer Prime AAVS1 region beyond Guamu AAVS-2f CTGGGGACTCTTTAAGGAAAGAAG (SEQ ID NO: 39), thereby obtaining a DNA fragment of 0.76kb for HR or MMEJ) were individually amplified. PCR products were presented on agarose gels. The non-specific band in the HR sample is the result of only a single round of PCR and is likely to be resolved by performing additional rounds.

DETAILED DESCRIPTION The present disclosure describes a T cell expressing a human chimeric antigen receptor (CAR) using a piggyBac ™ Transposon System and a stem cell memory T cell (T _SCM ) having potent CAR activity (herein, S _SCM ) , Referred to as CAR-T _SCM ) is provided. A composition comprising CAR-T _SCM made using the methods of the present disclosure comprises ≧ 60% CAR-T _SCM and exhibits a distinct functional profile consistent with this T cell subset. Other T cell subsets found in the compositions of the present disclosure include, but are not limited to, central memory CAR-T cells (CAR-T _CM ), effector memory CAR-T cells (CAR-T _EM ), effector Examples include CAR-T cells (CAR-T _E ) and well-differentiated effector CAR-T cells (CAR-T _TE ). Linear pathways of differentiation, such as _TN is the parental progenitor cell, which directly produces T _SCM, which in turn produces T _CM directly: naïve T cells (T _N )> T _SCM > T _CM > T _EM > T _E > T _TE may be involved in the generation of these cells. A composition comprising CAR-T _SCM , CARTYrin-T _SCM and / or VCAR-T _SCM of the present disclosure may comprise one or more of each parent CAR-T cell subset, with CAR-T _SCM being most abundant (For example, T _SCM > T _CM > T _EM > T _E > T _TE ). The absolute abundance / abundance and relative proportion of each parental T cell subset can vary between the patient blood sample and the naturally occurring cell population, where the naturally occurring cell population is abundance of _TSCM . And the composition of the present disclosure comprising CAR-T _SCM, which may be high in proportion and / or not naturally occurring in the treatment of patients for disease and cancer, is more potent and effective.

Immunotherapy using chimeric-antigen receptor (CAR) -T cells is emerging as an exciting therapeutic approach for cancer therapy. Autologous CAR-modified T cells that target tumor-associated antigen (Ag) can result in robust tumor killing, and in some cases, complete remission of CD19 ⁺ hematologic malignancies. Unlike conventional biologics and chemotherapeutic drugs, CAR-T cells have the ability to regenerate rapidly when Ag is recognized, thereby potentially eliminating the need for repeated treatments. To accomplish this, CAR-T cells are not only initially driven to destroy the tumor, but are also maintained as a stable population of memory T cells that can survive in the patient to prevent potential cancer recurrence. There must be. Therefore, focused efforts to develop CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, and CAR-T products that contain early memory cells, particularly stem cell memory (T _SCM ) The focus is on. Stem cell-like CAR-T exhibits maximum capacity for self-renewal and multipotency to elicit central memory T cells (T _CM ), effector memory T cells (T _EM ) and effector T cells (T _E ), thereby Good tumor eradication and prolonged CAR-T engraftment occur.

_{CAR-T SCM} of the present disclosure is referred to as CARTyrin, it may include a CAR based on Senchirin (hence, the cell may be referred to as _{CARTyrin-T SCM).} Sentilin is an alternative scaffold molecule based on the human consensus stenisin FN3 domain, which is smaller than the scFv molecule, can be selected for monomericity favoring stability, and also demonstrates the possibility of tonic signaling in the CAR molecule Reduce. The CARTYrin of the present disclosure is a plasmid DNA transposon encoding CARTYrin sandwiched between two cis-regulatory insulator elements to help stabilize CARTYrin expression by blocking inappropriate gene activation or silencing Can be used to introduce into T cells.

The CAR-T _SCM of the present disclosure may include a VHH-based CAR, referred to as VCAR (thus, the cell may be referred to as VCAR-T _SCM ). The VCAR of the present disclosure is a plasmid DNA transposon encoding VHH sandwiched between two cis-regulatory insulator elements to help stabilize VHH expression by blocking inappropriate gene activation or silencing. Can be used to introduce into T cells.

In certain embodiments of the disclosed methods, a single electroporation reaction is used to stably incorporate antigen-specific CARTYrin or VCAR (including cancer antigen-specific CARTYrin or VCAR) into dormant pan-T cells. In which the transposon is co-delivered with the mRNA transposase enzyme (although the transposon and transposase are contained in separate compositions until they are introduced into the cell), they are referred to as Super piggyBac ™ (SPB). PiggyBac ™ (PB) Transposon System can be used. Delivery of piggyBac ™ transposon to intact resting primary human pan-T cells resulted in 20-30% of cells with stable integration and expression of the gene delivered by PB. Unexpectedly, the majority of these modifications CARTyrin expressing T cells were positive for expression of CD62L and CD45RA, a marker associated with a general stem memory T cells _{(stem memory T-cell) (} T SCM cells). To confirm that this phenotype was retained upon stimulation and expansion of CAR-T cells, modified CARTYrin-expressing T cells positive for CD62L and CD45RA expression were activated by stimulation with CD3 and CD28. As a result of stimulation with CD3 and CD28,> 60% of CARTYrin + T cells showed a stem cell memory phenotype. Furthermore, these cells that expressed CARTYrin specific for the cancer antigen were sufficiently capable of expressing a powerful anti-tumor effector function.

Compare the phenotype of CAR-T cells generated by either PB transfer or lentiviral (LV) transduction to determine whether the PB system contributes directly to enhanced expression of stem-like markers did. To do this, a new vector was constructed by subcloning the CARTYrin transgene into a common LV construct for virus production. After CARTYrin was introduced into intact resting T cells by PB transposition or LV transduction, CARTYrin ⁺ cells were expanded and then returned to resting state. Various phenotypic and functional properties, including kinetic analysis of memory and exhaustion-related markers, secondary proliferation in response to homeostatic cytokines or tumor-related Ag, cytokine production, and lytic capacity in response to target tumor cells It was measured. Unlike CARTyrin ⁺ T cells were transferred by PB, the CARTyrin ⁺ T cells were transduced by LV, strengthening memory phenotype did not show. In addition, cells that had undergone metastasis with PB showed comparable or greater capacity for secondary growth and killing of target tumor cells. Taken together, these data demonstrate that CAR-T cells generated by PB metastasis are primarily _TSCM cells, a highly desirable product phenotype in the CAR-T field. In addition, these CARTYrin ⁺ T cells show strong antitumor activity and are longer in vivo due to the use of centrin-based CAR, which may be less prone to tonic signaling and functional exhaustion Persistent cells can be generated.
Chimeric antigen receptor

  The disclosure provides: (a) an ectodomain 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) providing a chimeric antigen receptor (CAR) comprising an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region may comprise two sequences that each specifically bind to the antigen, such that bispecific or tandem CARs occur. In certain embodiments, the antigen recognition region may comprise three sequences that each specifically bind to the antigen, such that a trispecific CAR occurs. In certain embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain. The sequences that each specifically bind to an antigen are not limited to this, but include single chain antibodies (eg, scFv), sequences that include one or more fragments of antibodies (eg, VHH, referred to as VCAR for CAR). Antibody mimics, and sentin (referred to as CARTYrin for CAR).

  In certain embodiments of the CAR of the present disclosure, the signal peptide comprises a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. obtain. In certain embodiments of the CAR of the present disclosure, the signal peptide may comprise a sequence that encodes a human CD8α signal peptide. The human CD8α signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide has at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising MALPVTALLLPLALLLLHAARP (SEQ ID NO: 8) or to an amino acid sequence comprising MALPVTALLLPLALLLLHAARP (SEQ ID NO: 8) A sequence may be included. The human CD8α signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagaca (SEQ ID NO: 9).

  In certain embodiments of the CAR of the present disclosure, the transmembrane domain encodes a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain Can be included. In certain embodiments of the CAR of the present disclosure, the transmembrane domain may comprise a sequence that encodes a human CD8α transmembrane domain. The CD8α transmembrane domain is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising IYIWAPLAGTCCGVLLLSLVITLYC (SEQ ID NO: 10) or an amino acid sequence comprising IYIWAPLAGTCCGVLLLSLVITLYC (SEQ ID NO: 10) A sequence may be included. The CD8α transmembrane domain can be encoded by a nucleic acid sequence comprising atctacattttgggcaccactggccgggacctgtgtggagtgctgctgctgagcctggtcatactactgtactgc (SEQ ID NO: 11).

  In certain embodiments of the CAR of the present disclosure, the endodomain may comprise a human CD3ζ endodomain.

  In certain embodiments of the CAR of the present disclosure, the at least one costimulatory domain may comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CAR of the present disclosure, the at least one costimulatory domain may comprise a CD28 and / or 4-1BB costimulatory domain. CD28 costimulation domain, at least 70% amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR amino acid sequence or RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12) (SEQ ID NO: 12), 80%, 90%, 95%, or 99% identity to A sequence may be included. CD28 costimulatory domain, cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgca It can be encoded by nucleic acid sequences comprising Gcactgcctccaagg (SEQ ID NO: 13). The 4-1BB costimulatory domain comprises an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) or KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14 to at least 95; Can be included. The 4-1BB co-stimulatory domain is encoded by the aagagaggcaggaagaaactgctgttatattttcaaaaaccccccttcatgcgccccgtgcagaactacccaggaggagagagacgggtgctcgtgagtgagtgaggag sequence The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

  In certain embodiments of the CAR of the present disclosure, the hinge may comprise a sequence derived from human CD8α, IgG4, and / or CD4 sequences. In certain embodiments of the CAR of the present disclosure, the hinge can comprise a sequence derived from a human CD8α sequence. The hinge has at least 70%, 80%, 90%, 95%, or 95% identical to a human CD8α amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTLGLDFACD (SEQ ID NO: 16) or an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) Can be included. The human CD8α hinge amino acid sequence is the actaccacaccacgaccaccacaccaccactacccccaccactacccatcgcgagtcagcccctgagtctgagacctgag sequence that contains the sequence 17

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

  The present disclosure provides a transposon comprising the CAR of the present disclosure. The transposons of the present disclosure are maintained as episomes or integrated into the genome of the recombinant / modified cell. The transposon can be part of a two-component piggyBac system that utilizes a transposon and a transposase to enhance non-viral gene transfer.

  The transposons of the present disclosure can include a selection gene for identifying, enriching and / or isolating cells that express the transposon. Exemplary selection genes encode any gene product (eg, transcript, protein, enzyme) essential for cell viability and survival. An exemplary selection gene is any that is essential to confer resistance to attack by drugs that are sensitive to cells (or can be lethal to cells) in the absence of the gene product encoded by the selection gene. Gene products (eg, transcripts, proteins, enzymes). An exemplary selection gene is any gene essential for viability and / or survival in cell media lacking one or more nutrients essential for cell viability and / or survival in the absence of the selection gene. Encodes a product (eg, transcript, protein, enzyme). Exemplary selection genes include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, confers resistance to methotrexate), TYMS (encodes thymidylate synthetase) , MGMT (encodes O (6) -methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (RAD51 paralog C ), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 homeobox 2).

  A transposon of the present disclosure can include, for example, at least one self-cleaving peptide located between one or more of the sequences that specifically bind to an antigen and a selection gene of the present disclosure. The at least one self-cleaving peptide can comprise, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

  The transposon of the present disclosure may comprise a first self-cleaving peptide and a second self-cleaving peptide, for example of a sequence that specifically binds to an antigen of the present disclosure. Located one or more upstream, the second self-cleaving peptide is located, for example, one or more downstream of a sequence that specifically binds an antigen of the present disclosure. The first and / or second self-cleaving peptide is, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide Can be included. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 023) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

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

  The transposon of the present disclosure may include a piggyBac transposon. The transposase enzyme of the present disclosure may include a piggyBac transposase or a compatible enzyme.

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

  Viral vectors of the present disclosure can include sequences isolated from or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors can include sequences isolated from or derived from adeno-associated virus (AAV). Viral vectors can include recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include two or more terminal inversion sequences located in cis next to one or more of the sequences that specifically bind antigen. (ITR) sequence. Exemplary and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, all serotypes (eg, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Not. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include self-complementary AAV (scAAV) and AAV hybrids containing one serotype genome and another serotype capsid (eg, AAV2 / 5, Including but not limited to AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, rAAV-LK03.

  Viral vectors of the present disclosure can include a selection gene. A selection gene may encode a gene product essential for cell viability and survival. A selection gene can encode a gene product essential for cell viability and survival upon challenge with selective cell culture conditions. Selective cell culture conditions can include compounds that are detrimental to cell viability or survival, and gene products confer resistance to such compounds. Exemplary selection genes of the present disclosure include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, confers resistance to methotrexate), TYMS (contains thymidylate synthetase). MGMT (encodes O (6) -methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes) RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2) or any combination thereof.

  Viral vectors of the present disclosure can include at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located upstream of the CAR, and the second self-cleaving peptide is CAR Located downstream of. The self-cleaving peptide can include, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

  The present disclosure provides a vector comprising the CAR of the present disclosure. In certain embodiments, the vector is an mRNA vector. The vector can be a recombinant mRNA vector. The T cells of the present disclosure can be expanded prior to contacting the T cells with an mRNA vector comprising the CAR of the present disclosure. Subsequently, T cells containing mRNA vectors and modified T cells can be administered to the subject.

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

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

  Nanoparticle vectors of the present disclosure can include at least one self-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the nanoparticle. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located upstream of the CAR and the second self-cleaving peptide is , Located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle, and the second self-cleaving peptide The cleavable peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle, and the second self-cleaving peptide The cleaving peptide is located downstream of the CAR, eg, between the CAR and the selection gene. The self-cleaving peptide can include, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

The present disclosure provides a composition comprising a vector of the present disclosure.
CARTYrin

  The disclosure provides: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one sentin; (b) a transmembrane domain, and (c) at least one costimulatory domain A chimeric antigen receptor (CAR) comprising an endodomain comprising As used throughout this disclosure, CARs containing sentin are referred to as CARTYrin. In certain embodiments, the antigen recognition region may include two sentinels so that bispecific or tandem CARs occur. In certain embodiments, the antigen recognition region may comprise three sentinels so that a trispecific CAR occurs. In certain embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.

  The disclosure provides (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one protein scaffold or antibody mimic; (b) a transmembrane domain, and (c) at least A chimeric antigen receptor (CAR) comprising an endodomain comprising one costimulatory domain is provided. In certain embodiments, the antigen recognition region may comprise two protein scaffolds or antibody mimetics such that bispecific or tandem CARs occur. In certain embodiments, the antigen recognition region may comprise three protein scaffolds or antibody mimetics such that a trispecific CAR occurs. In certain embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.

  In certain embodiments of the CAR of the present disclosure, the signal peptide comprises a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. obtain. In certain embodiments of the CAR of the present disclosure, the signal peptide may comprise a sequence that encodes a human CD8α signal peptide. The human CD8α signal peptide may comprise an amino acid sequence comprising MALPVTALLLPLALLLLHAARP (SEQ ID NO: 8). The human CD8α signal peptide has at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising MALPVTALLLPLALLLLHAARP (SEQ ID NO: 8) or to an amino acid sequence comprising MALPVTALLLPLALLLLHAARP (SEQ ID NO: 8) A sequence may be included. The human CD8α signal peptide may be encoded by a nucleic acid sequence comprising atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagaca (SEQ ID NO: 9).

  In certain embodiments of the CAR of the present disclosure, the transmembrane domain encodes a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain Can be included. In certain embodiments of the CAR of the present disclosure, the transmembrane domain may comprise a sequence that encodes a human CD8α transmembrane domain. The CD8α transmembrane domain is at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising IYIWAPLAGTCCGVLLLSLVITLYC (SEQ ID NO: 10) or an amino acid sequence comprising IYIWAPLAGTCCGVLLLSLVITLYC (SEQ ID NO: 10) A sequence may be included. The CD8α transmembrane domain can be encoded by a nucleic acid sequence comprising atctacattttgggcaccactggccgggacctgtgtggagtgctgctgctgagcctggtcatactactgtactgc (SEQ ID NO: 11).

  In certain embodiments of the CAR of the present disclosure, the endodomain may comprise a human CD3ζ endodomain.

  In certain embodiments of the CAR of the present disclosure, the at least one costimulatory domain may comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CAR of the present disclosure, the at least one costimulatory domain may comprise a CD28 and / or 4-1BB costimulatory domain. CD28 costimulation domain, at least 70% amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR amino acid sequence or RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12) (SEQ ID NO: 12), 80%, 90%, 95%, or 99% identity to A sequence may be included. CD28 costimulatory domain, cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgca It can be encoded by nucleic acid sequences comprising Gcactgcctccaagg (SEQ ID NO: 13). The 4-1BB costimulatory domain comprises an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) or KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14 to at least 95; Can be included. The 4-1BB co-stimulatory domain is encoded by the aagagaggcaggaagaaactgctgttatattttcaaaaaccccccttcatgcgccccgtgcagaactacccaggaggagagagacgggtgctcgtgagtgagtgaggag sequence The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

  In certain embodiments of the CAR of the present disclosure, the hinge may comprise a sequence derived from human CD8α, IgG4, and / or CD4 sequences. In certain embodiments of the CAR of the present disclosure, the hinge can comprise a sequence derived from a human CD8α sequence. The hinge has at least 70%, 80%, 90%, 95%, or 95% identical to a human CD8α amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTLGLDFACD (SEQ ID NO: 16) or an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) Can be included. The human CD8α hinge amino acid sequence is the actaccacaccacgaccaccacaccaccactacccccaccactacccatcgcgagtcagcccctgagtctgagacctgag sequence that contains the sequence 17

Sentilin of the present disclosure can include a protein scaffold, which can specifically bind to an antigen. Sentilin of the present disclosure can comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, wherein the scaffold is capable of specifically binding to an antigen. At least one fibronectin type III (FN3) domain can be derived from a human protein. The human protein can be tenascin-C. Consensus sequence may include ErupiEipikeienuerubuibuiesuibuitiidiesueruarueruesudaburyutieiPidieieiefudiesuefueruaikyuwaikyuiesuikeibuijiieiaienuerutibuiPijiesuiaruesuwaidierutijierukeipijiTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) or EmueruPiEipikeienuerubuibuiesuibuitiidiesueruarueruesudaburyutieiPidieieiefudiesuefueruaikyuwaikyuiesuikeibuijiieiaienuerutibuiPijiesuiaruesuwaidierutijierukeipijiTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). Consensus sequence can be encoded by a nucleic acid sequence comprising a Eitijishitijishishitijishieishishieieieijieieishishitijijitijijitijitishitishieitijitijieishieijieijijieitieijitijishishieijieishitijitishieitijijieishitijishitishishishijieishijishieijishishititishijieitieijititititieitishieitishijitijitieishishijijijieijieieishieitishijieieieishishijijishijieijijishishieititijitishishitijieishieijitijishishieijijijitishishijieieishijishitishititieitijieishishitijieishieijieitishitijieieijishishishijijieieishitijieijitieishitieitijitijishieijieitishijishishijijishijitishieieieijijieijijishieieitieitishieijishititiccctctgtccgcaatcttcaccaca (SEQ ID NO: 3). The consensus sequence comprises (a) an AB loop comprising or consisting of amino acid residues TEDS at positions 13-16 of the consensus sequence; (b) comprising or from amino acid residues TAPDAAF at positions 22-28 of the consensus sequence A BC loop comprising: (c) a CD loop comprising or consisting of amino acid residues SEKVGE at positions 38-43 of the consensus sequence; (d) comprising amino acid residues GSER at positions 51-54 of the consensus sequence; Or a DE loop consisting thereof; (e) an EF loop comprising or consisting of amino acid residues GLKPG at positions 60 to 64 of the consensus sequence; (f) amino acid residues KGGHRSN at positions 75 to 81 of the consensus sequence; An FG loop comprising or consisting of; or (g) any combination of (a)-(f) It may have been modified at one or more locations. Sentilin of the present disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type III (FN3) domains. The scaffold is less than or equivalent to 10 ⁻⁹ M or less than 10 ⁻¹⁰ M or less, or less than or equal to 10 ⁻¹¹ M, or less than or equal to 10 ⁻¹² M. ^10-13 equivalent less or and its than ^M, may be attached at ^{10 -14} comparable small or and its than M, and ^{10 -15} at least one affinity selected from comparable _{K D} of less or with it than M. _The K _D can be determined by surface plasmon resonance.

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

  The present disclosure provides a transposon comprising the CAR of the present disclosure. The transposons of the present disclosure are maintained as episomes or integrated into the genome of the recombinant / modified cell. The transposon can be part of a two-component piggyBac system that utilizes a transposon and a transposase to enhance non-viral gene transfer.

  The transposons of the present disclosure can include a selection gene for identifying, enriching and / or isolating cells that express the transposon. Exemplary selection genes encode any gene product (eg, transcript, protein, enzyme) essential for cell viability and survival. An exemplary selection gene is any that is essential to confer resistance to attack by drugs that are sensitive to cells (or can be lethal to cells) in the absence of the gene product encoded by the selection gene. Gene products (eg, transcripts, proteins, enzymes). An exemplary selection gene is any gene essential for viability and / or survival in cell media lacking one or more nutrients essential for cell viability and / or survival in the absence of the selection gene. Encodes a product (eg, transcript, protein, enzyme). Exemplary selection genes include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, confers resistance to methotrexate), TYMS (encodes thymidylate synthetase) , MGMT (encodes O (6) -methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (RAD51 paralog C ), GCS (encoding glucosylceramide synthase), and NKX2.2 (encoding NK2 homeobox 2).

  A transposon of the present disclosure may comprise, for example, at least one self-cleaving peptide located between one or more of the protein scaffolds of the present disclosure, sentin or CARTYrin and a selected gene of the present disclosure. The at least one self-cleaving peptide can include, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

  The transposon of the present disclosure may comprise a first self-cleaving peptide and a second self-cleaving peptide, wherein the first self-cleaving peptide is, for example, one of the protein scaffolds of the present disclosure, sentin or CARTYrin. Alternatively, the plurality of upstream, second self-cleaving peptides are located, for example, one or more downstream of the disclosed protein scaffold, sentin or CARTYrin. The first and / or second self-cleaving peptide is, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide Can be included. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

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

  The transposon of the present disclosure may include a piggyBac transposon. The transposase enzyme of the present disclosure may include a piggyBac transposase or a compatible enzyme.

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

  Viral vectors of the present disclosure can include sequences isolated from or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors can include sequences isolated from or derived from adeno-associated virus (AAV). Viral vectors can include recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure are two or more terminal inversion sequences located in cis next to sequences encoding the protein scaffolds, sentinulin or CARTYrin of the present disclosure. (ITR) sequence. Exemplary and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, all serotypes (eg, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Not. Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include self-complementary AAV (scAAV) and AAV hybrids containing one serotype genome and another serotype capsid (eg, AAV2 / 5, Including but not limited to AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the present disclosure include, but are not limited to, rAAV-LK03.

  Viral vectors of the present disclosure can include a selection gene. A selection gene may encode a gene product essential for cell viability and survival. A selection gene can encode a gene product essential for cell viability and survival upon challenge with selective cell culture conditions. Selective cell culture conditions can include compounds that are detrimental to cell viability or survival, and gene products confer resistance to such compounds. Exemplary selection genes of the present disclosure include, but are not limited to, neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, confers resistance to methotrexate), TYMS (contains thymidylate synthetase). MGMT (encodes O (6) -methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes) RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2) or any combination thereof.

  Viral vectors of the present disclosure can include at least one self-cleaving peptide. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the selection gene. In some embodiments, the vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located upstream of the CAR and the second self-cleaving peptide is CAR Located downstream of. The self-cleaving peptide can include, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

  The present disclosure provides a vector comprising the CAR of the present disclosure. In certain embodiments, the vector is an mRNA vector. The vector can be a recombinant mRNA vector. The T cells of the present disclosure can be expanded prior to contacting the T cells with an mRNA vector comprising the CAR of the present disclosure. Subsequently, T cells containing mRNA vectors and modified T cells can be administered to the subject.

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

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

  Nanoparticle vectors of the present disclosure can include at least one self-cleaving peptide. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the self-cleaving peptide is located between the CAR and the nanoparticle. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located upstream of the CAR and the second self-cleaving peptide is , Located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle, and the second self-cleaving peptide The cleavable peptide is located downstream of the CAR. In some embodiments, the nanoparticle vector may comprise at least one self-cleaving peptide, wherein the first self-cleaving peptide is located between the CAR and the nanoparticle, and the second self-cleaving peptide The cleaving peptide is located downstream of the CAR, eg, between the CAR and the selection gene. The self-cleaving peptide can include, for example, a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. The T2A peptide comprises an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising EGRGSLTTCGDVEENGPP (SEQ ID NO: 18) obtain. The GSG-T2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGEGRGSLLTCGDVEENGPP (SEQ ID NO: 19), or an amino acid sequence comprising GSGEGRGSLTTCGDVEENPGP (SEQ ID NO: 19) Can be included. The GSG-T2A peptide may comprise a nucleic acid sequence comprising ggactgggagagggagaggggaagcctgctgacctgtggagagtgtgaggagaaaaccccagacca (SEQ ID NO: 20). The E2A peptide comprises an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising QCTNYALLKLAGDVESPNPGP (SEQ ID NO: 21) obtain. The GSG-E2A peptide has at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) or an amino acid sequence comprising GSGQCTNYALLKLAGDVESPNPGP (SEQ ID NO: 22) Can be included. The F2A peptide comprises an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising VKQTLNFDLLKLAGDVESPNPGP (SEQ ID NO: 23) obtain. The GSG-F2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGVKQTLNFLLKLGAGDVESPNP (SEQ ID NO: 24) or an amino acid sequence comprising GSGVKQTLNFDLKLLAGDVESPNPGP (SEQ ID NO: 24) Can be included. The P2A peptide comprises an amino acid sequence comprising ATNFSLLKQAGDVEENGPP (SEQ ID NO: 25), or a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising ATNFFSLKQAGDVEENGPP (SEQ ID NO: 25) obtain. A GSG-P2A peptide is a sequence having at least 70%, 80%, 90%, 95% or 99% identity to an amino acid sequence comprising GSGATNFSLLKQAGDVEENGPP (SEQ ID NO: 26) or to an amino acid sequence comprising GSGATNFSLKLKAGGDVEENGPP (SEQ ID NO: 26) Can be included.

The present disclosure provides a composition comprising a vector of the present disclosure.
Scaffold protein

  Sentilin is one example of a protein scaffold of the present disclosure. The antigen recognition region of the CAR of the present disclosure may comprise at least one protein scaffold.

  Protein scaffolds of the present disclosure derive from fibronectin type III (FN3) repeat proteins, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods of making and using them Can do. In a preferred embodiment, the protein scaffold is composed of multiple FN3 domain consensus sequences from human tenascin-C (hereinafter “tenascin”). In a further preferred embodiment, the protein scaffold of the invention is a consensus sequence of 15 FN3 domains. The protein scaffolds of the present disclosure can be designed to bind to various molecules, eg, cell target proteins. In a preferred embodiment, the protein scaffold of the present disclosure can be designed to bind to an epitope of the wild type and / or variant form of the antigen.

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

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

  The disclosure includes (a) a protein scaffold and / or encoding nucleic acid based on the consensus sequence of a number of FN3 domains described herein; and (b) an appropriate and / or pharmaceutically acceptable carrier or diluent. At least one composition comprising is provided.

  The present disclosure provides a method for generating a library of protein scaffolds based on a fibronectin type III (FN3) repeat protein, preferably a consensus sequence of multiple FN3 domains, more preferably a consensus sequence of multiple FN3 domains from human tenascin To do. A library is formed by creating successive generations of scaffolds by altering (by mutation) certain parts of the scaffold, for example, the amino acids or number of amino acids in the molecule within the loop region. The Libraries can be generated by changing the amino acid composition of a single loop or simultaneously changing additional portions of multiple loops or scaffold molecules. The loop to be changed can be appropriately extended or shortened. Such a library can be generated to include all possible amino acids at each position, or a subset of amino acids can be designed. Library members can be used for screening by displays such as in vitro or CIS display (DNA, RNA, ribosome display, etc.), yeast display, bacterial display, and phage display.

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

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

  The protein scaffolds of the present disclosure provide advantages over conventional therapeutic agents, such as being able to be administered topically, orally, or across the blood brain barrier; can be expressed in E. coli, thereby increasing the expression of proteins in response to the expression capacity of mammalian cells, engineered to bind multiple targets or multiple epitopes of the same target The ability to be specific or tandem molecules, to be conjugated with drugs, polymers and probes, to be formulated at high concentrations, and to be able to effectively penetrate such tissues and tumors.

  Furthermore, protein scaffolds have many of the properties of antibodies in the context of their folding that mimics the variable region of antibodies. This orientation allows the FN3 loop to be exposed in the same manner as the antibody complementarity determining region (CDR). Protein scaffolds should be able to bind to cellular targets and can modify loops, eg, affinity maturation, to improve certain binding or related properties.

  Three of the six loops of the protein scaffold of the present disclosure correspond topologically to the complementarity determining regions (CDRs 1-3) of the antibody, ie, the antigen binding region, while the remaining three loops are antibody CDRs. Exposed on the surface in the same manner as. These loops are residues 13-16 or near residues 13-16 of SEQ ID NO: 1, residues 22-28 or residues 22-28, residues 38-43 or residues 38-43, residues Covers 51-54 or residue 51-54, residue 60-64 or residue 60-64, and residue 75-81 or residue 75-81. Loop regions near residues 22-28 or residues 22-28, residues 51-54 or residues 51-54, and residues 75-81 or residues 75-81 with respect to binding specificity and affinity It is preferable to change. One or more of these loop regions are added to the library, randomized with other loop regions and / or other strands that maintain their sequences as backbone parts, and have high affinity for specific protein targets Strong binding substances can be selected from the library. One or more of the loop regions can interact with the target protein as well as the interaction of antibody CDRs with the protein.

  Scaffolds of the present disclosure can include single chain antibodies (eg, scFv). A single chain antibody of the present disclosure may comprise three light chain and three heavy chain CDRs of the antibody. In certain embodiments, a single chain antibody of the present disclosure comprises three light chain and three heavy chain CDRs of the antibody, and the complementarity determining region (CDR) of the single chain antibody is a human sequence. The disclosure provides (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one single chain antibody (eg, scFv); (b) a transmembrane domain, and (c ) Providing a chimeric antigen receptor (CAR) comprising an endodomain comprising at least one costimulatory domain; In certain embodiments, the antigen recognition region may comprise two single chain antibodies (eg, two scFvs) so that bispecific or tandem CAR occurs. In certain embodiments, the antigen recognition region may comprise three single chain antibodies (eg, three scFv) so that a trispecific CAR occurs. In certain embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.

  Scaffolds of the present disclosure can include a sequence (eg, VHH) that includes one or more fragments of an antibody. A sequence comprising one or more fragments of an antibody of the present disclosure may comprise the two heavy chain variable regions of the antibody. In certain embodiments, the sequence comprises two heavy chain variable regions of the antibody, and the VHH complementarity determining region (CDR) is a human sequence. Scaffolds of the present disclosure can include a sequence (eg, VHH) that includes one or more fragments of an antibody. The disclosure provides (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one sequence (eg, VHH) comprising one or more fragments of an antibody; (b Provided is a chimeric antigen receptor (CAR) comprising a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the antigen recognition region can comprise two sequences (eg, two VHHs) comprising one or more fragments of an antibody so that bispecific or tandem CARs occur. In certain embodiments, the antigen recognition region may comprise three sequences (eg, three VHHs) comprising one or more fragments of the antibody so that a trispecific CAR occurs. In certain embodiments, the ectodomain can further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.

  Scaffolds of the present disclosure can include antibody mimetics.

  This non-covalent linkage can include, for example, an antibody mimic. As used herein, the term “antibody mimetic” is intended to describe an organic compound that specifically binds a target sequence and has a structure distinct from naturally occurring antibodies. Antibody mimetics can include proteins, nucleic acids, or small molecules. The target sequence to which the antibody mimic of the present disclosure specifically binds can be an antigen. Antibody mimetics provide superior properties over antibodies, including but not limited to excellent solubility, tissue penetration, heat and enzyme stability (eg, resistance to enzymatic degradation) and lower production costs Can do. Exemplary antibody mimics include Affibody, Affilin, Affimer, Affitin, Alphabody, Anticarin, and Avimer (also known as avidity multimer), DARPin (Designed Ankyrin Repeat Protein), Finomer , Kunitz domain peptides, as well as monobodies.

  Affibody molecules of the present disclosure comprise protein scaffolds comprising or consisting of one or more alpha helices without any disulfide bridges. Preferably, the Affibody molecule of the present disclosure comprises or consists of three alpha helices. For example, an Affibody molecule of the present disclosure can comprise an immunoglobulin binding domain. An Affibody molecule of the present disclosure may comprise a Z domain of protein A.

  Affilin molecules of the present disclosure include, for example, protein scaffolds produced by modification of exposed amino acids of either gamma-B crystallin or ubiquitin. Affinin molecules functionally mimic the affinity of an antibody for an antigen but do not structurally mimic an antibody. In any protein scaffold used to make affilin, amino acids accessible to solvents or possible binding partners in appropriately folded protein molecules are considered exposed amino acids. Any one or more of these exposed amino acids can be modified to specifically bind to a target sequence or antigen.

  Affymer molecules of the present disclosure include protein scaffolds comprising highly stable proteins engineered to display peptide loops that provide high affinity binding sites for specific target sequences. Exemplary affimer molecules of the present disclosure include protein scaffolds based on cystatin protein or its tertiary structure. Exemplary affimer molecules of the present disclosure may share a common tertiary structure including an alpha-helix present on top of the antiparallel beta-sheet.

  Affitin molecules of the present disclosure include an artificial protein scaffold, the structure of which can be derived from, for example, a DNA binding protein (eg, DNA binding protein Sac7d). Affitin of the present disclosure selectively binds a target sequence that can be all or part of an antigen. Exemplary affitin of the present disclosure is produced by randomizing one or more amino acid sequences on the binding surface of a DNA binding protein and subjecting the resulting protein to ribosome display and selection. The target sequences of the disclosed affitin can be found, for example, in the genome or on the surface of peptides, proteins, viruses or bacteria. In certain embodiments of the present disclosure, an aphitin molecule can be used as a specific inhibitor of an enzyme. Affitin molecules of the present disclosure may comprise a thermostable protein or derivative thereof.

  The alpha body molecules of the present disclosure may also be referred to as cell permeable alpha bodies (CPAB). The alphabody molecules of the present disclosure include small proteins (typically less than 10 kDa) that bind to various target sequences (including antigens). Alpha body molecules are able to reach and bind to intracellular target sequences. Structurally, the alpha body molecules of the present disclosure comprise artificial sequences that form a single chain alpha helix (similar to a naturally occurring coiled-coil structure). An alpha body molecule of the present disclosure may comprise a protein scaffold comprising one or more amino acids modified to specifically bind to a target protein. Regardless of the binding specificity of the molecule, the alphabody molecules of the present disclosure maintain precise folding and thermal stability.

  Anticalin molecules of the present disclosure include artificial proteins that bind to target sequences or sites in either proteins or small molecules. An anticalin molecule of the present disclosure may comprise an artificial protein derived from human lipocalin. The anticalin molecules of the present disclosure can be used in place of, for example, monoclonal antibodies or fragments thereof. Anticarin molecules may demonstrate better tissue penetration and thermal stability than monoclonal antibodies or fragments thereof. An exemplary anticalin molecule of the present disclosure may comprise about 180 amino acids with a mass of approximately 20 kDa. Structurally, the anticalin molecules of the present disclosure comprise a barrel structure comprising antiparallel beta-strands connected in pairs by loops and associated alpha helices. In a preferred embodiment, an anticalin molecule of the present disclosure comprises a barrel structure comprising eight antiparallel beta-strands connected in pairs by loops and associated alpha helices.

  The avimer molecules of the present disclosure include engineered proteins that specifically bind to a target sequence, which may be an antigen. An avimer of the present disclosure may recognize multiple binding sites within the same target or within separate targets. If an avimer of the present disclosure recognizes more than one target, the avimer mimics the function of a bispecific antibody. The engineered protein avimer can comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides can be connected via one or more linker peptides. The amino acid sequence of one or more of the avimer peptides can be derived from the A domain of the membrane receptor. Avimers have a rigid structure that may optionally contain disulfide bonds and / or calcium. The avimers of the present disclosure can demonstrate higher thermal stability compared to antibodies.

  DARPins (designed ankyrin repeat proteins) of the present disclosure include genetically engineered, recombinant, or chimeric proteins that have high specificity and high affinity for target sequences. In certain embodiments, a DARPin of the present disclosure is derived from an ankyrin protein and optionally includes at least three repeat motifs (also referred to as repeating structural units) of the ankyrin protein. Ankyrin proteins mediate high affinity protein-protein interactions. The DARPin of the present disclosure includes a large target interaction surface.

  The finomers of the present disclosure comprise a small binding protein (approximately 7 kDa) derived from the human Fyn SH3 domain and engineered to bind to target sequences and molecules with equal affinity and equal specificity as antibodies.

  The Kunitz domain peptides of the present disclosure include a protein scaffold that includes a Kunitz domain. The Kunitz domain contains an active site for inhibiting protease activity. Structurally, the Kunitz domain of the present disclosure includes a disulfide-rich alpha + beta folding structure (fold). This structure is illustrated by a bovine pancreatic trypsin inhibitor. Kunitz domain peptides recognize specific protein structures and function as competitive protease inhibitors. The Kunitz domain of the present disclosure may comprise ecarantide (derived from a human lipoprotein associated coagulation inhibitor (LACI)).

The monobodies of the present disclosure are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable to single chain antibodies. These genetically engineered proteins specifically bind target sequences containing antigens. The monobodies of the present disclosure can specifically target one or more distinct proteins or target sequences. In a preferred embodiment, a monobody of the present disclosure comprises a protein scaffold that mimics the structure of human fibronectin, more preferably the structure of the tenth extracellular type III domain of fibronectin. The 10th extracellular type III domain of fibronectin, as well as its monobody mimics, were exposed to three beta sheets forming the barrel and three complementarity determining regions (CDRs) on each side corresponding to the three antibodies. Contains a loop. In contrast to the structure of antibody variable domains, monobodies lack any binding sites for metal ions as well as a central disulfide bond. Multispecific monobodies can be optimized by modifying loops BC and FG. The monobodies of the present disclosure may include adnectin.
Production and production of scaffold proteins

  At least one scaffold protein of the present disclosure can optionally be produced by cell lines, mixed cell lines, immortalized cells or clonal populations of immortalized cells well known in the art. For example, edited by Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. NY, N .; Y. (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N .; Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N .; Y. (1989); Edited by Colligan et al., Current Protocols in Immunology, John Wiley & Sons, Inc. , NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N .; Y. , (1997-2001).

  Amino acids from the scaffold protein are altered, added and / or deleted to reduce immunogenicity, or binding, affinity, association rate (on-rate), dissociation rate (off-rate) , Binding activity, specificity, half-life, stability, solubility or any other suitable property known in the art can be reduced, enhanced or modified.

Optionally, scaffold proteins that retain high affinity for antigen and other favorable biological properties can be engineered. To achieve this goal, optionally, scaffold proteins can be obtained from a variety of conceptual engineered products using a parent sequence and a three-dimensional model of the parent sequence and engineered sequence. It can be prepared by an analysis process. Three-dimensional models are generally available and are well known to those skilled in the art. Computer programs are available that can illustrate and display putative three-dimensional conformational structures of selected candidate sequences and measure potential immunogenicity (eg, Xencor, Inc., Monrovia) Calif., Immunofilter program). Examining these displays allows analysis of the potential role of residues in the functionality of the candidate sequence, ie, analysis of residues that affect the ability of the candidate scaffold protein to bind to its antigen. . In this way, residues can be selected and combined from the parental sequence and the reference sequence so that the desired property, such as affinity for the target antigen (s), is achieved. Other suitable engineering operating methods may be used instead of or in addition to the above procedure.
piggyBac transposon system

The method of the present disclosure results in the modified _{TSCM of the} present disclosure, regardless of the method used to introduce the antigen receptor into the primary human T cells of the present disclosure. In the disclosed method, the modified T _{SCM of the} present disclosure with greater efficacy and / or the piggyBac transposon system is used to introduce the antigen receptor or therapeutic protein of the present disclosure into primary human T cells. A greater abundance, proportion, yield of the disclosed modified _TSCM is provided. The piggyBac transposon system of the present disclosure can include a piggyBac transposon comprising an antigen receptor of the present disclosure. Primary human T cells may be simultaneously (or very close in time, eg, primary human T cells) before the piggyBac transposon comprising the antigen receptor of the present disclosure and the transposase of the present disclosure are introduced into the cell. Preferably, the transposon and transposase are in contact with the same container (eg, contained in a cuvette, etc.), but they cannot interact in the absence of cells. Primary human T cells are preferably contacted simultaneously with the transposon and transposase before the piggyBac transposon comprising the antigen receptor of the present disclosure and the Super pigBac ™ (SPB) transposase of the present disclosure are introduced into the cell. In certain preferred embodiments, the Super piggyBac ™ (SPB) transposase is an mRNA sequence encoding the Super piggyBac ™ (SPB) transposase.

  Additional disclosures regarding the piggyBac transposon and the Super piggyBac ™ (SPB) transposase are disclosed in International Patent Publication No. WO 2010/099296, US Pat. No. 8,399,643, the entire contents of each of which are hereby incorporated by reference. U.S. Patent No. 9,546,382, U.S. Patent No. 6,218,185, U.S. Patent No. 6,551,825, U.S. Patent No. 6,962,810, and U.S. Patent No. 7,105,343. Can be seen in the issue.

  The present disclosure provides a method for introducing a polynucleotide construct comprising a DNA sequence into a host cell. Preferably, the introducing step is mediated by the piggyBac transposon system.

  In certain embodiments of the disclosed method, the transposon is a plasmid DNA transposon having a sequence 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 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.

In certain embodiments of the disclosed method, the transposase enzyme is a piggyBac ™ (PB) transposase enzyme. The piggyBac (PB) transposase enzyme is
May comprise or consist of at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage amino acid sequence therebetween.

In certain embodiments of the disclosed methods, the transposase enzyme is a sequence.
A piggyBac ™ (PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538.

  In certain embodiments, the transposase enzyme comprises or consists of an amino acid sequence having amino acid substitutions at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4 piggyBac ™ (PB) transposase enzyme. In certain embodiments, the transposase enzyme comprises or consists of an amino acid sequence having amino acid substitutions at 3 or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 4 piggyBac ™ (PB) transposase enzyme. In certain embodiments, the transposase enzyme comprises or consists of a piggyBac ™ comprising an amino acid sequence having amino acid substitutions at positions 30, 165, 282, and 538, respectively, of the sequence of SEQ ID NO: 4 (PB) Transposase enzyme. In certain embodiments, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 4 is a substitution of valine (V) with isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 4 is a substitution of serine (S) with glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 4 is a substitution of valine (V) with methionine (M). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 4 is a substitution of lysine (K) with asparagine (N).

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

  In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ or Super piggyBac ™ transposase enzyme is 3rd, 46th, 82th, 103th, 119th, 125th, 177th, 180th, 185th, 187th, 200th, 207th, 209th of the sequence of SEQ ID NO: 4 or 5 226th, 235th, 240th, 241st, 243th, 258th, 296th, 298th, 311th, 315th, 319th, 327th, 328th, 340th, 421st, 436th, 456th It may further comprise amino acid substitutions at one or more of positions 470, 486, 503, 552, 570 and 591In certain embodiments, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282 and / or 538, the piggyBac ™ or Super piggyBac ™ transposase enzyme is located at positions 46, 119 125th, 177th, 180th, 185th, 187th, 200th, 207th, 209th, 226th, 235th, 240th, 241st, 243rd, 296th, 298th, 298th, 311st, It may further comprise amino acid substitutions at one or more of positions 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570 . In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of asparagine (N) with serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of serine (S) with alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of threonine (T) with alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tryptophan (W) with isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of proline (P) with serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of proline (P) with arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alanine (A) with cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with cysteine (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of histidine (H) by tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of 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 a 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 a 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 a 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 a 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 a 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 a 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 a substitution of phenylalanine (F) with methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of arginine (R) with leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of serine (S) with asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tryptophan (W) with leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tyrosine (Y) with leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of phenylalanine (F) with leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alanine (A) with methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of valine (V) with methionine (M). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of isoleucine (I) with proline (P). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of valine with proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with arginine (R). In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of glycine (G) with threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of arginine (R) with tyrosine (Y). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of valine (V) by tyrosine (Y). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of glycine (G) with cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of histidine (H) with aspartic acid (D). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of isoleucine (I) with valine (V). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of tyrosine (Y) with methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of phenylalanine (F) with leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of leucine (L) with methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of isoleucine (I) with methionine (M). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of lysine (K) with valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of threonine (T) with alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of proline (P) with glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of arginine (R) with glutamine (Q).

  In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ transposase enzyme is SEQ ID NO: 4 or An amino acid substitution in one or more of 103, 194, 372, 375, 450, 509 and 570 of the sequence of number 5 may be included, and the Super piggyBac ™ transposase enzyme may further include this. In certain embodiments of the disclosed methods, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the piggyBac ™ transposase enzyme is SEQ ID NO: 4 or Amino acid substitutions at 2, 3, 4, 5, 6 or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of number 5. The Super piggyBac ™ transposase enzyme may further comprise this. In certain embodiments, including embodiments where the transposase comprises the above mutations at positions 30, 165, 282 and / or 538, the piggyBac ™ transposase enzyme is of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5 The amino acid substitutions at positions 103, 194, 372, 375, 450, 509 and 570 may be included, 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 a substitution of alanine (A) with lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of asparagine (N) with aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of glycine (G) with serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of serine (S) with asparagine (N). In certain embodiments, the piggyBac ™ transposase enzyme may comprise a substitution of valine (V) with methionine (M) at position 194 of SEQ ID NO: 4. In certain embodiments, including the embodiment where the piggyBac ™ transposase enzyme may include a substitution of valine (V) by methionine (M) at position 194 of SEQ ID NO: 4, the piggyBac ™ transposase enzyme comprises SEQ ID NO: It may further comprise amino acid substitutions at positions 372, 375 and 450 of the sequence of 4 or SEQ ID NO: 5. In certain embodiments, the piggyBac ™ transposase enzyme replaces valine (V) with methionine (M) at position 194 of SEQ ID NO: 4, arginine (R) of alanine (A) at position 372 of SEQ ID NO: 4. And a substitution of alanine (A) at position 375 of SEQ ID NO: 4 with lysine (K). In certain embodiments, the piggyBac ™ transposase enzyme replaces valine (V) with methionine (M) at position 194 of SEQ ID NO: 4, arginine (R) of alanine (A) at position 372 of SEQ ID NO: 4. Substitution of alanine (A) at position 375 of SEQ ID NO: 4 with lysine (K) and substitution of asparagine (N) at position 450 of SEQ ID NO: 4 with aspartic acid (D).

  “Introducing” intends to bring a polynucleotide construct into a plant in such a way that the construct enters the interior of the host cell. The methods of the present invention are independent of a particular method as long as the polynucleotide construct enters the interior of one cell of the host with respect to introducing the polynucleotide construct into the host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals include, but are not limited to, those skilled in the art, including stable transformation methods, transient transformation methods, and virus-mediated methods. Known in the art.

  The term “endogenous” as used throughout this disclosure refers to a nucleic acid or protein sequence that is naturally associated with a target gene or host cell into which it is introduced.

  By “stable transformation” is intended that a polynucleotide construct introduced into a plant can be integrated into the genome of the host and inherited to progeny.

  By “transient transformation” is intended that the polynucleotide construct introduced into the host is not integrated into the host's genome.

In a preferred embodiment, the exogenous sequence to generate the modified _{T SCM} or _{T CM} was introduced by stable transformation into primary human T cells, using a piggyBac transposon system.
Additional transposon system

  In certain embodiments of the disclosed 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 an overactive Sleeping Beauty transposase (SB100X).

The present disclosure provides a method for producing modified stem memory T cells (T _SCM ) or modified central memory T cells (T _CM ), the method comprising: (a) an antigen receptor for producing modified T cells; Or a transposon composition comprising a transposon comprising a therapeutic protein, and (b) introducing a transposase or a transposase composition comprising a transposase-encoding sequence into a primary human T cell, wherein the modified T cell is modified Express one or more cell surface markers of stem memory T cells (T _SCM ) or modified central memory T cells (T _CM ), thereby modifying modified stem memory T cells (T _SCM ) or modified central memory T cells (T _CM ). The present disclosure provides a method of generating a plurality of modified stem memory T cells (T _SCM ) or a plurality of modified central memory T cells (T _CM ), the method comprising generating a plurality of modified T cells, Introducing a transposon composition comprising a transposon comprising an antigen receptor, and (b) a transposase or a transposase composition comprising a transposase-encoding sequence into a plurality of primary human T cells, wherein At least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75% of modified T cells, 80%, 85%, 90%, 95%, 99% or any percentage in between are stem memory T cells (T _SCM ) or central memory T cells (T _CM ) Express one or more of the cell surface markers, thereby creating a plurality of modified stem memory T cells (T _SCM ) or a plurality of modified central memory T cells (T _CM ).

  In certain embodiments of the disclosed 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 an overactive Sleeping Beauty transposase (SB100X).

In certain embodiments of the disclosed methods, the Sleeping Beauty transposase enzyme is
At least 75%, 80%, 85%, 90%, 95%, 99% or any percentage amino acid sequence between them.

In certain embodiments of the disclosed methods, the overactive Sleeping Beauty (SB100X) transposase enzyme comprises:
At least 75%, 80%, 85%, 90%, 95%, 99% or any percentage amino acid sequence between them.

In certain embodiments of the disclosed methods, the transposase is a Helitron transposase. The Helitron transposase moves the Helraiser transposon, an ancient element from the bat genome that was active about 3000-36 million years ago. Exemplary Helraiser transposons of the present disclosure include:
Helibat1 containing a nucleic acid sequence containing

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

Exemplary Helitron transposases of the present disclosure are:
An amino acid sequence comprising

  In the Helitron transition, a hairpin near the 3 'end of the transposon functions as a terminator. However, this hairpin can be bypassed by a transposase, thereby leading to transduction of adjacent sequences. Furthermore, the Helraiser transition results in a cyclic intermediate that is covalently closed. Furthermore, Helitron transfer may lack target site duplication. In the Helraiser sequence, the transposase is flanked by left and right terminal sequences called LTS and RTS. These sequences terminate with a conserved 5'-TC / CTAG-3 'motif. The 19 bp palindromic sequence that has the potential to form a hairpin termination structure is located 11 nucleotides upstream of RTS and consists of the sequence GTGCACGAATTTCGTGCACCGGGCACTAG (SEQ ID NO: 29).

In certain embodiments of the disclosed method, the transposase is a Tol2 transposase. Tol2 transposons can be isolated or derived from the medaka genome and can be similar to hAT family transposons. An exemplary Tol2 transposon of the present disclosure contains a gene encoding a Tol2 transposase that is encoded by a sequence comprising about 4.7 kilobases and contains four exons. An exemplary Tol2 transposase of the present disclosure is:
An amino acid sequence comprising

Exemplary Tol2 transposons of the present disclosure including inverted repeats, near-terminal sequences and Tol2 transposase are:
Is encoded by a nucleic acid sequence comprising
Homologous recombination

In certain embodiments of the methods of the present disclosure, is made by a modified CAR-T _SCM or CAR-T _CM of the present disclosure, the introduction by homologous recombination of antigen receptor primary human T cells of the present disclosure. In certain embodiments of the present disclosure, homologous recombination is induced by single or double strand breaks induced by the genome editing composition or construct of the present disclosure. Homologous recombination methods of the present disclosure provide for the use of a genome editing composition or construct of the present disclosure to induce at least one truncation in the sequence and to provide an entry point for an exogenous donor sequence composition within the genomic sequence. Contacting the genomic sequence. The donor sequence composition of the present disclosure is integrated into the genomic sequence by the cell's native DNA repair mechanism at the induced entry point.

  In certain embodiments of the disclosed methods, homologous recombination introduces a sequence encoding the antigen receptor of the present disclosure and / or a donor sequence composition into a “genome safe harbor” site. In certain embodiments, the mammalian genomic sequence comprises a genome safe harbor site. In certain embodiments, the primate genomic sequence comprises a genome safe harbor site. In certain embodiments, the human genome sequence comprises a genome safe harbor site.

  Genome-safe harbor sites ensure the incorporation of new genetic material to ensure that newly inserted genetic elements function (eg, expressed at therapeutically effective levels of expression) and pose a risk to the host organism Can be adapted in a manner that ensures that no harmful changes to the host genome are caused. Potential genomic safe harbors include, but are not limited to, human albumin gene, adeno-associated virus site 1 (AAVS1), naturally occurring AAV virus integration site on chromosome 19, chemokine (CC motif) reception Examples include the intron sequences of the body 5 (CCR5) gene site and the human orthologue site of the mouse Rosa26 locus.

  In certain embodiments of the disclosed methods, homologous recombination allows the antigen-encoding receptor encoding and / or donor sequence composition to be transferred to an endogenous T cell receptor or major histocompatibility gene complex ( Introduced into a sequence encoding one or more components of MHC). In certain embodiments, the endogenous gene is disrupted by inducing homologous recombination within the genomic sequence encoding the endogenous T cell receptor or MHC, and optionally one of the coding sequences of the endogenous gene. Part is replaced with the donor sequence composition of the present disclosure. In certain embodiments, the endogenous gene is disrupted by inducing homologous recombination within the genomic sequence encoding the endogenous T cell receptor or MHC, and optionally the entire coding sequence of the endogenous gene. Replace with the donor sequence composition of the present disclosure. In certain embodiments of the disclosed methods, the antigen receptor is operably linked to an endogenous T cell promoter by introduction of a sequence encoding an antigen receptor or donor sequence composition of the present disclosure by homologous recombination. The In certain embodiments of the disclosed methods, the antigen receptor or therapeutic protein acts on a transcriptional or translational regulatory element by homologous recombination of the sequence encoding the antigen receptor or donor sequence composition of the present disclosure. Connected as possible. In certain embodiments of the disclosed methods, introduction of a sequence encoding an antigen receptor or donor sequence composition of the present disclosure by homologous recombination allows the antigen receptor or therapeutic protein to be operative to a transcriptional regulatory element. Connected. In certain embodiments, the transcriptional regulatory element comprises an endogenous T cell 5'UTR.

  In certain embodiments of the introducing step involving homologous recombination, the genome editing composition is contacted with the genomic sequence of at least one primary T cell of the plurality of T cells. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition is contacted with the genomic sequence of a portion of the primary T cell of the plurality of T cells. In certain embodiments, the portion of primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25 of the total number of primary T cells in the plurality of T cells. %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or Any percentage between these. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition is contacted with the genomic sequence of each primary T cell of the plurality of T cells. In certain embodiments of the introducing step involving homologous recombination, single-strand breaks are induced by the genome editing composition. In certain embodiments of the introducing step involving homologous recombination, double-strand breaks are induced by the genome editing composition. In certain embodiments of the introducing step involving homologous recombination, the introducing step further comprises a donor sequence composition. In certain embodiments, the donor sequence composition includes a sequence encoding an antigen receptor. In certain embodiments, the donor sequence composition comprises a sequence encoding an antigen receptor, a 5 ′ genomic sequence and a 3 ′ genomic sequence, wherein the 5 ′ genomic sequence is derived by a genome editing composition. Homologous or identical to the genomic sequence of the primary T cell, 5 ′ to the breakpoint, and the 3 ′ genome sequence is 3 ′ to the breakpoint induced by the genome editing composition. Homologous or identical to the T cell genomic sequence. In certain embodiments, the 5 ′ genomic sequence and / or the 3 ′ genomic sequence is at least 50 bp, 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 1100 bp, at least 1200 bp, at least 1300 bp, at least 1400, or at least 1500 bp, at least 1600 bp, at least 1700 bp, at least 1800 bp, at least 1900 bp, at least 2000 bp in length or any base pair in between (including the end point) bp). In certain embodiments of the introducing step involving homologous recombination, the genomic editing composition and donor sequence composition are contacted simultaneously or sequentially with the genomic sequence. In certain embodiments of the introduction step involving homologous recombination, the genome editing composition and the donor sequence composition are sequentially contacted with the genome sequence, resulting in the genome editing composition first. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease domain. In certain embodiments of the introducing step involving homologous recombination, the genome editing composition comprises a DNA binding domain and a nuclease domain. In certain embodiments of the genome editing composition, the DNA binding domain comprises a guide RNA (gRNA). In certain embodiments of the genome editing composition, the DNA binding domain comprises a TALEN DNA binding domain. In certain embodiments of the genome editing composition, the DNA binding domain comprises a ZFN DNA binding domain. In certain embodiments of the genome editing composition, the nuclease domain comprises a Cas9 nuclease or sequence thereof. In certain embodiments of the genome editing composition, the nuclease domain comprises inactive Cas9 (SEQ ID NO: 33, substitution of alanine (A) at position 10, aspartic acid (D) (D10A) and alanine (A) at position 840. Of histidine (H) (including H840A). In certain embodiments of the genome editing composition, the nuclease domain comprises short inactive Cas9 (SEQ ID NO: 32, substitution of alanine (A) at position 10 with aspartic acid (D) (D10A) and alanine at position 540 (A ) With asparagine (N) (including N540A). In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises a type IIS endonuclease. In certain embodiments of the genome editing composition, the type IIS endonuclease is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, MboII, MboII, Mbo1I PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsGE Contains BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments, the Type IIS endonuclease comprises Clo051. In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises TALEN or a nuclease domain thereof. In certain embodiments of the genome editing composition, the nuclease domain comprises or further comprises ZFN or a nuclease domain thereof. In certain embodiments of the introduction step involving homologous recombination, the genomic editing composition induces cleavage in the genomic sequence and the primary T cell's endogenous DNA repair machinery is used to insert the 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.

  In certain embodiments of the method of homologous recombination of the present disclosure, the nuclease domain of the genome editing composition or construct is capable of introducing a cleavage at a defined location within the genome sequence of a primary human T cell. And may further comprise, consist essentially of, or consist of a homodimer or heterodimer. In certain embodiments, the nuclease is an endonuclease. Effector molecules, including effector molecules comprising homodimers or heterodimers, can comprise, consist essentially of, or consist of a Cas9, Cas9 nuclease domain or fragment thereof. In certain embodiments, Cas9 is catalytically inactive or “inactivated” Cas9 (dCas9). In certain embodiments, Cas9 is catalytically inactive or an “inactivated” nuclease domain of Cas9. In certain embodiments, dCas9 is encoded by a shorter sequence derived from Cas9 inactivated as a full-length catalyst, referred to herein as “small molecule” dCas9 or dSaCas9.

In certain embodiments, the inactivated small molecule Cas9 (dSaCas9) is operably linked to an active nuclease. In certain embodiments, the disclosure is a fusion protein comprising, consisting essentially of, or consisting of a DNA binding domain and a molecular nuclease, wherein the nuclease comprises a small molecule inactivated Cas9 (dSaCas9), Provide a fusion protein. In certain embodiments, dSaCas9 of the present disclosure includes mutations D10A and N580A (underlined and bold) that inactivate the catalytic site. In certain embodiments, the dSaCas9 of the present disclosure is
Of the amino acid sequence.

In certain embodiments, dCas9 of the present disclosure comprises dCas9 isolated or derived from Staphylococcus pyogenes. In certain embodiments, dCas9 comprises dCas9 having substitutions at positions 10 and 840 of the amino acid sequence of dCas9, which inactivates the catalytic site. In certain embodiments, these substitutions are D10A and H840A. In certain embodiments, the amino acid sequence of dCas9 is
Contains an array of

In certain embodiments of the present disclosure, the nuclease domain may comprise, consist essentially of, or consist of dCas9 or dSaCas9 and type IIS endonuclease. In certain embodiments of the present disclosure, the nuclease domain is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, P1 AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EcI Examples include, but are not limited to, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. It may include have dSaCas9 and type IIS endonuclease, may be made may consist essentially, or from. In certain embodiments of the present disclosure, the nuclease domain may comprise, consist essentially of, or consist of dSaCas9 and Clo051. An exemplary Clo051 nuclease domain is:
Or may consist essentially of, or may consist of:

An exemplary dCas9-Clo051 nuclease domain is:
Comprising, consisting essentially of, or consisting of the amino acid sequence of (Clo051 sequence is underlined, the linker is in bold italic, and the dCas9 sequence is in italic) obtain.

  In certain embodiments, the nuclease capable of introducing a cleavage at a defined location in the genomic DNA of primary human T cells comprises, consists essentially of, or consists of a homodimer or a heterodimer, or It can consist of it. A nuclease domain of a genome editing composition or construct of the present disclosure comprises, consists essentially of, a nuclease domain that is single-stranded, derived or recombined from a transcriptional activator-like effector nuclease (TALEN), or It can consist of it. TALEN is a transcription factor with a programmable DNA binding domain that provides a means to create a designer protein that binds to a given DNA sequence or individual nucleic acid. A modular DNA binding domain has been identified in a transcriptional activator-like (TAL) protein, or more specifically, a transcriptional activator-like effector nuclease (TALEN), thereby synthesizing to bind to the DNA sequence of interest. It allows the creation of new transcription factors and, if desired, allows a second domain present on the protein or polypeptide to exhibit DNA-related activity. The TAL protein has been extracted from the organisms Xanthomonas and Ralstonia.

  In certain embodiments of the present disclosure, the nuclease domain of the genome editing composition or construct may comprise a nuclease domain isolated from, or derived from, TALEN and IIS type endonucleases. , May consist essentially of, or may consist of. In certain embodiments of the present disclosure, the type IIS endonuclease is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsE SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051 may be included and essential It may be made to, or may be made from. In certain embodiments of the present disclosure, the Type IIS endonuclease may comprise, consist essentially of, or consist of Clo051 (SEQ ID NO: 34).

  In certain embodiments of the present disclosure, the nuclease domain of the genome editing composition or construct is a nuclease domain isolated from, or derived from, zinc finger nuclease (ZFN) and type IIS endonuclease May consist of, may consist essentially of, or may consist of. In certain embodiments of the present disclosure, the type IIS endonuclease is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsE SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051 may be included and essential It may be made to, or may be made from. In certain embodiments of the present disclosure, the Type IIS endonuclease may comprise, consist essentially of, or consist of Clo051 (SEQ ID NO: 34).

In certain embodiments of the disclosed genome editing compositions or constructs, the DNA binding domain and the nuclease domain may be covalently linked. For example, the fusion protein may comprise a DNA binding domain and a nuclease domain. In certain embodiments of the genome editing compositions or constructs of the present disclosure, the DNA binding domain and the nuclease domain may be operably linked by non-covalent linkage.
Secreted protein from modified T cells

  In certain embodiments of the compositions and methods of the present disclosure, the modified T cells express a therapeutic protein. The therapeutic proteins of the present disclosure include secreted proteins. For treatment, the therapeutic protein is preferably a human protein including a secreted human protein. When expressed or secreted by CAR-T cells of the present disclosure, combinations comprising CAR-T cells and therapeutic proteins secreted therefrom can be considered monotherapy. However, the CAR-T cells of the present disclosure can also be administered as a combination therapy with a second agent. A database of human secreted proteins that can be expressed or secreted by the modified T cells of the present disclosure is the proteinatlas.com, the contents of which are incorporated herein by reference. org / search / protein_class: can be found in Predicted% 20 secreted% 20proteins. Exemplary human secreted proteins are presented in, but not limited to, human secreted proteins in Table 1.

  In some embodiments of the present disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the method of disorder, a composition comprising CAR-T cells modified to express or secrete human proteins is used to treat a coagulation disorder. Blood clotting occurs through a multi-step process known as the coagulation cascade. In the exogenous pathway, tissue factor (also known as factor III or thromboplastin) is in contact with factor VII to form an activated VIIa complex. This initiates the coagulation protease cascade, converting inactive factor X to active protease factor Xa, which, together with activated factor V, produces thrombin (IIa) from prothrombin (II). In the intrinsic pathway, collagen forms a complex with high molecular weight kininogen, prekallikrein and factor XII, thereby leading to the conversion of factor XII to factor XIIa. Factor XIa converts Factor XI to Factor XIa, Factor XI activates Factor IX to produce Factor IXa, Factor IXa forms a tenase complex with FVIIIa, and Factor X is activated, which aids in the conversion of prothrombin (II) to thrombin (IIa). This time thrombin leads to the conversion of fibrinogen (I) to fibrin, which together with factor XIIIa forms a cross-linked fibrin clot. Many clotting disorders are the result of low levels of secreted proteins in the blood involved in the clotting cascade. Coagulopathy can severely increase the amount of blood that exits the body at the time of injury, or cause bleeding in the subcutaneous or vital organs. These disorders are often genetic. Exemplary but non-limiting diseases caused by coagulation factor deficiency include hemophilia, von Willebrand disease and antithrombin III, protein C or protein S deficiency. Hemophilia A and B are X-linked and are caused by insufficient levels of coagulation factor VIII and factor IX (FIX), respectively. Hemophilia C is caused by insufficient factor XI. Loss of factor II, factor VII, factor X or factor XII also causes bleeding disorders. Von Willebrand disease results from low levels of von Willebrand coagulation factors in the blood. In some cases, deficiencies in blood proteins that regulate clotting can lead to excessive clotting. Factor V Leiden is a genetic disorder in which factor V Leiden protein reacts excessively, thereby causing blood to clot too often or excessively. A deficiency in antithrombin III, protein C or protein S that helps control bleeding can also cause excessive clotting. Currently, coagulation disorders such as haemophilia are treated using blood transfusions or infusion of missing coagulation factors (replacement therapy). However, complications of replacement therapy include the generation of antibodies to clotting factors, the incidence of viral infections from blood-derived products, and damage to the joints. Therefore, additional therapies are needed.

  In some embodiments of the present disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the method of disorder, a composition comprising CAR-T cells modified to express or secrete human proteins is used for enzyme replacement therapy. Enzyme replacement therapy generally involves intravenous infusion of a composition containing an enzyme that provides a balance to the underlying enzyme deficiency causing disease symptoms in a therapeutically effective amount. Thus, the missing enzyme activity is thus supplied exogenously. Exemplary diseases that can be treated by the modified T cells of the present disclosure include, but are not limited to, lysosomal storage diseases, Gaucher disease (glucocerebrosidase enzyme), Fabry disease, mucopolysaccharidosis I (MPS I), Mucopolysaccharidosis I (MPS II, or caused by Hunter syndrome, iduronic acid-2-sulfatase deficiency), mucopolysaccharidosis VI (caused by MPS VI, arylsulfatase B deficiency) and Pompe disease (or glycogenosis type II) , Caused by deficiency of acid alpha-glucosidase). Additional diseases that can be treated with enzyme replacement therapy include but are not limited to adenosine deaminase (ADA) deficiency, hepatic enzyme N-acetylglutamate synthetase (NAGS) deficiency, Examples include phosphataseemia, lysosomal acid lipase deficiency, Morquio syndrome type A, Wolman LAL lysosomal acid lipase deficiency, A1AT (alpha 1-antitrypsin) deficiency and urea cycle abnormality. Enzymes supplied to patients during enzyme replacement therapy include, but are not limited to, alpha 1-antitrypsin, beta-glucocerebrosidase, adenosine deaminase, alpha-galactosidase A, alpha-L-iduronidase, iduronic acid- Examples include 2-sulfatase, N-acetylgalactosamine-6 sulfatase, -acetylgalactosamine-4 sulfatase, and lysosomal acid lipase.

In some embodiments of the present disclosure, T cells are modified to express therapeutic proteins, including secreted proteins and secreted human proteins. In some embodiments of the method of disorders, a human antibody is made using a composition comprising CAR-T cells modified to express or secrete a human protein. In some embodiments, the disease treated by modified T cells that express secreted proteins is a disease that can be treated by intravenous infusion or injection of an antibody or antibody fragment. In addition to cancer, antibody-based therapies are used to treat many types of diseases, including arthritis and asthma, and immune-based diseases such as infection, as well as other diseases. An exemplary but non-limiting list of diseases that can be treated using the modified T cells of the present disclosure includes platelet aggregation, Clostridium difficile infection, rheumatoid arthritis, Crohn's disease, psoriasis vulgaris, psoriatic arthritis, Ankylosing spondylitis, juvenile idiopathic arthritis, Alzheimer's disease, sepsis, multiple sclerosis, hypercholesterolemia, systemic lupus erythematosus, prevention of organ transplant rejection, viral infection, asthma, severe allergic disorder, retinopathy, Osteoporosis, inflammatory bowel disease, inflammatory disease, influenza A, paroxysmal nocturnal hemoglobinuria, sepsis caused by Gram-negative bacteria, psoriasis, invasive Candida infection, ulcerative colitis, hypocholesterolemia, respiratory condition Endoplasmic reticulum virus infection, localized segmental glomerulosclerosis, graft-versus-host disease, ankylosing spine Inflammation, HIV infection, ulcerative colitis, autoimmune disease, chronic asthma, reduced scar formation after glaucoma surgery, hypercholesterolemia, leukocyte disease, systemic scleroderma, respiratory syncytial virus (prevention), Lupus erythematosus, type 1 diabetes, inflammation, Pseudomonas aeruginosa infection, macular degeneration, anthrax, cytomegalovirus infection, inflammation of the respiratory tract, skin and gastrointestinal tract, systemic lupus erythematosus, rheumatic disease, uveitis, cytomegalovirus infection Dermatomyositis, polymyositis, fibrosis, choroidal and retinal neovascularization, muscular dystrophy, Staphylococcus aureus infection, lupus nephritis, follicular lymphoma, chronic hepatitis B and ulcerative colitis.
Modified cell injection as an adoptive cell therapy

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising the modified cells of the present disclosure is between 2 × 10 ⁵ and 5 × 10 ⁸ per kg patient body weight per administration, Or any range, value or fraction of cells.

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising the modified cells of the present disclosure is between 0.2 × 10 ⁶ and 20 × 10 ⁶ per kg patient body weight per administration. Contains cells. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 0.2 × 10 ⁶ cells per kg patient body weight per administration, 2 × 10 ⁶ cells per kg body weight, 20 × 10 ⁶ cells per kg patient weight per dose, or cells per kg patient weight per any dose in between.

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising the modified cells of the present disclosure is 1 × 10 ⁶ cells or about 1 × 10 ⁶ cells per kg patient body weight per administration. Including cells.

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 3 × 10 ⁶ cells or about 3 × 10 ⁶ cells per kg patient body weight per administration. Including cells.

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising the modified cells of the present disclosure is from 0.7 × 10 ⁶ to 6.7 × 10 ⁶ per kg patient body weight per dose. Cells. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 0.7 × 10 ⁶ cells per kg patient body weight per administration, 6.7 × 10 ⁶ cells per kg body weight or cells per kg patient weight per any dose in between.

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising the modified cells of the present disclosure is between 0.7 × 10 ⁶ and 16 × 10 ⁶ per kg patient body weight per administration. Contains cells. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 0.7 × 10 ⁶ cells per kg patient body weight per administration, 2 × 10 ⁶ cells per kg body weight, 6 × 10 ⁶ cells per kg patient weight per dose, 10.7 × 10 ⁶ cells per kg patient weight per dose, per kg patient weight per dose 16 x 10 ⁶ cells or cells per kg patient body weight per any administration in between.

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising the modified cells of the present disclosure is from 1.2 × 10 ⁶ to 7.1 × 10 ⁶ per kg patient body weight per dose. Cells. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 1.2 × 10 ⁶ cells per kg patient body weight per administration, Contains 7.1 × 10 ⁶ cells per kg body weight or cells per kg patient body weight per any number of doses. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure provides 2 × 10 ⁶ to 3 × 10 ⁶ cells per kg patient body weight per dose. Including.

In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure provides between 1106 × 10 ⁶ and 2106 × 10 ⁶ cells per kg patient body weight per administration. Including. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 1106 × 10 ⁶ cells per kg patient body weight per dose, 1 kg patient weight per dose. Includes 2106 × 10 ⁶ cells per cell or cells per kg body weight of the patient per any dose in between. In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 0.7 × 10 ⁶ to 1.3 × 10 ⁶ per kg patient body weight per dose. Cells. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure is 0.7 × 10 ⁶ cells per kg patient body weight per administration, Includes 1.3 × 10 ⁶ cells per kg body weight or cells per kg body weight of the patient per any dose in between.

  In certain embodiments of the present disclosure, the modified cells of the present disclosure are delivered to a patient by injection or intravenous infusion. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure comprises a single dose or multiple doses. In certain embodiments, a therapeutically effective dose of a composition comprising the disclosed or modified cells of the present disclosure comprises divided doses. In certain embodiments, a therapeutically effective dose of a composition of the present disclosure or a composition comprising a modified cell of the present disclosure comprises an initial dose and a maintenance dose.

In certain embodiments of the present disclosure, the modified cells are T cells, which respond to T cell markers prior to either in vitro expansion or formulation with a pharmaceutically acceptable carrier. Can be selected. In some embodiments, modified T cells can be sorted using CD8 + and / or CD4 + markers.
Nucleic acid molecule

  Nucleic acid molecules of the present disclosure that encode protein scaffolds may be in the form of RNA, such as, but not limited to, mRNA, hnRNA, tRNA or any other form, obtained by cDNA and cloning or made synthetically It may be in the form of DNA, including genomic DNA to be processed, or any combination thereof. The DNA may be triple stranded, double stranded or single stranded, or any combination thereof. An arbitrary part of at least one strand of DNA or RNA may be a coding strand known as a sense strand or a non-coding strand also called an antisense strand.

  An isolated nucleic acid molecule of the present disclosure optionally comprises one or more introns, such as, but not limited to, an open reading frame (ORF) having at least one designated portion of at least one protein scaffold. A nucleic acid molecule comprising a protein scaffold or loop region coding sequence that binds to a target protein; and a nucleotide sequence substantially different from that described above, but due to the degeneracy of the genetic code, A nucleic acid molecule that still encodes a protein scaffold, sentinlin, CAR, CARTYrin, transposon, and / or transposase described in the literature and / or known in the art. Of course, the genetic code is well known in the art. Thus, the generation of such degenerate nucleic acid variants encoding a particular protein scaffold of the invention will be routine to those skilled in the art. See, eg, Ausubel et al., Supra. Such nucleic acid variants are also encompassed by the present invention.

As shown herein, nucleic acid molecules of the present disclosure, including, but not limited to, nucleic acids encoding protein scaffolds, sentin, CAR, CARTYrin, transposons, and / or transposases include, but are not limited to, protein scaffolds, sentinels, A nucleic acid that alone encodes the amino acid sequence of a CAR, CARtyrin, transposon, and / or transposase fragment; CAR, CARTYrin, transposon, and / or transposase, fragment or portion coding sequences, and without limitation, transcription, splicing and With additional non-coding sequences including non-coding 5 'and 3' sequences such as transcribed non-translated sequences that play a role in mRNA processing (eg, mRNA ribosome binding and stability) including polyadenylation signals Additional sequences, such as coding sequences for at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences such as at least one intron; additions such as those that provide additional functionality Mention may be made of additional coding sequences encoding typical amino acids. Thus, purification of a fused protein scaffold, centiline, CAR, CARTYrin, transposon, and / or transposase containing a protein scaffold fragment or portion comprising a sequence encoding a protein scaffold, sentin, CAR, CARTYrin, transposon, and / or transposase. It can be fused to a marker sequence, such as a sequence encoding a peptide to facilitate.
Nucleic acid construction

  Isolated nucleic acids of the present disclosure are made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as is well known in the art. be able to.

  A nucleic acid may conveniently comprise a sequence in addition to a polynucleotide of the invention. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, translatable sequences can be inserted to assist in the isolation of translated polynucleotides of the present disclosure. For example, a hexa-histidine marker sequence provides a convenient means for purifying the proteins of the present disclosure. The nucleic acids of the present disclosure, excluding the coding sequence, are optionally vectors, adapters, or linkers for cloning and / or expression of the polynucleotides of the present disclosure.

Additional to such cloning and / or expression sequences to optimize function in cloning and / or expression, to assist in the isolation of the polynucleotide, or to improve the introduction of the polynucleotide into the cell Sequences can be added. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, eg, Ausubel, supra; or Sambrook, supra).
Recombination methods for constructing nucleic acids

The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, use any number of cloning methodologies known to those skilled in the art from biological sources. Can be obtained. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the invention under stringent conditions are used to identify desired sequences in a cDNA or genomic DNA library. RNA isolation and construction of cDNA and genomic libraries are well known to those skilled in the art (see, eg, Ausubel, supra; or Sambrook, supra).
Vectors and host cells

  The present disclosure provides for the generation of a vector comprising an isolated nucleic acid molecule of the present disclosure, a host cell that is genetically engineered using a recombinant vector, and at least one protein scaffold by recombinant techniques well known in the art. Also related. See, for example, Sambrook et al., Supra; Ausubel et al., Supra, each fully incorporated herein by reference.

For example, a PB-EF1a vector can be used. The vector includes the following nucleotide sequence:

  The polynucleotide can optionally be conjugated to a vector containing a selectable marker for growth in the host. Generally, a plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

  The DNA insert should be operably linked to a suitable promoter. The expression construct further contains sites for transcription initiation, termination, and ribosome binding sites for translation within the transcription region. The coding portion of the mature transcript expressed by the construct preferably includes a translation initiation codon first and a termination codon (eg, UAA, UGA or UAG) appropriately positioned at the end of the translated mRNA. UAA and UAG are preferred for expression in animals or eukaryotic cells.

  The expression vector preferably includes at least one selectable marker, but is optional. Examples of such markers include, but are not limited to, for eukaryotic cell culture, ampicillin resistance gene, zeocin resistance gene (Sh bla gene), puromycin resistance gene (pac gene), hygromycin B resistance gene ( hygB gene), G418 / geneticin resistance gene (neo gene), mycophenolic acid resistance gene, or glutamine synthetase resistance gene (GS, US Pat. Nos. 5,122,464; 5,770,359; , 827, 739), a blasticidin resistance gene (bsd gene), and E. coli. E. coli and other bacterial or prokaryotic cultures, ampicillin resistance gene, zeocin resistance gene (Sh bla gene), puromycin resistance gene (pac gene), hygromycin B resistance gene (hygB gene), G418 / geneticin resistance gene (Neo gene), kanamycin resistance gene, spectinomycin resistance gene, streptomycin resistance gene, carbenicillin resistance gene, bleomycin resistance gene, erythromycin resistance gene, polymyxin B resistance gene, or tetracycline resistance gene. Fully incorporated by reference). Appropriate culture media and conditions for the above-described host cells are known in the art. Appropriate vectors will be readily apparent to those skilled in the art. Introduction of the vector construct into the host cell can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other known methods. Such methods include, for example, Sambrook, supra, chapters 1-4 and 16-18; Ausubel, supra, chapters 9, 9, 13, 15, 16, etc. It is described in the technical field.

  While it is preferred that the expression vector comprises at least one selectable cell surface marker for isolating cells modified by the compositions and methods of the present disclosure, it is optional. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or groups of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker is one that distinguishes cells that have been modified by the composition or method of the present disclosure from cells that have not been modified by the composition or method of the present disclosure. Such cell surface markers include, but are not limited to, for example, “cluster of design” or “classification determining” protein (often abbreviated as “CD”), eg, a truncated or full-length form of CD19. , CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al., Blood., August 21, 2014; 124 (8): 1277-87).

  While it is preferred that the expression vector includes at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure, it is optional. Selectable drug resistance markers of the present disclosure can include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

  At least one protein scaffold of the present disclosure can be expressed in a modified form, such as a fusion protein, and can include additional heterologous functional regions as well as secretion signals. For example, additional amino acids, particularly a region of charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells during purification or subsequent handling and storage. . A peptide moiety can also be added to the protein scaffold of the present disclosure to facilitate purification. Such regions can be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods include many such as Sambrook, supra, chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, chapters 16, 17 and 18. It is described in the standard laboratory manual.

  Those of skill in the art are knowledgeable of the numerous expression systems available for expressing nucleic acids encoding the proteins of the present disclosure. Alternatively, a nucleic acid of the present disclosure can be expressed in a host cell by turning it on (by manipulation) in a host cell that contains endogenous DNA encoding a protein scaffold of the present disclosure. Such methods are described, for example, in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5, which are fully incorporated herein by reference. , 733,761 are well known in the art.

  Examples of cell cultures useful for the production of protein scaffolds, designated portions or variants thereof are bacterial cells, yeast cells, and mammalian cells known in the art. Mammalian cell lines are often in the form of a monolayer of cells, although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including COS-1 cell lines (eg, ATCC CRL 1650), COS-7 cell lines ( For example, ATCC CRL-1651), HEK293 cell line, BHK21 cell line (eg ATCC CRL-10), CHO cell line (eg ATCC CRL 1610) and BSC-1 cell line (eg ATCC CRL-26), Cos -7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells and the like, for example, American Type Culture Collection, Manassas, Va. (Www.atcc.org) is readily available. Preferred host cells include cells of lymphoid origin such as myeloma cells and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851). In a particularly preferred embodiment, the recombinant cells are P3X63Ab8.653 cells or SP2 / 0-Ag14 cells.

  Expression vectors for these cells include the following expression control sequences such as, but not limited to, the origin of replication; promoter (eg, late or early SV40 promoter, CMV promoter (US Pat. No. 5,168,062; 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1 alpha promoter (US Pat. No. 5,266,491), at least one human promoter; enhancer, and / or It may contain one or more of processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (eg, SV40 large T Ag poly A addition site), and transcription terminator sequences. Sambrook et al., Supra.Other cells useful for the production of the nucleic acids or proteins of the invention are known and / or, for example, American Type Culture Collection Catalog Lines and Hybridomas (www. atcc.org) or other known or commercial sources.

When eukaryotic host cells are used, polyadenylation or transcription terminator sequences are generally incorporated into the vector. An example of a terminator sequence is a polyadenylation sequence derived from a bovine growth hormone gene. Sequences for precise splicing of transcripts can also be included. An example of a splicing sequence is the VP1 intron derived from SV40 (Sprague et al., J. Virol. 45: 773-781 (1983)). In addition, gene sequences for controlling replication in the host cell can be incorporated into the vector as is known in the art.
Purification of protein scaffold

  Protein scaffolds include but are not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography. Can be recovered and purified from recombinant cell cultures by well-known methods including chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can be used for purification. See, eg, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N., which are each fully incorporated herein by reference. Y. (1997-2001), see, for example, Chapter 1, Chapter 4, Chapter 6, Chapter 8, Chapter 9, Chapter 10.

The protein scaffolds of the present disclosure include naturally purified products, products of chemical synthesis procedures, and including products produced by recombinant techniques from prokaryotic or eukaryotic hosts including E. coli, yeast, higher plants, insects and mammalian cells. Depending on the host used in the recombinant production procedure, the protein scaffolds of the present disclosure may be glycosylated or non-glycosylated. Such methods are described in Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16 which are fully incorporated herein by reference. It is described in many standard laboratory manuals, such as Chapters, Chapters 18 and 20, Colligan, Protein Science, above, Chapters 12-14.
variant

  The amino acids that make up the protein scaffold of the present disclosure are often abbreviated. Amino acid names can be indicated by representing the amino acid by its one-letter code, its three-letter code, name, or three-nucleotide codon (s) as well understood in the art (Alberts, B Et al., Molecular Biology of The Cell, 3rd edition, Garland Publishing, Inc., New York, 1994). The protein scaffolds of the present disclosure can include one or more amino acid substitutions, deletions or additions derived from either natural mutation or human manipulation, as defined herein. The amino acids of the protein scaffolds of the present disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, eg, Ausubel, supra, 8th. Chapter, Chapter 15; Cunningham and Wells, Science, 244: 1081-1085 (1989)). In the latter procedure, a single alanine mutation is introduced for each residue in the molecule. The resulting mutant molecule is then tested for biological activity such as, but not limited to, at least one neutralizing activity. Sites critical for protein scaffold binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol., 224: 899-904). (1992) and de Vos et al., Science, 255: 306-312 (1992)).

  The term “substantially complementary” as used throughout this disclosure means that the first sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, when the first sequence is complementary to the complement of the second sequence. 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% over a region of 270, 360, 450, 540 or more nucleotides or amino acids Or 99% identical, or that two sequences hybridize under stringent hybridization conditions.

  As used throughout this disclosure, the term “substantially identical” means that the first and second sequences are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 450, 540 or more nucleotides or amino acids Being or referring to nucleic acid refers to the case where the first sequence is substantially complementary to the complement of the second sequence.

  The term “variant” as used throughout this disclosure, when used to describe a nucleic acid, (i) a portion or fragment of a reference nucleotide sequence; (ii) a reference nucleotide sequence or the complement of a portion thereof; (Iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto. .

  The term “vector” as used throughout this disclosure refers to a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector may be a DNA vector or an RNA vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid.

  The term “variant” as used throughout this disclosure, when used to describe a peptide or polypeptide, differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but at least one organism. Refers to a peptide or polypeptide that retains a biological activity. Variant can also mean a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

  It is generally recognized in the art that conservative substitutions of amino acids, ie, replacing amino acids with different amino acids having similar properties (eg, hydrophilicity, charge region extent and distribution) are generally accompanied by minor changes. ing. These minor changes can be identified, in part, by considering the hydropathic index of amino acids understood in the art (Kyte et al., J. Mol. Biol., 157: 105- 132 (1982)). The hydropathic index of amino acids is based on hydrophobicity and charge considerations. Amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, an amino acid with a hydropathic index of ± 2 is substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins that retain biological function. For peptides, consideration of the hydrophilicity of the amino acid makes it possible to calculate the maximum local average hydrophilicity of the peptide, which has been reported to correlate well with antigenicity and immunogenicity. It is a useful measure. U.S. Pat. No. 4,554,101, which is fully incorporated herein by reference.

  Substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, eg, immunogenicity. Substitutions can be performed with amino acids whose hydrophilicity values are within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this finding, amino acid substitutions that are compatible with biological function are evident in the side chains of amino acids, particularly those amino acids, as evidenced by hydrophobicity, hydrophilicity, charge, size, and other properties. It is understood that it depends on relative similarity.

  As used herein, “conservative” amino acid substitutions can be defined as described in Tables A, B, or C below. In some embodiments, the fusion polypeptide and / or nucleic acid encoding such a fusion polypeptide includes conservative substitutions introduced by modification of a polynucleotide encoding a polypeptide of the invention. Amino acids can be classified according to physical properties and contributions to protein secondary and tertiary structure. A conservative substitution is the replacement of another amino acid with similar properties with one amino acid. Exemplary conservative substitutions are listed in Table A.

  Alternatively, conservative amino acids are described in Lehninger, (Biochemistry, 2nd edition; Worth Publishers, Inc. NY, NY (1975), pages 71-77, as described in Table B. They can be grouped as they are.

  Alternatively, exemplary conservative substitutions are those described in Table C.

  A polypeptide of the present disclosure is a polypeptide having one or more insertions, deletions or substitutions of amino acid residues, or any combination thereof, as well as modifications other than insertions, deletions or substitutions of amino acid residues It should be understood that it is intended to include. A polypeptide or nucleic acid of the present disclosure may contain one or more conservative substitutions.

  As used throughout this disclosure, the term “more than one” of the above amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9 of the amino acid substitutions described. Refers to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. The term “more than one” may refer to 2, 3, 4, or 5 of the amino acid substitutions described.

  The polypeptides and proteins of the present disclosure can be non-naturally occurring, either in their entire sequence, or any part thereof. The polypeptides and proteins of the present disclosure may contain one or more mutations, substitutions, deletions or insertions that are not naturally occurring and render the entire amino acid sequence non-naturally occurring. The polypeptides and proteins of the present disclosure can contain one or more overlapping, inverted or repetitive sequences, whereby the resulting sequence is non-naturally occurring and the entire amino acid sequence is non-naturally occurring. The polypeptides and proteins of the present disclosure may contain modified, artificial, or synthetic amino acids that do not occur in nature and render the entire amino acid sequence non-natural.

  “Sequence identity” as used throughout this disclosure is a stand-alone implementation for blasting two sequences that can be retrieved using the default parameters from the National Center for Biotechnology Information (NCBI) ftp site. Can be determined by using the possible BLAST engine program (bl2seq) (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety) ). The term “identical” or “identity”, when used with respect to two or more nucleic acid or polypeptide sequences, refers to the specified percentage of residues that are the same across each specified region of the sequence. . The percentage aligns the two sequences optimally, compares the two sequences over a specified region, determines the number of positions where identical residues exist in both sequences, and obtains the number of matching positions; It can be calculated by dividing the number of matching positions by the total number of positions in the specified region and multiplying the result by 100 to obtain the percentage of sequence identity. If two sequences differ in length or alignment results in one or more sticky ends, and the specified comparison region contains only a single sequence, the single sequence residue is the calculated denominator. Is included, but not included in the molecule. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be done manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

Example 1
Generation of Stem-Like Modified T Cells The following is one protocol for modifying a T cell to express a chimeric antigen receptor (CAR) under conditions that induce or preserve the desired stem-like properties of the T cell. This is an illustrative but non-limiting example.

  Day 0: Nucleofection of T cells

ImmunoCult ™ -XF T cell expansion medium (Stemcell Technologies, Cat No. 10981) is pre-warmed in an incubator at 37 ° C., 5% CO ₂ , high humidity. For 5 × 10 ⁶ T cells per reaction (cuvette size 100 μL), warm 3 mL of medium per reaction in a single well of a 6-well plate. For 25 × 10 ⁶ T cells per reaction (cuvette size 100 μL), 20 mL of medium per reaction is warmed in G-Rex10 (Wilson Wolf, Cat number: 80040S).

  Warm P3 primary cell solution (Lonza, Cat number: PBP3-02250) to room temperature and add replenisher if necessary.

  The core unit (Lonza, Cat number: AAF-1002B) of the 4D-Nucleofector ™ System is turned on, thereby controlling the X-unit (Lonza, Cat number: AAF-1002X). Program the number of nucleofections that require the use of P3 buffer. Program EO-210.

  The cuvette is labeled and the transfer pipette (supplied with the Lonza P3 kit) is pre-opened and a suitable dilution of the nucleic acid is prepared before working with the cells.

For the transposon plasmid, make a 0.5 μg / μL solution in H ₂ O without nuclease.

  Collected CD14-depleted, CD56-depleted, and CD19-depleted cells are counted using CliniMACs Prodigy and the volume required for the number of cells required is calculated.

  T cells are centrifuged in a Heraeus Multifuge X3R benchtop centrifuge (Thermofisher Scientific) for 10 minutes with a brake at 90 g, 7. If multiple reactions are performed using the same number of cells / reaction, all necessary cells are centrifuged in a single centrifuge tube. Depending on the volume, either 15 mL (Fisher, Cat number: 14-959-49B) or 50 mL (Fisher, Cat number: 14-959-49A) conical tubes can be used. During centrifugation, the nucleic acid is added directly to the bottom of the cuvette with the P3 primary cell solution box (Lonza, Cat number: PBP3-02250). Add 2 μL of the 0.5 μg / μL transposon plasmid solution made for 1 μg total transposon in step 4 to one corner of the bottom of the cuvette. Add 5 μg Super piggyBac ™ (SPB) transposase mRNA to the other corner of the cuvette.

  Since mRNA can degrade rapidly, it is optimal to minimize the time of contact with other nucleic acid solutions and cells prior to electroporation because of the potential for RNase to be present. For this reason, for example, transposons and transposases are delivered to the opposite corners of the cuvette to prevent mixing. Furthermore, it is optimal to keep the total volume of nucleic acid below 10 μL (10%) relative to the total reaction volume.

Both the amount of transposon (1 μg) and the amount of transposase (5 μg) remain the same regardless of the number of cells per reaction. The transfer efficiency remains unchanged between 5 × 10 ⁶ cells per 100 μL reaction to 25 × 10 ⁶ cells per 100 μL reaction.

  After centrifugation, the medium is completely aspirated off without disturbing the cell pellet.

  The cell pellet is suspended in 100 μL of room temperature P3 buffer containing supplementary material per reaction.

  Transfer 100 μL of cells in P3 buffer to a cuvette containing the appropriate nucleic acid, taking care not to introduce any air bubbles into the solution optimally. It is recommended that only a maximum of two cuvettes should be loaded with cells at a time. It is optimal to work immediately after adding the cells to the cuvette, and it is efficient to reduce the contact time between the cells and the mRNA before nucleofection. Although no reduction in metastasis efficiency has been observed for cells resting in P3 buffer for up to 10 minutes, it is recommended to minimize the time that cells remain in P3.

  Mix the contents of the cuvette by flicking several times and load up to two cuvettes into the 4D-Nucleofector ™ X-unit.

  Cells are pulsed with program EO-210 to ensure that there are no errors recorded by the machine.

  Nucleofected cells are immediately transferred to either a 6-well plate or G-Rex10 using a transfer pipette supplied by the Lonza P3 kit. To transfer cells, a small amount of pre-warmed media is first drawn from a 6-well plate or G-Rex flask into a transfer pipette. The medium is then pipetted into the cuvette and the entire contents of the cuvette are transferred to the final culture dish using the pipette. It is recommended not to pipette cells up and down in the cuvette or in the final culture dish.

  For any remaining reaction, the protocol goes from transferring the cells in P3 buffer to a cuvette containing the appropriate nucleic acid, to mixing, pulsing and transferring the nucleated cells into 6-well plates or G-Rex10. repeat.

Place the cells in a 37 ° C., 5% CO ₂ , high humidity incubator.

  Day 2: T cell activation

  ImmunoCult ™ human CD3 / CD28 / CD2 T cell activator (Stemcell Technologies, Cat No: 10970) 25 μL / mL is added to the nucleated cells.

  Mix cells gently by pipetting.

Return cells to 37 ° C., 5% CO ₂ , high humidity incubator.

  For cells grown in G-Rex flasks: it is essential not to disturb the culture until a visible cell clump is observed. Therefore, it is recommended to separate media addition and exchange from cell disruption / mixing / pipetting.

  Culture media note: In order to grow cells in G-Rex flasks, media addition and / or exchange should be based on the level of glucose and other metabolites. If the glucose level (or another indicator metabolite) drops to a critical level (eg, glucose about 100 mg / dL), use pre-warmed ImmunoCult ™ XF T cell expansion medium to Is supplemented by doubling and / or changing medium halfway. Medium addition should be performed slowly and care should be taken not to destroy the cells as much as possible. The medium half change should occur at least 12 hours after mechanical disruption of the cell culture in order to allow the cells to settle completely at the bottom of the culture flask.

  Cell sampling and destruction: Cells should remain undisturbed for the majority of the culture period.

  The first disruption of the cell culture after addition of the activating reagent should be done once large visible cell aggregates have formed (the aggregates are 3 of the lattice that can be seen on the G-Rex membrane). ~ 4 squares x 3-4 squares).

  Once the cell aggregates have reached the required size, they may be mechanically broken using a 10 mL serum pipette. This time point can be days 11-14 depending on donor and transfer efficiency. In certain situations, such as when using manual cassettes, large volumes and / or large cell numbers for nucleofection, this point in time can be closer to day 14 than day 11. Cell sampling should be collected at this point for cell counting, viability, and flow analysis. Ideally, the volume of culture medium at this point should not exceed twice the initial volume used (200 mL for G-Rex100). It is recommended to collect all of the required cells at once so that the cells do not need to be disturbed again.

  Once the cells are destroyed, they are left undisturbed for 12 hours in the same volume of medium as the starting. The cells should have reaggregated at this point, but the aggregates are smaller and more numerous. These agglomerates should be 1-2 squares × 1-2 squares of the lattice of the G-Rex film.

  Three days after the first disruption of the cells (days 14-17 depending on the culture), the cells can be pipetted a second time. Samples should be taken again for cell counting, viability, and flow cytometry. Again, the cells should be left undisturbed for at least 12 hours after sampling. It is recommended to collect all of the required cells at once so that the cells do not need to be disturbed again.

  After this second disruption, the cells do not form any clumps and the growth rate of the cells can be considerably slower.

  Cell harvesting should be performed 3 days after the second disruption of cells between days 17-20 of culture.

  Flow cytometry

  Flow should be performed on day 5, D-Day, D-Day + 3, and D-Day + 6.

  On day 5, for D-Day and D-Day + 3, use the CD45, CD4, CD8, and CARTYrin flow panels

For D-Day + 6, there are three target panels:
a. Panel 1: CD3, CD8, CD4, CARTYrin, CD45RA, CD45RO, CD62L
b. Panel 2: CD3, CD8, CD4, CARTYrin, CD25, CXCR4, PD-1
c. Panel 3: CD45, CD14, CD20, CD56, CD8, CD4, CD3
(Example 2)
Functional characterization of CARTYrin + stem memory T cells

  The CARTYrin of the present disclosure is a plasmid DNA transposon encoding CARTYrin sandwiched between two cis-regulatory insulator elements to help stabilize CARTYrin expression by blocking inappropriate gene activation or silencing Can be used to introduce into T cells.

In certain embodiments of the disclosed methods, in order to stably incorporate antigen-specific (including cancer antigen-specific) CARTYrin into dormant pan-T cells, the transposon is mRNA transposase in a single electroporation reaction. A piggyBac ™ (PB) Transposon System, called Super piggyBac ™ (SPB), which is co-delivered with the enzyme can be used. Delivery of piggyBac ™ transposon to intact resting primary human pan-T cells resulted in 20-30% of cells with stable integration and expression of the gene delivered by PB. Unexpectedly, the majority of these modifications CARTyrin expressing T cells were positive for expression of CD62L and CD45RA, a marker associated with a general stem memory T cells _{(T SCM} cells). To confirm that this phenotype was retained upon stimulation and expansion of CAR-T cells, modified CARTYrin-expressing T cells positive for CD62L and CD45RA expression were activated by stimulation with CD3 and CD28. As a result of stimulation with CD3 and CD28,> 60% of CARTYrin + T cells showed a stem cell memory phenotype. Furthermore, these cells that expressed CARTYrin specific for the cancer antigen were sufficiently capable of expressing a powerful anti-tumor effector function.

Compare the phenotype of CAR-T cells generated by either PB transfer or lentiviral (LV) transduction to determine whether the PB system contributes directly to enhanced expression of stem-like markers did. To do this, a new vector was constructed by subcloning the CARTYrin transgene into a common LV construct for virus production. After CARTYrin was introduced into intact resting T cells by PB transposition or LV transduction, CARTYrin ⁺ cells were expanded and then returned to resting state. Various phenotypic and functional properties, including kinetic analysis of memory and exhaustion-related markers, secondary proliferation in response to homeostatic cytokines or tumor-related Ag, cytokine production, and lytic capacity in response to target tumor cells It was measured. Unlike CARTyrin ⁺ T cells were transferred by PB, the CARTyrin ⁺ T cells were transduced by LV, strengthening memory phenotype did not show. In addition, cells that had undergone metastasis with PB showed similar or greater capacity for secondary growth and killing of target tumor cells. Taken together, these data demonstrate that CAR-T cells generated by PB metastasis are primarily _TSCM cells, a highly desirable product phenotype in the CAR-T field. In addition, these CARTYrin ⁺ T cells show strong antitumor activity and are longer in vivo due to the use of centrin-based CAR, which may be less prone to tonic signaling and functional exhaustion Persistent cells can be generated.
(Example 3)
Transfer by Sleeping Beauty mainly yields _TSCM phenotype

In the transition by Sleeping Beauty (SB100x), the method of the present disclosure is used and the T _SCM phenotype is mainly obtained. As shown in FIG. 10, human pan-T cells were metastasized using either 1 μg of Sleeping Beauty transposon plasmid or piggyBac transposon plasmid, and SB100x or SPB mRNA, respectively. After metastasis, cells were expanded ex vivo and all non-metastatic cells were depleted using a drug selection system. After culturing for 18 days, the cells were stained with the phenotypic markers CD4, CD8, CD45RA, and CD62L. The stem cell memory phenotype (T _SCM ) is defined by CD45RA and CD62L double positive cells and constitutes> 65% of the cells in all samples.
Example 4
Expression of factor IX in modified T cells

  The genetic deficiency of factor IX (FIG. 11) leads to a life-threatening disease called hemophilia B. Hemophilia B is a rare disease that affects 1 in 25,000 to 1 in 30,000. Current hemophilia B treatment involves injecting recombinant factor IX protein every 2-3 days, and costs about $ 250,000 per year.

Stem memory T cells ( _TSCM cells) have been maintained in humans for decades and thus do not require frequent blood transfusions and secrete factor IX, which is missing in hemophilia B patients An ideal vehicle to supply. T cells were transformed with PiggyBac to secrete factor IX. Transgenic T cells encoding the human factor IX transgene were tested for T cell markers and T _SCM cell markers using FACS and approximately 80% of all cells showed a T _SCM phenotype (FIG. 12). ). These modified T cells were able to secrete human factor IX (FIG. 13A) and this secreted factor IX resulted in clotting activity (FIG. 13B).
Include by reference

All documents cited herein, including any cross-referenced or related patents or applications, are hereby incorporated by reference in their entirety, unless expressly excluded or otherwise limited. Incorporated. Citation of any document is either prior art with respect to any invention disclosed or claimed herein, or by itself or with any other reference (s) It is not an admission that any such invention is taught, suggested or disclosed in any combination. In addition, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document Shall dominate.
Other embodiments

  While particular embodiments of the present disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the present disclosure. The scope of the appended claims includes all such changes and modifications within the scope of this disclosure.

Claims (307)

A method for producing modified stem memory T cells ( _TSCM ) comprising:
To generate modified T cells, primary human T cells encode (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding it, and (b) a transposase or transposase. Introducing a transposase composition comprising a sequence;
The modified T cells express one or more cell surface markers of stem memory T cells ( _TSCM );
A method whereby modified stem memory T cells ( _TSCM ) are produced. A method for producing a plurality of modified stem memory T cells ( _TSCM ) comprising:
To generate a plurality of modified T cells, a plurality of primary human T cells are divided into (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding it, and (b) a transposase or Introducing a transposase composition comprising a sequence encoding the transposase,
At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of modified T cells is one or more of stem memory T cells (T _SCM ) Express cell surface markers,
A method whereby a plurality of modified stem memory T cells ( _TSCM ) are generated. 3. The method of claim 2, 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 ). A method of making a modified central memory T cells _{(T CM),}
To generate modified T cells, primary human T cells encode (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding it, and (b) a transposase or transposase. Introducing a transposase composition comprising a sequence;
Said modified T cells express one or more cell surface markers of central memory T cells (T _CM );
A method whereby modified central memory T cells (T _CM ) are produced. A method for producing a plurality of modified central memory T cells (T _CM ) comprising:
To generate a plurality of modified T cells, a plurality of primary human T cells are divided into (a) a transposon composition comprising a transposon comprising an antigen receptor, a therapeutic protein or a sequence encoding it, and (b) a transposase or Introducing a transposase composition comprising a sequence encoding the transposase,
At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of modified T cells are one or more of the central memory T cells (T _CM ) Express cell surface markers,
A method whereby a plurality of modified central memory T cells (T _CM ) are produced. At least 60% of said plurality of modified T cells express one or more cell surface markers of central memory T cells (T _CM), The method of claim 5.   7. The transposon 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. the method of.   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 a piggyBac transposase.   10. The method of claim 9, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4.   11. The piggyBac transposase is an overactive variant, wherein the overactive variant comprises an amino acid substitution at one or more of positions 30, 165, 282, and 538 of SEQ ID NO: 4. the method of.   The method according to claim 11, wherein the amino acid substitution at position 30 of SEQ ID NO: 4 is substitution of valine (V) with isoleucine (I) (I30V).   The method according to claim 11, wherein the amino acid substitution at position 165 of SEQ ID NO: 4 is substitution of serine (S) with glycine (G) (G165S).   The method according to claim 11, wherein the amino acid substitution at position 282 of SEQ ID NO: 4 is substitution of valine (V) with methionine (M) (M282V).   The method according to claim 11, wherein the amino acid substitution at position 538 of SEQ ID NO: 4 is substitution of lysine (K) with asparagine (N) (N538K).   The method according to any one of claims 1 to 15, wherein the transposase is a Super piggyBac (SPB) transposase.   17. The method of claim 16, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5.   The method according to any one of claims 1 to 17, wherein the sequence encoding the transposase is an mRNA sequence.   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 a Sleeping Beauty transposase or an overactive Sleeping Beauty transposase (SB100X).   The method according to claim 1, wherein the transposon is a Helraiser transposon.   22. A method according to any one of claims 1 to 6 or 21 wherein the transposase is a Helitron transposase.   The method according to any one of claims 1 to 6, wherein the transposon is a Tol2 transposon.   24. A method according to any one of claims 1 to 6 or 23, wherein the transposase is a Tol2 transposase.   7. A method according to any one of claims 1 to 6, wherein the transposon is derived from or is recombined from any species.   26. A method according to any one of claims 1 to 6 or 25, wherein the transposon is a synthetic transposon.   27. A method according to any one of claims 1 to 26, wherein the antigen receptor is a T cell receptor.   28. The method of claim 27, wherein the T cell receptor is naturally occurring.   28. The method of claim 27, wherein the T cell receptor is not naturally occurring.   30. The method of claim 29, wherein the T cell receptor comprises one or more mutations compared to a wild type T cell receptor.   31. The method of 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).   34. The method of claim 32, wherein the CAR is CARTYrin.   35. The method of claim 32, wherein the CAR comprises one or more VHH sequences.   35. The method of claim 34, wherein the CAR is a VCAR.   In order to produce a modified T cell capable of expressing a therapeutic protein, the step of (c) introducing a composition comprising a second transposon comprising a sequence encoding the therapeutic protein into the primary human T cell. The method according to any one of claims 1 to 32, further comprising:   34. The method of claim 33, wherein the therapeutic protein is a secreted protein or a secreted protein.   35. The method of claim 33 or 34, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.   36. The method of claim 35, wherein the sequence encoding the therapeutic protein is a DNA sequence.   37. The method according to any one of claims 33 to 36, wherein the transposase composition of (b) moves the transposon of (a) and the second transposon of (c). Further comprising the step of (d) introducing a second transposase composition comprising a transposase or a sequence encoding said transposase into said primary human T cells,
The second transposase of (d) can transfer the transposon of (c);
The second transposase composition of (d) and the transposase composition of (b) are not identical;
38. A method according to any one of claims 1-37.   39. The method according to claim 38, wherein the transposon of (a) is moved by the transposase composition of (b), and the transposon of (c) is moved by the transposase composition of (d). A method for producing modified stem memory T cells ( _TSCM ) comprising:
(A) introducing an antigen receptor, a therapeutic protein or a composition containing a sequence encoding the same into a primary human T cell to produce a modified T cell, the antigen receptor or the therapeutic The protein is not contained in the transposon, and
(B) In order to produce an activated modified T cell, an antibody complex of the modified T cell and an anti-human CD3 monospecific tetramer, an anti-human CD28 monospecific tetramer Contacting a T cell activator composition comprising one or more of an antibody complex and an activation supplement,
The activated modified T cells express one or more cell surface markers of stem memory T cells ( _TSCM );
A method whereby modified stem memory T cells ( _TSCM ) are produced. A method for producing a plurality of modified stem memory T cells ( _TSCM ) comprising:
(A) introducing an antigen receptor, a therapeutic protein, or a composition containing a sequence encoding the same into a plurality of primary human T cells to produce a plurality of modified T cells, Or the therapeutic protein is not contained in the transposon, and
(B) In order to generate a plurality of activated modified T cells, the plurality of modified T cells, an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific Contacting a T cell activator composition comprising one or more of a tetrameric antibody complex and an activation supplement,
At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ) Or express multiple cell surface markers,
A method whereby a plurality of modified stem memory T cells ( _TSCM ) are generated. _42. The method of claim 41, wherein at least 60% of the plurality of activated modified T cells express one or more cell surface markers of stem memory T cells (T _SCM ). A method of making a modified central memory T cells _{(T CM),}
(A) introducing an antigen receptor, a therapeutic protein or a composition containing a sequence encoding the same into a primary human T cell to produce a modified T cell, the antigen receptor or the therapeutic The protein is not contained in the transposon, and
(B) In order to produce an activated modified T cell, an antibody complex of the modified T cell and an anti-human CD3 monospecific tetramer, an anti-human CD28 monospecific tetramer Contacting a T cell activator composition comprising one or more of an antibody complex and an activation supplement,
The activated modified T cells express one or more cell surface markers of central memory T cells (T _CM),
A method whereby modified central memory T cells (T _CM ) are produced. A method for producing a plurality of modified central memory T cells (T _CM ) comprising:
(A) introducing an antigen receptor, a therapeutic protein, or a composition containing a sequence encoding the same into a plurality of primary human T cells to produce a plurality of modified T cells, Or the therapeutic protein is not contained in the transposon, and
(B) In order to generate a plurality of activated modified T cells, the plurality of modified T cells, an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific Contacting a T cell activator composition comprising one or more of a tetrameric antibody complex and an activation supplement,
At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T cells is one of central memory T cells (T _CM ) Or express multiple cell surface markers,
A method whereby a plurality of modified central memory T cells (T _CM ) are produced. At least 60% of said plurality of activated modified T cells express one or more cell surface markers of central memory T cells (T _CM), The method of claim 44.   46. The method of any one of claims 40 to 45, wherein a viral vector comprises the antigen receptor or therapeutic protein.   48. The method of claim 46, wherein the viral vector comprises a sequence isolated from or derived from a lentivirus.   48. The method of claim 46, wherein the viral vector comprises a sequence isolated from or derived from a retrovirus.   49. The method of claim 48, wherein the retrovirus is a gamma retrovirus.   47. The method according to any one of claims 40 to 46, wherein the viral vector comprises a sequence isolated from or derived from an adeno-associated virus (AAV).   46. A method according to any one of claims 40 to 45, wherein a nucleic acid vector comprises the antigen receptor or therapeutic protein.   52. The method of claim 51, wherein an mRNA vector comprises the antigen receptor or therapeutic protein.   46. A method according to any one of claims 40 to 45, wherein a nanoparticle vector comprises the antigen receptor or therapeutic protein.   46. The method according to any one of claims 40 to 45, wherein the introducing step comprises homologous recombination.   The homologous recombination comprises contacting the antigen receptor or the therapeutic protein, the composition comprising a genome editing construct, and the genomic sequence of at least one primary human T cell of the plurality of primary human T cells. 55. The method of claim 54.   56. The method of claim 55, wherein the vector comprises the antigen receptor or therapeutic protein.   57. The method of claim 56, wherein the vector is an adeno-associated vector (AAV).   58. The genome editing construct of any one of claims 54 to 57, comprising a guide RNA and a clustered and regularly arranged short palindromic repeat (CRISPR) associated protein 9 (Cas9) DNA endonuclease. the method of.   59. The method of claim 58, wherein the genome editing construct comprises a DNA binding domain and a type IIS endonuclease.   60. The method of claim 59, wherein the genome editing construct encodes a fusion protein.   60. The genome editing construct encodes the DNA binding domain and the type IIS endonuclease, wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked. The method described.   63. A method according to any one of claims 58 to 62, wherein the genome editing construct comprises a sequence derived from a Cas9 endonuclease.   64. The method of claim 62, wherein the sequence derived from Cas9 endonuclease is a DNA binding domain.   64. The method of claim 62 or 63, wherein the sequence derived from the Cas9 endonuclease encodes inactive Cas9.   65. The method of claim 64, wherein the sequence derived from the Cas9 endonuclease comprises an amino acid substitution (H840A) of histidine (H) with alanine (A) at position 840.   66. The method of any one of claims 62 to 65, wherein the sequence derived from the Cas9 endonuclease encodes a truncated Cas9.   68. The method of claim 66, wherein the sequence derived from the Cas9 endonuclease comprises an amino acid substitution (N580A) of asparagine (N) with alanine (A) at position 580.   68. The method of any one of claims 62 to 67, wherein the sequence derived from the Cas9 endonuclease comprises an amino acid substitution (D10A) of aspartic acid (D) with alanine (A) at position 10.   62. A method according to any one of claims 58 to 61, wherein the genome editing construct comprises a sequence derived from a transcriptional activator-like effector nuclease (TALEN).   70. The method of claim 69, wherein the sequence derived from TALEN is a DNA binding domain.   59. The method of claim 58, wherein the genome editing construct comprises TALEN.   62. The method according to any one of claims 58 to 61, wherein the genome editing construct comprises a sequence derived from zinc finger nuclease (ZFN).   73. The method of claim 72, wherein the sequence derived from ZFN is a DNA binding domain.   59. The method of claim 58, wherein the genome editing construct comprises a zinc finger nuclease (ZFN).   75. A method according to any one of claims 58 to 74, wherein the genome editing construct targets a safe harbor site on a mammalian chromosome.   75. The method of any one of claims 58 to 74, wherein the genome editing construct targets a safe harbor site on a human chromosome.   77. The method of claim 75 or 76, wherein the chromosome is in vivo, in situ, ex vivo or in vitro.   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. 78. A method according to any one of 58 to 77.   59. 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. 78. The method according to any one of items 1 to 77.   80. The method according to any one of claims 40 to 79, wherein the antigen receptor is a T cell receptor.   81. The method of claim 80, wherein the T cell receptor is naturally occurring.   81. The method of claim 80, wherein the T cell receptor is not naturally occurring.   83. The method of claim 82, wherein the T cell receptor comprises one or more mutations compared to a wild type T cell receptor.   84. The method of claim 82 or 83, wherein the T cell receptor is a recombinant T cell receptor.   80. A method according to any one of claims 40 to 79, wherein the antigen receptor is a chimeric antigen receptor (CAR).   86. The method of claim 85, wherein the CAR comprises one or more sentin sequences.   90. The method of claim 86, wherein the CAR is CARTYrin.   86. The method of claim 85, wherein the CAR comprises one or more VHH sequences.   90. The method of claim 88, wherein the CAR is a VCAR.   90. The method of claim 40, further comprising introducing a composition comprising a sequence encoding the therapeutic protein into the primary human T cells to generate a modified T cell capable of expressing the therapeutic protein. The method as described in any one of.   The method according to any one of claims 40 to 90, wherein the therapeutic protein is a secreted protein or a secreted protein.   92. The method of claim 90 or 91, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.   94. The method of claim 92, wherein the sequence encoding the therapeutic protein is a DNA sequence.   94. The method according to any one of claims 90 to 93, wherein the introducing step comprises homologous recombination.   95. The method of any one of claims 40 to 94, wherein the T cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. (C) To produce a plurality of augmented modified T cells, the activated modified T cells are treated with human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmented supplement Contacting with a T cell expansion composition comprising one or more of:
At least 2% of the plurality of augmented modified T cells express one or more cell surface markers of stem memory T cells (T _SCM );
96. A method according to any one of claims 40 to 42 or 46 to 95. At least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70 of the plurality of increased modified T cells 97.%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, expresses a cell surface marker of stem memory T cells (T _SCM ). the method of. _98. The method of claim 97, wherein at least 60% of the plurality of augmented modified T cells express stem memory T cell (T _SCM ) cell surface markers. (C) To produce a plurality of augmented modified T cells, the activated modified T cells are treated with human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmented supplement Contacting with a T cell expansion composition comprising one or more of:
Wherein at least 2% of the plurality of increased modified T cells express one or more cell surface markers of central memory T cells (T _CM),
96. A method according to any one of claims 43 to 95. At least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70 of the plurality of increased modified T cells %, 75%, 80%, 85%, 90%, 95%, any percentage between 99% or those, express cell surface markers central memory T cells _{(T CM),} according to claim 99 the method of. Wherein at least 60% of the plurality of increased modified T cells express the cell surface markers of central memory T cells (T _CM), The method of claim 99. (D) at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% modified T cells expressing cell surface markers of stem memory T cells (T _SCM ), To make a composition comprising 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween 104. The method of any one of claims 40-42 or 46-101, further comprising the step of enriching a plurality of increased modified T cells. (D) further comprising enriching said plurality of augmented modified T cells to produce a composition comprising at least 60% modified T cells expressing a cell surface marker of stem memory T cells (T _SCM ). A method according to any one of claims 40 to 42 or 46 to 101. (D) at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of modified T cells expressing a cell surface marker of central memory T cells (T _CM ), To make a composition comprising 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween 102. The method of any one of claims 43 to 101, further comprising enriching a plurality of increased modified T cells. (D) further comprising enriching said plurality of augmented modified T cells to produce a composition comprising at least 60% modified T cells expressing a cell surface marker of central memory T cells (T _CM ). 102. A method according to any one of claims 43 to 101. The enriching step comprises isolating modified T cells that express one or more cell surface markers of stem memory T cells (T _SCM ) from the plurality of enriched modified T cells. 104. The method according to item 102 or 103. In order for the enrichment step to produce a plurality of increased enriched modified _TSCM , the isolated modified _TSCM is _transformed into human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, 107. The method of claim 106, further comprising contacting a T cell expansion composition comprising Iskov MDM and one or more augmentation supplements. 105. The enriching step comprises isolating modified T cells that express one or more cell surface markers of central memory T cells ( _SCM ) from the plurality of enriched modified T cells. Or the method according to 105. Wherein said step of enriching is to produce a modified T _CM that a plurality of increased enriched, isolated modified T _CM, human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove 109. The method of claim 108, further comprising contacting the MDM and a T cell expansion composition comprising one or more of augmentation supplements.   The T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzene. 41. Further comprising one or more of a sulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic hydrazide, oleamide, sterol and alkane. 110. The method according to any one of items 109 to 109.   110. The method of any one of claims 40 to 109, wherein the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and sterol.   The T cell expansion composition comprises octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including endpoints; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoints; 0.2 mg including endpoints Linoleic acid at a concentration of / 20-20 mg / kg; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; and 1 of sterol at a concentration of approximately 0.1 mg / kg to 10 mg / kg including the endpoint 111. The method of claim 111, further comprising one or more.   The T cell expansion composition comprises 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 about 1 mg. 112. The method of claim 111, further comprising one or more of sterols at a concentration of / kg.   The T-cell-enhancing composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 0.75 μmol including endpoints Linoleic acid at a concentration of / 75 to 75 μmol / kg; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; one of the sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint 112. The method of claim 111, further comprising a plurality.   The T cell expansion composition comprises 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, olein at a concentration of about 7.5 μmol / kg. 111. The method of claim 111, further comprising one or more of an acid and a sterol at a concentration of about 2.5 [mu] mol / kg. A method for producing modified stem memory T cells ( _TSCM ) comprising:
(A) introducing a composition comprising an antigen receptor or a therapeutic protein into primary human T cells to produce modified T cells, wherein the transposon comprises said antigen receptor;
(B) In order to produce an activated modified T cell, an antibody complex of the modified T cell and an anti-human CD3 monospecific tetramer, an anti-human CD28 monospecific tetramer Contacting a T cell activator composition comprising one or more of an antibody complex and an activation supplement,
The activated modified T cells express one or more cell surface markers of stem memory T cells ( _TSCM );
A method whereby modified stem memory T cells ( _TSCM ) are produced. A method for producing a plurality of modified stem memory T cells ( _TSCM ) comprising:
(A) introducing a composition comprising an antigen receptor or a therapeutic protein into a plurality of primary human T cells to produce a plurality of modified T cells, wherein the transposon comprises the antigen receptor When,
(B) In order to generate a plurality of activated modified T cells, the plurality of modified T cells, an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific Contacting a T cell activator composition comprising one or more of a tetrameric antibody complex and an activation supplement,
At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T cells is one of stem memory T cells (T _SCM ) Or express multiple cell surface markers,
A method whereby a plurality of modified stem memory T cells ( _TSCM ) are generated. _118. The method of claim 117, wherein at least 60% of the plurality of activated modified-T cells express one or more cell surface markers of stem memory T cells (T _SCM ). A method of making a modified central memory T cells _{(T CM),}
(A) introducing a composition comprising an antigen receptor or a therapeutic protein into primary human T cells to produce modified T cells, wherein the transposon comprises said antigen receptor;
(B) In order to produce an activated modified T cell, an antibody complex of the modified T cell and an anti-human CD3 monospecific tetramer, an anti-human CD28 monospecific tetramer Contacting a T cell activator composition comprising one or more of an antibody complex and an activation supplement,
The activated modified T cells express one or more cell surface markers of central memory T cells (T _CM),
A method whereby modified central memory T cells (T _CM ) are produced. A method for producing a plurality of modified central memory T cells (T _CM ) comprising:
(A) introducing a composition comprising an antigen receptor or a therapeutic protein into a plurality of primary human T cells to produce a plurality of modified T cells, wherein the transposon comprises the antigen receptor When,
(B) In order to generate a plurality of activated modified T cells, the plurality of modified T cells, an anti-human CD3 monospecific tetramer antibody complex, an anti-human CD28 monospecific Contacting a T cell activator composition comprising one or more of a tetrameric antibody complex and an activation supplement,
At least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T cells is one of central memory T cells (T _CM ) Or express multiple cell surface markers,
A method whereby modified central memory T cells (T _CM ) are produced. At least 60% of said plurality of activated modified -T cells express one or more cell surface markers of central memory T cells (T _CM), The method of claim 120.   122. The method of any one of claims 116 to 121, wherein the T cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. (C) To produce a plurality of augmented modified T cells, the activated modified T cells are treated with human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmented supplement Contacting with a T cell expansion composition comprising one or more of:
At least 2% of the plurality of augmented modified T cells express one or more cell surface markers of stem memory T cells (T _SCM );
119. A method according to any one of claims 116 to 118 or 122. At least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70 of the plurality of increased modified T cells 124.%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, expresses a cell surface marker of stem memory T cells ( _TSCM ). the method of. _124. The method of claim 123, wherein at least 60% of the plurality of expanded modified T cells express a cell surface marker of stem memory T cells (T _SCM ). (C) To produce a plurality of augmented modified T cells, the activated modified T cells are treated with human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmented supplement Contacting with a T cell expansion composition comprising one or more of:
At least 2% of the plurality of augmented modified T cells express one or more cell surface markers of stem memory T cells (T _SCM );
122. A method according to any one of claims 119 to 122. At least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70 of the plurality of increased modified T cells 127.%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, expresses a cell surface marker of stem memory T cells (T _SCM ). the method of. 127. The method of claim 126, wherein at least 60% of the plurality of expanded modified T cells express a cell surface marker of stem memory T cells (T _SCM ). (D) at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% modified T cells expressing cell surface markers of stem memory T cells (T _SCM ), To make a composition comprising 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween 119. The method of any one of claims 116-118 or 122-128, further comprising enriching a plurality of increased modified T cells. (D) further comprising enriching said plurality of augmented modified T cells to produce a composition comprising at least 60% modified T cells expressing a cell surface marker of stem memory T cells (T _SCM ). 119. A method according to any one of claims 116 to 118 or 122 to 128. (D) at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% modified T cells expressing cell surface markers of stem memory T cells (T _SCM ), To make a composition comprising 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween 129. The method of any one of claims 119 to 128, further comprising enriching a plurality of increased modified T cells. (D) further comprising enriching said plurality of augmented modified T cells to produce a composition comprising at least 60% modified T cells expressing a cell surface marker of stem memory T cells (T _SCM ). 119. A method according to any one of claims 119 to 128. The enriching step comprises isolating modified T cells that express one or more cell surface markers of stem memory T cells (T _SCM ) from the plurality of enriched modified T cells. 129. The method according to 129 or 130. In order for the enrichment step to produce a plurality of increased enriched modified _TSCM , the isolated modified _TSCM is _transformed into human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, 143. The method of claim 133, further comprising contacting a T cell expansion composition comprising Iskov MDM and one or more augmentation supplements. The enriching step comprises isolating modified T cells that express one or more cell surface markers of stem memory T cells (T _SCM ) from the plurality of enriched modified T cells. 131. The method according to 131 or 132. In order for the enrichment step to produce a plurality of increased enriched modified T _SCMs , the isolated modified T _SCM is _transformed into human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov, 138. The method of claim 135, further comprising contacting the T cell expansion composition comprising MDM and one or more of augmentation supplements.   The T cell expansion composition comprises octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzene. 117. Further comprising one or more of a sulfonamide, 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic hydrazide, oleamide, sterol and alkane. 136. The method according to any one of 1 to 136.   138. The method of any one of claims 116-137, wherein the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterol.   The T cell expansion composition comprises octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including endpoints; palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg including endpoints; 0.2 mg including endpoints Linoleic acid at a concentration of / 20-20 mg / kg; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint; and 1 of sterol at a concentration of approximately 0.1 mg / kg to 10 mg / kg including the endpoint 140. The method of claim 138, further comprising one or more.   The T cell expansion composition comprises 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 about 1 mg. 139. The method of claim 138, further comprising one or more of sterols at a concentration of / kg.   The T-cell-enhancing composition comprises octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg including endpoints; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg including endpoints; 0.75 μmol including endpoints Linoleic acid at a concentration of / 75 to 75 μmol / kg; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg including the endpoint; one of the sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint 142. The method of claim 138, further comprising: or a plurality.   The T cell expansion composition comprises 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, olein at a concentration of about 7.5 μmol / kg. 138. The method of claim 138, further comprising one or more of an acid and a sterol at a concentration of about 2.5 [mu] mol / kg.   In order to produce a modified T cell capable of expressing a therapeutic protein, the step of (c) introducing a composition comprising a second transposon comprising a sequence encoding the therapeutic protein into the primary human T cell. 143. The method according to any one of claims 116 to 142, further comprising:   145. The method of claim 143, wherein the therapeutic protein is a secreted protein or a secreted protein.   145. The method of claim 143 or 144, wherein the sequence encoding the therapeutic protein is a nucleic acid sequence.   145. The method of claim 143 or 144, wherein the sequence encoding the therapeutic protein is a DNA sequence.   147. The method of any one of claims 143 to 146, wherein the transposase composition of (b) moves the transposon of (a) and the second transposon of (c). Further comprising the step of (d) introducing a second transposase composition comprising a transposase or a sequence encoding said transposase into said primary human T cells,
The second transposase of (d) can transfer the transposon of (c);
The second transposase composition of (d) and the transposase composition of (b) are not identical;
148. A method according to any one of claims 143 to 147.   149. The method of any one of claims 1 to 148, wherein the introducing step further comprises a composition comprising a genome editing construct.   149. The genome editing construct comprises guide RNA and clustered regularly interspersed short palindromic repeats (CRISPR) -related protein 9 (Cas9) DNA endonuclease. the method of.   156. The method of claim 150, wherein the genome editing construct comprises a DNA binding domain and a type IIS endonuclease.   153. The method of claim 151, wherein the genome editing construct encodes a fusion protein.   152. The genome editing construct encodes the DNA binding domain and the type IIS endonuclease, wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked. The method described.   154. The method of any one of claims 149 to 153, wherein the genome editing construct comprises a sequence derived from a Cas9 endonuclease.   157. The method of claim 154, wherein said sequence derived from Cas9 endonuclease is a DNA binding domain.   165. The method of claim 154 or 155, wherein said sequence derived from Cas9 endonuclease encodes inactive Cas9.   157. The method of claim 156, wherein said sequence derived from Cas9 endonuclease comprises an amino acid substitution (H840A) of alanine (A) with histidine (H) at position 840.   158. The method of any one of claims 154 to 157, wherein the sequence derived from a Cas9 endonuclease encodes a truncated Cas9.   159. The method of claim 158, wherein said sequence derived from Cas9 endonuclease comprises an amino acid substitution (N580A) of alanine (A) with asparagine (N) at position 580.   160. The method according to any one of claims 154 to 159, wherein said sequence derived from Cas9 endonuclease comprises an amino acid substitution (D10A) of alanine (A) with aspartic acid (D) at position 10.   154. The method of any one of claims 149 to 153, wherein the genome editing construct comprises a sequence derived from a transcriptional activator-like effector nuclease (TALEN).   164. The method of claim 161, wherein the sequence derived from TALEN is a DNA binding domain.   149. The method of claim 149, wherein the genome editing construct comprises TALEN.   154. The method of any one of claims 149 to 153, wherein the genome editing construct comprises a sequence derived from zinc finger nuclease (ZFN).   166. The method of claim 164, wherein the sequence derived from ZFN is a DNA binding domain.   149. The method of claim 149, wherein the genome editing construct comprises a zinc finger nuclease (ZFN).   154. The method of any one of claims 149 to 153, further comprising introducing into the primary human T cells a composition comprising a sequence encoding a therapeutic protein.   166. The method of claim 167, wherein the therapeutic protein is a secreted protein or a secreted protein.   169. The method of claim 168, wherein the therapeutic protein is an intracellular protein.   169. The method of claim 168, wherein the therapeutic protein is a cytoplasmic protein.   169. The method of claim 168, wherein the therapeutic protein is a membrane bound protein.   169. The method of claim 168, wherein the therapeutic protein is a transmembrane protein. The cell surface markers of the modified _{T SCM} comprises CD62L and CD45RA, method according to any one of claims 1 to 172. Cell surface marker of said modified _{T SCM} is, CD62L, CD45RA, CD28, CCR7 , CD127, CD45RO, CD95, CD95 and one or more of IL-2R beta, according to any one of claims 1 to 172 the method of. Cell surface marker of said modified _{T SCM} is, CD45RA, CD95, IL-2Rβ , CR7, and CD62L includes one or more of The method according to any one of claims 1 to 172. The cell surface markers of the modified _{T CM} is, CD45RO, CD95, IL-2Rβ , including one or more of CCR7 and CD62L, method according to any one of claims 1 to 172. 179. The plurality of augmented modified T cells comprises naive T cells (modified _TN ), and the cell surface marker of CAR- _TN comprises one or more of CD45RA, CCR7 and CD62L. The method according to any one of the above. The includes a plurality of increased modified T cells Central memory T cells (modified _{T CM),} cell surface markers of _{CAR-T CM,} including CD45RO, CD95, IL-2Rβ, CCR7, and one or more of CD62L 177. A method according to any one of claims 96 to 176. Wherein the plurality of increased modified T cells include effector memory T cells (modified _{T EM),} cell surface markers of _{CAR-T EM, CD45RO, CD95} , and includes one or more of IL-2R beta, claim 96 176. A method according to any one of 176 to 176. 97. The plurality of augmented modified T cells comprises effector T cells (modified T _EFF ), and the cell surface marker for CAR-T _EFF comprises one or more of CD45RA, CD95, and IL-2Rβ. 176. The method according to any one of up to 176.   40. Any one of claims 1-39 or 116-180, wherein the transposon is a plasmid DNA transposon having a sequence encoding the antigen receptor or therapeutic protein adjacent to two cis-regulatory insulator elements. The method according to item.   184. The method of claim 181, wherein the introducing step further comprises a composition comprising an mRNA sequence encoding a transposase.   183. The method of claim 181 or 182 wherein the transposon is a piggyBac transposon.   184. The method according to any one of claims 181 to 183, wherein the transposase is a piggyBac transposase.   185. The method of claim 184, wherein the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 4.   186. The piggyBac transposase is an overactive variant, wherein the overactive variant comprises amino acid substitutions at one or more of positions 30, 165, 282, and 538 of SEQ ID NO: 4. Method.   187. The method of 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).   187. The method of 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).   187. The method of claim 186, wherein the amino acid substitution at position 282 of SEQ ID NO: 4 is a substitution of valine (V) with methionine (M) (M282V).   187. The method of 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).   191. The method according to any one of claims 183 to 190, wherein the transposase is a Super piggyBac (SPB) transposase.   191. The method of claim 191, wherein the Super piggyBac (SPB) transposase comprises an amino acid sequence comprising SEQ ID NO: 5.   The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is a Sleeping Beauty transposon.   196. The method of claim 193, wherein the transposase is a Sleeping Beauty transposase or an overactive Sleeping Beauty transposase (SB100X).   The method according to any one of claims 1-39 or 116-180, wherein the transposon is a Helraiser transposon.   196. The method of claim 195, wherein the transposase is a Helitron transposase.   The method according to any one of claims 1-39 or 116-180, wherein the transposon is a Tol2 transposon.   199. The method of claim 197, wherein the transposase is a Tol2 transposase.   198. The method of any one of claims 1-39 or 116-198, wherein the sequence encoding the transposase is an mRNA sequence.   The method according to any one of claims 1 to 39 or 116 to 180, wherein the transposon is derived from or recombined from any species.   The method according to any one of claims 1-39 or 116-180, wherein the transposon is a synthetic transposon.   140. The method of any one of claims 1-39 or 116-180, wherein the transposon further comprises a selection gene.   203. The method of claim 202, wherein the T cell expansion composition further comprises a selection agent.   204. The method according to any one of claims 1 to 203, wherein the antigen receptor is a T cell receptor.   205. The method of claim 204, wherein the T cell receptor is naturally occurring.   205. The method of claim 204, wherein the T cell receptor is not naturally occurring.   207. The method of claim 206, wherein the T cell receptor comprises one or more mutations compared to a wild type T cell receptor.   207. The method of claim 206 or 207, wherein the T cell receptor is a recombinant T cell receptor.   204. The method according to any one of claims 1 to 203, wherein the antigen receptor is a chimeric antigen receptor (CAR).   212. The method of claim 209, wherein the CAR comprises one or more sentin sequences.   213. The method of claim 210, wherein the CAR is CARTYrin.   209. The method of claim 209, wherein the CAR comprises one or more VHH sequences.   223. The method of claim 212, wherein the CAR is a VCAR.   220. The method of any one of claims 1-39 and 116-213, wherein the introducing step comprises electroporation or nucleofection. The introducing step comprises nucleofection, wherein the nucleofection comprises:
(A) contacting a transposon composition, a transposase composition, and a composition comprising a plurality of primary human T cells in a cuvette;
(B) applying one or more electrical pulses to the cuvette;
(C) T cell expansion comprising a composition comprising said plurality of primary human T cells comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iskov MDM, and augmentation supplement The method of any one of claims 1-39 and 116-213, comprising incubating at 37 ° C in a composition comprising the composition.   218. The method of claim 215, wherein the transposon is a first transposon or a second transposon.   219. The method of claim 215 or 216, wherein the transposase composition is a first transposase composition or a second transposase composition.   218. To obtain 1 μg of transposon, the transposon composition is a 0.5 μg / μl solution containing nuclease-free water, and the cuvette contains 2 μl of the transposon composition. The method according to one item.   219. The method of claim 218, wherein the transposon composition comprises a piggyBac transposon.   219. The method of claim 219, wherein the transposon composition comprises a Sleeping Beauty transposon.   223. The method of claim 219 or 220, wherein the transposase composition comprises 5 [mu] g transposase.   223. The method of claim 221, wherein the transposase composition comprises a Super piggyBac (SPB) transposase.   223. The method of claim 221, wherein the transposase composition comprises an overactive Sleeping Beauty (SB100X) transposase.   219. The method of claim 219, wherein the transposon comprises a Helraiser transposon.   224. The method of claim 224, wherein the transposase composition comprises a Helitron transposase.   219. The method of claim 219, wherein the transposon comprises a Tol2 transposon.   227. The method of claim 226, wherein the transposase composition comprises Tol2 transposase.   228. The composition of any one of claims 215 to 227, wherein the composition comprising 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. The method described in 1.   229. The method of claim 228, wherein the buffer maintains or enhances the cell viability level and / or stem-like phenotype of the primary human T cells prior to the nucleofection.   229. The method of claim 228, wherein the buffer maintains or enhances the cell viability level and / or stem-like phenotype of the primary human T cells during the nucleofection.   229. The method of claim 228, wherein the buffer maintains or enhances the level of cell viability and / or stem-like phenotype of the primary human T cells after the nucleofection.   234. The method according to any one of claims 228 to 231 wherein the buffer comprises a P3 primary cell solution. It said buffer, _KCl, MgCl 2, ClNa, one or more of glucose and Ca _{(NO 3) 2} containing in any absolute or relative abundance or concentration, either of claims 228 to 231 one The method according to item.   229. The method of claim 228, wherein the buffer further comprises a replenisher selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. It said buffer, KCl of 5 mM, 15 mM of MgCl _2, 90 mM of ClNa, Ca _{(NO 3)} of 10mM glucose and 0.4mM containing ₂ The method of claim 228 or 229.   236. The method of claim 235, wherein the buffer further comprises a supplement comprising 20 mM HEPES and 75 mM Tris / HCl. Said buffer, _Na of _{40mM 2 HPO 4 / NaH 2 PO} 4, further comprising a supplement containing pH 7.2, The method of claim 236.   241. The method of claim 215 or 228-237, wherein the composition comprising primary human T cells is depleted of cells expressing CD14, CD56, and / or CD19. 239. The method of any one of claims 215 to 238, wherein the composition comprising primary human T cells comprises 100 [mu] l of the buffer and 5 x 10 < ⁶ > to 25 x 10 < ⁶ > cells.   240. The method according to any one of claims 215 to 239, wherein the method is performed simultaneously in one or more cuvettes.   245. The method of any one of claims 215 to 240, wherein the incubating comprises incubating the composition comprising the plurality of primary human T cells in a pre-warmed T cell expansion composition. Method.   242. The method according to any one of claims 215 to 241 wherein the duration of the incubation step is 2 days.   243. A method according to any one of claims 40 to 242, wherein the activation supplement comprises one or more cytokines.   244. The method of claim 243, wherein the one or more cytokines comprises IL-2.   253. The method of any one of claims 96 to 244, wherein the augmentation supplement comprises one or more cytokines.   254. The method of claim 245, wherein the one or more cytokines comprise IL-2. Modifying further comprises a T _SCM cell or introducing a composition into the modified T _CM cells containing genomic editing construct, method according to any one of claims 1 to 246.   247. The genomic editing construct of claim 247, comprising guide RNA and clustered regularly interspersed short palindromic repeats (CRISPR) -related protein 9 (Cas9) DNA endonuclease. the method of.   249. The method of claim 248, wherein the genome editing construct comprises a DNA binding domain and a type IIS endonuclease.   249. The method of claim 249, wherein the genome editing construct encodes a fusion protein.   249. The genome editing construct encodes the DNA binding domain and the type IIS endonuclease, wherein the expressed DNA binding domain and the expressed type IIS endonuclease are non-covalently linked. The method described.   254. The method of any one of claims 247-251, wherein the genome editing construct comprises a sequence derived from a Cas9 endonuclease.   253. The method of claim 252, wherein the sequence derived from Cas9 endonuclease is a DNA binding domain.   254. The method of claim 253, wherein the sequence derived from Cas9 endonuclease comprises an amino acid substitution (H840A) of alanine (A) with 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 truncated Cas9.   259. The method of claim 255, wherein said sequence derived from Cas9 endonuclease comprises an amino acid substitution (N580A) of alanine (A) with asparagine (N) at position 580.   259. The method of any one of claims 252 to 256, wherein the sequence derived from Cas9 endonuclease comprises an amino acid substitution (D10A) of alanine (A) with aspartic acid (D) at position 10.   254. The method of any one of claims 247 to 251 wherein the genome editing construct comprises a sequence derived from a transcriptional activator-like effector nuclease (TALEN).   259. The method of claim 258, wherein the sequence derived from TALEN is a DNA binding domain.   248. The method of claim 247, wherein the genome editing construct comprises TALEN.   254. The method of any one of claims 247-251, wherein the genome editing construct comprises a sequence derived from zinc finger nuclease (ZFN).   268. The method of claim 261, wherein the sequence derived from ZFN is a DNA binding domain.   248. The method of claim 247, wherein the genome editing construct comprises a zinc finger nuclease (ZFN).   268. The method of any one of claims 1-263, wherein the primary human T cells express one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. . The primary human T cells is a naive T cells _{(T N),} the _{T N} is CD45RA, express CCR7 and one or more of CD62L, according to any one of claims 1 to 263 Method. The primary human T cell is a T memory stem cell (T _SCM ), wherein the T _SCM expresses one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L. 264. A method according to any one of items 1 to 263. The primary human T cells is a central memory T cells _{(T CM),} wherein _{T CM} is, CD45RO, CD95, IL-2Rβ , CCR7, and expressing one or more of CD62L, Claims 1 to 263 The method as described in any one of. The primary human T cells is a effector memory T cells _{(T EM),} wherein _{T EM} is CD45RO, CD95, and express one or more of IL-2R beta, any of claims 1 to 263 one The method according to item. 268. The one of claims 1 to 263, wherein the primary human T cell is an effector T cell (T _EFF ) and the T _EFF expresses one or more of CD45RA, CD95, and IL-2Rβ. The method described in 1.   275. The method according to any one of claims 1 to 269, wherein the primary human T cells express CD4 and / or CD8. 270. A composition comprising a modified _TSCM made by the method of any one of claims _1-270 . Composition comprising a modified T _CM produced by the method according to any one of claims 1 to 270.   The use of a composition according to claim 271 or 272 for the manufacture of a medicament for treating a subject in need thereof. Said modified _{T SCM} or modified _{T CM} is autologous use according to claim 273. It said modified _{T SCM} or modified _{T CM} are allogenic Use according to claim 274.   275. Use according to any one of claims 273 to 275, wherein the antigen receptor is a T cell receptor.   276. Use according to claim 276, wherein the T cell receptor is naturally occurring.   276. Use according to claim 276, wherein the T cell receptor is not naturally occurring.   294. The use of claim 278, wherein the T cell receptor comprises one or more mutations compared to a wild type T cell receptor.   279. Use according to claim 278 or 279, wherein the T cell receptor is a recombinant T cell receptor.   276. Use according to any one of claims 273 to 275, wherein the antigen receptor is a chimeric antigen receptor (CAR).   289. The use of claim 281, wherein the CAR comprises one or more sentin sequences.   283. Use according to claim 282, wherein the CAR is CARTYrin.   284. The method of claim 283, wherein the CAR comprises one or more VHH sequences.   285. The method of claim 284, wherein the CAR is a VCAR.   275. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of claim 271 or 272. It said modified _{T SCM} or modified _{T CM} is autologous A method according to claim 286. It said modified _{T SCM} or modified _{T CM} is allogeneic The method of claim 286.   290. The method of any one of claims 286 to 288, wherein the antigen receptor is a T cell receptor.   290. The method of claim 289, wherein the T cell receptor is naturally occurring.   290. The method of claim 289, wherein the T cell receptor is not naturally occurring.   292. The method of claim 291, wherein the T cell receptor comprises one or more mutations compared to a wild type T cell receptor.   293. The method of claim 291 or 292, wherein the T cell receptor is a recombinant T cell receptor.   290. The method according to any one of claims 286 to 288, wherein the antigen receptor is a chimeric antigen receptor (CAR).   295. The method of claim 294, wherein the CAR comprises one or more sentin sequences.   294. The method of claim 295, wherein the CAR is CARTYrin.   295. The method of claim 294, wherein the CAR comprises one or more VHH sequences.   297. The method of claim 297, wherein the CAR is a VCAR.   298. The method of any one of claims 286 to 298, wherein the disease or disorder is cancer and the antigen receptor specifically targets a cancer antigen.   298. 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, bacterial, yeast or microbial antigen. The method described in 1.   The disease or disorder is a disease or disorder characterized by a lack of secreted protein activity or a low abundance, or the disease or disorder reduces the activity or abundance of the secreted protein. 298. The method of any one of claims 286 to 298, wherein the disease or disorder is treated by increasing.   302. The method of claim 301, wherein the secreted protein comprises a coagulation factor VIII protein or a coagulation factor IX protein.   331. The method of claim 301 or 302, wherein the abundance of the secreted protein is determined at a local site. The local site is accessible by the modified _{T SCM} cells or modified _{T CM} cells, The method of claim 303.